HPr prevents FruR-mediated facilitation of RNA polymerase binding to the fru promoter in Vibrio cholerae.

Phosphorylation state-dependent interactions of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) components with transcription factors play a key role in carbon catabolite repression (CCR) by glucose in bacteria. Glucose inhibits the PTS-dependent transport of fructose and is preferred over fructose in Vibrio cholerae, but the mechanism is unknown. We have recently shown that, contrary to Escherichia coli, the fructose-dependent transcriptional regulator FruR acts as an activator of the fru operon in V. cholerae and binding of the FruR-fructose 1-phosphate (F1P) complex to an operator facilitates RNA polymerase (RNAP) binding to the fru promoter. Here we show that, in the presence of glucose, dephosphorylated HPr, a general PTS component, binds to FruR. Whereas HPr does not affect DNA-binding affinity of FruR, regardless of the presence of F1P, it prevents the FruR-F1P complex from facilitating the binding of RNAP to the fru promoter. Structural and biochemical analyses of the FruR-HPr complex identify key residues responsible for the V. cholerae-specific FruR-HPr interaction not observed in E. coli. Finally, we reveal how the dephosphorylated HPr interacts with FruR in V. cholerae, whereas the phosphorylated HPr binds to CcpA, which is a global regulator of CCR in Bacillus subtilis and shows structural similarity to FruR.

A pGpG-specific phosphodiesterase regulates cyclic di-GMP signaling in Vibrio cholerae.

The bacterial second messenger bis-(3'-5')-cyclic diguanylate monophosphate (c-di-GMP) controls various cellular processes, including motility, toxin production, and biofilm formation. c-di-GMP is enzymatically synthesized by GGDEF domain-containing diguanylate cyclases and degraded by HD-GYP domain-containing phosphodiesterases (PDEs) to 2 GMP or by EAL domain-containing PDE-As to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). Since excess pGpG feedback inhibits PDE-A activity and thereby can lead to the uncontrolled accumulation of c-di-GMP, a PDE that degrades pGpG to 2 GMP (PDE-B) has been presumed to exist. To date, the only enzyme known to hydrolyze pGpG is oligoribonuclease Orn, which degrades all kinds of oligoribonucleotides. Here, we identified a pGpG-specific PDE, which we named PggH, using biochemical approaches in the gram-negative bacteria Vibrio cholerae. Biochemical experiments revealed that PggH exhibited specific PDE activity only toward pGpG, thus differing from the previously reported Orn. Furthermore, the high-resolution structure of PggH revealed the basis for its PDE activity and narrow substrate specificity. Finally, we propose that PggH could modulate the activities of PDE-As and the intracellular concentration of c-di-GMP, resulting in phenotypic changes including in biofilm formation.

Effect of Clostridium butyricum on High-Fat Diet-Induced Intestinal Inflammation and Production of Short-Chain Fatty Acids.

BACKGROUND/AIMS: A high-fat diet (HFD) can cause intestinal inflammation and alter the gut microbiota; probiotics, however, are known to have anti-inflammatory effects. This study aimed to investigate the response of rat colon to HFD and the effect of Clostridium butyricum on HFD-induced intestinal inflammation and production of short-chain fatty acids (SCFAs) according to sex. METHODS: Male and female 6-week-old Fischer-344 rats were fed a chow diet or HFD for 8 weeks, and Biovita or three different concentrations of C. butyricum were orally gavaged. The levels of tight junction proteins (TJPs), inflammatory markers in the ascending colonic mucosa, and bile acids (BAs) and SCFAs in stool were measured. RESULTS: HFD significantly increased the histological inflammation scores and fat proportions. Fecal BA levels were higher in the HFD group than in the control group, with a more prominent increase in deoxycholic acid/cholic acid after probiotics administration in females; however, no statistically significant differences were observed. TJPs showed an opposite response to HFD depending on sex, and tended to increase and decrease after HFD in males and females, respectively. The HFD-reduced TJPs were recovered by probiotics, with some statistical significance in females. HFD-decreased butyric acid in stools appeared to be recovered by probiotics in males, but not in females. The expression of inflammatory markers (TNF-α) was increased by HFD in males and decreased with medium-concentration probiotic supplementation. The opposite was observed in females. MPO was increased by HFD in both sexes and decreased by probiotic supplementation. CONCLUSIONS: The probiotic C. butyricum improved indicators of HFD-induced colonic inflammation such as levels of inflammatory markers and increased the production of SCFAs and the expression of TJPs. These effects tended to be more pronounced in male rats, showing sex difference.

