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The intracellular pathogen Chlamydia temporally regulates the expression of its genes, but the upstream signals that control transcription are not known. The best-studied regulatory pathway is a partner-switching mechanism that involves an anti-sigma factor, RsbW, which inhibits transcription by binding and sequestering the sigma subunit of RNA polymerase. RsbW is itself regulated by an anti-anti-sigma factor, RsbV, whose phosphorylation state is controlled by the phosphatase RsbU. In this study, we showed that Chlamydia trachomatis RsbU requires manganese or magnesium as a cofactor and dephosphorylates RsbV1 and RsbV2, which are the two chlamydial paralogs of RsbV. The gene for RsbU is adjacent to the enolase gene in a number of Chlamydia genomes, and we showed that eno and rsbU are cotranscribed from the same operon. In other bacteria, there is no known functional connection between the Rsb pathway and enolase, which is an enzyme in the glycolytic pathway. We found, however, that Chlamydia RsbU phosphatase activity was inhibited by phosphoenolpyruvate (PEP), the product of the enolase reaction, but not by 2-phosphoglycerate (2PGA), which is the substrate. These findings suggest that the enolase reaction and, more generally, glucose metabolism, may provide an upstream signal that regulates transcription in Chlamydia through the RsbW pathway. IMPORTANCE The RsbW pathway is a phosphorelay that regulates gene expression in Chlamydia, but its upstream signal has not been identified. We showed that RsbU, a phosphatase in this pathway, is inhibited by phosphoenolpyruvate, which is the product of the enolase reaction. As enolase is an enzyme in the glycolytic pathway, these results reveal an unrecognized link between glucose metabolism and gene regulation in chlamydiae. Moreover, as these intracellular bacteria acquire glucose from the infected host cell, our findings suggest that glucose availability may be an external signal that controls chlamydial gene expression.

Toll-like receptor (TLR) stimulation induces a pronounced shift to increased glycolytic metabolism in mammalian macrophages. We observed that bone marrow-derived macrophages (BMMs) increase glycolysis in response to infection with Legionella pneumophila, but the role of host macrophage glycolysis in terms of intracellular L. pneumophila replication is not currently understood. Treatment with 2-deoxyglucose (2DG) blocks L. pneumophila replication in mammalian macrophages but has no effect on bacteria grown in broth. In addition, we found that 2DG had no effect on bacteria grown in amoebae. We used a serial enrichment strategy to reveal that the effect of 2DG on L. pneumophila in macrophages requires the L. pneumophila hexose-phosphate transporter UhpC. Experiments with UhpC-deficient L. pneumophila revealed that mutant bacteria are also resistant to growth inhibition following treatment with phosphorylated 2DG in broth, suggesting that the inhibitory effect of 2DG on L. pneumophila in mammalian cells requires 2DG phosphorylation. UhpC-deficient L. pneumophila replicates without a growth defect in BMMs and protozoan host cells and also replicates without a growth defect in BMMs treated with 2DG. Our data indicate that neither TLR signaling-dependent increased macrophage glycolysis nor inhibition of macrophage glycolysis has a substantial effect on intracellular L. pneumophila replication. These results are consistent with the view that L. pneumophila can employ diverse metabolic strategies to exploit its host cells.IMPORTANCE We explored the relationship between macrophage glycolysis and replication of an intracellular bacterial pathogen, Legionella pneumophila Previous studies demonstrated that a glycolysis inhibitor, 2-deoxyglucose (2DG), blocks replication of L. pneumophila during infection of macrophages, leading to speculation that L. pneumophila may exploit macrophage glycolysis. We isolated L. pneumophila mutants resistant to the inhibitory effect of 2DG in macrophages, identifying a L. pneumophila hexose-phosphate transporter, UhpC, that is required for bacterial sensitivity to 2DG during infection. Our results reveal how a bacterial transporter mediates the direct antimicrobial effect of a toxic metabolite. Moreover, our results indicate that neither induction nor impairment of host glycolysis inhibits intracellular replication of L. pneumophila, which is consistent with a view of L. pneumophila as a metabolic generalist.

