Catabolic Repression Research Articles

Hydrogenesis in hyperthermophilic microorganisms: Implications for biofuels

Hydrothermal microbiotopes are characterized by the consumption and production of molecular hydrogen. Heterotrophic hyperthermophilic microorganisms (growth T opt⩾80 °C) actively participate in the production of H 2 in these environments through the fermentation of peptides and carbohydrates. Hyperthermophiles have been shown to approach the theoretical (Thauer) limit of 4 mol of H 2 produced per mole of glucose equivalent consumed, albeit at lower volumetric productivities than observed for mesophilic bacteria, especially enterics and clostridia. Potential advantages for biohydrogen production at elevated temperatures include fewer metabolic byproducts formed, absence of catabolic repression for growth on heterogeneous biomass substrates, and reduced loss of H 2 through conversion to H 2S and CH 4 by mesophilic consortia containing sulfate reducers and methanogens. To fully exploit the use of these novel microorganisms and their constituent hydrogenases for biohydrogen production, development of versatile genetic systems and improvements in current understanding of electron flux from fermentable substrates to H 2 in hyperthermophiles are needed.

Nitrogen catabolic repression controls the release of volatile thiols bySaccharomyces cerevisiaeduring wine fermentation

Volatile thiols such as 4-methyl-4-sulfanylpentan-2-one (4MSP) and 3-sulfanylhexan-1-ol (3SH) are aromatic molecules having an important organoleptic impact on white wines. These components are produced from inodorous nonvolatile cysteinylated precursors by Saccharomyces cerevisiae metabolic activity during alcoholic fermentation. Here we provide a new insight into the genetic determinism of the production of volatile thiols by yeast. Using a gene deletion approach, we investigated the role of three yeast beta-lyases and demonstrate that Irc7p, a putative cystathionine beta-lyase, is one of the main proteins catalyzing the 4MSP and 3SH release under enological conditions. Moreover, we demonstrate that Ure2p/Gln3p proteins mainly control the bioconversion of volatile thiols by the transcriptional regulation of the IRC7 gene through the general mechanism of nitrogen catabolic repression. Finally, our findings suggest that the enantiomer balance of 3SH may be modulated by activating specifically stereoselective enzymes such as Irc7p.

Sulphur and nitrogen regulation of the protease-encoding ACP1 gene in the fungus Botrytis cinerea: correlation with a phospholipase D activity

Sulphur and nitrogen catabolic repressions are regulations that have long been recognized in fungi, but whose molecular bases remain largely elusive. This paper shows that catabolic repression of a protease-encoding gene correlates with the modulation of a phosphatidylethanolamine (PE)-specific phospholipase D (PLD) activity in the pathogenic fungus Botrytis cinerea. Our results first demonstrate that the ACP1 gene is subject to sulphur catabolic repression, with sulphate and cysteine inhibiting its expression. Sulphate and cysteine also cause a decrease of the total cellular PLD activity and, reciprocally, the two PLD inhibitors AEBSF [4-(2-aminoethyl)benzenesulphonyl fluoride] and curcumin negatively affect ACP1 expression in vivo. Cysteine moreover inhibits the PE-specific PLD activity in cell extracts. ACP1 is regulated by nitrogen, but here we show that this regulation does not rely on the proximal AREA binding site in its promoter, and that glutamine does not play a particular role in the process. A decrease in the total cellular PLD activity is also observed when the cells are fed ammonia, but this effect is smaller than that produced by sulphur. RNA-interference experiments finally suggest that the enzyme responsible for the PE-specific PLD activity is encoded by a gene that does not belong to the known HKD gene family of PLDs.

Production of -mannanase by Bacillus amylolequifaciens 10A1 cultured on potato peels

Different Bacillus species were screened for the production of 1,4-
- mannanase on solid media. Bacillus amyloliquefaciens was selected as the most potent in producing enzyme of high activity. Utilization of various agro-industrial residues on mannanase production was evaluated. Potato peels at a concentration of 14 g/l was found to be the most effective carbon source. Maximum enzyme activity (61.5 U/mg protien) was obtained with ammonium nitrate as a nitrogen source. The optimum incubation temperature and pH for enzyme production were 35oC and 7, respectively. The influence of inoculum size was so remarkable that, at optimum, a crude filtrate with an enzyme activity of 105.7 U/mg protien was obtained and appeared among the highest levels reported for mannanase titre. Addition of simple carbon source to media containing potato peels cause catabolic repression of mannanase synthesis.

