Showing papers on "Catabolite repression published in 2010"
TL;DR: This review summarizes the regulatory mechanisms responsible for CCR in the bacteria of the genus Pseudomonas, which can live in many different habitats and has implications in the optimization of biotechnological processes such as biotransformations or bioremediation strategies.
Abstract: Metabolically versatile free-living bacteria have global regulation systems that allow cells to selectively assimilate a preferred compound among a mixture of several potential carbon sources. This process is known as carbon catabolite repression (CCR). CCR optimizes metabolism, improving the ability of bacteria to compete in their natural habitats. This review summarizes the regulatory mechanisms responsible for CCR in the bacteria of the genus Pseudomonas, which can live in many different habitats. Although the information available is still limited, the molecular mechanisms responsible for CCR in Pseudomonas are clearly different from those of Enterobacteriaceae or Firmicutes. An understanding of the molecular mechanisms underlying CCR is important to know how metabolism is regulated and how bacteria degrade compounds in the environment. This is particularly relevant for compounds that are degraded slowly and accumulate, creating environmental problems. CCR has a major impact on the genes involved in the transport and metabolism of nonpreferred carbon sources, but also affects the expression of virulence factors in several bacterial species, genes that are frequently directed to allow the bacterium to gain access to new sources of nutrients. Finally, CCR has implications in the optimization of biotechnological processes such as biotransformations or bioremediation strategies.
448 citations
TL;DR: Surprisingly, untargeted metabolite profiling of Mtb growing on ¹³C-labeled carbon substrates revealed that Mtb could catabolize multiple carbon sources simultaneously to achieve enhanced monophasic growth.
315 citations
TL;DR: By exploiting CCR-negative phenotypes, various integrated processes have been developed that incorporate both enzyme hydrolysis of lignocellulosic material and mixed sugar fermentation, thereby enabling greater productivity and fermentation efficacy.
Abstract: Lignocellulosic biomass is an attractive carbon source for bio-based fuel and chemical production; however, its compositional heterogeneity hinders its commercial use. Since most microbes possess carbon catabolite repression (CCR), mixed sugars derived from the lignocellulose are consumed sequentially, reducing the efficacy of the overall process. To overcome this barrier, microbes that exhibit the simultaneous consumption of mixed sugars have been isolated and/or developed and evaluated for the lignocellulosic biomass utilization. Specific strains of Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis have been engineered for simultaneous glucose and xylose utilization via mutagenesis or introduction of a xylose metabolic pathway. Other microbes, such as Lactobacillus brevis, Lactobacillus buchneri, and Candida shehatae possess a relaxed CCR mechanism, showing simultaneous consumption of glucose and xylose. By exploiting CCR-negative phenotypes, various integrated processes have been developed that incorporate both enzyme hydrolysis of lignocellulosic material and mixed sugar fermentation, thereby enabling greater productivity and fermentation efficacy.
272 citations
TL;DR: This study is an example of how a systems biology approach allowed to propose a new regulation mechanism for overflow metabolism in E. coli shown by proteomic, transcriptomic and metabolomic levels coupled to two-phase acetate accumulation.
Abstract: Background
The biotechnology industry has extensively exploited Escherichia coli for producing recombinant proteins, biofuels etc. However, high growth rate aerobic E. coli cultivations are accompanied by acetate excretion i.e. overflow metabolism which is harmful as it inhibits growth, diverts valuable carbon from biomass formation and is detrimental for target product synthesis. Although overflow metabolism has been studied for decades, its regulation mechanisms still remain unclear.
204 citations
TL;DR: This study offers a genetic modification strategy for improving simultaneous utilization of mixed sugars by Clostridium, which is essential for commercial exploitation of lignocellulose for the production of solvents and biofuels.
156 citations
TL;DR: This work characterized a second hierarchy in E. coli, that between arabinose and xylose, and found that this repression is AraC dependent and involves a mechanism where arabinoses-bound AraC binds to the xyloses promoters and represses gene expression.
Abstract: Bacteria such as Escherichia coli will often consume one sugar at a time when fed multiple sugars, in a process known as carbon catabolite repression. The classic example involves glucose and lactose, where E. coli will first consume glucose, and only when it has consumed all of the glucose will it begin to consume lactose. In addition to that of lactose, glucose also represses the consumption of many other sugars, including arabinose and xylose. In this work, we characterized a second hierarchy in E. coli, that between arabinose and xylose. We show that, when grown in a mixture of the two pentoses, E. coli will consume arabinose before it consumes xylose. Consistent with a mechanism involving catabolite repression, the expression of the xylose metabolic genes is repressed in the presence of arabinose. We found that this repression is AraC dependent and involves a mechanism where arabinose-bound AraC binds to the xylose promoters and represses gene expression. Collectively, these results demonstrate that sugar utilization in E. coli involves multiple layers of regulation, where cells will consume first glucose, then arabinose, and finally xylose. These results may be pertinent in the metabolic engineering of E. coli strains capable of producing chemical and biofuels from mixtures of hexose and pentose sugars derived from plant biomass.
