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Is casein hydrolysate the same thing as casamino acids?

Is casein hydrolysate the same thing as casamino acids?


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Probably a dumb question, but these seem identical. If a protocol calls for one, can I use the other instead?


Roughly, yes they are similar, but there are processing differences- the real difference is filtering. A product containing simply casein hydrosylate will only be roughly filtered with lots of peptides (like body builder products). Whereas casamino acids should be primarily clean/free amino acids (like nutrients for bacteria).

The actual manufacturing methods will be proprietary for the manufacturer… but you can trust that they are basically doing whatever works best to obtain the individual amino acids and/or whatever is cheapest for obtaining a useable product.

I have used bodybuiling and several other grades of casein products for growing bacteria. I have found that although various products may get the job done there are some serious drawbacks to casein products not intended for growing bacteria.

Differences include clarity of media, cleaning of glassware/fermenter/bioreactor, sudzing of aerated media, and the microorganism. Some microorganisms won't grow very well, and generally, those that can may still not grow quite as well or as fast… so that extra 10% or 20% cfus may be important (or maybe not since the price for quality may be 10 times higher).

The quality-features vs price must be weighed by the grower. Personally, I sometimes autoclave a cheap casein product in one (easy to clean) vessel then rag-filter it into another (then autoclave it again) for growing bacteria… but that's because I have about a thousand pounds of rough casein hydrosylate, and only a kg or so of casamino acids (for particular studies). This method does not eliminate all of the problems but it does reduce some of them.


Isolation and screening of actinomycetes producing antimicrobial substances from an extreme Moroccan biotope

1 Team of Biotechnology and Environment, Natural Resources and Environment Laboratory, Department of Biology Chemistry Geology, Polydisciplinary Faculty of Taza, Sidi Mohamed Ben Abdellah University, Fez 30050, Morocco

Soumia Ait Assou

2 Team of Microbial Biotechnology, Biotechnology Laboratory, Faculty of Sciences Dhar El Mahraz, Department of Biology, Sidi Mohamed Ben Abdellah University, Fez 30050, Morocco

Mohammed El Hassouni

2 Team of Microbial Biotechnology, Biotechnology Laboratory, Faculty of Sciences Dhar El Mahraz, Department of Biology, Sidi Mohamed Ben Abdellah University, Fez 30050, Morocco


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This list of vaccine ingredients comes from the CDC. This is what people mean when they say do your own research.
CDC document bombshell reveals list of all vaccine excipients, including “African Green Monkey Kidney Cells” and fibroblast cells from aborted human fetuses … see the complete list
Monday, March 06, 2017 by: Mike Adams
Tags: African Monkey Kidney Cells, CDC, excipients, Fetal cells, ingredients, toxins, vaccines
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Image: CDC document bombshell reveals list of all vaccine excipients, including “African Green Monkey Kidney Cells” and fibroblast cells from aborted human fetuses … see the complete list

(Natural News) Almost no one has any real idea what’s found in vaccines. When they allow themselves to be injected with vaccines, they’re oblivious to the fact that they are being injected with aborted human fetus cell lines or African Green Monkey kidney pus cells harvested from infected, disease primates. (See proof from the CDC, below.)

Yet, astonishingly, the CDC openly admits to all this (and more). In a PDF posted on the CDC website entitled “Vaccine Excipient & Media Summary,” the CDC lists all the excipients currently used in vaccines being injected into adults and children across the United States. The CDC’s list, current as of January 6, 2017, was “extracted from manufacturers’ package inserts,” according to the CDC.

The complete list is found in this CDC document (PDF). In case the CDC removes it — because they’ve been known to suddenly “memory hole” documents they don’t want the public to see — we’ve also posted a copy at the Natural News servers (PDF).

The WI-38 cell line is widely known to be “derived from lung tissue of an aborted white (caucasian) female fetus,” as even the pro-vaccine Wikipedia website admits. As the Coriell Institute for Medical Research explains about the MRC-5 cell line / WI-38:

The MRC-5 cell line was developed in September 1966 from lung tissue taken from a 14 week fetus aborted for psychiatric reason from a 27 year old physically healthy woman. The cell morphology is fibroblast-like. The karyotype is 46,XY normal diploid male. Cumulative population doublings to senescence is 42-48. G6PD isoenzyme is type B.

The human fetal tissue cells have become such an issue of outrage that even the Vatican has issued a statement concerning their use, in which they address, “vaccines containing live viruses which have been prepared from human cell lines of fetal origin, using tissues from aborted human fetuses as a source of such cells.” You can find the Vatican’s response at this link, in which they discuss the moral and ethical issues of “The principle of licit cooperation in evil.”

Below, you’ll find the complete list published by the CDC, de-duplicated and sorted alphabetically. Notice that these ingredients include toxic metals (aluminum salts), bizarre animal cells from humans, monkeys, cows, pigs and chickens, ingredients derived from GMOs, the radioactive element barium, artificial coloring chemicals, excitotoxins such as glutamate, chemical cleansing agents (Triton X-100), dangerous bacterial strains (E.coli), toxic chemicals such as glutaraldehyde, thimerosal (mercury) and much more.

No one can refute any of this because it’s admitted by the CDC itself.

More analysis of the toxicity of these ingredients will be published at Vaccines.news and Natural News.

Here’s what happens to some children when they’re injected with these toxins:

The complete list of vaccine excipients published by the CDC, current as of January 6, 2017


Selective Utilization of Exogenous Amino Acids by Dehalococcoides ethenogenes Strain 195 and Its Effects on Growth and Dechlorination Activity

Fig. 1 . Mass fraction of unlabeled proteinogenic amino acids (M0 fraction) of D. ethenogenes strain 195 (▩) and E. coli K-12 MG1655 (□).

Effects of amino acids on physiological traits of D. ethenogenes strain 195.

Fig. 2 . Dechlorination profiles by D. ethenogenes strain 195 amended with 0 (AA0), 1 (AA1), 4 (AA4), or 20 (AA20) amino acids after five subcultures. The data shown are the mean values from triplicate biotic experiments, with error bars representing one standard deviation. Fig. 3 . Growth curves of D. ethenogenes strain 195 amended with 0 (AA0), 1 (AA1), 4 (AA4), or 20 (AA20) amino acids after five subcultures. The data shown are the mean values from triplicate biotic experiments, with error bars representing one standard deviation.

Bypass of genetic constraints during mutator evolution to antibiotic resistance

Genetic constraints can block many mutational pathways to optimal genotypes in real fitness landscapes, yet the extent to which this can limit evolution remains to be determined. Interestingly, mutator bacteria elevate only specific types of mutations, and therefore could be very sensitive to genetic constraints. Testing this possibility is not only clinically relevant, but can also inform about the general impact of genetic constraints in adaptation. Here, we evolved 576 populations of two mutator and one wild-type Escherichia coli to doubling concentrations of the antibiotic cefotaxime. All strains carried TEM-1, a β-lactamase enzyme well known by its low availability of mutational pathways. Crucially, one of the mutators does not elevate any of the relevant first-step mutations known to improve cefatoximase activity. Despite this, both mutators displayed a similar ability to evolve more than 1000-fold resistance. Initial adaptation proceeded in parallel through general multi-drug resistance mechanisms. High-level resistance, in contrast, was achieved through divergent paths with the a priori inferior mutator exploiting alternative mutational pathways in PBP3, the target of the antibiotic. These results have implications for mutator management in clinical infections and, more generally, illustrate that limits to natural selection in real organisms are alleviated by the existence of multiple loci contributing to fitness.

1. Introduction

A striking insight gained over the past decade of research is that epistatic interactions are common in experimental fitness landscapes [1]. Of particular interest is the observation of a special type of epistasis termed ‘sign epistasis’ [2], according to which mutations are beneficial or deleterious depending on the presence or absence of others [3–8]. Under sign epistasis, neighbouring mutation combinations display contrasting fitness values, introducing ‘ruggedness’ in the surface of the fitness landscape. This feature can have at least two important evolutionary consequences. First, it generates mutational pathways whose intermediate steps are no longer arranged in ascending order. Therefore, the number of pathways accessible by natural selection decreases [2] and successful adaptation becomes contingent upon the identity of first-step mutations [7]. A second, more drastic consequence is the emergence of multiple fitness peaks, which requires a particularly extreme form of epistasis known as reciprocal sign epistasis [9]. Overall, these observations led to the conjecture that natural selection may be limited by pervasive genetic constraints, contributing to explain the surprising degree of repeatability observed in many microbial evolution experiments [10,11].

One case presumably well suited to investigate the influence of genetic constraints on adaptation is that of bacterial mutators. Bacteria with a high mutation rate are frequently isolated in clinical settings [12,13] and in laboratory evolution experiments [14,15]. Their emergence is an eventual consequence of the genetic structure of asexuals, which allows mutator alleles to hitchhike with beneficial mutations occurring in the same genome [16,17]. Mutators pose a serious concern in clinical infections because they can readily evolve further adaptations, such as those promoting evasion of the immune system [18], increasing resistance to antibiotics [19] or alleviating the resistance fitness cost [20]. Different mutators, however, display a characteristic tendency to elevate only some types of transitions, transversions or frameshifts. This is because mutator phenotypes arise from alterations in specific mutation-avoidance mechanisms, whose malfunctioning permits the accumulation of particular mutational classes [21].

Here, we hypothesized that such mutational idiosyncrasy could render mutators particularly sensitive to genetic constraints. Each mutator genotype is expected to heighten the occurrence rate of only an arbitrary subset of beneficial first-step mutations [22]. If such a subset does not include the best possible mutations, mutator populations could be compelled to follow suboptimal paths—either leading to the same or a different fitness peak. Furthermore, in cases where the initial steps were drastically limited, mutators failing to raise the relevant first-step mutations may not be more able to adapt than their wild-type counterpart. Such stringent circumstances could happen when adaptation requires very specific multiple amino acid substitutions, as is observed in some cases of antibiotic resistance [8,23].

To study these possibilities, we characterized the evolution of wild-type and mutator strains of Escherichia coli towards increased resistance to the third-generation β-lactam antibiotic cefotaxime (CTX). Our experimental design sought to track mutations in the gene coding for the β-lactamase enzyme TEM-1 (blaTEM-1). The original allele, well adapted to hydrolyse early β-lactams, diversified naturally through the accumulation of point mutations into hundreds of variants with greater activity against later compounds of this family [24]. This natural evolution has been extensively emulated in a variety of laboratory experiments, yielding TEM-1 as one of the best-characterized models for the study of molecular evolution [7,25–27].

In the specific case of mutations conferring CTX resistance, there are several dozens of substitutions known to increase the resistance phenotype [26]. Despite the potentially huge number of combinations among these, only a small fraction are observed repeatedly both in nature and in laboratory evolution experiments—a signature of epistatic constraints [7,24]. Interestingly, we realized that the most frequent CTX resistance substitutions observed in clinical isolates arise from G : C → A : T transitions, one of the two dominant nucleotide changes in the spectrum of mismatch repair-deficient strains, the most prevalent type of strong bacterial mutators [28]. These substitutions include the top-ranked G238S and E104K, which appear combined in many notorious naturally occurring TEM alleles, and substitutions R164H and A237T [24,26]. In addition, the other transition characteristic of these mutators (A : T → G : C) also generates clinically relevant substitutions, such as M182T, H153R and I173V [24,26]. We next examined the case of MutT-deficient mutators, the second most prevalent type of strong mutator bacteria [28]. In sharp contrast, the elevation of A : T → C : G transversions peculiar to this background does not match any of the common CTX resistance mutations found in natural isolates [26]. Substitutions of intermediate to low effect, nonetheless, have been described (S268R, E240A, E104A and F72V), yet they have exclusively been observed in laboratory settings [26].

We reasoned that CTX resistance evolution could be a suitable model to examine the extent to which limited availability of mutational pathways can hinder the evolution of bacterial mutators. Studying this possibility is doubly relevant not only because of the clinical importance of mutators, but because it can also inform about the more general question of the impact of genetic constraints in evolution. We thus serially propagated 192 independent populations of a ΔmutT mutator, a mismatch repair-deficient ΔmutH mutator and wild-type E. coli in the presence of periodically increasing concentrations of CTX. Crucially, we expected that the performance of the ΔmutT mutator would depend on its ability to exploit rare mutational pathways compatible with its narrow mutational spectrum. We also conjectured that any spectrum-dependent differences would be levelled out by a general increase in the supply of beneficial mutations. The rationale for this is that such an increase will favour that the best mutations are available for selection, irrespective of the genetic background. To test this, in the experimental set-up, half of the populations for each strain carry a single chromosomal copy of blaTEM-1, whereas the remaining half contain approximately 18 plasmidic copies of this allele [29]. Overall, we established six experimental settings (three mutational spectra with two gene dosages), for a total of 576 experimental populations.