Vibrio cholerae FruR facilitates binding of RNA polymerase to the fru promoter in the presence of fructose 1-phosphate.

In most bacteria, efficient use of carbohydrates is primarily mediated by the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), which concomitantly phosphorylates the substrates during import. Therefore, transcription of the PTS-encoding genes is precisely regulated by transcriptional regulators, depending on the availability of the substrate. Fructose is transported mainly through the fructose-specific PTS (PTSFru) and simultaneously converted into fructose 1-phosphate (F1P). In Gammaproteobacteria such as Escherichia coli and Pseudomonas putida, transcription of the fru operon encoding two PTSFru components, FruA and FruB, and the 1-phosphofructokinase FruK is repressed by FruR in the absence of the inducer F1P. Here, we show that, contrary to the case in other Gammaproteobacteria, FruR acts as a transcriptional activator of the fru operon and is indispensable for the growth of Vibrio cholerae on fructose. Several lines of evidence suggest that binding of the FruR-F1P complex to an operator which is located between the -35 and -10 promoter elements changes the DNA structure to facilitate RNA polymerase binding to the promoter. We discuss the mechanism by which the highly conserved FruR regulates the expression of its target operon encoding the highly conserved PTSFru and FruK in a completely opposite direction among closely related families of bacteria.

Phosphotransferase system sugars immediately induce mutations of Cra in an Escherichia coli ptsH mutant.

Most bacteria use the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) to catalyse coupled transport and phosphorylation of sugars. The PTS consists of several sugar-specific components (enzyme IIs) and two general components: enzyme I, encoded by ptsI, and HPr, encoded by ptsH, which are common to most PTS carbohydrates. Although both enzyme I and HPr are believed to be required to utilize these PTS sugars, an E. coli ptsH mutant has been reported to exhibit a leaky growth phenotype on these sugars. Here, we show that this phenomenon occurs because the ptsH mutant undergoes adaptive mutations in the presence of PTS sugars within a few generation times. The ptsH mutant cells once exposed to a PTS sugar showed a growth rate similar to that of the wild-type strain when transferred to fresh medium supplemented with the same PTS sugar, suggesting the acquisition of additional genetic variations. Genome sequencing revealed that the PTS sugar-adapted variants harboured loss-of-function mutations in cra, which increased expression of the fruBKA operon. Our results suggest that the presence of a PTS sugar can exert a strong selective pressure when a general PTS component is defective.

Evidence for reciprocal evolution of the global repressor Mlc and its cognate phosphotransferase system sugar transporter.

Because the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is involved in the regulation of various physiological processes in addition to carbohydrate transport, its expression is precisely regulated in response to the availability of PTS sugars. The PTS consists of enzyme I and histidine phosphocarrier protein, and several sugar-specific enzymes II. In Escherichia coli, genes for enzymes II specific for glucose and related sugars are co-regulated by the global repressor Mlc, and glucose induction of the Mlc regulon genes is achieved by its interaction with glucose-specific enzyme II (EIIGlc ). In this study, we revealed that, in Vibrio species, which are phylogenetically older than Enterobacteriaceae, the membrane sequestration of Mlc and thereby the induction of its regulon genes is mediated by N-acetylglucosamine (NAG)-specific EII. While Vibrio Mlc interacts only with the EIIB domain of EIINag , E. coli Mlc interacts with the EIIB domain of both EIIGlc and EIINag . The present data suggest that EIINag may be the primordial regulator of Mlc, and EIIGlc has evolved to interact with Mlc since an EIIA domain was fused to EIINag in Enterobacteriaceae. Our findings provide insight into the coevolutionary dynamics between a transcription factor and its cognate regulator according to long-term resource availability in the bacterial natural habitat.

Functional dissection of the phosphotransferase system provides insight into the prevalence of Faecalibacterium prausnitzii in the host intestinal environment.