150 citations
TL;DR: Sugar-specific induction of many transport and metabolism genes indicates that these processes are regulated at the transcriptional level and are subject to carbon catabolite repression, and sugar-specific transport roles are suggested for various transporters of the PTS, the ABC superfamily and members of the major facilitator superfamily.
Abstract: Transcriptional analysis was performed on Clostridium acetobutylicum with the goal of identifying sugar-specific mechanisms for the transcriptional regulation of transport and metabolism genes. DNA microarrays were used to determine transcript levels from total RNA isolated from cells grown on media containing eleven different carbohydrates, including two pentoses (xylose, arabinose), four hexoses (glucose, mannose, galactose, fructose), four disaccharides (sucrose, lactose, maltose, cellobiose) and one polysaccharide (starch). Sugar-specific induction of many transport and metabolism genes indicates that these processes are regulated at the transcriptional level and are subject to carbon catabolite repression. The results show that C. acetobutylicum utilizes symporters and ATP-binding cassette (ABC) transporters for the uptake of pentose sugars, while disaccharides and hexoses are primarily taken up by phosphotransferase system (PTS) transporters and a gluconate : H+ (GntP) transporter. The transcription of some transporter genes was induced by specific sugars, while others were induced by a subset of the sugars tested. Sugar-specific transport roles are suggested, based on expression comparisons, for various transporters of the PTS, the ABC superfamily and members of the major facilitator superfamily (MFS), including the GntP symporter family and the glycoside-pentoside-hexuronide (GPH)-cation symporter family. Additionally, updates to the C. acetobutylicum genome annotation are proposed, including the identification of genes likely to encode proteins involved in the metabolism of arabinose and xylose via the pentose phosphate pathway.
142 citations
TL;DR: This first description of genes involved in acrylate catabolism in any organism shows that the relationship between the catabolic pathways of acrylated and DMSP differs from that which had been suggested in other bacteria.
Abstract: A bacterium in the genus Halomonas that grew on dimethylsulfoniopropionate (DMSP) or acrylate as sole carbon sources and that liberated the climate-changing gas dimethyl sulfide in media containing DMSP was obtained from the phylloplane of the macroalga Ulva. We identified a cluster that contains genes specifically involved in DMSP catabolism (dddD, dddT) or in degrading acrylate (acuN, acuK) or that are required to break down both substrates (dddC, dddA). Using NMR and HPLC analyses to trace 13C- or 14C-labelled acrylate and DMSP in strains of Escherichia coli with various combinations of cloned ddd and/or acu genes, we deduced that DMSP is imported by the BCCT-type transporter DddT, then converted by DddD to 3-OH-propionate (3HP), liberating dimethyl sulfide in the process. As DddD is a predicted acyl CoA transferase, there may be an earlier, unidentified catabolite of DMSP. Acrylate is also converted to 3HP, via a CoA transferase (AcuN) and a hydratase (AcuK). The 3HP is predicted to be catabolized by an alcohol dehydrogenase, DddA, to malonate semialdehyde, thence by an aldehyde dehydrogenase, DddC, to acyl CoA plus CO2. The regulation of the ddd and acu genes is unusual, as a catabolite, 3HP, was a co-inducer of their transcription. This first description of genes involved in acrylate catabolism in any organism shows that the relationship between the catabolic pathways of acrylate and DMSP differs from that which had been suggested in other bacteria.
123 citations
TL;DR: It is demonstrated that malate is a second preferred carbon source for B. subtilis, which is rapidly co-utilized with glucose and strongly represses the uptake of alternative substrates, and concludes that both substrates might act through different molecular mechanisms.
118 citations
TL;DR: A genetic analysis of the tagatose-6-phosphate (lac) and Leloir (gal) pathways revealed a remarkably high degree of complexity in the regulation of lactose/galactose catabolism.