Menstrual toxic shock syndrome (mTSS) is a rare but life-threatening disease associated with the use of high-absorbency tampons. The production of the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) superantigen is involved in nearly all cases of mTSS and is tightly controlled by regulators responding to the environment. In the prototypic mTSS strain S. aureus MN8, the major repressor of TSST-1 is the carbon catabolite protein A (CcpA), which responds to glucose concentrations in the vaginal tract. Healthy vaginal Lactobacillus species also depend on glucose for both growth and acidification of the vaginal environment through lactic acid production. We hypothesized that interactions between the vaginal microbiota [herein referred to as community state types (CSTs)] and S. aureus MN8 depend on environmental cues and that these interactions subsequently affect TSST-1 production. Using S. aureus MN8 ΔccpA growing in various glucose concentrations, we demonstrate that the supernatants from different CSTs grown in vaginally defined medium (VDM) could significantly decrease tst expression. When co-culturing CST species with MN8 ΔccpA, we show that Lactobacillus jensenii completely inhibits TSST-1 production in conditions mimicking healthy menstruation or mTSS. Finally, we show that growing S. aureus in "unhealthy" or "transitional" CST supernatants results in higher interleukin 2 (IL-2) production from T cells. These findings suggest that dysbiotic CSTs may encourage TSST-1 production in the vaginal tract and further indicate that the CSTs are likely important for the protection from mTSS. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus chronically colonizes up to 30% of the human population on the skin or mucous membranes, including the nasal tract or vaginal canal. While colonization is often benign, this bacterium also has the capability to cause serious infections. Menstrual toxic shock syndrome (mTSS) is a serious toxinosis associated with improper use of tampons, which can induce an environment that is favorable to the production of the superantigen known as toxic shock syndrome toxin-1 (TSST-1). To better understand environmental signaling that influences TSST-1 production, we analyzed expression in the prototype mTSS strain S. aureus MN8. Using transcriptional and protein-based analysis in two niche-related media, we observed that TSST-1 expression was significantly higher in synthetic nasal medium (SNM) than in vaginally defined medium (VDM). One major divergence in medium composition was high glucose concentration in VDM. The glucose-dependent virulence regulator gene ccpA was deleted in MN8, and, compared with wild-type MN8, we observed increased TSST-1 expression in the Δ ccpA mutant when grown in VDM, suggesting that TSST-1 is repressed by catabolite control protein A (CcpA) in the vaginal environment. We were able to relieve CcpA-mediated repression by modifying the glucose level in vaginal conditions, confirming that changes in nutritional conditions contribute to the overexpression of TSST-1 that can lead to mTSS. We also compared CcpA-mediated repression to other key regulators of tst , finding that CcpA regulation is dominant compared to other characterized regulatory mechanisms. This study underlines the importance of environmental signaling for S. aureus pathogenesis in the context of mTSS. IMPORTANCE Menstrual toxic shock syndrome (mTSS) is caused by strains of Staphylococcus aureus that overproduce a toxin known as toxic shock syndrome toxin-1 (TSST-1). This work studied how glucose levels in a model vaginal environment could influence the amount of TSST-1 that is produced by S. aureus. We found that high levels of glucose repress TSST-1 production, and this is done by a regulatory protein called catabolite control protein A (CcpA). The research also demonstrated that, compared with other regulatory proteins, the CcpA regulator appears to be the most important for maintaining low levels of TSST-1 in the vaginal environment, and this information helps to understand how changes in the vaginal environmental can lead to mTSS.

Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon's density shift relative to that taxon's density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in 18O and 13C composition after exposure to [18O]water or [13C]glucose. The addition of glucose increased the assimilation of 18O into DNA from [18O]water. However, the increase in 18O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing. [ABSTRACT FROM AUTHOR]

Corynebacterium glutamicum, a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions to L-lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD600) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO2 is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology of C. glutamicum under anaerobic conditions. [ABSTRACT FROM AUTHOR]

Microbial metabolism is often considered modular, but metabolic engineering studies have shown that transferring pathways, or modules, between organisms is not always straightforward. The Thi5-dependent pathway(s) for synthesis of the pyrimidine moiety of thiamine from Saccharomyces cerevisiae and Legionella pneumophila functioned differently when incorporated into the metabolic network of Salmonella enterica. Function of Thi5 from Saccharomyces cerevisiae ( Sc Thi5) required modification of the underlying metabolic network, while Lp Thi5 functioned with the native network. Here we probe the metabolic requirements for heterologous function of Sc Thi5 and report strong genetic and physiological evidence for a connection between alpha-ketoglutarate (αKG) levels and Sc Thi5 function. The connection was built with two classes of genetic suppressors linked to metabolic flux or metabolite pool changes. Further, direct modulation of nitrogen assimilation through nutritional or genetic modification implicated αKG levels in Thi5 function. Exogenous pyridoxal similarly improved Sc Thi5 function in S. enterica. Finally, directly increasing αKG and PLP with supplementation improved function of both Sc Thi5 and relevant variants of Thi5 from Legionella pneumophila ( Lp Thi5). The data herein suggest structural differences between Sc Thi5 and Lp Thi5 impact their level of function in vivo and implicate αKG in supporting function of the Thi5 pathway when placed in the heterologous metabolic network of S. enterica. IMPORTANCE Thiamine biosynthesis is a model metabolic node that has been used to extend our understanding of metabolic network structure and individual enzyme function. The requirements for in vivo function of the Thi5-dependent pathway found in Legionella and yeast are poorly characterized. Here we suggest that αKG modulates function of the Thi5 pathway in S. enterica and provide evidence that structural variation between Sc Thi5 and Lp Thi5 contributes to their functional differences in a Salmonella enterica host.