2. Results

(a) Dynamics of adaptation to increasing antibiotic concentrations

To test whether different mutational spectra confer disparate ability to evolve high-level, multi-step CTX resistance, we serially passaged mutator and wild-type bacteria to doubling concentrations of the drug starting from 1/4 to 1024 times the minimal inhibitory concentration (MIC) of the ancestral strain. In general terms, the results show that an increase in mutational supply, either through a mutator phenotype or by multiple plasmidic copies of blaTEM-1, translates into a greater fraction of replicates surviving at the end of the experiment (figure 1). More quantitatively, we found a strong linear correlation between the final number of survivors and the estimated product of mutation rate and blaTEM-1 copy number (Pearson's r = 0.87, p = 0.026 electronic supplementary material, figure S1). This indicates that the ΔmutT genotype, despite not elevating mutagenesis to the typical CTX resistance blaTEM-1 mutations, is not broadly hindered to evolve high levels of resistance.

Figure 1. Experimental evolution of wild-type and mutator E. coli to CTX resistance. The figure shows the number of surviving lineages against antibiotic concentration, which was doubled every 2 days. The different curves correspond to wild-type (black squares), ΔmutH (red circles) and ΔmutT (blue triangles) lineages carrying either one (a) or multiple (b) copies of the gene coding for the β-lactamase TEM-1. Overall, an increase either in mutation rate or in blaTEM-1 copy number translates into a higher number of final survivors. Note that the anticipated advantage of the ΔmutH over the ΔmutT mutator was not realized, despite being the only one elevating mutagenesis to the most-beneficial known substitutions in TEM-1 (see text for details).

In the blaTEM-1 single-copy setting, the anticipated advantage of the ΔmutH over the ΔmutT genotype was not observed (figure 1a). Indeed, the final number of survivors was greater in the ΔmutT than in the ΔmutH background (9/96 versus 3/96), although the values are not statistically different (Fisher's exact test, p = 0.1332). It is worth noting that 18 ΔmutH populations went prematurely extinct at 2 mg l −1 of CTX, a significant drop in the survival curve (log-rank test, p < 0.001) which may reflect an early evolutionary dead-end associated with its mutational spectrum. Regarding the blaTEM-1 multiple-copy setting, we found no discrepancies between the two mutator backgrounds (figure 1b), neither in terms of final number of survivors (Fisher's exact test, p = 0.5354) nor in survival curves (log-rank test, p = 0.808).

As pointed out in the Introduction, the most common blaTEM-1 CTX resistance substitutions arise mainly from transitions, which are only elevated in the ΔmutH genotype [21]. Taking this into account, it is revealing to compare the performance of the plasmid-harbouring wild-type (figure 1b) with that of both mutator populations carrying a single-copy blaTEM-1 (figure 1a). An increased copy number confers a capacity to reach high resistance similar to hypermutability (7/96 versus 9/96 and 3/96 final survivors Fisher's exact test, p > 0.3), although the wild-type's survival curve is notably unique (log-rank test, p < 0.0001). This suggests that the evolutionary paths followed by the different strains are not simply the result of accumulating the well-known blaTEM-1 CTX resistance substitutions. If that were the case, the wild-type would be expected to perform better than the ΔmutT strain, because the latter is effectively non-mutator for these typical substitutions whereas the former should produce them at an approximately 18-fold higher rate [29]. In addition, the ΔmutH genotype should enjoy the best outcome, because it elevates approximately 100- to 300-fold the required transitions [30,31].

At least two non-mutually exclusive possibilities might explain these results. First, both ΔmutT and the wild-type strains could be substituting non-canonical blaTEM-1 CTX resistance mutations. The wild-type may thus profit from the fact that an increase in copy number elevates not only the two transitions, but also all the six possible base substitutions. Although the effect on each mutation type is moderate, the overall elevation in mutational supply becomes comparable to that of the ΔmutH background. Additionally, the greater allelic diversity generated may be fuelling evolution through homologous interplasmid recombination [32] (although the electronic supplementary material, figure S1 gives no clear support for this hypothesis). In turn, the ΔmutT strain elevates only one type of base substitution (A : T → C : G transversions), but it does so by approximately 500- to 10 000-fold [30,33] a huge increment that may allow this genotype to accumulate rare blaTEM-1 CTX resistance substitutions.

A second possibility is that other chromosomal loci played a substantial role in the acquisition of high-resistance phenotypes. Expanding the mutational target size should, in principle, mitigate spectrum-dependent differences, which could explain the lack of impairment observed in the chromosomally blaTEM-1-encoding ΔmutT genotype. Candidate loci include genes controlling multi-drug resistance determinants, such as efflux pumps and porin channels and those coding for the penicillin-binding proteins, the target of β-lactam antibiotics [34].

(b) Phenotypic characterization of evolved lineages

To gain insights into putative non-β-lactamase-mediated resistance mechanisms, we assembled a collection of 160 clonal isolates and tested them against a panel of chemicals, antibiotics and bacteriophage viruses. The largest part of the collection consisted in endpoint isolates from: (i) lineages that survived at the end of the experiment (n = 50), and (ii) the highest resistant lineages within each setting that went extinct before the end of the experiment (n = 57). To compensate that most mutator populations went extinct very late, the collection was completed with 43 clones from random lineages isolated at low CTX concentrations (less than or equal to 0.5 mg l −1 ). The battery of assays (see Material and methods) was designed to examine the status of AcrAB, the main efflux pump involved in β-lactam export [35] and the outer membrane porins OmpF and OmpC, the primary routes of entry of these drugs [36]. To assess the validity of the tests, the loci acrA, acrB, acrR, envZ, ompR, ompF and ompC were sequenced in two independent strains positive for efflux and porin alterations. Both strains confirmed non-synonymous point mutations in several of these genes: acrB and acrR in both cases, together with envZ and ompC in one case and ompR in the other (see the electronic supplementary material, table S1).

The collection exhibited a high frequency of altered porin phenotypes (79%) and, to a lesser extent, increased efflux phenotypes (43% figure 2)—a combination usually observed in multi-drug-resistant isolates [37]. Intriguingly, we found that 20% of the strains exhibit hypersusceptibility to erythromycin, a well-known substrate of AcrAB [38]. This phenotype could be a by-product of mutations improving the specificity of AcrAB for CTX, at the cost of reducing erythromycin extrusion [39]. Alternatively, it could emerge from mutations in the peptidoglycan synthesizing apparatus (including but not necessarily limited to PBP3, the molecular target of CTX), with pleiotropic effects in the organization of the outer membrane that result in enhanced passive diffusion of hydrophobic agents such as erythromycin [40]. Whatever the mechanism, we reported this phenotype owing to its prevalence, which might well be an underestimation given the high frequency of increased efflux phenotypes observed. Only 11% of the isolates showed no apparent alteration in any of these phenotypes.

Figure 2. Phenotypic profiles of representative evolved clones. Bars represent the frequency of isolates displaying alterations in permeability (black), efflux (dark grey) and erythromycin hypersusceptibility (light grey). White bars correspond to isolates without alterations. (a) Phenotypic profiles of wild-type and mutator strains. Only differences in erythromycin hypersusceptibility were found to be statistically significant (see main text). (b) Phenotypic profiles according to the antibiotic concentration from which the clones were isolated. No statistically significant differences between early and late clones were observed. These results suggest that general multi-drug resistance evolved early and in parallel across the different backgrounds, and therefore is not sufficient to explain the final survival patterns.

No significant profile differences were detected among the three genotypes (Fisher's exact test, p > 0.15 figure 2a). The only exception is erythromycin hypersusceptibility, at least 10 times more frequent in either mutator than in the wild-type background (Fisher's exact test, p = 0.00014) suggesting that this phenotype may be arising only from a few, very specific point mutations. We also found no significant differences among clones isolated from early and late CTX concentrations (less than or equal to 0.5 versus more than or equal to 16 mg l −1 , Fisher's exact test, p > 0.3 figure 2b). Taken together, the results indicate that efflux and permeability adaptations were acquired early and in parallel across the different genotypes, and therefore are not sufficient to explain the final survival patterns.

(c) Genotypic characterization of evolved lineages

Given the evidence provided above, we reasoned that high-level resistance may presumably be the result of either non-canonical blaTEM-1 substitutions or the combination of blaTEM-1 substitutions with alterations in ftsI, the gene coding for the main target of CTX (the transpeptidase PBP3). To explore these possibilities, we sequenced both loci from a subset of 70 strains from the collection of isolates used for the phenotypic assays (see the electronic supplementary material, table S2).

We found 21 out of 70 strains to be positive for non-synonymous blaTEM-1 substitutions, all of which belonged to the multiple-copy setting (n = 44 see the electronic supplementary material, table S2). Loss or insufficiency of expression of the chromosomal bla gene in the single-copy setting (n = 26) was discarded because all the analysed isolates retained inhibitor-susceptible resistance to ampicillin (data not shown). In total, we identified 27 point mutations corresponding to just five amino acid changes (E104K, H153R, R164S, M182T and G238S figure 3a). These substitutions gave rise to five different alleles, including the double-mutant TEM15 and TEM112 alleles, and the triple-mutant TEM52 allele (see the electronic supplementary material, tables S2 and S3). All these substitutions and alleles have already been described both in clinical and experimental studies, and details about them can be read elsewhere [24]. The most frequent amino acid change in our dataset was G238S (20/27), in agreement with its prevalence in previous reports [7,41]. As anticipated, most of the mutations arose from either G : C → A : T or A : T → G : C transitions. The only exception was R164S, arising from a C : G → A : T transversion, detected just in one wild-type isolate. In consequence, it is not surprising that ΔmutH was the background where the greatest portion of substitutions were observed (16/27 figure 3a), and also that exhibited the highest average number of substitutions per sequence (1.25 electronic supplementary material, figure S2).

Figure 3. Substitutions in TEM-1 and PBP3 generated by wild-type and mutator strains. The histogram shows abundance of different amino acid substitutions among 70 isolates of wild-type (black), ΔmutH (red) and ΔmutT (blue) populations. (a) Substitutions in TEM-1. (b) Substitutions in PBP3, grouped by matching of the underlying nucleotide change with the mutational spectrum of ΔmutT (A : T → C : G, left), ΔmutH (G : C → A : T and A : T → G : C, centre) and wild-type (others, right) backgrounds. Note the contrasting levels of molecular idiosyncrasy observed in both proteins, indicating that mutational pathways are more constrained in TEM-1 than in PBP3.

Examination of the ftsI locus revealed 54 out of 70 strains displaying non-synonymous substitutions, with a remarkably high degree of polymorphism. We identified 92 point mutations (34 unique), affecting 31 positions, accounting for a total of 39 different alleles (figure 3b and electronic supplementary material, tables S2 and S3). Some of these variants have been already associated with resistance to β-lactam antibiotics, either in laboratory strains of E. coli (A257V and N361S) [42,43] or in related species such as Salmonella enterica (P311S) [44] and Haemophilus influenzae (L369F) [45]. All the substitutions but one mapped within the transpeptidase module (D237–V577) of the PBP3 protein, clustered in the surroundings of the three catalytic motifs (STVK310, SSN361 and KTG496) [46]. The sole mutation observed in the non-catalytic module (G57-D237), R167C, affected the first position of the conserved motif RYYPSG172. This module is thought to function as an intramolecular chaperone, and thus mutations in it could contribute to resistance by influencing the folding or stability of the whole protein [46]. Mutations in this module, indeed, have been reported in E. coli to confer resistance to ceftazidime, a β-lactam antibiotic structurally very close to CTX [47].

In contrast to what was observed in the bla locus, both mutators accumulated highly idiosyncratic substitutions in ftsI, including double- and triple-mutant alleles where all mutations correspond to each specific mutational spectrum (figure 3 and electronic supplementary material, tables S2 and S3). Regarding to this, only 7 out of 83 substitutions found in both mutators did not arise from spectrum-specific base changes, suggesting that these are mutations with strong beneficial effects. Interestingly, the ΔmutT background was the one where the greatest portion of substitutions were observed (52/92), exhibiting the highest average number of substitutions per isolate (2.08 electronic supplementary material, figure S2) and the highest total number of triple mutant alleles (eight in total, six unique see the electronic supplementary material, tables S2 and S3).

Two lines of evidence indicate that these observed polymorphisms in the loci bla and ftsI are likely to explain high-level resistance patterns. On the one hand, all sequenced strains contained substitutions in at least one of these two genes, with the unsurprising exception of five clones from the wild-type background isolated at low concentrations (less than or equal to 0.5 mg l −1 electronic supplementary material, table S2). On the other hand, although some isolates exhibited alterations in both TEM and PBP3 (12/70), there was a statistically significant tendency to lack changes in PBP3 when TEM substitutions are already present (Fisher's exact test, p < 0.0001). Actually, whenever a triple-mutant allele was observed at either loci, alterations at the other locus were completely absent (electronic supplementary material, table S2).

3. Discussion

This work was motivated by the question of whether limits to natural selection posed by genetic constraints could become particularly apparent in mutator bacteria. To gain insights into this issue, we monitored the evolution of two distinct mutators in an experimental system where only one of them elevates known beneficial mutations. Under such conditions, the performance of the a priori inferior mutator would depend on its ability to exploit secondary mutational pathways, if available. In the extreme case, we hypothesized that its performance could be as limited as that of the wild-type.