Abstract: Abundant in milk and other dairy products, lactose is considered to have an important role in oral microbial ecology and can contribute to caries development in both adults and young children. To better understand the metabolism of lactose and galactose by Streptococcus mutans, the major etiological agent of human tooth decay, a genetic analysis of the tagatose-6-phosphate (lac) and Leloir (gal) pathways was performed in strain UA159. Deletion of each gene in the lac operon caused various alterations in expression of a PlacA-cat promoter fusion and defects in growth on either lactose (lacA, lacB, lacF, lacE, and lacG), galactose (lacA, lacB, lacD, and lacG) or both sugars (lacA, lacB, and lacG). Failure to grow in the presence of galactose or lactose by certain lac mutants appeared to arise from the accumulation of intermediates of galactose metabolism, particularly galatose-6-phosphate. The glucose- and lactose-PTS permeases, EIIMan and EIILac, respectively, were shown to be the only effective transporters of galactose in S. mutans. Furthermore, disruption of manL, encoding EIIABMan, led to increased resistance to glucose-mediated CCR when lactose was used to induce the lac operon, but resulted in reduced lac gene expression in cells growing on galactose. Collectively, the results reveal a remarkably high degree of complexity in the regulation of lactose/galactose catabolism.
105 citations
TL;DR: DNA microarrays and real-time RT-PCR were used to study gene expression during growth on D-glucose, D-xylose mixtures on a defined minimal medium together with monitoring substrate consumption and product formation and identified a CRE site in the catabolite repressed operon but not in the operon that was not subject to catabolic repression.
TL;DR: It is shown that under selective pressure, S. aureus Newman generates proline-prototrophic variants at a frequency of 3 x 10(-6), introducing frameshift and missense mutations in ccpA or IS1811 insertions in ptsH, two regulatory genes that carry out carbon catabolite repression (CCR) in staphylococci and other Gram-positive bacteria.
Abstract: Human clinical isolates of Staphylococcus aureus, for example, strains Newman and N315, cannot grow in the absence of proline, albeit their sequenced genomes harbor genes for two redundant proline synthesis pathways. We show here that under selective pressure, S. aureus Newman generates proline-prototrophic variants at a frequency of 3 x 10(-6), introducing frameshift and missense mutations in ccpA or IS1811 insertions in ptsH, two regulatory genes that carry out carbon catabolite repression (CCR) in staphylococci and other Gram-positive bacteria. S. aureus Newman variants with mutations in rocF (arginase), rocD (ornithine aminotransferase), and proC (Delta(1)-pyrroline 5-carboxylate [P5C] reductase) are unable to generate proline-prototrophic variants, whereas a variant with a mutation in ocd (ornithine cyclodeaminase) is unaffected. Transposon insertion in ccpA also restored proline prototrophy. CcpA was shown to repress transcription of rocF and rocD, encoding the first two enzymes, but not of proC, encoding the third and final enzyme in the P5C reductase pathway. CcpA bound to the upstream regions of rocF and rocD but not to that of proC. CcpA's binding to the upstream regions was greatly enhanced by phosphorylated HPr. The CCR-mediated proline auxotrophy was lifted when nonpreferred carbohydrates were used as the sole carbon source. The ccpA mutant displayed reduced staphylococcal load and replication in a murine model of staphylococcal abscess formation, indicating that carbon catabolite repression presents an important pathogenesis strategy of S. aureus infections.
TL;DR: The data confirm the important role of mannose PTS in class IIa bacteriocin sensitivity and demonstrate its importance involving global carbon catabolite control.
Abstract: The class IIa bacteriocin, pediocin PA-1, has clear potential as food preservative and in the medical field to be used against Gram negative pathogen species as Enterococcus faecalis and Listeria monocytogenes. Resistance towards class IIa bacteriocins appear in laboratory and characterization of these phenotypes is important for their application. To gain insight into bacteriocin resistance we studied mutants of E. faecalis V583 resistant to pediocin PA-1 by use of transcriptomic analyses. Mutants of E. faecalis V583 resistant to pediocin PA-1 were isolated, and their gene expression profiles were analyzed and compared to the wild type using whole-genome microarray. Significantly altered transcription was detected from about 200 genes; most of them encoding proteins involved in energy metabolism and transport. Glycolytic genes were down-regulated in the mutants, but most of the genes showing differential expression were up-regulated. The data indicate that the mutants were relieved from glucose repression and putative catabolic responsive elements (cre) could be identified in the upstream regions of 70% of the differentially expressed genes. Bacteriocin resistance was caused by reduced expression of the mpt operon encoding the mannose-specific phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), and the same transcriptional changes were seen in a mptD-inactivated mutant. This mutant also had decreased transcription of the whole mpt operon, showing that the PTS is involved in its own transcriptional regulation. Our data confirm the important role of mannose PTS in class IIa bacteriocin sensitivity and we demonstrate its importance involving global carbon catabolite control.