Effect of moisture content in polyurethane foams as support for solid-substrate fermentation of Lecanicillium lecanii on the production profiles of chitinases

Finely minced (MPUF) and roughly cut (CPUF) polyurethane foam was used as inert support for the growth and chitinases production of Lecanicillium lecanii by solid-substrate fermentation. L. lecanii growths on CPUF produced loose and disperse mycelia throughout the polymer honeycomb, but MPUF resulted in dense aggregates at the ends of the polymer branches. Despite similar growth rates, μ, and maximum biomass concentration, X max, there were significant differences in the enzyme production. Highest enzyme titers ( e max) without glucose supplementation showed best results for >85% moisture content. e max, of exo-chitinase was 45-fold higher in CPUF than MPUF. Endochitinases, e max, were similar for CPUF and MPUF. Catabolic repression of enzyme production depended on moisture level, being stronger for lower moisture contents for exo-chitinases and milder or insignificant for endo-chitinases. Biomass yield coefficients of enzymes, Y e/ x , were higher with MPUF than with CPUF for endo-chitinases, but the reverse was found for exo-chitinases.

Mineralization of 2-aminobenzenesulfonate by a bacterial consortium

A variety of environmental inocula were tested for the development of 2-aminobenzenesulfonate (2-ABS, Orthanilic acid) degrading bacterial enrichment. A bacterial consortium (BC), which could utilize 2-ABS as the sole carbon and energy source, could only be developed from the sludge derived from a wastewater treatment unit of a large chemical industry manufacturing nitro and aminoaromatics. BC consisted of two bacterial strains. Based on 16S rDNA sequence analysis, these strains were identified to be belonging to the genus, Acinetobacter and Flavobacterium. The consortium could degrade 1,000 mg l−1 2-ABS within 40 h. Evidence for the extensive mineralization of 2-ABS, during the growth of BC, was derived from U.V-spectral and total organic carbon analysis. BC was highly specific for 2-ABS, as other benzene sulfonates tested in this study, including other ABS isomers, were not utilized as growth substrates. 2-ABS removal pattern in the presence of glucose was significantly influenced by acclimation characteristics of the culture. Consortium adapted to 2-ABS/glucose demonstrated the concomitant removal of both substrates, whereas glucose exerted catabolic repression on 2-ABS removal with glucose adapted culture. Presence of chloramphenicol inhibited 2-ABS degradation by cells, pregrown on succinate, indicating that the 2-ABS degrading enzymes are inducible in nature. Thus the presence of 2-ABS is essential for maintaining the high degradation potential. This enrichment culture can find an application in the treatment of 2-ABS containing wastewaters.

Saccharification of cellulosic biomass: Regulation of cellulase and xylanase activities under catabolic repression conditions

Saccharification of cellulosic biomass: Regulation of cellulase and xylanase activities under catabolic repression conditions

Identification of regulatory network topological units coordinating the genome-wide transcriptional response to glucose in Escherichia coli