We found that both mutators were indistinguishable in terms of their ability to evolve high-level CTX resistance. In view of the above considerations, a reasonable explanation for these results relies on the seemingly large size of the global mutational target for CTX resistance evolution. Such large size provides multiple alternative evolutionary routes and, consequently, increases the likelihood that a particular mutational spectrum raises fitting beneficial mutations. The causes for this large mutational target size are at least twofold. First, adaptation can proceed via changes at loci other than bla, most notably at ftsI. Second, the mutational target within the ftsI locus is apparently very broad in itself. In this respect, it is worth mentioning the high proportion of PBP3 substitutions observed only once (22/34 electronic supplementary material, table S2), which points at the existence of many other possible mutations that just went unsampled [48]. This is consistent with the fact that more than 30 PBP3 substitutions not detected here have been described elsewhere to confer resistance to a variety of β-lactams, including CTX [42–45,49].

A recent study with Pseudomonas aeruginosa reported minor differences between hypermutability and stress-induced mutagenesis in promoting adaptation, a result akin to ours in that both experiments involved different mutational spectra [50]. However, the negligible effect on evolvability observed here is particularly revealing, because our experimental design purposely involved both a narrow-spectrum mutator and a well-characterized model of constrained molecular evolution. A comparison with the literature lead us to believe that this result is probably not peculiar to our specific conditions, but rather it could be fairly general. While our system, in fact, exhibited substantial flexibility in evolutionary trajectories, this flexibility is similar to what is typically observed in many bacterial evolution experiments [51]: hard adaptive constraints are rarely observed at the nucleotide level [52], and even in the examples that reported the highest degree of molecular convergence, adaptation involved more than one loci and featured a considerable amount of intralocus diversity [53–55]. Therefore, given that mutational target sizes seem not strictly limited, we expect spectrum effects on evolvability to be modest at best in most cases.

In a broader evolutionary context, this work contributes to the question to what extent the ruggedness of fitness landscapes can hinder natural selection. Several reports have documented both inter- [4,5,56] and intralocus [27,57,58] sign epistasis in different model systems. These observations promoted the idea that genetic constraints could be prevalent and hence adaptation could proceed through very few mutational paths to optimal genotypes [8,11]. This possibility implies that evolution may be largely repeatable and, perhaps, even predictable [11]. It has been argued, however, that adaptive limitations can be particular to low-dimensional fitness landscapes, which to date have been the only ones amenable to empirical examination [11]. The rationale for this is that, because increasing dimensionality implies that genotypes have more neighbours, what might look like an isolated fitness peak at some scale can actually be connected at higher dimensions [59]. An example along these lines can be found in the case of substitution G238S in TEM-1 β-lactamase. In vitro experiments showed that this substitution is the first-step mutation that confers the highest resistance to CTX [26]. Most interestingly, they revealed that once it becomes fixed, epistatic interactions strongly restrict which other mutations can be substituted next [7]. In contrast to these limitations, our results uncovered the existence of multiple alternative pathways emerging from the combination of G238S with various substitutions in PBP3 (electronic supplementary material, table S2) illustrating how high dimensionality facilitates the bypass of local adaptive constraints in real fitness landscapes.

Apart from evolvability, there are other possible long-term consequences to mutational spectrum differences. Mutational biases forcibly impose a high level of divergence at the molecular level. This idiosyncratic variability, although perhaps equivalent in fitness under the selective conditions, might confer distinct properties in other environments. In our experiment, each mutator exhibited a marked preference for accumulating amino acid changes either in TEM or in PBP3 (figure 4). It is easy to imagine different long-term advantages and disadvantages for both resistance mechanisms. Some of the observed TEM alleles, for instance, are able to hydrolyse β-lactams not primarily targeting PBP3, thus conferring resistance to a broader range of compounds [60]. In addition, given that these enzymes are commonly plasmid-encoded in natural isolates, they could recombine at a higher rate and also be more readily transmissible through horizontal-gene transfer [61]. In turn, being an essential gene, PBP3 alleles are likely to entail fitness costs in other conditions. Such costs have already been reported in some cases, including reduced growth at high temperatures [42] and impaired biofilm formation [62]. Alterations in the cell wall structure, however, could also have an adaptive value: a recent report showed that PBP3 substitutions can increase the ability of H. influenza to invade epithelial cells [63].

Figure 4. Mutational spectrum effects on divergence of mutational pathways. Bars represent number of isolates with different combinations of substitutions in TEM-1 and PBP3. Data from plasmid-carrying lineages surviving at the end of the experiment (MIC more than or equal to 64 mg l −1 ). (a) ΔmutH background. (b) ΔmutT background. To aid visualization, shades of grey are proportional to the number of isolates. Note how the mutational spectrum directs populations through divergent adaptive paths each mutator preferentially accumulating substitutions either in TEM-1 or PBP3.

While these divergent prospects are specific to β-lactam resistance evolution, it seems reasonable to expect similar effects in other systems provided that adaptation can proceed through a sufficiently large mutational target. From an applied perspective, this adds an extra layer of complexity to the management of mutators in clinical infections, already recognized as a risk factor for antibiotic resistance development [64]. Future studies should consider the long-term consequences of molecular divergence when evaluating the risks of antibiotic therapy in the presence of mutators, including aspects such as cross-resistance, virulence, transmissibility or fitness in other environments.

4. Material and methods

(a) Strains and media

All strains are derivative of E. coli AB1157, obtained from the laboratory collection of Dr Bruce R. Levin. This strain naturally carries the gene coding for the β-lactamase AmpC, which was deleted to prevent interference with the experimental system. Knockouts of the genes ampC, mutH and mutT were done by P1 transduction and subsequent pCP20-mediated removal of the kanamycin resistance cassette from the appropriate Keio clone [65,66]. The single-copy blaTEM-1 strains were generated by transducing the galK::cfp/yfp, blaTEM-1 cassette from an E. coli MC4100 kindly provided by Dr Roy Kishony [67]. The multiple-copy blaTEM-1 strains were obtained by transformation with plasmid pBRACI a pBR322 derivative which, in an attempt to reduce the fitness burden of carrying the plasmid [68], was digested with enzymes AvaI and PstII to excise the tetracycline resistance cassette. Note that these strains do not carry the blaTEM-1chromosomal copy. All genotypes were confirmed by PCR and gel electrophoresis. Bacteria were cultured in a modified version of M9 minimal medium, optimized to support maximal growth in the experimental conditions [66]. The modification consists of supplementing M9 salts with 1% glucose (G8270, Sigma-Aldrich) and 1% acid hydrolysate of casein (casamino acids, Becton Dickinson).

(b) Evolution experiment

For each of the six genotypes used here, a total of 96 parallel lineages were serially propagated in the presence of increasing concentrations of CTX (Claforan, Sanofi-Aventis). The populations were grown without agitation at 37°C in 96-well microtiter plates (1.1 ml Deep Well, Axygen), covered with loose-fitting plastic lids to allow aeration. To minimize cross-contamination, populations were arranged in a chessboard pattern across each plate, such that half of the wells contained sterile medium [67]. The fraction of these control wells that got contaminated on a daily basis was below 0.5%. Every 24 h, 16 μl of each culture was transferred into fresh 800 μl of medium, allowing a minimum of approximately 5.7 generations per day (note that this estimate represents a lower limit, owing to drops in final population density along the course of the experiment). Serial passage was conducted during 28 days. The minimal inhibitory concentration of cefotaxime (MIC) in the experimental medium for all parental strains was determined to be 0.064 mg l −1 by the microdilution method (CLSI, 2006), irrespective of plasmid carriage. The first two passages were done in the absence of antibiotic. At day three, the populations were exposed to 0.016 mg l −1 of CTX (1/4 × MIC). The antibiotic concentration was subsequently doubled every 48 h, until a final concentration of 64 mg l −1 CTX (1024 × MIC) was reached. Throughout the course of the experiment, the number of surviving populations was estimated by visual examination, and the plates were regularly stored at −80°C.

(c) Phenotypic characterization

After completion of the evolution experiment, single clones from 160 representative lineages were isolated by streaking onto Luria broth (LB) agar plates. These isolates were transferred to 96-well microtiter plates, incubated overnight and stored at −80°C for later analysis. All incubations were conducted in LB broth at 37°C without agitation. The phenotypic characterization measured susceptibility to a collection of chemicals, antibiotics and bacteriophage viruses. Assays were performed in triplicate after overnight growth of a replica of the frozen collection plates. These overnight cultures were 1 : 100 diluted and re-grown for 4 h. A 96 pin replicator was then used to transfer aliquots to square Petri dishes containing LB agar supplemented with varying concentrations of each specific agent. To examine the status of the AcrAB pump, the major pump extruding β-lactams, we estimated the MIC of three well-known substrates such as acriflavine, tetracycline and erythromycin (all purchased from Sigma-Aldrich) [35]. Altered-efflux mutants were identified using a conservative criterion: either the MIC of one them was elevated more than or equal to fourfold, or the MIC of at least two rose more than equal to twofold. To study alterations in porins OmpF and OmpC, the major route of entry of the antibiotic into the cell [36], we assessed resistance to the bacteriophages Tu1a and Tu1b, which use these porins, respectively, as their attachment site [69]. The resistance criterion was growth on a multiplicity of infection sufficient to prevent growth of the ancestor strain. As positive controls, Keio-derived knockout mutants of the genes acrR, ompF and ompC were routinely included in the aforementioned assays.

(d) Genetic characterization

PCR amplification and Sanger sequencing of the chromosomal bla gene was performed with oligonucleotides 5′-TGAACA TTCCGA AATGCG C-3′ and 5′-CCTTCG TTCACC GTCTTC A-3′. Regarding the plasmidic bla gene, pBRACI extraction and purification were performed using the Qiagen plasmid mini kit, and sequencing was performed with oligonucleotides 5′-GCTCAG GACTGG TCTAAC-3′ and 5′-CTTTGC GGTTAG ACTGGT C-3′. Amplification and sequencing of the ftsI gene was carried out with oligonucleotides 5′-GCCCAG CATGTT TCACAA GATG-3′ and 5′-CGAGCA GAGATG CTGCGA A-3′. The internal oligonucleotide 5′-CATCGT GCCCTA ACAACA ACC-3′ was also employed for sequencing, owing to the large size of the gene (approx. 1.8 kb). Sequencing services were provided by Secugen (www.secugen.es), sequence analysis were carried out with Ridom T race E dit (www.ridom.de/traceedit) and sequence alignments were performed with MAFFT v. 6 (mafft.cbrc.jp/alignment/software).


Discussion

As observed in our previous work, ΔnuoNΔnqrF1 was unable to grow under oxic conditions in M5 minimal medium with either D,L-lactate or NAG as the substrate (Duhl et al., 2018). However, we have now observed that addition of 0.1% (w/v) tryptone to the medium allowed ΔnuoNΔnqrF1 to grow. The major component of tryptone is free amino acids and peptides, suggesting that the mutant strain requires amino acid supplementation and cannot make sufficient amino acids de novo to support growth. Together with accumulation of acetate and pyruvate the requirement for tryptone suggests reduced TCA cycle activity because some TCA reactions are required for de novo amino acid synthesis (Kanehisa and Goto, 2000). However, there are caveats to using tryptone because it is an undefined tryptic digest of casein and could contain other nutrients. We observed that other sources of amino acids, including casamino acids (acid-hydrolyzed casein) or defined amino acids did not rescue growth of the mutant. However, we propose that the rescue was caused by peptides in the tryptone, not by other nutrients. While there are minor differences in carbohydrate and mineral content between tryptone and casamino acids, these differences are small in comparison to the total mineral and carbohydrate content of the overall medium recipe (BD Biosciences, 2006). Further, previous work indicated that S. oneidensis MR-1 is incapable of using individual amino acids as carbon sources but is capable a wide variety of defined dipeptides (Serres and Riley, 2006). This suggests that S. oneidensis MR-1 is much more efficient in peptide uptake than free amino acid uptake, which would explain why tryptone rescues growth of the mutant, while casamino acids do not.

Along with the growth defect observed in the NADH dehydrogenase knockout strain, another finding of this study was that total NAD(H) pool sizes differed significantly between WT and ΔnuoNΔnqrF1. With either D,L-lactate or NAG as carbon sources, we observed roughly 2-fold increases in the total NAD(H) pools. We hypothesize that the increased NAD(H) pool size is caused by NAD + synthesis to counteract the increase in [NADH] within the cell caused by the limitation of NADH dehydrogenase activity. In S. oneidensis MR-1, NAD + synthesis is regulated by the repressor NrtR. When [NAD + ] decreases, NrtR is released from promotors to allow expression of NAD + synthesis related genes (Rodionov et al., 2008). Because we observed increased NAD(H) pool sizes in the ΔnuoNΔnqrF1 mutant strain, we propose that excess [NADH] and limited [NAD + ] led to overexpression of genes involved in NAD + synthesis.