Bioluminescence in Vibrio fischeri ES114 is activated by autoinducer pheromones, and this regulation serves as a model for bacterial cell-cell signaling. As in other bacteria, pheromone concentration increases with cell density; however, pheromone synthesis and perception are also modulated in response to environmental stimuli. Previous studies suggested that expression of the pheromone-dependent bioluminescence activator LuxR is regulated in response to glucose by cyclic AMP (cAMP) receptor protein (CRP) (P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 164:45-50,1985; P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 170: 4040-4046,1988; P. V. Dunlap, J. Bacteriol. 171:1199-1202,1989; and W. F. Friedrich and E. P. Greenberg, Arch. Microbiol. 134:87-91,1983). Consistent with this model, we found that bioluminescence in V. fischeri ESI 14 is modulated by glucose and stimulated by cAMP. In addition, a Δcrp mutant was ~100-fold dimmer than ES114 and did not increase luminescence in response to added cAMP, even though cells lacking crp were still metabolically capable of producing luminescence. We further discovered that CRP regulates not only luxR but also the alternative pheromone synthase gene ainS. We found that His-tagged V. fischeri CRP could bind sequences upstream of both luxR and ainS, supporting bioinformatic predictions of direct regulation at both promoters. Luminescence increased in response to cAMP if either the ainS or luxR system was under native regulation, suggesting cAMP-CRP significantly increases luminescence through both systems. Finally, using transcriptional reporters in trans-genic Escherichia coli, we elucidated two additional regulatory connections. First, LuxR-independent basal transcription of the luxI promoter was enhanced by CRP. Second, the effect of CRP on the ainS promoter depended on whether the V. fischeri regulatory gene litR was also introduced. These results suggest an integral role for CRP in pheromone signaling that goes beyond sensing cell density. [ABSTRACT FROM AUTHOR]

Rhizobium leguminosarum bv. trifolii pssA encodes a glucosyl-isoprenylphosphate (IP)-transferase involved in the first step of exopolysaccharide (EPS) synthesis. It was found that the pssA gene is an important target for regulation of this biosynthetic pathway. The data of this study indicate that pssA transcription is a very complex and mainly positively regulated process. A detailed analysis of a 767-bp-long pssA upstream region revealed the presence of several sequence motifs recognized by regulatory proteins that are associated with phosphate-, carbon-, and iron-dependent regulation. In addition, numerous inverted repeats of different lengths have been identified in this region. pssA transcription is directed from two distal P1 and proximal P3 promoters whose sequences demonstrate a significant identity to promoters recognized by RNA polymerase sigma factor σ70. Among rhizobial proteins, RosR seems to be a primary regulator that positively affects pssA expression. This protein binds to RosR box 1 located downstream of the P1 promoter. In addition, phosphate and the carbon source strongly affect pssA transcription. A significantly lower level of pssA expression was observed in both the wild-type strain growing under phosphate-rich conditions and the phoB mutant. In this regulation, the PhoB protein and Pho box 2 located upstream of the P3 promoter were engaged. pssA transcription is also significantly affected by glucose. Transcriptional analysis of a set of pssA-lacZ fusions expressed in Escherichia coli wild-type and cyaA and crp mutants confirmed that cyclic AMP (cAMP) receptor protein (CRP) and two cAMP-CRP boxes located upstream of the P1 are required for this upregulation. Moreover, the production of EPS was totally abolished in R. leguminosarum bv. trifolii mutant strains 4440 and 1012 containing a Tn5 insertion downstream of the P3 promoter and downstream of the P3 -35 hexamer, respectively. [ABSTRACT FROM AUTHOR]

During growth under conditions of glucose and oxygen excess, Staphylococcus aureus predominantly accumulates acetate in the culture medium, suggesting that the phosphotransacetylase-acetate kinase (Pta-AckA) pathway plays a crucial role in bacterial fitness. Previous studies demonstrated that these conditions also induce the S. aureus CidR regulon involved in the control of cell death. Interestingly, the CidR regulon is comprised of only two operons, both encoding pyruvate catabolic enzymes, suggesting an intimate relationship between pyruvate metabolism and cell death. To examine this relationship, we introduced ackA and pta mutations in S. aureus and tested their effects on bacterial growth, carbon and energy metabolism, cid expression, and cell death. Inactivation of the Pta-AckA pathway showed a drastic inhibitory effect on growth and caused accumulation of dead cells in both pta and ackA mutants. Surprisingly, inactivation of the Pta-AckA pathway did not lead to a decrease in the energy status of bacteria, as the intracellular concentrations of ATP, NAD+, and NADH were higher in the mutants. However, inactivation of this pathway increased the rate of glucose consumption, led to a metabolic block at the pyruvate node, and enhanced carbon flux through both glycolysis and the tricarboxylic acid (TCA) cycle. Intriguingly, disruption of the Pta-AckA pathway also induced the CidR regulon, suggesting that activation of alternative pyruvate catabolic pathways could be an important survival strategy for the mutants. Collectively, the results of this study demonstrate the indispensable role of the Pta-AckA pathway in S. aureus for maintaining energy and metabolic homeostasis during overflow metabolism. [ABSTRACT FROM AUTHOR]