BackgroundGlucose is the preferred carbon and energy source for Escherichia coli. A complex regulatory network coordinates gene expression, transport and enzyme activities in response to the presence of this sugar. To determine the extent of the cellular response to glucose, we applied an approach combining global transcriptome and regulatory network analyses.ResultsTranscriptome data from isogenic wild type and crp- strains grown in Luria-Bertani medium (LB) or LB + 4 g/L glucose (LB+G) were analyzed to identify differentially transcribed genes. We detected 180 and 200 genes displaying increased and reduced relative transcript levels in the presence of glucose, respectively. The observed expression pattern in LB was consistent with a gluconeogenic metabolic state including active transport and interconversion of small molecules and macromolecules, induction of protease-encoding genes and a partial heat shock response. In LB+G, catabolic repression was detected for transport and metabolic interconversion activities. We also detected an increased capacity for de novo synthesis of nucleotides, amino acids and proteins. Cluster analysis of a subset of genes revealed that CRP mediates catabolite repression for most of the genes displaying reduced transcript levels in LB+G, whereas Fis participates in the upregulation of genes under this condition. An analysis of the regulatory network, in terms of topological functional units, revealed 8 interconnected modules which again exposed the importance of Fis and CRP as directly responsible for the coordinated response of the cell. This effect was also seen with other not extensively connected transcription factors such as FruR and PdhR, which showed a consistent response considering media composition.ConclusionThis work allowed the identification of eight interconnected regulatory network modules that includes CRP, Fis and other transcriptional factors that respond directly or indirectly to the presence of glucose. In most cases, each of these modules includes genes encoding physiologically related functions, thus indicating a connection between regulatory network topology and related cellular functions involved in nutrient sensing and metabolism.

Staphylococcus aureus ArcR Controls Expression of the Arginine Deiminase Operon

We identified a single open reading frame that is strongly similar to ArcR, a member of the Crp/Fnr family of bacterial transcriptional regulators, in all sequenced Staphylococcus aureus genomes. The arcR gene encoding ArcR forms an operon with the arginine deiminase (ADI) pathway genes arcABDC that enable the utilization of arginine as a source of energy for growth under anaerobic conditions. In this report, we show that under anaerobic conditions, S. aureus growth is subject to glucose catabolic repression and is enhanced by arginine. Likewise, glucose and arginine have reciprocal effects on the transcription of the arcABDCR genes. Furthermore, we show using a mutant deleted for arcR that the transcription of the arc operon under anaerobic conditions depends strictly on a functional ArcR. These findings are supported by proteome analyses, which showed that under anaerobic conditions the expression of the ADI catabolic proteins depends on ArcR. Bioinformatic analysis of S. aureus ArcR predicts an N-terminal nucleotide binding domain and a C-terminal helix-turn-helix DNA binding motif. ArcR binds to a conserved Crp-like sequence motif, TGTGA-N(6)-TCACA, present in the arc promoter region and thereby activates the expression of the ADI pathway genes. Crp-like sequence motifs were also found in the regulatory regions of some 30 other S. aureus genes mostly encoding anaerobic enzymatic systems, virulence factors, and regulatory systems. ArcR was tested and found to bind to the regulatory regions of four such genes, adh1, lctE, srrAB, and lukM. In one case, for lctE, encoding l-lactate dehydrogenase, ArcR was able to bind only in the presence of cyclic AMP. These observations suggest that ArcR is likely to play an important role in the expression of numerous genes required for anaerobic growth.

Improvement of Bacillus thuringiensis delta-endotoxin production by overcome of carbon catabolite repression through adequate control of aeration

Over-production of delta-endotoxin by Bacillus thuringiensis, strain BNS3, entomopathogenic towards lepidoptera through overcome of catabolic repression of its synthesis, was investigated into full-controlled 3 l fermenter using simple and complex media. Equilibrium between density of the vegetative cells and their ability to synthesize toxins during sporulation was shown to be necessary to take into account. By application of various dissolved oxygen profiles during fermentation, it was clear that the level of dissolved oxygen saturation in the fermentation broth affected cell density as well as delta-endotoxin synthesis. Indeed, the adequate dissolved oxygen profiles to be followed throughout the fermentation for high production of bioinsecticides were determined using the two different media. It was found out that aeration rates corresponding to 60% and 70% oxygen saturation during the first 6 h of fermentation should be applied into 15 g l −1 glucose-based medium and 42 g l −1 gruel-based one, respectively. Then, 40% oxygen saturation should be ensured up to the end of fermentation, independently of the carbon source origin. With higher oxygen saturation values, cell densities were increased, but delta-endotoxin synthesis yields were strongly reduced. Cells produced with low aeration into the medium acquired higher capacities to synthesize toxins during sporulation. Interestingly, by following adequate oxygen profiles, it was possible to use increased initial glucose or gruel concentrations, without significant decrease of delta-endotoxin production yields. Consequently, the adequate control of dissolved oxygen in the culture media of B. thuringiensis allowed at least partial overcome of the carbon catabolite repression which seemed to be an apparent regulation, mostly due to the rate of use of energy generated during a first step of high growth of B. thuringiensis cells. These results are of great importance from practical point of view, since it could be possible to produce large quantities of insecticidal crystal proteins during large-scale fermentation, contributing to the reduction of B. thuringiensis insecticides production cost. Interestingly, in bioinsecticides produced with adequate aeration profiles, spores have lower yield, which is in favor of the dissemination of less spores into the environment.