The TCA cycle and upstream reactions are also affected by changes in internal redox state and NAD(H) pool size. NADH is an inhibitor of citrate synthase, which converts acetyl-CoA and oxaloacetate to citrate to bring carbon into the TCA cycle (Weitzman and Jones, 1968 Stokell et al., 2003). This suggests that increased NADH/NAD + may inhibit TCA cycle function by affecting citrate synthase activity. Furthermore, reactions upstream of the TCA cycle maybe affected, because pyruvate dehydrogenase (Pdh) activity is also regulated by NADH/NAD + and acetyl-CoA concentrations in S. oneidensis MR-1 (Pinchuk et al., 2011 Novichkov et al., 2013). Reduced citrate synthase activity would increase acetyl-CoA concentrations, which may inhibit Pdh function, together with increased NADH/NAD + . It also has been previously shown that Pdh activity can be affected by both NADH/NAD + and NAD(H) pool size (Shen and Atkinson, 1970), meaning activity is slowed when NADH/NAD + ratios within the cell increase. Metabolic analysis of the mutant strain supports the hypothesis that pyruvate oxidation and TCA cycle activity were inhibited in ΔnuoNΔnqrF1 and further explains previous data observed in single-knockouts of Nqr1 and Nuo (Duhl et al., 2018). We observed excretion of high levels of pyruvate and acetate by ΔnuoNΔnqrF1, which would be expected if flux through the TCA cycle is blocked (Figure 8). It is also important to note that all four of the NADH dehydrogenases have not been knocked out of S. oneidensis MR-1 in this study. This study sought to understand the roles of the two aerobically expressed NADH dehydrogenases (Pinchuk et al., 2010 Duhl et al., 2018), even though the other NADH dehydrogenases may play a role in redox state regulation in the ΔnuoNΔnqrF1 mutant strain.

Figure 8. Proposed effect of knocking out NADH dehydrogenases in S. oneidensis MR-1 and the subsequent effects on NADH oxidation, redox state, and TCA cycle inhibition within the cell.

To better understand the effects of NADH dehydrogenase knockouts on the physiological redox state in S. oneidensis MR-1 throughout growth, we used the Rex-based redox sensing system developed by Liu et al. (2019). One of the major limitations to standard NADH and NAD + quantification assays is the need to remove the bacteria from their growth environment to conduct extractions. We found that the cell harvest and extraction procedure may cause shifts in the cells' redox state. Prior to quenching with acid or base solution in the protocol, shaking of the cultures is ceased and samples are transferred to 15 mL conical tubes (Kern et al., 2014), likely leading to oxygen limitation. We have shown that the cells likely deplete all oxygen within the medium during the centrifugation step, creating an oxygen limited environment that can influence and equalize redox state in both S. oneidensis strains. Conversely, the Rex-based redox sensor directly interacts with intracellular NADH and NAD + and allows real-time, qualitative measurements of NADH/NAD + via fluorescent reporter output (Liu et al., 2019). This allowed us to assess NADH/NAD + without processing the cells in a way that would influence redox state. Our data show that the sensor works as expected in S. oneidensis and that ΔnuoNΔnqrF1 exhibits increased fluorescence output compared to WT.

While the sensor did influence growth of the strains, we were still able to gain qualitative measurements of the internal redox state. It is not clear why the addition of the Rex sensor influenced growth in S. oneidensis MR-1. In E. coli, the Rex sensor did not appear to influence growth (Liu et al., 2019). It is possible that the metabolic burden generated from carrying the redox sensing system caused the changes, and that use of different plasmid backbones or promoters would reduce the effects of the sensor. However, we believe that the output of the sensor is still a valuable source of information for this study for multiple reasons the sensor generated the expected output for an aerobic to anaerobic transition and the overall phenotypes and differences between WT and the mutant remained similar with the sensor. I.e., the mutant strain still grew to a lower final OD600 and at a slower rate than the WT when the sensor was present (Figure 6 and Table 3). Further, differences in substrate consumption and acetate and pyruvate accumulation remained similar when the sensor was present, with the mutant strain failing to utilize all available carbon source and accumulating acetate and pyruvate. WT with the sensor was able to consume all available substrate and did not accumulate acetate or pyruvate. Although the sensor appeared to affect WT more than the mutant, the general effect on both strains appears similar, and the essential phenotypes remain the same therefore, we believe that the sensor output reflects real differences in intracellular NADH/NAD + between the strains.

Altogether, our data indicate that deletion of NADH dehydrogenases affected NAD(H) pool sizes, NADH/NAD + , and upstream metabolic activities, specifically by inhibiting the TCA cycle and blocking amino acid synthesis. Because we have shown that deleting NADH dehydrogenases from S. oneidensis MR-1 led to increased NADH/NAD + levels, larger NAD(H) pool sizes, and metabolic shifts within the cell, NADH dehydrogenase knockouts may provide an avenue for metabolic engineering. When engineering pathways in bacteria to generate products that are redox cofactor-dependent, it is advantageous to make modifications to that organism to generate higher levels of NADH (Berrios-Rivera et al., 2002). If enzyme concentrations no longer limit the rate of product formation, then the availability of redox cofactors may become limiting (Berrios-Rivera et al., 2002 Balzer et al., 2013). For example, it was necessary to eliminate pathways that compete for NADH in E. coli to improve 1-butanol production (Atsumi et al., 2008). Increasing NADH generation has also been used to enhance electric current production by S. oneidensis MR-1 (Li et al., 2018). These studies show the importance of NADH availability when engineering redox cofactor-dependent pathways. We have shown that knocking out NADH dehydrogenases and eliminating a competing pathway for NADH in S. oneidensis MR-1 increases the availability of NADH, which provides a possible background strain for metabolic engineering in S. oneidensis MR-1. With additional NADH available in these NADH dehydrogenase mutant strains, we could direct NADH into synthetic pathways for product formation, even when oxygen is present, as indicated by the high levels of pyruvate and acetate accumulation by the mutant strain.


Discussion

Transmembrane Protein Transport

Roles of Transmembrane Proteins and Their Associated Ions

As shown in the green and orange sections of Figure 4, when L. helveticus utilized proteins such as casein, it first hydrolyzed them into polypeptides. The DEPs of the ABC transporter were significantly upregulated. The ABC or ATP-binding cassette transporter is a type of transmembrane protein that uses energy from ATP hydrolysis to absorb essential nutrients. In contrast, exporters carry substrates out of the cell by active transport (Theodoulou and Kerr, 2015). The low Mn 2+ concentration in experimental group B induced cell envelope proteinase (CEP) in L. helveticus (Guo et al., 2009). Research on the growth of L. helveticus CNRZ32 in skim milk media revealed upregulation of CEPs such as PrtH, PrtH2, and PrtM, the novel proteinases PrtH3 and PrtH5, the Dtp transport system, and peptidases such as pep O2 compared to CNRZ32 grown in MRS (Smeianov et al., 2007). A comparison of L. helveticus CRL1062 and CRL974 growth in media containing casitone, casamino acids, or β-casein disclosed that both strains showed the highest growth rates on casitone and the lowest PrtH activity levels in peptide-rich media (Hebert et al., 2000). Research on the caseinolytic properties of six L. helveticus strains in cheese revealed that after incubation in milk, all strains produced CEPs that could directly hydrolyze casein. In contrast, strains incubated in MRS did not have this ability (Jensen et al., 2009). Proteolytic genes were upregulated in vitamin-free media with casein acid hydrolysate compared to those with basic amino acids. This finding is consistent with those of previous reports.

Figure 4. Diagram of the mechanism of the utilization of amino acids (A) and proteins (B) by Lactobacillus helveticus CICC22171. The orange color represents the membrane structure, which includes the phospholipid bilayer, the passive and active transport transporters, and the ATP-binding cassette (ABC) transporters. The boldness of the arrows and the addition of substrates, process enzymes, and products indicate the intensification of the reaction. Green represents the transport of nitrogen sources, spherical represents amino acids, and green ellipse represents proteins. Blue is the central rule. The blue helix is DNA, the straight strand is mRNA, and the blue ellipse is protein. Red indicates carbon source metabolism, ATP transport and amino acid production. The yellow color shows the Fe ion conversion of the electron respiratory chain.

Upregulation of DEGs associated with transmembrane transport is rarely seen. In fact, transmembrane transport is usually downregulated. Substitution of nitrogen-derived amino acids with proteins decreases relative transmembrane transport and the metabolic processes associated with it. Thus, casein is transported into cells via ABC transporter upregulation and Mn 2+ -associated CEP downregulation.

ATP and Energy Utilization Reduction During Transport

Figure 4 shows that the Mg 2+ concentration was downregulated in B. Therefore, protein utilized by L. helveticus strengthens translation. Iron and heme play vital roles in the electron transfer chain (ETC). Low Fe 2+ concentrations explain a decreased requirement for aerobic respiration. ATP catabolism and phosphorylation and synthesis- and hydrolysis-coupled proton transport were all downregulated. The DEPs regulated ATP synthesis and energy metabolism and included H + -ATPase, ATP synthase, and nucleotide-binding activity related to ATP synthesis. Low Mn 2+ concentrations weaken glycosyltransferase and phosphoenolpyruvate (PEP) carboxykinase activity (Park et al., 1999).

Energy and carbon source metabolism requirements were reduced and these processes were attenuated. Glucose is often the main carbon and energy source for bacterial growth. As a heterotrophic microorganism, L. helveticus CICC22171 uses the glycolytic pathway (EMP) as its main energy source. Here, the EMP and PTS family mannose porter genes were downregulated in media containing casein. Thus, casein inhibits glucose utilization. The downregulated DEPs of the mannose PTS include a special transmembrane protein (Reizer and Saier, 1997) called the PTS family mannose porter. Its IID component identifies, transports, and phosphorylates carbohydrates. The PTS family mannose porter may operate in glucose and fructose transport systems (Saulnier et al., 2007). More sugar transport systems (including the PTS family and the mannose or fructose PTS family porter) were upregulated in L. casei Zhang grown on milk than in those cultured on soymilk. Hence, numerous carbohydrates could sustain bacterial growth (Wang et al., 2013). The alternate carbon source for L. lactis replaced glucose when the content of the latter had decreased because the enzymes involved in rapid carbon metabolism were downregulated (Redon et al., 2005). Hence, lactic acid bacteria could utilize alternate carbon sources by regulating the associated genes. Lactic acid bacteria require energy from glycometabolism to assist their respiration. Hence, bacterial respiration is strongly influenced by environmental conditions (Pedersen et al., 2012). Further studies should be conducted on the respiratory chains of bacteria in various media to optimize L. helveticus CICC22171 growth and metabolism.

The present study showed that bacterial energy metabolism declines with active transport and the respiratory ETC. Glycolysis generates amino acid carbon skeletons. The observed reduction in sugar utilization indicates that protein as a nitrogen source reduced carbon skeleton biosynthesis in Lactobacillus strains and compensated for the amino acid deficiency in the A medium.

Intracellular Protein Hydrolysis

DNA Transcription and Protein Translation in Preparation for Hydrolysis

The blue section of Figure 4 shows that transcription was the first step in gene expression and the sigma factor was vital to this process. The sigma factor activates initial transcription by reversibly binding the active site of RNA polymerase (Helmann and Chamberlim, 1988). Phage integrase-recombinase is a site-specific recombinase and includes the tyrosine and serine families (Stoll et al., 2002). It mediates DNA recombination by covalent interactions. The tyrosine family acts on the DNA skeleton via the tyrosine hydroxyl and binds the DNA chain after breakage (Groth and Calos, 2004). Uracil-DNA glycosylase effects DNA repair by preventing and correcting mispairing. It recognizes and hydrolyzes the N-glycosidic linkages between deoxyribose and the wrong base (Sakumi and Sekiguchi, 1990). Transcription factors functionally couple sigma factor regulons under harsh environments and substantially modulate transcription (Binder et al., 2016). Elongation factors Tu (EF-Tu) were the most abundant proteins in translation. They play vital roles in protein synthesis elongation in prokaryotes, mitochondria, plastids, and plasmids (Fu et al., 2012). Here, the sigma factor was upregulated after the nitrogen source in the medium was replaced with acid-hydrolyzed casein. This finding aligned with the observed upregulation of various enzymes involved in transcription. Therefore, transcription may be induced when the bacteria utilize casein.

The putative pre-16S rRNA nuclease is in the ribosome biogenesis subprocess under the GO classification 𠇋iological process.” It might have hydrolase activity on the 5′ end of pre-16S rRNA and attack ester bonds. Pyrimidine-nucleoside phosphorylase is mainly involved in nucleic acid systems. It might catalyze pyrimidine nucleoside hydrolysis and add phosphate groups to the receptor. The putative elongation factor Tu is related to the translational elongation stage. Low Mg 2+ concentrations promote the synthesis of large and small ribosome subunits and assist in protein translation (Chakraborty et al., 2019). Here, the Mg 2+ level was decreased in B. This discovery confirmed that the protein utilized by L. helveticus could stimulate protein translation. A few studies examined how casein influences gene expression during L. helveticus growth (Chen et al., 2019 Zhao et al., 2019). However, most of them concluded with analyses of the expression levels of the enzymes involved in bacterial hydrolysis and did not address the changes occurring in transcription or translation. Hence, the results of the present work could lay a foundation for further investigations into the influences of casein media on protein synthesis in L. helveticus CICC22171 and the regulatory mechanisms of the enzymes and factors involved in transcription and translation.

Proteomics here disclosed that phage-integrase recombinase was upregulated in strain CICC22171 cultured on casein media. This enzyme participates in DNA recombination. A previous study reported that several phage-related genes, the small and large phage terminase subunits, and phage proteins were upregulated in L. helveticus CNRZ32 grown in milk media (Smeianov et al., 2007). However, no genes encoding phage cytase were confirmed for strain CICC22171. Thus, further study is required to validate prophage induction in DNA recombination and autolysis in L. helveticus CICC22171.