TL;DR: It is demonstrated in this study that the degree of xylanase-encoding gene induction strictly depends on the concentration of d-xylose, which was found to be optimal from 0.5 to 1 mM for 3 h of cultivation.
Abstract: For Hypocrea jecorina (anamorph Trichoderma reesei), a filamentous fungus used for hydrolase production in different industries, it has been a long-term practice to use d-xylose as an inducing substance. We demonstrate in this study that the degree of xylanase-encoding gene induction strictly depends on the concentration of d-xylose, which was found to be optimal from 0.5 to 1 mM for 3 h of cultivation. At higher concentrations of d-xylose, a reduced level of xylanase gene expression was observed. In the present study, we also provide evidence that the d-xylose concentration-dependent induction is antagonized by carbon catabolite repressor 1. This repressor mediates its influence on d-xylose indirectly, by reducing the expression of xylanase regulator 1, the main activator of most hydrolase-encoding genes. Additionally, a direct influence of the repressor on xylanase 1 expression in the presence of d-xylose was found. Furthermore, we show that d-xylose reductase 1 is needed to metabolize d-xylose to achieve full induction of xylanase expression. Finally, a strain which expresses xylanase regulator 1 at a constant level was used to partially overcome the negative influence exerted by carbon catabolite repressor 1 on d-xylose.
TL;DR: It is proposed that Crc controls TOL genes in two ways: first, Crc inhibits the translation of the XylR and XylS regulators, thereby reducing the transcription of all TOL pathway genes, and second, CrC inhibits thetranslation of specific structural genes of the pathway, acting mainly on proteins involved in the first steps of toluene assimilation.
TL;DR: Analysis of S. mutans lrg and cid mutants revealed a role for these operons in autolysis, biofilm formation, glucosyltransferase expression and oxidative stress tolerance, and its expression is controlled by two major environmental signals, oxygen and glucose.
Abstract: The tight control of autolysis by Streptococcus mutans is critical for proper virulence gene expression and biofilm formation. A pair of dicistronic operons, SMU.575/574 (lrgAB) and SMU.1701/1700 (designated cidAB), encode putative membrane proteins that share structural features with the bacteriophage-encoded holin family of proteins, which modulate host cell lysis during lytic infection. Analysis of S. mutans lrg and cid mutants revealed a role for these operons in autolysis, biofilm formation, glucosyltransferase expression and oxidative stress tolerance. Expression of lrgAB was repressed during early exponential phase and was induced over 1000-fold as cells entered late exponential phase, whereas cidAB expression declined from early to late exponential phase. A two-component system encoded immediately upstream of lrgAB (LytST) was required for activation of lrgAB expression, but not for cid expression. In addition to availability of oxygen, glucose levels were revealed to affect lrg and cid transcription differentially and significantly, probably through CcpA (carbon catabolite protein A). Collectively, these findings demonstrate that the Cid/Lrg system can affect several virulence traits of S. mutans, and its expression is controlled by two major environmental signals, oxygen and glucose. Moreover, cid/lrg expression is tightly regulated by LytST and CcpA.
TL;DR: This study highlights the importance of the genetic background in the processes of glucose signaling and regulation, contributes novel evidence on the overlap between the classical glucose repression pathway and the cAMP/PKA signaling pathway, and might have the potential to resolve some of the conflicting findings existing in the field.
Abstract: Under aerobic, high glucose conditions, Saccharomyces cerevisiae exhibits glucose repression and thus a predominantly fermentative metabolism. Here, we show that two commonly used prototrophic representatives of the CEN.PK and S288C strain families respond differently to deletion of the hexokinase 2 (HXK2) - a key player in glucose repression: In CEN.PK, growth rate collapses and derepression occurs on the physiological level, while the S288C descendant FY4 Deltahxk2 still grows like the parent strain and shows a fully repressed metabolism. A CEN.PK Deltahxk2 strain with a repaired adenylate cyclase gene CYR1 maintains repression but not growth rate. A comparison of the parent strain's physiology, metabolome, and proteome revealed higher metabolic rates, identical biomass, and byproduct yields, suggesting a lower Snf1 activity and a higher protein kinase A (PKA) activity in CEN.PK. This study highlights the importance of the genetic background in the processes of glucose signaling and regulation, contributes novel evidence on the overlap between the classical glucose repression pathway and the cAMP/PKA signaling pathway, and might have the potential to resolve some of the conflicting findings existing in the field.
TL;DR: In S. mutans, serine‐phosphorylated HPr functions in concert with particular PTS permeases to prioritize carbohydrate utilization by modulating sugar transport and transcription of catabolic operons.