The Gram-positive Corynebacterium glutamicum efficiently metabolizes maltose by a pathway involving maltodextrin and glucose formation by 4-α-glucanotransferase, glucose phosphorylation by glucose kinases, and maltodextrin degradation via maltodextrin phosphorylase and α-phosphoglucomutase. However, maltose uptake in C. glutamicum has not been investigated. Interestingly, the presence of maltose in the medium causes increased expression of ptsG in C. glutamicum by an unknown mechanism, although the ptsG-encoded glucose-specific EII permease of the phosphotransferase system itself is not required for maltose utilization. We identified the maltose uptake system as an ABC transporter encoded by musK (cg2708; ATPase subunit), musE (cg2705; substrate binding protein), musF (cg2704; permease), and musG (cg2703; permease) by combination of data obtained from characterization of maltose uptake and reanalyses of transcriptome data. Deletion of the mus gene cluster in C. glutamicum Δmus abolished maltose uptake and utilization. Northern blotting and reverse transcription-PCR experiments revealed that musK and musE are transcribed monocistronically, whereas musF and musG are part of an operon together with cg2701 (musI), which encodes a membrane protein of unknown function with no homologies to characterized proteins. Characterization of growth and [14C] maltose uptake in the musI insertion strain C. glutamicum IMcg2701 showed that musI encodes a novel essential component of the maltose ABC transporter of C. glutamicum. Finally, ptsG expression during cultivation on different carbon sources was analyzed in the maltose uptake-deficient strain C. glutamicum Amus. Indeed, maltose uptake by the novel ABC transport system MusEFGK2I is required for the positive effect of maltose on ptsG expression in C. glutamicum. [ABSTRACT FROM AUTHOR]

Recently, the S-layer protein of Sulfolobus acidocaldarius was shown to be N-linked with a tribranched hexasaccharide, composed of Man2GIc,GlcNAc2 and a sulfated sugar called sulfoquinovose. To identify genes involved in the biosynthesis and attachment of this glycan, markerless in-frame deletions of genes coding for predicted glycosyltransferases were created. The successful deletion of agl16, coding for a glycosyltransferase, resulted in the S-layer protein and archaellins having reduced molecular weights, as visualized by Coomassie staining or immunoblotting. This analysis indicated a change in the N-glycan composition. Nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses confirmed that the glycan of the S-layer protein from the agl16 deletion mutant was a pentasaccharide, which was missing a terminal hexose residue. High-performance liquid chromatography (HPLC) analyses of the hydrolyzed N-glycan indicated that the missing hexose is a glucose residue. A physiological characterization of the agll6 deletion mutant revealed a significant effect on the growth at elevated salt concentrations. At 300 mM NaCl, the doubling time of the Δagl16 mutant was increased 2-fold compared to that of the background strain. Furthermore, the incomplete glycan structure of the Δagl16 deletion strain affected the assembly and function of the archaellum, as exemplified by semisolid Gelrite plate analysis, in which the motility is decreased according to the N-glycan size. [ABSTRACT FROM AUTHOR]

In Escherichia coli several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase system (glucose PTS), but also the mannose PTS and the galactose and maltose transporters can translocate glucose. Mutant strains which lack the enzyme IIBC (EIIBC) protein of the glucose PTS have been previously investigated because their lower rate of acetate formation offers advantages in industrial applications. Nevertheless, a systematic study to analyze the impact of the different glucose uptake systems has not been undertaken. Specifically, how the bacteria cope with the deletion of the major glucose uptake system and which alternative transporters react to compensate for this deficit has not been studied in detail. Therefore, a series of mutant strains were analyzed in aerobic and anaerobic batch cultures, as well as glucose limited continuous cultivations. Deletion of EIIBC disturbs glucose transport severely in batch cultures; cyclic AMP (cAMP)-cAMP receptor protein (CRP) levels rise, and induction of the mgl operon occurs. Nevertheless Mgl activity is not essential for growth of these mutants, as deletion of this transporter did not affect growth rate; the activities of the remaining transporters seems to be sufficient. Under conditions of glucose limitation, mgl is 23-fold up-regulated compared to growth under glucose excess. Despite the strong induction of mgl upon glucose limitation, deletion of this transport-system did not lead to further changes. Although the galactose transporters are often regarded as important for glucose-uptake at micromolar concentrations, the glucose- as well as mannose PTS might be sufficient for growth at this relatively low dilution rate. [ABSTRACT FROM AUTHOR]