Protein Activity Was Enhanced to Promote Intracellular Hydrolysis

Proteolysis was induced to acquire the essential nutrients needed to sustain bacterial growth in media deficient in free amino acids. Proteins were hydrolyzed into peptides which were then transported to the cells. The endopeptidase Clp complex in the bacteria generated low-molecular-weight peptides and liberated amino acids from the casein matrix. The L. helveticus proteolytic system is very efficient and comprises numerous enzymes with various functions. Protein synthesis may vary with medium composition. The present study verified that the bacterial media primarily influenced protein metabolism.

The medium constituents also influenced the expression of the genes encoding protein transport and intracellular proteolysis. A study on L. helveticus CNRZ32 demonstrated that expression of the peptidase pepI was higher in MRS than skim milk media, whereas the opposite was true for the peptidases pep N, pep X, and pep R. However, the expression levels of the peptidase pepC were the same in both media (Smeianov et al., 2007). An investigation of L. helveticus CRL1062 and CRL974 revealed that peptidase pep N was not influenced by the nitrogen source in the medium as its expression levels were similar in both MRS and casitone (Hebert et al., 2000). Therefore, the impact of nitrogen source on peptidase activity varies with bacterial strain. In contrast, an analysis of L. casei Zhang cultured in bovine milk and soymilk revealed that its proteasome, oligopeptide transport, and peptidase systems were upregulated in soymilk. For this reason, the soymilk contained sufficient free amino acids to support bacterial growth. The L. helveticus membrane had a major influence, while that of its cytoplasm was minor. Thus, there was an evident aerobic effect. However, gene transcription, RNA translation, related genetics, and transmembrane metabolism were promoted, while energy metabolism was inhibited. ABC transporter upregulation may move the hydrolysates out of the cell so that protein metabolism may be completed.


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Is casein hydrolysate the same thing as casamino acids? - Biology

1 Departamento de Genética, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

* Corresponding Author: [email protected]

2 Phoneutria Biotecnologia e Serviços Ltda, Belo Horizonte, Brazil

Received 1 December 2012 revised 31 December 2012 accepted 8 January 2013

Keywords: Lipase Esterase Wastewater Sewage Treatment Lipolytic Microorganisms Amylase Protease Cellulase

The main organic contaminants in municipal wastewater are proteins, polysaccharides, and lipids, which must be hydrolyzed to smaller units. A high concentration of oil and grease in wastewater affects biological wastewater treatment processes by forming a layer on the water surface, which decreased the oxygen transfer rate into the aerobic process. Microbial proteases, lipases, amylases, and celullases should play essential roles in the biological wastewater treatment process. The present study aimed to isolate lipaseand other hydrolytic enzyme-producing microorganisms and assess their degradation capabilities of fat and oil wastewater in the laboratory. We also evaluated microbial interactions as an approach to enhance lipolytic activity. We place emphasis on lipase activity because oil and grease are not only environmental pollutants, but also form an undesirable tough crust on pipes of sewage treatment plants. Thirty-five lipolytic microorganisms from sewage were identified and assessed for hydrolytic enzyme profiles. Lipases were characterized in detail by quantification, chain length affinity, and optimal conditions for activity. The good stability of isolated lipases in the presence of chemical agents, thermal stability, wide range of pH activity and tolerance, and affinity for different lengths of ester chains indicates that some of these enzymes may be good candidates for the hydrolysis of organic compounds present in wastewater. A combination of enzymes and fermenting bacteria may facilitate the complete hydrolysis of triglycerides, proteins, and lignocellulose that normally occur in the wastes of industrial processes. This study identifies enzymes and microbial mixtures capable of digesting natural polymeric materials for facilitating the sewage cleaning process.

For more than a century, biological wastewater treatment has been used to minimize anthropogenic damage to the environment. Oil and Grease (O&G) are the major problems and contaminants in biological wastewater treatment processes. Because of their nature, O&G form a layer on the water surface and decrease the oxygen transfer rate into an aerobic process [1]. These contaminants are mainly discharged from restaurants, food industries, and households [1,2]. Proteins and polysaccharides must also be hydrolyzed to smaller units by extracellular enzymes in a municipal wastewater treatment plant [3,4].

The composition and activity of the microbial community within a wastewater treatment plant play a substantial role in the efficiency and robustness of the purification process [5]. The efficiency of conventional biological processes in wastewater treatment is reduced by the high concentrations of O&G in effluents [6]. In the activated sludge process, high levels of O&G lead to a reduction of biological activity of the flocs due to the difficulty of oxygen and substrate to penetrate the floc due to the oil film formation around it [7]. Moreover, in the case of anaerobic digestion, excessive amounts of O&G inhibit the action of acetogenic and methanogenic bacteria [6,8,9]. The Brazilian National Council on the Environment (CONAMA) established the maximum level of mineral oil concentration allowed for effluent in water bodies at 20 mg/l, and the maximum level of vegetable oils and animal fats to 50 mg/l in Article 34, Resolution number 357 established on March 17, 2005 [10].

Traditional approaches to treat oily effluents include gravity separation, dissolved air flotation (DAF), deemulsification, coagulation, and flocculation. Free oil is removed from wastewater by gravity separation however, this process cannot remove small oil droplets and emulsions. Oil that adheres to the surface of solid particles can be removed by particle settling [11]. DAF uses solubilized air to increase buoyancy of the smaller oil droplets and improve separation. In addition, emulsified oil is removed by chemical or thermal de-emulsifying processes, or both [11]. Wastewater containing emulsified oil is heated to reduce the viscosity, accentuate density differences, and weaken the interfacial films stabilizing the oil phase. Thereafter, acidification and the addition of a cationic polymer neutralize the negative charges, and elevation of pH to an alkaline level induces flocculation of inorganic salts. Flocs with adsorbed oil are separated and the sludge is dewatered [11]. In this context, the usage of lipolytic microorganisms in wastewater treatment could eliminate this pretreatment process [12]. Chigusa et al. [13] showed that the percentage of fat in wastewater treated with a mixed culture of nine lipase-producing yeast strains decreased by 94%.

Oily effluents can also be pretreated as an approach to conform with the CONAMA resolution, but the achievement of such pretreatment processes depends on the costs of the enzyme [14]. Many industrial processes require breakdown of solids and the prevention of fat blockage or filming in waste systems before the wastewater can be released into the sewage system. This can be accomplished 1) by degradation of organic polymers with a commercial mixture of lipase, cellulase, protease, amylase, and inorganic nutrients or 2) by sewage treatment, cleaning of holding tanks, septic tanks, grease traps, and other systems. WW07P is produced by Environmental Oasis Ltd. and contains a range of high-performance microorganisms adapted for use in the biological treatment of wastewater containing high fat and oils. It also contains surfactants capable of liquefying heavy fat deposits, thereby assisting in their biodegradation [15].

Lipases and esterases constitute a large category of ubiquitous enzymes expressed by many organisms. Carboxylesterases (EC 3.1.1.1) have broad substrate specificity toward esters and thioesters. Esterases that hydrolyze long-chain acylglycerols (containing more than 10 carbon atoms) are termed lipases (EC 3.1.1.3) and can be considered lipolytic and esterolytic enzymes [16]. Most lipases are water-soluble enzymes that hydrolyze ester bonds of water-insoluble substrates [17]. Therefore, lipases act at the interface between a substrate phase and an aqueous phase, in which the enzyme is dissolved [18]. It is often necessary to combine two or more lipases in order to release a glycerol molecule, since all three acyl chains of a triacylglycerol molecule are rarely released by a single lipase [17].

The α-amylases (E.C.3.2.1.1) are enzymes that hydrolyze starch molecules to generate progressively smaller polymers composed of glucose units [19]. Today, a large number of microbial amylases have almost completely replaced the chemical hydrolysis of starch. The main advantage of using microorganisms for the production of amylase is the ability to bulk produce the enzyme and the easy manipulation of microbes to achieve enzymes with desired characteristics. Moreover, the stability of microbial amylases are higher than those of plant and animal [20].

Microbial proteases are used in waste treatment from various food-processing industries and household activities to solubilize proteinaceous waste and reduce the biological oxygen demand of aquatic systems [21,22]. Hydrolytic enzymes, such as lipases, amylases, and proteases, have a promising application in wastewater treatment of candy, ice cream, dairy, and meat industries. Enzymes for wastewater treatment do not require purification and thus should present a low production cost [23]. These characteristics have led to an increasing interest in enzyme production technology and the search for new microorganisms with a diverse ability to produce enzymes [24-27].

Microbial cellulases are widely used in the paper, wine, animal feed, and textile industries as well as for biofuels production, food processing, olive oil and carotenoid extraction, and waste management [28]. The wastes generated from agricultural fields and agroindustries contain a large amount of unutilized cellulose, thereby causing environmental pollution. Today, these wastes are utilized to produce valuable products, such as enzymes, sugars, biofuels, chemicals, and others [29-33].

Biosurfactants are amphiphilic molecules that possess both polar and nonpolar domains that have effective surface-active properties. There are two main types of these molecules: 1) those that reduce surface tension at the air-water interface (biosurfactants), and 2) those that reduce the interfacial tension between immiscible liquids or at the solid-liquid interface (bioemulsifiers) [34,35]. Biosurfactants usually exhibit an emulsifying capacity, but bioemulsifiers do not necessarily reduce the surface tension [34,35]. These molecules are microbial synthesized, and the different types of biosurfactants include lipopeptides synthesized by many species of Bacillus, glycolipids synthesized by Pseudomonas and Candida sp., phospholipids synthesized by Thiobacillus thiooxidans, and polysaccharidelipid complexes synthesized by Acinetobacter sp. [36-38].

The emulsification of lipids through the breakdown of lipid droplets favors the occurrence of hydrolysis, since the water-soluble lipolytic enzymes have greater surface contact with the substrate to be hydrolyzed. Natural bio-surfactants exhibit low toxicity, biodegradability, and ecological acceptability, which provide an alternative to chemically-prepared conventional surfactants. They can be produced from various substrates but are often generated from renewable resources, such as vegetable oils as well as distillery and dairy wastes [39]. They are applicable for environmental protection and management, bioremediation of soil [40], crude oil recovery, cleanup of hydrocarbon contaminated groundwater, and enhanced oil recovery [41], antimicrobial agents in healthcare [36] or in a wide variety of industrial processes involving emulsification, foaming, detergency, wetting, dispersing, or solubilization [42].

Lipase-producing microorganisms are also found in fat and oil contaminated sources. Thus, they originate from dairy, household, and biotechnology industry wastewaters. The intense competition for limited carbon sources may result in the evolution of novel genes and/or novel biochemical pathways in the specialized environment of wastewaters [43]. Therefore, in this study we isolated lipase and other hydrolytic enzyme-producing microorganisms and assessed their fat and oil degradation capabilities.

2.1. Selection of Lipase-Producing Microorganisms Using Tributyrin as a Growth Substrate

Our group currently has a microbial stock composed of more than 1100 lipolytic microorganisms that were obtained randomly. Among this library, 35 strains were selected for this study that had been originally isolated from four different sewage tanks. Samples were obtained from wastewaters at 2 farm houses (A and C), a dairy industry (B), and a biotechnological industry (D). Samples or its dilutions in sterile water were spread over Spirit Blue agar (Himedia, Mumbai/India) supplemented with 3% (v/v) tributyrin emulsion (20% v/v tributyrin 0.2% v/v tween 80). After incubation at 25˚C for 1 - 7 d, representative lipolytic colonies of each morphological type were isolated and purified in the same media. The strains were maintained in LB medium (10.0 g/l peptone, 5.0 g/l yeast extract, and 5.0 g/l sodium chloride) supplemented with 50% (v/v) fetal bovine serum and kept at 󔽘˚C.

2.2. Determination of the Degradative Enzymatic Profile of Organic Compounds between the Lipolytic Selected Microorganisms

Lipolytic strains were inoculated in 2 ml of LB broth in a 96-well plate. After 24 h at 25˚C and 30 hz agitation, 2 µl of culture were inoculated in the follow media: 1) tributyrin-agarose [1% (w/v) agarose 50 mM Tris-HCl pH 6,8 1 mM CaCl2 0.6% (v/v) tributyrin emulsion—to detect lipase] 2) casein-agarose [1% (w/v) agarose, 1 mM CaCl2, 10% (v/v) of a casein solution in 1X PBS pH 7.4—to detect caseinase activity] 3) corn starch-agarose [1% (w/v) agarose 50 mM Tris-HCl pH 6.8, 1 mM CaCl2, 0.5% (w/v) corn starch—to detect amylase] 4) carboxymethylcellulose-agarose [1% (w/v) agarose, 50 mM Tris-HCl pH 6.8, 1 mM CaCl2, 0.5% (w/v) carboxymethylcellulose—to detect cellulase modified from Akhtar et al. [44] and 5) gelatin media [2 ml media/ assay tube: 50 mM Tris-HCl pH 6.8, 1 mM CaCl2, 10% (w/v) gelatin—to detect gelatin-specific proatease], the formulation of all those media were adapted from Vuong et al. [45]. After 24 h incubation at 25˚C, enzymatic activities were detected by assessing for the presence of a clear halo in the media tests listed above except for gelatin media (see below). For media 1, the result was obtained by direct observation. Media 2 - 4 required revelation prior to a final analysis of the results as follows: 2) 2 min incubation with 1 N HCl solution to precipitate remaining casein 3) 2 min incubation with 2% iodine solution to colorize remaining starch 4) 30 min incubation with Congo red [0.25% (w/v) in 0.1 M Tris-HCl, pH 8,0) followed by 5 minutes in a destaining solution (0.5 M NaCl, 0.1 M Tris-HCl, pH 8.0) [46]. Microorganisms that produced a gelatin-specific protease were capable of liquefying the gelatin medium in media 5.