Abstract: Carbohydrate catabolite repression (CCR) in Streptococcus mutans can be independent of catabolite control protein A (CcpA) and requires specific components of phosphoenolpyruvate-dependent sugar:phosphotransferase system (PTS) permeases. Here, the effects of various ptsH (HPr) and hprK (HPr kinase/phosphatase) mutations on growth and CCR were evaluated. An hprKV265F mutation, which enhanced Ser46 phosphorylation of HPr, inhibited growth on multiple PTS sugars. A ptsHS46A mutation reversed the effects of hprKV265F in most cases. A strain carrying a ptsHS46D mutation, which mimics HPr(Ser-P), presented with more severe growth defects than the hprKV265F mutant. The hprKV265F mutant enhanced CCR of the fruA and levD operons, a phenotype reversible by the ptsHS46A mutation. The effects of the hprKV265F mutation on fruA and levD expression were independent of CcpA, but dependent on ManL (IIAB(Man)) and, to a lesser extent, on FruI (IIABC(Fru)), in a carbohydrate-specific fashion. Expression of the Bacillus subtilis ptsG gene in the manL mutant did not restore CCR of the lev or fru operons. The hprKV265F mutation inhibited growth on cellobiose and lactose, but only the transcription of the cel operon was decreased. Thus, in S. mutans, serine-phosphorylated HPr functions in concert with particular PTS permeases to prioritize carbohydrate utilization by modulating sugar transport and transcription of catabolic operons.
TL;DR: The functional analysis of these two hexose transporters is the first step in elucidating the mechanisms of sugar metabolism and catabolite repression in P. pastoris.
Abstract: In this work, the identification and characterization of two hexose transporter homologs in the methylotrophic yeast Pichia pastoris, P. pastoris Hxt1 (PpHxt1) and PpHxt2, are described. When expressed in a Saccharomyces cerevisiae hxt-null mutant strain that is unable to take up monosaccharides, either protein restored growth on glucose or fructose. Both PpHXT genes are transcriptionally regulated by glucose. Transcript levels of PpHXT1 are induced by high levels of glucose, whereas transcript levels of PpHXT2 are relatively lower and are fully induced by low levels of glucose. In addition, PpHxt2 plays an important role in glycolysis-dependent fermentative growth, since PpHxt2 is essential for growth on glucose or fructose when respiration is inhibited. Notably, we firstly found that the deletion of PpHXT1, but not PpHXT2, leads to the induced expression of the alcohol oxidase I gene (AOX1) in response to glucose or fructose. We also elucidated that a sharp dropping of the sugar-induced expression level of Aox at a later growth phase is caused mainly by pexophagy, a degradation pathway in methylotrophic yeast. The sugar-inducible AOX1 promoter in an Δhxt1 strain may be promising as a host for the expression of heterologous proteins. The functional analysis of these two hexose transporters is the first step in elucidating the mechanisms of sugar metabolism and catabolite repression in P. pastoris.
TL;DR: The results show that ME is essential for l-malic acid utilization in L. casei and deletion of either the gene encoding the histidine kinase or the response regulator of the TC system resulted in the loss of the ability to grow on l- malic acid.
Abstract: Lactobacillus casei can metabolize l-malic acid via malolactic enzyme (malolactic fermentation [MLF]) or malic enzyme (ME). Whereas utilization of l-malic acid via MLF does not support growth, the ME pathway enables L. casei to grow on l-malic acid. In this work, we have identified in the genomes of L. casei strains BL23 and ATCC 334 a cluster consisting of two diverging operons, maePE and maeKR, encoding a putative malate transporter (maeP), an ME (maeE), and a two-component (TC) system belonging to the citrate family (maeK and maeR). Homologous clusters were identified in Enterococcus faecalis, Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus uberis. Our results show that ME is essential for l-malic acid utilization in L. casei. Furthermore, deletion of either the gene encoding the histidine kinase or the response regulator of the TC system resulted in the loss of the ability to grow on l-malic acid, thus indicating that the cognate TC system regulates and is essential for the expression of ME. Transcriptional analyses showed that expression of maeE is induced in the presence of l-malic acid and repressed by glucose, whereas TC system expression was induced by l-malic acid and was not repressed by glucose. DNase I footprinting analysis showed that MaeR binds specifically to a set of direct repeats [5′-TTATT(A/T)AA-3′] in the mae promoter region. The location of the repeats strongly suggests that MaeR activates the expression of the diverging operons maePE and maeKR where the first one is also subjected to carbon catabolite repression.