Faecalibacterium prausnitzii is a dominant member of healthy human colon microbiota, regarded as a beneficial gut bacterium due to its ability to produce anti-inflammatory substances. However, little is known about how F. prausnitzii utilizes the nutrients present in the human gut, influencing its prevalence in the host intestinal environment. The phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) is a widely distributed and highly efficient carbohydrate transport system found in most bacterial species that catalyses the simultaneous phosphorylation and import of cognate carbohydrates; its components play physiological roles through interaction with other regulatory proteins. Here, we performed a systematic analysis of the 16 genes encoding putative PTS components (2 enzyme I, 2 HPr, and 12 enzyme II components) in F. prausnitzii A2-165. We identified the general PTS components responsible for the PEP-dependent phosphotransfer reaction and the sugar-specific PTS components involved in the transport of two carbohydrates, N-acetylglucosamine and fructose, among five enzyme II complexes. We suggest that the dissection of the functional PTS in F. prausnitzii may help to understand how this species outcompetes other bacterial species in the human intestine.

Rsd balances (p)ppGpp level by stimulating the hydrolase activity of SpoT during carbon source downshift in Escherichia coli.

Bacteria respond to nutritional stresses by changing the cellular concentration of the alarmone (p)ppGpp. This control mechanism, called the stringent response, depends on two enzymes, the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT in Escherichia coli and related bacteria. Because SpoT is the only enzyme responsible for (p)ppGpp hydrolysis in these bacteria, SpoT activity needs to be tightly regulated to prevent the uncontrolled accumulation of (p)ppGpp, which is lethal. To date, however, no such regulation of SpoT (p)ppGpp hydrolase activity has been documented in E. coli In this study, we show that Rsd directly interacts with SpoT and stimulates its (p)ppGpp hydrolase activity. Dephosphorylated HPr, but not phosphorylated HPr, of the phosphoenolpyruvate-dependent sugar phosphotransferase system could antagonize the stimulatory effect of Rsd on SpoT (p)ppGpp hydrolase activity. Thus, we suggest that Rsd is a carbon source-dependent regulator of the stringent response in E. coli.

Polar landmark protein HubP recruits flagella assembly protein FapA under glucose limitation in Vibrio vulnificus.

How motile bacteria recognize their environment and decide whether to stay or navigate toward more favorable location is a fundamental issue in survival. The flagellum is an elaborate molecular device responsible for bacterial locomotion, and the flagellum-driven motility allows bacteria to move themselves to the appropriate location at the right time. Here, we identify the polar landmark protein HubP as a modulator of polar flagellation that recruits the flagellar assembly protein FapA to the old cell pole, thereby controlling its activity for the early events of flagellar assembly in Vibrio vulnificus. We show that dephosphorylated EIIAGlc of the PEP-dependent sugar transporting phosphotransferase system sequesters FapA from HubP in response to glucose and hence inhibits FapA-mediated flagellation. Thus, flagellar assembly and motility is governed by spatiotemporal control of FapA, which is orchestrated by the competition between dephosphorylated EIIAGlc and HubP, in the human pathogen V. vulnificus.

Noncanonical DNA-binding mode of repressor and its disassembly by antirepressor.

DNA-binding repressors are involved in transcriptional repression in many organisms. Disabling a repressor is a crucial step in activating expression of desired genes. Thus, several mechanisms have been identified for the removal of a stably bound repressor (Rep) from the operator. Here, we describe an uncharacterized mechanism of noncanonical DNA binding and induction by a Rep from the temperate Salmonella phage SPC32H; this mechanism was revealed using the crystal structures of homotetrameric Rep (92-198) and a hetero-octameric complex between the Rep and its antirepressor (Ant). The canonical method of inactivating a repressor is through the competitive binding of the antirepressor to the operator-binding site of the repressor; however, these studies revealed several noncanonical features. First, Ant does not compete for the DNA-binding region of Rep. Instead, the tetrameric Ant binds to the C-terminal domains of two asymmetric Rep dimers. Simultaneously, Ant facilitates the binding of the Rep N-terminal domains to Ant, resulting in the release of two Rep dimers from the bound DNA. Second, the dimer pairs of the N-terminal DNA-binding domains originate from different dimers of a Rep tetramer (trans model). This situation is different from that of other canonical Reps, in which two N-terminal DNA-binding domains from the same dimeric unit form a dimer upon DNA binding (cis model). On the basis of these observations, we propose a noncanonical model for the reversible inactivation of a Rep by an Ant.