Biodegradation of phenol by Ewingella americana: Effect of carbon starvation and some growth conditions

Ewingella americana was grown in batch cultures using a phenol-containing M9 minimal medium. Phenol was found to be the sole source for carbon and energy. Phenol was found to inhibit the growth rate with a maximum concentration of 1100 ppm, beyond which no growth occurred. The Haldane model was used to predict the specific growth rate-concentration data. The maximum growth rates on phenol (300 ppm) for starved and non-starved cells reached only 0.32 and 0.29 h −1, respectively. When the phenol-containing M9 minimal medium was supplemented with different C and N sources, different degradation rates (ppm/h) were obtained. Only fructose as the carbon source showed catabolic repression of the degradation activity; however, yeast extract, casein and glutamine caused the same effect, as did the fructose. The data showed that different initial (inocula) densities did not affect the induction time for phenol degradation. However, carbon-starvation minimized the acclimation period, accelerated the complete degradation achievement of phenol and affected the growth of cells differently based on the data obtained for growth phases. In the log phase, a higher growth rate was shown for starved cells with a shorter acclimation period, whereas in the stationary phase, a lower rate of growth was attained, compared with non-starved cells. Phenol degradation was optimally achieved at a 37 °C incubation temperature, a pH of 7.5 and an agitation rate of 200 rpm.

Cellulase production by Penicillium echinulatum on lactose

The inducer effect of lactose on cellulase activity in Penicillium echinulatum 9A02S1 was studied. Submerged cultivation was performed using different concentrations of lactose and cellulose, in which the pH, mycelial mass, soluble proteins, filter paper activity (FPA), and activity of beta-glucosidases were measured. The cultures containing lactose only presented low FPAs (0.1 FPU/ml). The cultures with associated cellulose and lactose and those containing cellulose only presented similar enzymatic activities (1.5 FPU/ml), suggesting the possibility of up to 75% reduction in the cellulose concentration. In relation to the beta-glucosidases, increasing the lactose/cellulose ratio results in a proportional increase of enzymatic activity. In the cultures using both inducers, there is a longer duration of the acid phase in relation to treatments using only cellulose or lactose, indicating diauxia and catabolic repression.

Inulinase-hyperproducing strains of Kluyveromyces sp. isolated from aguamiel (Agave sap) and pulque

Two strains of Kluyveromycesmarxianus (A1 and A2) isolated from ‘aguamiel’ (agave sap) and one strain of K. lactis var. lactis (P7) isolated from ‘pulque’ (its fermented product), were studied to make a survey of inulinase production. The strains of K. marxianus A1 and A2 were the best producers of inulinase, giving up to 2.5 times more enzyme than the control hyperproducing strain K. marxianus CDBB-L-278, and showed lower catabolic repression than this. One strain isolated from pulque was identified as K. lactis var. lactis and was also an excellent inulinase producer, being the first strain of this species reported as such. These strains were very good inulinase producers and they had low susceptibility to catabolic repression probably because the source from which they were isolated was rich in sucrose and oligofructans. They can be used in the transformation of inulin to produce fructose and/or oligofructans.