To evaluate the emulsifying capacity of the isolates, 200 µl of a culture with optical density (OD 600 nm) 0.5 were inoculated into 35 ml of LB broth supplemented with 2% soybean oil. The culture was then incubated for 35 d at 37˚C and 250 rpm agitation. When the emulsification of the oil took place in the media, it acquired a milky appearance and consistency.

2.3. Characterization of Extracellular Lipase

2.3.1. Production of Extracellular Enzymatic Extract

Cells (500 µl) were grown in LB medium for 24 h under 30 hz agitation at 25˚C and then seeded on the surface of a sterile dialysis membrane (12,000 Da) that had an equal diameter as a petri dish. The membrane was then placed on Spirit Blue agar containing 0.6% (v/v) tributyrin and incubated at 25˚C for a period of 1 - 7 d (see results section) [47]. The membranes were washed with 2 ml of buffer (10 mM Tris-HCl pH 8.4 and 40 mM NaCl). Thereafter, all experimental steps were conducted on ice. The cell suspension was centrifuged at 25,000 g for 20 min at 4˚C and filtered through a 0.22 µm filter. Then, 4 µl of enzyme extract was applied to the tributyrin-agarose (media one described previously) and incubated for 24 h at 25˚C in order to verify lipase activity.

2.3.2. Determination of Optimal ph and Temperature of Action

Optimal pH was tested using 2 ml of freshly prepared 0.3% tributyrin broth [50 mM Tris-HCl—evaluated at pH values of 4.3, 6.8, 9.8, and 12.3—1 mM CaCl2, and 1.5% (v/v) tributyrin emulsion] and 75 µl of enzymatic extract in a 1 cm cuvette. The optimal temperature was tested using 2 ml of the same broth at the optimal pH and 75 µl of enzymatic extract. Analyzed temperatures were 4˚C, 25˚C, 37˚C, and 50˚C. The reduction in OD was measured at 800 nm in a Shimadzu double beam spectrophotometer (UV-ISO-02, Kyoto, Japan) using a negative control as a standard at 0, 3, and 21 h of incubation.

2.3.3. Quantification of Lipase Activity in P-Nitrophenyl Esters

The lipase assay was performed by measuring the increase in the absorbance at 405 nm in a Thermo Plate microplate reader (TP-reader) caused by the release of p-nitrophenol after hydrolysis of p-nitrophenyl-butyrate (C4), decanoate (C10), and palmitate (C16) at 25˚C for 15 min at pH 8.0 as previously described [48], but modified by adding 10 mM CaCl2 to solution B. An enzymefree control was used as the reference. One unit of lipase (U) was defined as the amount of enzyme that releases 1 µmol p-nitrophenol per min under the assay conditions.

2.3.4. Test of Lipase Activity in the Presence of Chemical Agents and Thermal Resistance

For further characterization, thermal and chemical resistance was evaluated using p-NPB as a substrate. Solution B described above was added with one of following chemical compounds: 0.25% (v/v) H2O2, 0.1% (v/v) NaClO, 0.1% (v/v) liquid detergent, or 10 mM EDTA. For thermal resistance, extracellular enzyme extracts were incubated for 30 min at 50˚C and residual activity was also evaluated using p-NPB as a substrate.

2.4. Identification of Microorganisms

Genomic DNA was prepared from a loopful of cells grown in LB agar for 24 h. The cell pellet was resuspended in 250 µl of solution I (50 mM glucose, 25 mM Tris-HCl pH 8.0, and 10 mM EDTA). The cells were lysed by adding 25 µl of solution II [200 mM NaOH and 1% (w/v) SDS], and mixed for 5 min. Then, 500 µl of solution I and 2.5 µl of RNAse A (10 mg/ml) was added and incubated for 2 h at 37˚C. This methodology was adapted from alkaline lysis first described by Birnboim & Doly [49]. DNA was then purified with phenol-chloroform using a standard laboratory protocol and after precipitation, DNA was resuspended in 30 µl of TE (10 mM Tris-HCl pH 8.0 and 1 mM EDTA).

2.4.2. Ribosomal RNA Gene Amplification

Bacterial isolates were identified by sequencing rDNA. The PCR reaction was performed as previously described [50] using the primers 8F (5'-AGAGTTTGATYMTGGCTCAG-3') [51] and 907R (5'-CCGTCAATTCMTTTRAGTTT-3') [52]. Fungal identification was performed as previously described [53] by sequencing D1D2 of 26S rDNA using primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG) and NL4 (5'-GGTCCGTGTTTCAAGACGG). The sequences of PCR products were analyzed using standard protocols with a dideoxy nucleotide dye terminator (Big Dye vs. 3.1—Applied Biosystems, CA, USA) and Genetic Analyzer 3130 (Applied Biosystems, CA, USA). All 16S and 26S rRNA gene sequences were checked for quality, aligned, and analyzed with CodonCode Aligner v.3.7.1 (CodonCode Corp., Centerville, MA, USA). All sequences were compared with reference sequences in the Ribosomal Database Project (RDP) using Sequence Match and sequences in GenBank using BLASTN.

2.5. Induction of Lipase Production and Synergistic Effect between Different Strains

Pre-inoculum of five different isolates was induced with tributyrin or left untreated (group control) to evaluate if microbial metabolism is increased or not by this triglyceride (pre-induction). Then, 100 µl of pre-inoculum from each isolate were inoculated in LB broth containing 10 µl alamar blue in four different treatments groups: 1) no supplementation, 2) supplemented with 2% tributyrin, 3) supplemented with 2% soybean oil, or 4) supplemented with 2% soybean oil emulsified with sterile bacterial extract contained lipase to evaluate possible synergism between different strains. Monitoring the percentage of alamar blue reduction indicated the conditions in which the culture demonstrated higher metabolism. A calculation of standard deviation indicated the differences between the assessed values and the average. Twoway ANOVA with Bonferroni correction post test was performed using GraphPad Prism version 5.03 for Windows [54].

Our lipolytic microbial stock was selected by the presence of a halo around colonies when wastewaters were spread over Spirit Blue agar supplemented with 3% (v/v) tributyrin emulsion. Because of this triglyceride is formed by a glycerol and three four-carbon chains, esterases were selected preferably over lipases. However, because any lipase can also be classified as an esterase, some also showed lipolytic activity among the selected microorganisms.

The 16S/28S rDNA sequence analysis provides molecular identification of isolates. The microorganism collection site, identification, surfactant production [ability to emulsify 2% (v/v) soybean oil in culture medium], and enzymatic profile against agarose tributyrin, agarose casein, gelatin media, agarose corn starch, and agarose carboxymethylcellulose are shown in Table 1 . Collection

Table 1 . Collection site, identification, GenBank accession numbers and enzymatic profile of isolates.

(-) no enzymatic activity (+) low enzymatic activity (colony size/halo size * maximum experimental time was 36 days.

sites A and B presented a predominance of members of the Bacillaceae family and at sites C and D, we observed Enterobacteriaceae, Pseudomonas, and Bacillus spp. At site D, Pseudomonas sp. was prevalent.

The extracellular bacterial lipases are of commercial importance, as thousands of lipase units can be produced from only several liters of culture medium [55]. Bacterial lipases are mostly extracellular and are greatly influenced by nutritional and physicochemical factors, such as temperature, pH, nitrogen and carbon sources, presence of lipids, inorganic salts, agitation, and dissolved oxygen concentration [55]. Lipases are mostly inducible enzymes and are thus generally produced in the presence of a lipid source, or any other inducer, such as triacylglycerols, fatty acids, hydrolysable esters, tweens, bile salts, and glycerol [55-57]. However, their production is significantly influenced by other carbon sources, such as sugars, sugar alcohol, polysaccharides, whey, casamino acids, and other complex sources [58,59]. Therefore, 10 ml of extracellular extract were produced for lipase characterization of each of the 35 isolates. Despite the use of an inductor at this step, the lipase activity of two isolates (A9—Staphylococcus spp.- and B9—Bacillus spp.) could not be recovered, even with tributyrin induction ( Table 2 ). In a grease trap ecosystem, the coexistence of microbial strains could supply the nutritional needs due to partial degradation of other biopolymers naturally present in wastewater. Moreover, competition for nutrients and microbial interaction could also lead to the induction of lipase expression so that it may remains active during the first few subcultures. We found that each strain required a different incubation period to express extracellular lipase ( Table 2 ). The exact incubation period required to express lipase over the dialysis membrane was monitored by parallel incubation in spirit blue agar supplemented with tributyrin. It needs to be pointed out that the optimum growth condition was not determined for each strain separately.

Table 2 . Collection site, incubation period over dialysis membrane, optimal pH, optimal temperature, and quantification in lipase units with p-NPB (C4), p-NPD (C10), and p-NPP (C16).

* Range of values in which the extract retains more than 70% of its activity ** One unit of lipase (U) is defined as the amount of enzyme that releases 1 µmol p-nitrophenol per min, in the assay conditions *** Numbers between parentheses indicate the value in which the enzyme showed higher activity—it sometimes indicates a range of values.

After confirmation of lipase activity, the optimal pH and temperature of the extracts were defined ( Table 2 ). Among the extracts, mesophilic lipases with an optimal pH in the basic range were prevalent, but psychrophiles and acidophilus were also observed. In general, bacterial lipases have optimal activity at neutral or alkaline pH [60-63]. Lipases from Bacillus species are active over a broad pH range (pH 3-12) [64], and our findings indicated that lipase extracts produced by Bacillus species often presented more than one optimal pH value. However, in contrast to the findings of the previous study, lipases secreted by ours isolates belonging to the Bacillus genus showed less activity at high temperatures, with optimal activity at mesophilic temperatures. Only lipases secreted by B. cereus were more thermotolerant, reaching optimal activity at 50˚C. Some of our lipase extracts showed thermal stability up to 50˚C and retained more than 70% of activity after thermal treatment for 30 min, including A5 (Terribacillus sp.), A10 (family Flavobacteriaceae), B3, B4 and B5 (Lysinibacillus spp.), B7, B8, and C5 (Bacillus subtilis), B10 (Mutualistic association of Bacillus sp. and Staphylococcus epidermidis), B11 (Bacillus cereus), C2, D2, D3, D5, and D7 (Pseudomonas spp.), and D1 (Bacillus megaterium) ( Table 3 ). The thermal resistance of lipases from Bacillus and Pseudomonas has already been described [65-68].

Table 3 . Percentage of lipase residual activity after various treatments.

Results are shown for lipase activity after incubation in the presence of 1 0.25% (v/v) H2O2, 2 0.1% (v/v) NaClO, 3 0.1% (v/v) liquid detergent, 4 10 mM EDTA, or 5 after thermal treatment for 30 min at 50˚C. Hydrolysis of p-NPB (C4) was used for all measurements. Controls with no treatment were used for comparison.

Bacterial lipases generally have optimal activity in the temperature range of 30˚C - 60˚C however some reports have shown that bacterial lipases exist with optimal activity at both low and high temperature ranges [60,61,63, 69]. Lipases quantification using pNP-butyrate (C4), decanoate (C10), and palmitate (C16) was done in a fixed condition for all enzymes, at 25˚C and pH 8.0 ( Table 2 ). The majority of lipase extracts were active on short chain esters, which was expected since the selection was made with tributyrin. In addition, these findings are consistent with the fact that all lipases are also esterases and harbor esterolytic activity. However, isolates A2, A10, B4, B5, B6, C1, C2, D2, D3, D4, D5, D6, and D7 showed a greater affinity for long chain esters, therefore characterizing a “true” lipase extract, since a “true” lipase hydrolyses esters with more than 10 carbon atoms ( Table 2 ).

There are three categories of microbial lipases: nonspecific, regiospecific, and fatty acid-specific [55]. Nonspecific lipases act randomly on triacylglyceride molecules, which results in the complete breakdown tofatty acid and glycerol. Regiospecific lipases are 1, 3-specific lipases that hydrolyze only primary ester bonds, which is observed in lipases produced by some Bacillus species. The third group, fatty acid-specific lipases, comprise those with a pronounced fatty acid preference [55]. Despite showing activity on tributyrin-agarose, the isolated A7 (Bacillus pumillus) was inactive when evaluated for hydrolysis of pNP-ester with 4 and 16 carbons and exhibited poor activity for pNP-ester with 10 carbon atoms. This can be explained by the fact that some lipases have affinity for triacylglycerols, exhibiting no or little activity against monoand diglycerides [17,70]. Another possibility is that A7 isolate was originally capable of producing more than one type of esterase. However, over the course of the passages in vitro, the strain began to only express the esterase that hydrolyzes the ester of 10 carbon atoms. This behavior has been observed in some species in vitro. In addition, it is possible that our system was not sensitive enough to detect the reduced expression after several passages in culture media.