A mutant unable to utilize galactose was isolated in Sinorhizobium meliloti strain Rml021. The mutation was found to be in a gene annotated dgoK1, a putative 2-keto-3-deoxygalactonokinase. The genetic region was isolated on a complementing cosmid and subsequently characterized. Based on genetic and bioinformatic evidence, the locus encodes all five enzymes (galD, dgoK, dgoA, SMc00883, and ilvD1) involved in the De Ley-Doudoroffpathway for galactose catabolism. Although all five genes are present, genetic analysis suggests that the galactonase (SMc00883) and the dehydratase (ilvD!) are dispensable with respect to the ability to catabolize galactose. In addition, we show that the transport of galactose is partially facilitated by the arabinose transporter (AraABC) and that both glucose and galactose compete with arabinose for transport. Quantitative reverse transcription-PCR (qRT-PCR) data show that in a dgoKbackground, the galactose locus is constitutively expressed, and the induction of the ara locus seems to be enhanced. Assays of competition for nodule occupancy show that the inability to catabolize galactose is correlated with an increased ability to compete for nodule occupancy. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a versatile opportunistic pathogen whose success is driven by its ability to adapt to diverse environments and host-imposed stresses. Two-component signal transduction systems, such as ArlRS, often mediate these adaptations. Loss of ArlRS or the response regulator ArlR alone impairs the ability of S. aureus to respond to host-imposed manganese starvation and glucose limitation. As sensor histidine kinases and response regulators frequently work as pairs, it has been assumed that ArlS senses and activates ArlR in response to these stimuli. However, recent work suggests that the sensor histidine kinase GraS can also activate ArlR, calling the contribution of ArlS in responding to manganese and glucose availability into question. The results of current studies reveal that ArlS is necessary to activate ArlR in response to manganese sequestration by the host immune effector calprotectin and glucose limitation. Although the loss of ArlS does not completely eliminate ArlR activity, this response regulator is no longer responsive to manganese or glucose availability in the absence of its cognate histidine kinase. Despite the residual activity of ArlR in the absence of ArlS, ArlR phosphorylation by ArlS is required for S. aureus to resist calprotectin-imposed metal starvation. Cumulatively, these findings contribute to the understanding of S. aureus signal transduction in response to nutritional immunity and support the previous observation indicating that ArlRS is activated by a common signal derived from host-imposed manganese and glucose limitation. IMPORTANCE The ability of pathogens, including Staphylococcus aureus, to sense and adapt to diverse environments partially relies on two-component systems, such as ArlRS. Recent work revealed that the response regulator ArlR can be cross-activated by the sensor histidine kinase GraS, rendering the role of its cognate partner, ArlS, in response to manganese and glucose limitation uncertain. The results of this study reveal that ArlS is necessary for the activation of ArlR in response to calprotectin and glucose limitation. Although a low level of ArlR activity remains in the absence of ArlS, ArlS phosphotransfer to ArlR is required for S. aureus to overcome calprotectin-induced nutritional stress. Collectively, this study provides fundamental information to understand how ArlRS mediates staphylococcal adaptation during infection.

The pck (cg3169) gene of Corynebacterium glutamicum encodes a phosphoenolpyruvate carboxykinase (PEPCK). Here, a candidate transcriptional regulator that binds to the promoter region of pck was detected using a DNA affinity purification approach. An isolated protein was identified to be PckR (Cg0196), a GntR family transcriptional regulator which consists of 253 amino acids with a mass of 27 kDa as measured by peptide mass fingerprinting. The results of electrophoretic mobility shift assays verified that PckR specifically binds to the pck promoter. The putative regulator binding region extended from position -44 to -27 (an 18-bp sequence) relative to the transcriptional start point of the pck gene. We measured the expression of pck in a pcRR deletion mutant by using quantitative real-time reverse transcription-PCR. The expression level of pck in the pckR mutant was 7.6 times higher than that in wild-type cells grown in glucose. Comparative DNA microarray hybridizations and bioinformatic searches revealed the gene composition of the transcriptional regulon of C. glutamicum. Based on these results, PckR seemed to play an important role in the regulation of PEPCK in C. glutamicum grown in glucose. In particular, these assays revealed that PckR acts as a repressor of pck expression during glucose metabolism. [ABSTRACT FROM AUTHOR]

Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-l-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coil cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high Vmax in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (Vmax of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenoipyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium. [ABSTRACT FROM AUTHOR]

The glucose-phosphotransferase system (PTS) in Escherichia coli K-12 is a complex sensory and regulatory system. In addition to its central role in glucose uptake, it informs other global regulatory networks about carbohydrate availability and the physiological status of the cell. The expression of the ptsG gene encoding the glucose-PTS transporter EIICBGlc is primarily regulated via the repressor Mlc, whose inactivation is glucose dependent. During transport of glucose and dephosphorylation of EIICBGlc, Mlc binds to the B domain of the transporter, resulting in derepression of several Mlc-regulated genes. In addition, Mlc can also be inactivated by the cytoplasmic protein MtfA in a direct protein-protein interaction. In this study, we identified the binding site for Mlc in the carboxy-terminal region of MtfA by measuring the effect of mutated MtfAs on ptsG expression. In addition, we demonstrated the ability of MtfA to inactivate an Mlc super-repressor, which cannot be inactivated by EIICBGlc, by using in vivo titration and gel shift assays. Finally, we characterized the proteolytic activity of purified MtfA by monitoring cleavage of amino 4-nitroanilide substrates and show Mlc's ability to enhance this activity. Based on our findings, we propose a model of MtfA as a glucose-regulated peptidase activated by cytoplasmic Mlc. Its activity may be necessary during the growth of cultures as they enter the stationary phase. This proteolytic activity of MtfA modulated by Mlc constitutes a newly identified PTS output signal that responds to changes in environmental conditions. [ABSTRACT FROM AUTHOR]