Glucose Diffusion on Support for Solid State Fermentation and its Influence on Tannase Production Profiles

In this study, catabolic repression of tannase in solid-state culture of Aspergillus niger Aa-20 on polyurethane foam was investigated considering the diffusion of a repressor molecule as the main responsible factor. The fundamental hypothesis considers that if repressor molecule uptake rate is higher than repressor molecule diffusion rate on solid matrix, the catabolic repression phenomenon will be minimized. Results showed that addition of low glucose concentrations enhanced the extracellular and intracellular tannase activities (from 5.8 to 6.6 and from 0.96 to 1.76 IU). Besides the glucose uptake rate was higher (1.23 x 10-5 cm2/seg) than glucose diffusion rate on solid matrix (2.8 x 10-6 cm2/seg). On the contrary results at high glucose concentrations, showed that when extracellular tannase activity decreased from 6.6 to 1.87 IU, while intracellular tannase activity was not affected; and under these conditions the glucose diffusion rate governed the process. A particular behaviour was obtained in the repression ratio and the glucose uptake rate/glucose diffusion rate ratio, which permitted to know the sensitivity degree of the culture system, according to the glucose amount added. Substrate diffusion in solid-state culture can be a key factor in its capacity to minimize the catabolic repression of hydrolytic fungal enzymes.

Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: Production and optimization

A newly isolated haloalkaliphilic Bacillus sp., Ve1, produced substantial levels of extracellular alkaline protease. Enzyme production corresponded with growth and reached a maximum level (410 U/ml) during the early stationary phase along with a brick red pigmentation. Protease production was maximum (397 U/ml) in gelatin broth. While growth and protease production was optimum at 10% (w/v) NaCl, only marginal growth without enzyme production was evident in the absences of salt. Ve1 could grow and produce protease at pH 7–9, the optimum being at 8 and 9, respectively. Among the organic nitrogen sources used, growth was best supported by a combination of peptone and yeast extract, while the optimum protease production was with casamino acid followed by gelatin. Enzyme production was highly reduced in soya peptone, trader's protein and tryptone. Inorganic nitrogen sources proved to be less favourable. Strong catabolic repression on protease production was observed with glucose and ammonia. The study assumes significance in the light of increasing emphasis on biocatalysis under more than one extreme condition.

Multiple transcripts regulate glucose-triggered mRNA decay of the lactate transporter JEN1 from Saccharomyces cerevisiae

The Saccharomyces cerevisiae JEN1 gene encoding the lactate transporter undergoes strong catabolic repression at both transcriptional and post-transcriptional levels. JEN1 mRNA decay is greatly accelerated upon the addition of a pulse of glucose, fructose or mannose to induced cell cultures. Mapping of the 5′UTRs and 3′UTRs of JEN1 transcripts revealed multiple transcription start-sites located at position −51, +391 or +972, depending on the cell culture conditions. The presence of the JEN1(+391) transcript correlated with rapid glucose-triggered mRNA degradation of the JEN1(−51) transcript, whereas when the small transcript started at position +972, the JEN1(−51) mRNA turnover rate was unaffected. Overexpressed JEN1(+391) transcript accelerated JEN1(−51) mRNA decay in all conditions tested but was not translated. We propose that the JEN1(+391) transcript may have a “sensor-like” function, regulating glucose-triggered degradation of JEN1(−51) protein-coding mRNA.

Dynamics of a two-prey–one-predator system with predator switching regulated by a catabolic repression control-like mode

The strategy of alternative prey (switching), regulated by a catabolic repression control-like (CRCL) mode, in which the desirable prey acts as repressor on the predator's attack on the alternative prey, was studied in a two-prey–one-predator chemostat system. In this system, alternative prey has to survive predation and competition for nutrients from the desirable prey. Therefore, when the alternative prey has no competitive advantage for the common substrate over the desirable prey, its survival depends on the protection offered by the desirable prey via CRCL. In this case, CRCL allows the coexistence of both desirable and alternative prey and predator populations. However, when the alternative prey has the competitive advantage over the desirable prey, CRCL negatively affects both the state of survival of the desirable prey and the coexistence state.