TL;DR: The results suggest that glucose repression of the uptake system in L. monocytogenes possibly is by inducer exclusion, a mechanism not described so far in this organism.
Abstract: Background
In the environment as well as in the vertebrate intestine, Listeriae have access to complex carbohydrates like maltodextrins. Bacterial exploitation of such compounds requires specific uptake and utilization systems.
Methodology/Principal Findings
We could show that Listeria monocytogenes and other Listeria species contain genes/gene products with high homology to the maltodextrin ABC transporter and utilization system of B. subtilis. Mutant construction and growth tests revealed that the L. monocytogenes gene cluster was required for the efficient utilization of maltodextrins as well as maltose. The gene for the ATP binding protein of the transporter was located distant from the cluster. Transcription analyses demonstrated that the system was induced by maltose/maltodextrins and repressed by glucose. Its induction was dependent on a LacI type transcriptional regulator. Repression by glucose was independent of the catabolite control protein CcpA, but was relieved in a mutant defective for Hpr kinase/phosphorylase.
Conclusions/Significance
The data obtained show that in L. monocytogenes the uptake of maltodextrin and, in contrast to B. subtilis, also maltose is exclusively mediated by an ABC transporter. Furthermore, the results suggest that glucose repression of the uptake system possibly is by inducer exclusion, a mechanism not described so far in this organism.
TL;DR: By constructing protein sequence alignments a DNA binding motif at the N-terminal end, two PTS regulation domains (PRDs), and an EIIA- and EIIB-like domain were identified in the ManR sequence, indicating that ManR is a PRD-containing transcription activator of the mannose operon.
Abstract: The mannose operon of Bacillus subtilis consists of three genes, manP, manA, and yjdF, which are responsible for the transport and utilization of mannose. Upstream and in the same orientation as the mannose operon a regulatory gene, manR, codes for a transcription activator of the mannose operon, as shown in this study. Both mannose operon transcription and manR transcription are inducible by mannose. The presence of mannose resulted in a 4- to 7-fold increase in expression of lacZ from the manP promoter (PmanP) and in a 3-fold increase in expression of lacZ from the manR promoter (PmanR). The transcription start sites of manPA-yjdF and manR were determined to be a single A residue and a single G residue, respectively, preceded by −10 and −35 boxes resembling a vegetative σA promoter structure. Through deletion analysis the target sequences of ManR upstream of PmanP and PmanR were identified between bp −80 and −35 with respect to the transcriptional start site of both promoters. Deletion of manP (mannose transporter) resulted in constitutive expression from both the PmanP and PmanR promoters, indicating that the phosphotransferase system (PTS) component EIIMan has a negative effect on regulation of the mannose operon and manR. Moreover, both PmanP and PmanR are subject to carbon catabolite repression (CCR). By constructing protein sequence alignments a DNA binding motif at the N-terminal end, two PTS regulation domains (PRDs), and an EIIA- and EIIB-like domain were identified in the ManR sequence, indicating that ManR is a PRD-containing transcription activator. Like findings for other PRD regulators, the phosphoenolpyruvate (PEP)-dependent phosphorylation by the histidine protein HPr via His15 plays an essential role in transcriptional activation of PmanP and PmanR. Phosphorylation of Ser46 of HPr or of the homologous Crh protein by HPr kinase and formation of a repressor complex with CcpA are parts of the B. subtilis CCR system. Only in the double mutant with an HPr Ser46Ala mutation and a crh knockout mutation was CCR strongly reduced. In contrast, PmanR and PmanP were not inducible in a ccpA deletion mutant.
TL;DR: The simulation model simulated the production of glutamate by transient batch-cultivation using a model of Escherichia coli central metabolism and concluded that it could function as a new tool for the rational improvement and design of metabolic and regulatory networks.
TL;DR: An expression vector system based on the broad host range plasmid pBBR1MCS2 with the Escherichia coli rhamnose-inducible expression system for applications in Pseudomonas and other γ-proteobacteria is developed and observed no catabolite repression of the rhaPBAD promoter.