Some biochemical similarities can be observed between microorganisms with the same identification. Lysinibacillus spp. isolates showed many similarities, including the expression of active lipases within 48 h of incubation, having an optimal pH that was alkaline and an optimal temperature of 37˚C of higher, and the absence of gelatinase, caseinase, or amylase activity. Twenty strains belonged to the Bacillaceae family, of which 15 belonged to the Bacillus genus. All strains identified as B. megaterium were gelatin-specific protease producers and were unable to produce cellulase. They expressed extracellular lipase after 24 h of incubation over a dialysis membrane and their lipases had optimal activity at a pH in the alkaline range. In addition, these strains are mesophilic/psicrophilic and are more active against esters with only 4 carbon atoms. Among the five strains identified as B. subtilis, all were amylase and cellulase producers. Five were also capable of producing gelatinspecific proteases, but only 4 strains were able to produce casein-specific proteases as well. All five B. subtilis lipases had better activity at an alkaline pH and against 4 carbon esters. Among the five strains identified as Pseudomonas spp. all were amylase and cellulase producers, and 4 were still capable of producing gelatinand caseinspecific proteases All Pseudomonas lipases presented considerable activity at 37˚C and against 10 carbon atom esters, indicating the presence of at least one type of lipase (Tables 1 and 2).

For preliminary assessment of the potential use of lipases in sewage treatment, the thermal and chemical resistance of the enzymes was evaluated using p-NPB as a substrate ( Table 3 ). An abundance of cleaning products and other chemical compounds are released daily from grease traps. None of the extracts maintained more than 70% residual activity in all analyzed conditions. Isolate A2 (unclassified Saccharomycetales) exhibited the best results for all conditions combined and maintained more than 50% of residual activity in the presence of H2O2, NaClO, liquid detergent, EDTA, and thermal treatment. Other extracts that showed good results included B6 (from Bacillus cereus) and B12 (from Acetobacter pasteurianus). They showed residual activity in greater than 20% of all of the conditions. In addition to the A2 extract, a combination of extracts could also be potentially advantageous for the development of a biological method for cleaning O&G from grease traps and sewage treatment plant equipment. The strong inhibition caused by 0.1% (v/v) detergent could be due to inactivation of the enzyme as a result of a disruption of its tertiary structure. When dishes, clothes or floor are washed, high concentrations of detergents and soaps are released into the sewer over a short period of time. Therefore, the choice of a stable enzyme is an important aspect for sewage treatment. Therefore, we analyzed the ability of isolates to produce multiple degradative enzymes and found that isolates B2, B8, C4, C5, D1, D2, and D7 presented a wide range of ability in utilizing biopolymers commonly present in wastewater ( Table 3 ). These isolates all produced proteases and at least one other hydrolytic enzyme besides lipase. Among the 35 strains, more than 70% were able to produce at least two enzymes or more. This multiple approach allows for the use of a small number of isolates in sewage treatment, since each one individually has the ability to secrete more than one enzyme at a time. This can also reduce the requirement for nutritional supplementation of the system. Among the extracellular lipases produced from the 35 strains, approximately 45% were resistant to 0.25% (v/v) H2O2, approximately 20% were resistant to 0.1% (v/v) of NaClO or 10 mM EDTA, and more than 50% were resistant to 0.1% (v/v) liquid detergent ( Table 3 ).

Bioremediation techniques in situ include the introduction of different strains of live microorganisms to wastewater at various stages of its treatment. Almost all known methods for sludge treatment introduce microbial strains in the log phase of growth. These microbes are in active phase of multiplication, however their action requires time to degrade the substrats [71]. Estera et al. [72] previously developed a method for reducing the time for degradation, which includes first providing an enzyme mixture capable of digesting natural polymeric materials, and only the adding at least one species of fermenting bacteria to the system that is able to ferment the resulting suspension. Dash et al. [71] described a composition for the treatment of wastewater to remove pollutants that was comprised of a synergistic composition of microbes, enzymes, and cofactors/nutrients. The microbes in the composition were selected Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas desmolyticum, Coriolus versicolour, Lactobacillus sp., Bacillus subtilis, Bacillus cereus, Staphylococcus sp., and Phanerochaete chrysosporium, alone or in combination. The enzymes produced include proteases, lipases, amylases, glucose oxidases, and others. This composition exhibits synergy and effectively removes pollutants from the wastewater. Enzymes act by dissociating the molecules to simpler forms, and microbes utilize these intermediates in their metabolism, which results in the complete degradation of the pollutants in the wastewater. Microbes will grow faster due to the increasing availability of intermediates and therefore will produce more enzymes that can further degrade the pollutants. Thus, enzymes and microbes are interdependent and work together to facilitate faster degradation of the pollutant molecules [71].

Diverse microorganisms are able to hydrolyze different types of oil. However, the biodegradation process can be lengthy due to the low water solubility of oil [73]. In natural or induced conditions, many microorganisms are able to produce emulsifying agents, which minimizes the time required for biodegradation of O&G by enhancing hydrophobic substrate bioavailability [74]. Cell-bound esterase synthesis has been recently reported in association with the generation of surface active substances, indicating the coupled function of emulsification with lipolytic activity [75,76]. Biosurfactants increase the uptake of microorganisms when grown on insoluble substrates and also increase the efficiency of bioremediation [77].

As shown by Gautam et al. [78], Saharam et al. [79], and Pattanathu et al. [80], several species belonging to the genera Pseudomonas are capable of producing different classes of biosurfactants. In our study, we found that various isolates belonging to those genera and Bacilaceae class (A3, A4, A5, A6, A7, B3, B4, B5, B10, C2, C4, C5, D2, and D5) were able to emulsify 2% soybean oil, although we did not identify the class of biosurfactant produced ( Table 1 ).

Acetobacter pasteurianus is an acetogenic bacterial species normally associated with wine production and spoilage [81]. It produces acetic acid due to the incomplete oxidation of a carbon source into CO2 [81]. In our study, isolates B12 and B13 were associated with wastewater and lipase production. These enzymes preferentially hydrolyzed triglycerides with esters of 4 carbon atoms and were active in the basic pH range. Despite these similarities, they showed different chemical and thermal resistance, indicating that they are most likely different enzymes. In addition, these two strains were not able to produce any other type of hydrolytic enzyme or biosurfactant among those evaluated.

According to the literature, emulsification of lipids would favor its hydrolysis, since the water-soluble lipolytic enzymes have greater surface contact with the substrate to be hydrolyzed due to the breakdown of lipid droplets. To evaluate the behavior of some of our isolates, five strains with different emulsification and hydrolysis profiles were subjected to metabolic quantification by reduction of alamar blue, in four different conditions: 1) LB broth culture media, 2) LB broth with 2% tributyrin (triglyceride of 4 carbons), 3) LB with 2% soybean oil, and (iv) LB with 2% lipid emulsion ( Figure 1 ). The initial curve of alamar blue reduction indicated the best volume of pre-inoculum and the optimal period of incubation in the presence of the reagent for each strain. The graphs in Figure 1 show that the presence of an emulsified lipid in the culture medium did not increased the metabolic rates of any of the microorganisms, and rather the metabolic rate was reduced. However, previous induction of lipase production by the addition of tributyrin to pre-inoculum media proved effective in raising the metabolism in all experimental conditions for the A3 and B1 strains. The only situation in which the induction was not efficient was for strain B13, wherein the pre-inoculum that was not induced was more metabolically efficient in all experimental conditions. However, because lipase production may be influenced by the carbon source used in the induction process, the absence of lipase production for strain B13 in the presence of tributyrin is justifiable, since no other inducer was evaluated. The metabolism of strains B1 and B12 was markedly increased in presence of tributyrin compared to the presence of soybean oil, and these findings were in agreement with the hydrolysis of p-nitrophenol esters. Strains B12 and B13 were also more efficient at hydrolyzing

Figure 1 . Metabolic status of five different strains when induced or non-induced pre-inocula were challenged against a simple trigliceride, a complex mixture of triglicerides, or an oily emulsion of a complex mixture of triglicerides. Quantification of metabolism was performed by determining the percent reduction of 10% (v/v) alamar blue reagent.

p-nitrophenol butyrate and more metabolically active in LB media containing 2% tributyrin. Similar results were observed for strain D4, which was more efficient in hydrolyzing p-nitrophenol decanoate and more metabolically active in LB media containing 2% soybean oil ( Figure 1 ).

Cellulose is the most common organic polymer. It is the most prevalent material in waste from agriculture and the most abundant renewable biopolymer on Earth [82]. A promising strategy for utilization of this energetic renewable source is microorganism-mediated hydrolysis of discarded lignocellulose, followed by fermentation of the resulting compound, which produces the desired metabolites or biofuel [82]. Among our selected lipolytic microorganisms, 34.3% presented also hydrolytic activity against CM cellulose. Parmar et al. [83] showed that a mixture of hydrolytic enzymes, such as cellulases, proteases, and lipases, in equal proportion by weight, reduced total suspended solids (TSS) by 30% - 50% and improved sedimentation of solids in sludge. An increase in solid reduction was observed with increasing enzyme concentration. That reduction occurred due to the hydrolysis of residual polymers, proving that enzymatic synergism can effectively reduce the organic matter in industrial wastewater pretreatment plants.

Cultures isolated from the vast diversity of microorganisms provide a major source of biological material for industrial biocatalysts and other environmental applications. Lipases and esterases obtained in this study presented different resistances and affinities. Enzymes were characterized with the aim of identifying suitable candidates for use in wastewater treatment. The good stability of isolated lipases in the presence of chemical agents, thermal stability, wide range of pH activity and tolerance, and affinity for different lengths of ester chains indicates that some of these enzymes may be good candidates for the hydrolysis of organic compounds and polymers present in the wastewater of diverse industries. As bacterial enzymes are highly robust, being active over a wide range of pH and temperature and possessing a diverse range of substrate specificity, they could easily be used in pretreatment sludge processes, since they possess adequate resistance of some chemical elements and can be produced at a low cost. The absence of purification requirements contributes to the cost reduction of using these enzymes for sewage treatment. In addition, it is possible that a combination of two or more enzymes may facilitate the process of complete hydrolysis of triglycerides, proteins, and lignocellulose that normally occurs in the wastes of industrial processes. However, careful selection of the strains to be used in sewage treatment is essential, because it may be possible to use fewer strains to achieve the same purpose, since several strains showed the capability of producing two or more enzymes. This will ensure that the hydrolysis of all compounds commonly discarded in wastewater will be sufficiently achieved. In order to give a more real reflection of degradation capabilities of those microorganisms, further studies will use a simulation condition of common sewage as culture to test the degradation capabilities, and also focus on optimizing hydrolysis conditions with the aim of using combined enzyme/microbial strategies for improving industrial wastewater treatment processes.

We thank CNPQ for financial support, projects number: 580311/ 2008-2, 560912/2010-2, 551113/2011-1, 300721/2012-9.


Stimulatory Effects of Casein Hydrolysate and Proline in in vitro Callus Induction and Plant Regeneration from Five Deepwater Rice ( Oryza sativa L.)

Interactive effects of genotypes with callus induction and plant regeneration medium combinations on callus induction and plantlet regeneration response were studied for five deepwater Indica rice cultivars namely Habiganj Aman-1, Habiganj Aman-2, Habiganj Aman-8, Murabajal and Gheoch. Mature seed scutellums were cultured on MS and LS basal media supplemented with different combinations of 2,4-D, casein hydrolysate (CH) and proline. In cv. HA-8, basal medium combination of MS+2 mg L -1 2,4-D supplemented with 0.6% (w/v) CH was found to be the best for callogenesis where callusing frequency was 87%. The growth rate of callus was frequently increased by the addition of different concentrations of CH with callus induction media. On the other hand, when proline was supplemented in to callus induction media it had no residual effect on callus growth. Embryogenic calli were transferred on MS and LS based regeneration media supplemented with 2 mg L -1 BAP and also different concentrations of casein hydrolysate and proline. The highest regeneration frequency (80%) was observed in cv. HA-1 on LS basal media supplemented with only 2 mg L -1 BAP. However, present study demonstrated that plant regeneration media supplemented with proline is not inhibitory for plant regeneration but have a noticeable comparatively stimulating effect on regeneration from callus. Here combination of casein hydrolysate tremendously reduced plant regeneration . An over all analysis of variations of frequencies for callusing and plant regeneration revealed a contrasting interaction among the culture media and genotypes.

L. Khaleda and M. Al-Forkan , 2006. Stimulatory Effects of Casein Hydrolysate and Proline in in vitro Callus Induction and Plant Regeneration from Five Deepwater Rice ( Oryza sativa L.). Biotechnology, 5: 379-384.