Carbon catabolite repression (CCR) is a widespread phenomenon in many bacteria that is defined as the repression of catabolic enzyme activities for an unfavorable carbon source by the presence of a preferable carbon source. In Streptomyces, secondary metabolite production often is negatively affected by the carbon source, indicating the involvement of CCR in secondary metabolism. Although the CCR mechanism in Streptomyces still is unclear, glucokinase is presumably a central player in CCR. SgGIkA, a glucokinase from S. griseus, belongs to the ROK family glucokinases, which have two consensus sequence motifs (1 and 2). Here, we report the crystal structures of apo-SgGlkA, SgG1kA in complex with glucose, and SgG1kA in complex with glucose and adenylyl imidodiphosphate (AMPPNP), which are the first structures of an ROK family glucokinase. SgG1kA is divided into a small α/β domain and a large α+β domain, and it forms a dimer-of-dimer tetrameric configuration. SgG1kA binds a β-anomer of glucose between the two domains, and His157 in consensus sequence 1 plays an important role in the glucose-binding mechanism and anomer specificity of SgG1kA. In the structures of SgG1kA, His 157 forms an HC3-type zinc finger motif with three cysteine residues in consensus sequence 2 to bind a zinc ion, and it forms two hydrogen bonds with the C1 and C2 hydroxyls of glucose. When the three structures are compared, the structure of SgG1kA is found to be modified by the binding of substrates. The substrate-dependent conformational changes of SgG1kA may be related to the CCR mechanism in Streptomyces. [ABSTRACT FROM AUTHOR]

There are two closely related hik31 operons involved in signal transduction on the chromosome and the pSYSX plasmid in the cyanobacterium Synechocystis sp. strain PCC 6803. We studied the growth, cell morphology, and gene expression in operon and hik mutants for both copies, under different growth conditions, to examine whether the duplicated copies have the same or different functions and gene targets and whether they are similarly regulated. Phenotype analysis suggested that both operons regulated common and separate targets in the light and the dark. The chromosomal operon was involved in the negative control of autotrophic events, whereas the plasmid operon was involved in the positive control of heterotrophic events. Both the plasmid and double operon mutant cells were larger and had division defects. The growth data also showed a regulatory role for the chromosomal hik gene under high-CO2 conditions and the plasmid operon under low-O2 conditions. Metal stress experiments indicated a role for the chromosomal hik gene and operon in mediating Zn and Cd tolerance, the plasmid operon in Co tolerance, and the chromosomal operon and plasmid hik gene in Ni tolerance. We conclude that both operons are differentially and temporally regulated. We suggest that the chromosomal operon is the primarily expressed copy and the plasmid operon acts as a backup to maintain appropriate gene dosages. Both operons share an integrated regulatory relationship and are induced in high light, in glucose, and in active cell growth. Additionally, the plasmid operon is induced in the dark with or without glucose. [ABSTRACT FROM AUTHOR]

NAD kinase (NADK), which phosphorylates NAD to NADP, is one of the key enzymes regulating the cellular NADP(H) leve Synechocystis sp. strain PCC 6803, slr0400 and sll1415 were shown to encode NAD kinases. The NADP(H) pool in the cyanoterium was remarkably reduced by an sll1415-null mutation but slightly reduced by an slr0400-null mutation. The reduction the NADP(H) level in the sll1415 mutant led to a significant accumulation of glucose-6-phosphate and a loss of photohetero trophic growth. As the primary NADK gene, sll1415 was found to inhibit the transcription of genes involved in redox homeosta sis and to exert stronger effects on methyl viologen tolerance than slr0040. [ABSTRACT FROM AUTHOR]

The nonsulfur purple bacteria that exhibit unusual metabolic versatility can produce hydrogen gas (H2) using the electrons derived from metabolism of organic compounds during photoheterotrophic growth. Here, based on 13C tracer experiments, we identified the network of glucose metabolism and quantified intracellular carbon fluxes in Rhodobacter sphaeroides KD131 grown under H2-producing conditions. Moreover, we investigated how the intracellular fluxes in R. sphaeroides responded to knockout mutations in hydrogenase and poly-β-hydroxybutyrate synthase genes, which led to increased H2 yield. The relative contribution of the Entner-Doudoroff pathway and Calvin-Benson-Bassham cycle to glucose metabolism differed significantly in hydrogenase-deficient mutants, and this flux change contributed to the increased formation of the redox equivalent NADH. Disruption of hydrogenase and poly-β-hydroxybutyrate synthase resulted in a significantly increased flux through the phosphoenolpyruvate carboxykinase and a reduced flux through the malic enzyme. A remarkable increase in the flux through the tricarboxylic acid cycle, a major NADH producer, was observed for the mutant strains. The in vivo regulation of the tricarboxylic acid cycle flux in photoheterotrophic R. sphaeroides was discussed based on the measurements of in vitro enzyme activities and intracellular concentrations of NADH and NAD+. Overall, our results provide quantitative insights into how photoheterotrophic cells manipulate the metabolic network and redistribute intracellular fluxes to generate more electrons for increased H2 production. [ABSTRACT FROM AUTHOR]