Genetic Evidence that Catabolites of the Entner-Doudoroff Pathway Signal C Source Repression of the σ54PuPromoter ofPseudomonas putida

Glucose and other C sources exert an atypical form of catabolic repression on the sigma54-dependent promoter Pu, which drives transcription of an operon for m-xylene degradation encoded by the TOL plasmid pWW0 in Pseudomonas putida. We have used a genetic approach to identify the catabolite(s) shared by all known repressive C sources that appears to act as the intracellular signal that triggers downregulation of Pu. To this end, we reconstructed from genomic data the pathways for metabolism of repressor (glucose, gluconate) and nonrepressor (fructose) C sources. Since P. putida lacks fructose-6-phosphate kinase, glucose and gluconate appear to be metabolized exclusively by the Entner-Doudoroff (ED) pathway, while fructose can be channeled through the Embden-Meyerhof (EM) route. An insertion in the gene fda (encoding fructose-1,6-bisphosphatase) that forces fructose metabolism to be routed exclusively to the ED pathway makes this sugar inhibitory for Pu. On the contrary, a crc mutation known to stimulate expression of the ED enzymes causes the promoter to be less sensitive to glucose. Interrupting the ED pathway by knocking out eda (encoding 2-dehydro-3-deoxyphosphogluconate aldolase) exacerbates the inhibitory effect of glucose in Pu. These observations pinpoint the key catabolites of the ED route, 6-phosphogluconate and/or 2-dehydro-3-deoxyphosphogluconate, as the intermediates that signal Pu repression. This notion is strengthened by the observation that 2-ketogluconate, which enters the ED pathway by conversion into these compounds, is a strong repressor of the Pu promoter.

Prey–predator dynamics with predator switching regulated by a catabolic repression control mode

Since many predators can live under certain circumstances as saprophytes or consume more than one prey, and different enzymes are generally required for each prey or nutrient digestion, the predator must be sufficiently adaptive for effective utilization of the prey mass. Control modes as induction and repression, however, act at the level of genes and cause changes in the biosynthesis rate of these enzymes. In this work, an extension of the catabolic repression control mode from the level of genes to the level of the behavior of the predator is proposed, in order to model the balanced attack of the predator on the prey. It is demonstrated that, when the prey population has the competitive advantage over the predator (in using the common substrate), the catabolic repression mechanism favors the prey population, which dominates over the predator even at low specific dilution rate values, whereas, the stable steady or periodic coexistence state is not favored. When the predator has the competitive advantage at low substrate concentrations and the prey at high substrate concentrations, the introduction of the catabolic repression mechanism in the model again favors the stable steady state of the prey, while the coexistence region is dramatically reduced. Conversely, when the prey population has the competitive advantage at low and the predator at high substrate concentrations, dominance of prey and coexistence steady state could be favored by the catabolic repression mechanism. It is concluded that the catabolic repression control favors dominance of the prey population and, under certain circumstances, coexistence of both prey and predator populations.

Regulation of tetralin biodegradation and identification of genes essential for expression of thn operons.

The tetralin biodegradation genes of Sphingomonas macrogolitabida strain TFA are clustered in two closely linked and divergent operons. To analyze expression of both operons under different growth conditions, transcriptional and translational gene fusions of the first genes of each operon to lacZ have been constructed in plasmids unable to replicate in Sphingomonas and integrated by recombination into the genome of strain TFA. Expression analysis indicated that the transcription of both genes is induced in similar ways by the presence of tetralin. Gene expression in both operons is also subjected to overimposed catabolic repression. Two additional genes named thnR and thnY have been identified downstream of thnCA3A4 genes. ThnR is similar to LysR-type regulators, and mutational analysis indicated that ThnR is strictly required for expression of the thn operons. Unlike other LysR-type regulators, ThnR does not repress its own synthesis. In fact, ThnR activates its own expression, since thnR is cotranscribed with the thnCA3A4 genes. ThnY is similar to the ferredoxin reductase components of dioxygenase systems and shows the fer2 domain, binding a Cys4[2Fe-2S] iron sulfur center, and the FAD-binding domain, common to those reductases. However, it lacks the NAD-binding domain. Intriguingly, ThnY has a regulatory role, since it is also strictly required for expression of the thn operons. Given the similarity of ThnY to reductases and the possibility of its being present in the two redox states, it is tempting to speculate that ThnY is a regulatory component connecting expression of the thn operons to the physiological status of the cell.