From the pre historic era to the modern times, rice has been the most important source of human nutritions and has help sustain the increasing population and the development of human civilization. Rapid and remarkable recent advances in genetic engineering could result in genetically modified rice plants producing completely novel products. Rice would be the ideal for this purpose, especially deepwater rice sustains millions of subsistence farmer in South and Southeast Asia. It is endemic to Bangladesh. In vitro techniques for culture of the plant cells, tissues and organs have advanced dramatically during the last few decades due to the great advantage of plant biotechnology. The use of meristematic tissue such as immature and mature embryos, young inflorescences and the base of mature leaves at a defined stage of development provide the basis for most rice tissue culture systems. Plant regeneration from the major cultivated Indica rice varieties is generally poor (Abe and Futsuhara, 1984 Kavi Kishor and Reddy, 1986). An important trait of plants derived from somatic embryos, in their uniformity and genetic stability (Vasil, 1995). The different combinations of medium may influence the variations of callus formation and plant regeneration . In this investigation effect of supplementation of callus induction and plant regeneration media with Casein Hydrolysate (CH) and proline has been studied. An observation by Murashige and Skoog (1962) that the presence of CH allowed vigorous organ development over broader range of IAA and Kinetin level. Proline is one kind of amino acid . Amino acids provides plant cells with an immediately available source of nitrogen and uptake can be much more rapid than an organic constituent in the same medium (Thom, 1981). It has been reported that the enhancing effect of plant regeneration by a dehydration treatment of rice callus coincides with the decline of the proline content of the callus. Reproducible and efficient protocol of rice through somatic embryogenesis from callus has been developed (Al-Forkan et al ., 2005). The number, mass and morphology of the callus formed on the scutellum were dependent on the medium tested. National and International efforts have been made in the past for improving deepwater rice cultures. However, keeping in the mind, the magnitude of the problem related to cultivation of deepwater rice and continuing suffering of millions of farmers in Bangladesh, the necessity of research to develop cell to plant system in the deepwater rice has been given priority. The successful application of available gene transfer technology to rice will only be possible when reproducible regeneration systems are routinely available. In this study reproducible and efficient plant regeneration system has been established.

The experiment was conducted at Plant Tissue Culture Laboratory in the Department of Botany, University of Chittagong, Bangladesh during 2004-2005. Five deepwater rice ( O. sativa L.) cultivars namely Habiganj Aman-1, Habiganj Aman-2, Habiganj Aman-8, Murabajal and Gheoch were used in this study. The MS (Murashige and Skoog, 1962) and LS (Linsmaier and Skoog, 1965) based basal media with different hormonals and additives combinations with 30 g L -1 sucrose and 0.8% (w/v) agar solidified media were used for enhancing callus from mature seed scutellum of selected cultivars. MS and LS basal media were supplemented with different combinations of 2 mg L -1 2,4-D 2 mg L -1 2, 4-D+0.25 mg L -1 proline+0.1% (w/v) CH 2 mg L -1 2, 4-D+0.2% (w/v) CH, 2 mg L -1 2,4-D+0.4% (w/v) CH and 2 mg L -1 2,4-D+0.6% (w/v) CH. MS and LS based plant regeneration media were also supplemented with different combinations and concentrations of plant growth regulators and additives such as 2 mg L -1 BAP (RM-1, RM-4) 2 mg L -1 BAP+0.25 mg L -1 2,4-D+0.25 mg L -1 proline (RM-2, RM-5) and 2 mg L -1 BAP+0.1% (w/v) CH (RM-3, RM-6) were used for this purpose. In the parenthesis RM-1, RM-2 and RM-3 referred as MS based and later RM-4, RM-5 and RM-6 referred as LS based media, respectively.

Seeds were dehusked manually and sterilized by soaking seeds in 0.2% (w/v) HgCl 2 for 15 min and washed thoroughly (5-6 times) with sterile glass-distilled water. Then, seeds were placed in petri dish contained 20 ml callus induction medium and incubated at 26±2°C in dark. After 14 day the number of calli induced (from MSS) per petri-dish was counted, recorded and elongated shoots and roots were removed. Then seeds attached with scutella derived callus were cultured freshly on respective medium for another 14 day under the same growth condition as mentioned before. MSS derived embryogenic calli were transferred to regeneration medium where media were semi solidified with 1% (w/v) agar and incubated in the dark at 26±1°C for 10 days. After that calli were sub-cultured on the same medium except medium was semi-solidified with 0.8% (w/v) agar and kept under a 16 h photoperiod at 26±1°C for three weeks. The shoot regeneration frequencies were recorded 20 day after transfer of tissues to regeneration medium at the percentage of scutella derived calli each producing one or more shoots. Each shoot (2-3 cm in height) was detached from individual calli and each shoot was multiplied by transferring for 20-30 day on regeneration medium. After that regenerates were cultured on MS+1.5 mg L -1 NAA rooting medium. Well-developed plantlets were then transferred to the natural condition in pot.

Two types of calli formed at the scutella region of the cultured seeds. One type of callus was compact and nodular in structure and called embryogenic callus (Fig. 1a). The other type of callus was friable, translucent and slimy which never formed embryoids and called non-embryogenic callus. However, this type of callus was proliferated faster. The highest percentage of callus production 87 and 82% were obtained on MS and LS based media, respectively, where both the basal media supplemented with 2 mg L -1 2,4-D+0.6% (w/v) CH in cv. HA-8 (Table 1). Significant differences on percentage of callus production were recorded on among the media such as 2 mg L -1 2,4-D 2 mg L -1 2,4-D+0.25 mg L -1 proline+0.1% (w/v) CH and 2 mg L -1 2,4-D with 2-6% (w/v) CH.

Table 1: Comparison of callusing percentage on MS or LS based media supplemented with different combinations of plant growth regulators and additives

In general, calli from all the cultivars were compact, yellowish and big in size, well proliferated, some times dry, rooty and hardy. The supplementation of callus induction medium with CH tremendously increased callus production (Fig. 1a-c and 2a-c).

In casein hydrolysate free MS and LS based media, which consisted only 2 mg L -1 2,4-D (Fig. 1d), the frequency of calli formation were 36 and 33% which were comparatively lower than on MS and LS based media which were supplemented with 0.6% (w/v) CH where the percentage of calli were 85 and 80% in cv. HA-1. When proline was supplemented into the callus induction medium it had no residual effect on callus growth. Cultivar HA-2, cultured on MS and LS media supplemented with 2 mg L -1 2,4-D+0.25 mg L -1 proline+0.1% (w/v) CH were produced the lowest percentage of calli compared to other concentrations of CH added media (Fig. 2d). However, all the cultivars responded better on MS based callus induction medium compared to LS based callus induction medium (Table 1). Cultured callus of cv. HA-1 produced the highest percentage (50%) of plant on MS based media supplemented with 2 mg L -1 BAP (RM-1, Fig. 4). On the other hand, cv. Gheoch produced 25% of plant on the same medium. In contrast of that cv. HA-1 produced the highest percentage (80%) of plants on LS based RM-4 medium (Fig. 5). Interestingly, cv. Gheoch did not response on MS and LS based RM-3 and RM-6 medium, where media were supplemented with 2 mg L -1 BAP+0.1% (w/v) CH (Fig. 4). These results revealed that all the cultivars responded poorly in terms of plant regeneration on CH added RM-3 and RM-6 media. These types of calli were yellowish and watery (Fig. 3a). The shoot developed in CH supplemented media occasionally became brown and finally died. The addition of CH with MS and LS based regeneration media, the plant regeneration frequency tremendously reduced in all the cultivars.

Proline has a noticeable comparatively stimulating effect on plant regeneration from callus. Proline added in MS and LS based RM-2 and RM-5 media, cv. HA-1 produced comparatively higher percentage of plants 29% and 38% (Fig. 3b), respectively, than the CH added RM-3 and RM-6 media. On LS based proline supplemented medium (RM-5), cv. HA-1 produced deep green healthy plants with many shoots (Fig. 3d) whilst on medium RM-6 the same cultivars produced plants with fewer shoots (Fig. 3c). The percentage of plant regeneration varied genotype to genotype as well as different combination of medium.

Earlier studies have shown that embryogenesis and shoot regeneration are genetically determined in rice (Abe and Futsuhara, 1985). The present study has confirmed that the highest callus formation and plant regeneration are probably influenced by interaction of media components. It has been reported that the variation in embryogenic callus formation could be influenced by many other factors including differences in the media composition, concentrations of endogenous growth regulators and also addition of spermidine, casein enzymatic hydrolysate, proline, ascorbic acid and activated charcoal (Minhas et al., 1999). CH can be a source of calcium, several micronutrients, vitamins and most importantly a mixture of up to 18 amino acid s. Several investigators have concluded that CH itself is more effective for plant cultures than the addition of the major amino acid s, which it’s provide. This has led to speculation that CH might contain some unknown growth-promoting factor (Inoue and Maeda, 1982), which promoted callus growth. For cv. Murabajal, a higher frequency of plant regeneration was obtained on LS based RM-5 medium compared to RM-6 medium. This result demonstrates that proline is more relevant additive than CH. Many studies suggest that proline function in the intracellular osmotic adjustment between cytoplasm and vacuoles (Bandurska, 1993 Solomon et al., 1994) and intracellular structures (Van Rensburg et al ., 1993), a free radical scavenger (Smirnoff and Cumbes, 1989) or a strong compound of carbon and nitrogen for rapid recovery from stress (Jager and Meyer, 1977). Plant regeneration was also influenced by many other factors including the composition of the basal medium and nature of plant growth regulators added in the regeneration medium. A high frequency of plant regeneration was obtained from cv. HA-1 on RM-4 medium, which contained only 2 mg L -1 BAP as opposed to RM-6 medium, which contained 2 mg L -1 BAP+0.1% (w/v) CH. This demonstrates that the concentration of BAP is a more relevant growth regulator than a combination of BAP and 0.1% CH. Xie et al . (1995) reported that plant regeneration capability was dependent on the callus that was affected by the growth regulator combinations used in callus induction medium. In prepared mixture of amino acid s resembling those in CH, competitive inhibition between some of the constituents was often observed the percentage of CH decreased plant regeneration . In this investigation cv. Gheoch cultured on MS and LS based RM-3 and RM-6 media, which contained 2 mg L -1 BAP+0.1% (w/v) CH did not produce plantlets. Ansist and Northcote (1973) reported that, the brand of CH known as N-Z amine’ TM can produce toxic substances if concentrated solutions are heated or solutions are frozen and thawed several times. CH is therefore added in the media for shoot cultures, was found to hault plant regeneration and enhanced callus induction . This finding is contradict with the observation of Sheeja et al ., (2004) where callus induction was haulted but plant regeneration enhanced by addition of CH in the medium. The probable reason might be due to differences between monocotyledons and dicotyledons plant. By observing overall response, it can be concluded that proline is not inhibitory for plant regeneration but CH inhibited formation and proliferation of plant growth. Furthermore, the calli, which survived, regeneration process delayed by 1-2 week depending on the concentration of CH. However, these studies clearly demonstrate that the frequency of callus formation and efficient plant regeneration are mainly influenced by the plant genotype including composition of the culture media and plant growth regulators and additives. Furthermore, this experiment clearly demonstrated that CH could be added with callus induction medium for vigorous callus formation and proline could be added with plant regeneration medium for efficient plant regeneration . The protocol described in this study is recommended for high frequency of plant regeneration from deepwater rice cultivars as well as for production of transgenic rice plants with desired traits.

2: Abe, T. and Y. Futsuhara, 1985. Efficient plant regeneration by somatic embryogenesis from root callus tissues of rice (Oryza sativa L.). J. Plant Physiol., 121: 111-118.
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3: Al-Forkan, M., M.A. Rahim, T. Chowdhury, P. Akter and L. Khaleda, 2005. Development of highly in vitro callogenesis and regeneration system for some salt tolerant rice (Oryza sativa L.) cultivars of Bangladesh. Biotechnology, 4: 230-234.
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4: Anstis and Northcote, 1973. Plant Propagation by Tissue Culture. 2nd Edn., Exegetics Limited, UK., pp: 285.

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6: Inoue, M. and E. Maeda, 1982. Plant Propagation by Tissue Culture. 2nd Edn., Exegetics Limited, UK., pp: 285.

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9: Linsmaier, E.M. and F. Skoog, 1965. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant., 18: 100-127.
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10: Minhas, D., M.V. Raham and A. Grover, 1999. Maintenance of callus growth during sub-culturing is a genotype dependent response in rice: Mature seed-derived callus from IR 54 rice cultivar lacks culture ability. Curr. Sci., 77: 1410-1413.

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12: Sheeja, T.E., A.B. Mondal and R.K.S. Rathore, 2004. Efficient plantlet regeneration in tomato (Lycopersicon esculentum Mill.). Plant Tissue Cult., 14: 45-53.
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13: Smirnoff, N. and Q.J. Cumbes, 1989. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry, 28: 1057-1060.
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14: Solomon, A., S. Beer, Y. Waisel, G.P. Jones and L.G. Paleg, 1994. Effects of NaCl on the carboxylating activity of Rubisco from Tamarix jordanis in the presence and absence of proline-related compatible solutes. Physiol. Plant., 90: 198-204.
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15: Thom, 1981. Plant Propagation by Tissue Culture. 2nd Edn., Exegetics Limited, UK., pp: 285.


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