Abstract: We developed an expression vector system based on the broad host range plasmid pBBR1MCS2 with the Escherichia coli rhamnose-inducible expression system for applications in Pseudomonas For validation and comparison to E coli, enhanced green fluorescent protein (eGFP) was used as a reporter For further characterization, we also constructed plasmids containing different modifications of the rhaP(BAD) promoter Induction experiments after the successful transfer of these plasmids into Pseudomonas putida KT2440 wild-type and different knockout strains revealed significant differences In Pseudomonas, we observed no catabolite repression of the rhaP(BAD) promoter, and in contrast to E coli, the binding of cyclic adenosine monophosphate (cAMP) receptor protein (Crp)-cAMP to this promoter is not necessary for induction as shown by deletion of the Crp binding site The crp(-) mutant of P putida KT2440 lacked eGFP expression, but this is likely due to problems in rhamnose uptake, since this defect was complemented by the insertion of the L-rhamnose-specific transporter rhaT into its genome via transposon mutagenesis Other global regulators like Crc, PtsN, and CyoB had no or minor effects on rhamnose-induced eGFP expression Therefore, this expression system may also be generally useful for Pseudomonas and other gamma-proteobacteria
TL;DR: Interestingly, a double-deletion mutant showed impaired growth not only on sugars but also on nonfermentable nutrients, and growth on gluconeogenic carbon sources was strongly suppressed in the presence of glucose.
Abstract: Fungi contain several hexokinases, which are involved either in sugar phosphorylation or in carbon source sensing. Glucose and fructose phosphorylations appear to rely exclusively on glucokinase and hexokinase. Here, we characterized the catalytic glucokinase and hexokinase from the opportunistic human pathogen Aspergillus fumigatus and showed that both enzymes display different biochemical properties and play different roles during growth and development. Glucokinase efficiently activates glucose and mannose but activates fructose only to a minor extent. Hexokinase showed a high efficiency for fructose activation but also activated glucose and mannose. Transcript and activity determinations revealed high levels of glucokinase in resting conidia, whereas hexokinase was associated mainly with the mycelium. Consequentially, a glucokinase mutant showed delayed germination at low glucose concentrations, whereas colony growth was not overly affected. The deletion of hexokinase had only a minor impact on germination but reduced colony growth, especially on sugar-containing media. Transcript determinations from infected mouse lungs revealed the expression of both genes, indicating a contribution to virulence. Interestingly, a double-deletion mutant showed impaired growth not only on sugars but also on nonfermentable nutrients, and growth on gluconeogenic carbon sources was strongly suppressed in the presence of glucose. Furthermore, the glkA hxkA deletion affected cell wall integrity, implying that both enzymes contribute to the cell wall composition. Additionally, the absence of either enzyme deregulated carbon catabolite repression since mutants displayed an induction of isocitrate lyase activity during growth on glucose-ethanol medium. Therefore, both enzymes seem to be required for balancing carbon flux in A. fumigatus and are indispensable for growth under all nutritional conditions.
TL;DR: Catabolite repression control regulates a broad spectrum of genes in Pseudomonas, and some virulence traits such as alginate and rhamnolipid production also appear to be regulated by Crc, which links nutritional status cues with the regulation of virulent traits.
Abstract: Catabolite repression control (CRC) is an important global control system in Pseudomonas that fine tunes metabolism in order optimise growth and metabolism in a range of different environments. The mechanism of CRC in Pseudomonas spp. centres on the binding of a protein, Crc, to an A-rich motif on the 5' end of an mRNA resulting in translational down-regulation of target genes. Despite the identification of several Crc targets in Pseudomonas spp. the Crc regulon has remained largely unexplored. In order to predict direct targets of Crc, we used a bioinformatics approach based on detection of A-rich motifs near the initiation of translation of all protein-encoding genes in twelve fully sequenced Pseudomonas genomes. As expected, our data predict that genes related to the utilisation of less preferred nutrients, such as some carbohydrates, nitrogen sources and aromatic carbon compounds are targets of Crc. A general trend in this analysis is that the regulation of transporters is conserved across species whereas regulation of specific enzymatic steps or transcriptional activators are often conserved only within a species. Interestingly, some nucleoid associated proteins (NAPs) such as HU and IHF are predicted to be regulated by Crc. This finding indicates a possible role of Crc in indirect control over a subset of genes that depend on the DNA bending properties of NAPs for expression or repression. Finally, some virulence traits such as alginate and rhamnolipid production also appear to be regulated by Crc, which links nutritional status cues with the regulation of virulence traits. Catabolite repression control regulates a broad spectrum of genes in Pseudomonas. Some targets are genus-wide and are typically related to central metabolism, whereas other targets are species-specific, or even unique to particular strains. Further study of these novel targets will enhance our understanding of how Pseudomonas bacteria integrate nutritional status cues with the regulation of traits that are of ecological, industrial and clinical importance.
TL;DR: Results indicate that GlxR plays an important role in CCR as well as in acetate metabolism, particularly in glyoxylate bypass and carbon catabolite repression.