Escherichia coli K-12 suffers acetic acid stress during prolonged incubation in glucose minimal medium containing a limiting concentration of inorganic phosphate (0.1 mM Pi), which decreases the number of viable cells from 6 x 108 to ≤ 10 CFU/ml between days 6 and 14 of incubation. Here we show that following two serial transfers into P≤i-limiting medium, evolved mutants survived prolonged incubation (≈107 CFU/ml on day 14 of incubation). The evolved strains that overtook the populations were generally PhnE+, whereas the ancestral K-12 strain carries an inactive phnE allele, which prevents the transport of phosphonates. The switching in phnE occurred with a high frequency as a result of the deletion of an 8-bp repeated sequence. In a mixed culture starved for P≤ that contained the K-12 ancestral strain in majority, evolved strains grew through PhnE-dependent scavenging of probably organic phosphate esters (not phosphonates or P≤) released by E. coli K-12 between days 1 and 3, before acetic acid excreted by E. coli K-12 reached toxic levels. The growth yield of phnE+ strains in mixed culture was dramatically enhanced by mutations that affect glucose metabolism, such as an rpoS mutation inactivating the alternative sigma factor RpoS. The long-term viability of evolved populations was generally higher when the ancestral strain carried an inactive rather than an active phnE allele, which indicates that cross-feeding of phosphorylated products as a result of the phnE polymorphism may be essential for the spread of mutants which eventually help populations to survive under P≤ starvation conditions. [ABSTRACT FROM AUTHOR]

Caulobacter crescentus differentiates from a motile, foraging swarmer cell into a sessile, replication-competent stalked cell during its cell cycle. This developmental transition is inhibited by nutrient deprivation to favor the motile swarmer state. We identify two cell cycle regulatory signals, ppGpp and polyphosphate (polyP), that inhibit the swarmer-to-stalked transition in both complex and glucose-exhausted media, thereby increasing the proportion of swarmer cells in mixed culture. Upon depletion of available carbon, swarmer cells lacking the ability to synthesize ppGpp or polyp improperly initiate chromosome replication, proteolyze the replication inhibitor CtrA, localize the cell fate determinant DivJ, and develop polar stalks. Furthermore, we show that swarmer cells produce more ppGpp than stalked cells upon starvation. These results provide evidence that ppGpp and polyp are cell-type-specific developmental regulators. [ABSTRACT FROM AUTHOR]

Glucose is a carbon source that is capable of modulating the level of cyclic AMP (cAMP)-regulated genes. In the present study, we found that the stability of ompA mRNA was reduced in Escherichia coli when glucose (40 mM) was present in Luria-Bertani (LB) medium. This effect was associated with a low level of cAMP induced by the glucose. The results were confirmed with an adenylyl cyclase mutant with low levels of cAMP that are not modulated by glucose. Northern blot and Western blot analyses revealed that the host factor I (Hfq) (both mRNA and protein) levels were downregulated in the presence of cAMP. Furthermore, we showed that a complex of cAMP receptor protein (CRP) and cAMP binds to a specific P3hfq promoter region of hfq and regulates hfq expression. The regulation of the hfq gene was confirmed in vivo using an hfq-deficient mutant transformed with an exogenous hfq gene containing the promoter. These results demonstrated that expression of hfq was repressed by the CRP-cAMP complex. The presence of glucose resulted in increased Hfq protein levels, which decreased ompA mRNA stability. An additional experiment showed that cAMP also increased the stability of fur mRNA. Taken together, these results suggested that the repression of Hfq by cAMP may contribute to the stability of other mRNA in E. coli. [ABSTRACT FROM AUTHOR]

Xylose is rarely described as a component of bacterial glycans. UDP-xylose is the nucleotide-activated form necessary for incorporation of xylose into glycans and is synthesized by the decarboxylation of UDP-glucuronic acid (UDP-GlcA). Enzymes with UDP-GlcA decarboxylase activity include those that lead to the formation of UDP-xylose as the end product (Uxs type) and those synthesizing UDP-xylose as an intermediate (ArnA and RsU4kpxs types). In this report, we identify and confirm the activities of two Uxs-type UDP-GlcA decarboxylases of Bacteroides fragilis, designated BfUxs1 and BfUxs2. Bfuxs1 is located in a conserved region of the B. fragilis genome, whereas Bfuxs2 is in the heterogeneous capsular polysaccharide F (PSF) biosynthesis locus. Deletion of either gene separately does not result in the loss of a detectable phenotype, but deletion of both genes abrogates PSF synthesis, strongly suggesting that they are functional paralogs and that the B. fragilis NCTC 9343 PSF repeat unit contains xylose. UDP-GlcA decarboxylases are often annotated incorrectly as NAD-dependent epimerases/dehydratases; therefore, their prevalence in bacteria is underappreciated. Using available structural and mutational data, we devised a sequence pattern to detect bacterial genes encoding UDP-GlcA decarboxylase activity. We identified 826 predicted UDP-GlcA decarboxylase enzymes in diverse bacterial species, with the ArnA and RsU4kpxs types confined largely to proteobacterial species. These data suggest that xylose, or a monosaccharide requiring a UDP-xylose intermediate, is more prevalent in bacterial glycans than previously appreciated. Genes encoding BfUxs1-like enzymes are highly conserved in Bacteroides species, indicating that these abundant intestinal microbes may synthesize a conserved xylose-containing glycan. [ABSTRACT FROM AUTHOR]

Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maitodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in ΔmalP (encoding maltodextrin phosphorylase), ΔmalQ (encoding amylomaltase), ΔglgA (encoding glycogen synthase), and ΔglgA ΔmalP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the ΔmalP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the ΔmalP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The ΔmalQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the ΔglgA and ΔglgA ΔmalP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro, MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase. [ABSTRACT FROM AUTHOR]

Sulfolobus acidocaidarius utilizes glucose and xylose as sole carbon sources, but its ability to metabolize these sugars simultaneously is not known. We report the absence of diauxie during growth of S. acidocaldarius on glucose and xylose as co-carbon sources. The presence of glucose did not repress xylose utilization. The organism utilized a mixture of 1 g/liter of each sugar simultaneously with a specific growth rate of 0.079 h-1 and showed no preference for the order in which it utilized each sugar. The organism grew faster on 2 g/liter xylose (0.074 h-1) as the sole carbon source than on an equal amount of glucose (0.022 h-1). When grown on a mixture of the two carbon sources, the growth rate of the organism increased from 0.052 h-1 to 0.085 h-1 as the ratio of xylose to glucose increased from 0.25 to 4. S. acidocaldarius appeared to utilize a mixture of glucose and xylose at a rate roughly proportional to their concentrations in the medium, resulting in complete utilization of both sugars at about the same time. Gene expression in cells grown on xylose alone was very similar to that in cells grown on a mixture of xylose and glucose and substantially different from that in cells grown on glucose alone. The mechanism by which the organism utilized a mixture of sugars has yet to be elucidated. [ABSTRACT FROM AUTHOR]

Corynebacterium glutamicum R has two (i-glucoside phosphoenolpyruvate, carbohydrate phosphotransferase systems (PTS) encoded by bglF and bglF2 located in the respective clusters, bglF-bgJA-bglG and bglF2-bglA2-bglG2. Previously, we reported that whereas p-glucoside-dependent induction of bglF is strongly repressed by glucose, glucose repression of bglF2 is very weak. Here, we reveal the mechanism behind the different effects of glucose on the two bgl genes. Deletion of the ribonucleic antiterminator sequence and transcriptional terminator located upstream of the translation initiation codon of bglF markedly relieved the glucose repression of a bglF-lacZ fusion, indicating that glucose affects the antitermination mechanism that is responsible for the P-glucoside-dependent induction of the bglF cluster. The glucose repression ofbglF mRNA was also relieved by introducing a multicopy plasmid carrying the bglG gene encoding an antiterminator of the bglF cluster. Moreover, replacement of the GUG translation initiation codon otbglG with AUG was effective in relieving the glucose repression of bglF and bglG. Inversely, expression of bglF2 and bglG2 was subject to strict glucose repression in a mutant strain in which the AUG translation initiation codon of bglG2 encoding antiterminator of the bglF2 cluster was replaced with GUG. These results suggest that the translation initiation efficiency of the antiterminator proteins, at least in part, determines whether the target genes are subject to glucose repression. We also found that bglF expression was induced by glucose in the BglG-overexpressing strains, which may be explained by the ability of BglF to transport glucose. [ABSTRACT FROM AUTHOR]

Accumulation of certain nonmetabolizable sugar-phosphates (including α-methyl glucoside-6-phosphate) in Escherichia coli is growth inhibitory and elicits the glucose-phosphate stress response. The transcription factor SgrR activates transcription of the small RNA SgrS under stress conditions. SgrS represses translation of mRNAs encoding sugar transporters. The sgrR and sgrS genes are located directly upstream of setA, and this gene organization is conserved in numerous enteric species, prompting the hypothesis that SetA contributes to the glucose-phosphate stress response. SetA is a proton motive force-driven efflux pump capable of transporting various sugars and sugar analogs in vitro. This study demonstrates that setA expression is induced in response to glucose-phosphate stress, and this requires SgrR. Under stress conditions, setA is cotranscribed with sgrS from the sgrS promoter. A setA mutant exhibits a growth defect under stress conditions that can be complemented by setA in trans, suggesting that SetA contributes to the optimal cellular recovery from stress. Despite previous in vitro evidence that SetA can promote efflux of the stress-causing glucose analog a-methyl glucoside, in vivo data in this study indicate that SetA is not the major efflux pump responsible for removal of α-methyl glucoside under stress conditions. [ABSTRACT FROM AUTHOR]

Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes L-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S L-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of L-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C1 units and interferes with cell wall synthesis. We suggest that at high concentrations, L-serine decreases synthesis of UDP-N-acetylmuramate- L-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high L-serine is overcome in several ways: by a large concentration of L-alanine, by overproducing MurC together with a low concentration of t.-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide. [ABSTRACT FROM AUTHOR]

N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ~4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 µM in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in deuterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. L-[ethyl-²H4]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose. [ABSTRACT FROM AUTHOR]

Beneficial mutations in diversifying glucose-limited Escherichia coli populations are mostly unidentified. The genome of an evolved isolate with multiple differences from that of the ancestor was fully assembled. Remarkably, a single mutation in hfq was responsible for the multiple benefits under glucose limitation through changes in at least five regulation targets. [ABSTRACT FROM AUTHOR]