Abstract: Recently, a cyclic AMP receptor protein homologue, GlxR, was reported to bind to the upstream regions of several genes involved in the regulation of diverse physiological processes in Corynebacterium glutamicum. However, the function of GlxR has not yet been explored in C. glutamicum in vivo using a glxR deletion mutant. Therefore, this study examines the role of GlxR as a repressor in glyoxylate bypass and carbon catabolite repression (CCR) using a deletion mutant. The disruption of glxR resulted in a severe growth defect, but growth was restored by complementation with the glxR and crp genes from C. glutamicum and Streptomyces coelicolor, respectively. The production of isocitrate lyase (ICL) and malate synthase (MS) was significantly increased in the glxR mutant. The specific activities of both enzymes were increased in the glxR mutant, regardless of the carbon source. In accordance, the promoter activities of ICL and MS using lacZ fusion were derepressed in the glxR mutant. In addition, the glxR mutant exhibited derepression of the gluA gene for glutamate uptake in the presence of glucose, thereby relieving CCR by glucose. These results indicate that GlxR plays an important role in CCR as well as in acetate metabolism.
TL;DR: The recombinant C. glutamicum engineered to express the xylose reductase gene XYL1 of Pichia stipitis was able to efficiently produce xylitol from d -xylose using glucose as source of reducing equivalents, and the molar yield ofxylitol to energy source during the biotransformation reached approximately 1.6 mol of xylOSE/mol of glucose.
TL;DR: The results presented extend the understanding both of CCR and of the effects of Crc for all aromatic degradative pathways of A. baylyi and increase the number of operons known to be controlled by two additional mechanisms, cross-regulation and vertical regulation.
Abstract: The degradation of many structurally diverse aromatic compounds in Acinetobacter baylyi is accomplished by the β-ketoadipate pathway. In addition to specific induction of expression by certain aromatic compounds, this pathway is regulated by complex mechanisms at multiple levels, which are the topic of this study. Multiple operons feeding into the β-ketoadipate pathway are controlled by carbon catabolite repression (CCR) caused by succinate plus acetate. The pathways under study enable the catabolism of benzoate (ben), catechol (catA), cis,cis-muconate (catB,C,I,J,F,D), vanillate (van), hydroxycinnamates (hca), dicarboxylates (dca), salicylate (sal), anthranilate (ant) and benzyl esters (are). For analysis of CCR at the transcriptional level a luciferase reporter gene cassette was introduced into the operons. The Crc (catabolite repression control) protein is involved in repression of all operons (except for catA), as demonstrated by the analysis of respective crc strains. In addition, cross-regulation was demonstrated for the vanA,B, hca and dca operons. The presence of protocatechuate caused transcriptional repression of the vanA,B- and hca-encoded funnelling pathways (vertical regulation). Thus the results presented extend the understanding both of CCR and of the effects of Crc for all aromatic degradative pathways of A. baylyi and increase the number of operons known to be controlled by two additional mechanisms, cross-regulation and vertical regulation.
TL;DR: A reverse genetic approach was employed to explore the role of the SNF1 ortholog as a potential regulator of CWDE gene expression in Ustilago maydis and deleted snf1 from the fungal genome, finding that, contrary to several other fungal species, U. maydis Snf1 was not required for utilization of alternative carbon sources.
Abstract: Many fungal plant pathogens are known to produce extracellular enzymes that degrade cell wall elements required for host penetration and infection. Due to gene redundancy, single gene deletions generally do not address the importance of these enzymes in pathogenicity. Cell wall degrading enzymes (CWDEs) in fungi are often subject to carbon catabolite repression at the transcriptional level such that, when glucose is available, CWDE-encoding genes, along with many other genes, are repressed. In Saccharomyces cerevisiae, one of the main players controlling this process is SNF1, which encodes a protein kinase. In this yeast, Snf1p is required to release glucose repression when this sugar is depleted from the growth medium. We have employed a reverse genetic approach to explore the role of the SNF1 ortholog as a potential regulator of CWDE gene expression in Ustilago maydis. We identified U. maydis snf1 and deleted it from the fungal genome. Consistent with our hypothesis, the relative expression of an endoglucanase and a pectinase was higher in the wild type than in the Δsnf1 mutant strain when glucose was depleted from the growth medium. However, when cells were grown in derepressive conditions, the relative expression of two xylanase genes was unexpectedly higher in the Δsnf1 strain than in the wild type, indicating that, in this case, snf1 negatively regulated the expression of these genes. Additionally, we found that, contrary to several other fungal species, U. maydis Snf1 was not required for utilization of alternative carbon sources. Also, unlike in ascomycete plant pathogens, deletion of snf1 did not profoundly affect virulence in U. maydis.