Phosphotransferase System Components Research Articles

Novel motif associated with carbon catabolite repression in two major Gram-positive pathogen virulence regulatory proteins.

Carbon catabolite repression (CCR) is a widely conserved regulatory process that ensures enzymes and transporters of less-preferred carbohydrates are transcriptionally repressed in the presence of a preferred carbohydrate. This phenomenon can be regulated via a CcpA-dependent or CcpA-independent mechanism. The CcpA-independent mechanism typically requires a transcriptional regulator harboring a phosphotransferase regulatory domain (PRD) that interacts with phosphotransferase system (PTS) components. PRDs contain a conserved histidine residue that is phosphorylated by the PTS-associated HPr-His15~P protein. PRD-containing regulators often harbor additional domains that resemble PTS-associated EIIB protein domains with a conserved cysteine residue that can be phosphorylated by cognate PTS components. We noted that Mga, the PRD-containing central virulence regulator of Streptococcus pyogenes, has an EIIBGat domain containing a cysteine that, based on the presence of a similar motif in glycerol kinase, could be a target for phosphorylation. Using site-directed mutagenesis, we constructed phospho-ablative and phospho-mimetic substitutions of this cysteine and found that these substitutions modify the CCR of the Rgg2/3 quorum-sensing system. Moreover, we provide genetic evidence that the phospho-donor of this cysteine residue is likely to be ManL, the EIIA/B subunit of the mannose PTS system. Interestingly, a structurally distinct virulence gene regulator, PrfA of Listeria monocytogenes, harbors a similar cysteine-containing motif, and phospho-ablative and phospho-mimetic substitutions of the cysteine-altered CCR of PrfA-dependent virulence gene expression. Collectively, our data suggest that phosphorylation of a cysteine within the shared novel motif in Mga and PrfA may be a heretofore missing link between cellular metabolism and virulence.IMPORTANCEIn this study, we identified a novel cysteine-containing motif within the amino acid sequence of two structurally distinct transcriptional regulators of virulence in two Gram-positive pathogens that appears to link carbon metabolism with virulence gene expression. The results also highlight the potential post-translational modification of cysteine in bacterial species, a rare and understudied modification.

Catabolite control protein C contributes to virulence and hydrogen peroxide-induced oxidative stress responses in Listeria monocytogenes.

Listeria monocytogenes causes listeriosis, an infectious and potentially fatal disease of animals and humans. A diverse network of transcriptional regulators, including LysR-type catabolite control protein C (CcpC), is critical for the survival of L. monocytogenes and its ability to transition into the host environment. In this study, we explored the physiological and genetic consequences of deleting ccpC and the effects of such deletion on the ability of L. monocytogenes to cause disease. We found that ccpC deletion did not impact hemolytic activity, whereas it resulted in significant reductions in phospholipase activities. Western blotting revealed that the ΔccpC strain produced significantly reduced levels of the cholesterol-dependent cytolysin LLO relative to the wildtype F2365 strain. However, the ΔccpC mutant displayed no significant intracellular growth defect in macrophages. Furthermore, ΔccpC strain exhibited reduction in plaque numbers in fibroblasts compared to F2365, but plaque size was not significantly affected by ccpC deletion. In a murine model system, the ΔccpC strain exhibited a significantly reduced bacterial burden in the liver and spleen compared to the wildtype F2365 strain. Interestingly, the deletion of this gene also enhanced the survival of L. monocytogenes under conditions of H2O2-induced oxidative stress. Transcriptomic analyses performed under H2O2-induced oxidative stress conditions revealed that DNA repair, cellular responses to DNA damage and stress, metalloregulatory proteins, and genes involved in the biosynthesis of peptidoglycan and teichoic acids were significantly induced in the ccpC deletion strain relative to F2365. In contrast, genes encoding internalin, 1-phosphatidylinositol phosphodiesterase, and genes associated with sugar-specific phosphotransferase system components, porphyrin, branched-chain amino acids, and pentose phosphate pathway were significantly downregulated in the ccpC deletion strain relative to F2365. This finding highlights CcpC as a key factor that regulates L. monocytogenes physiology and responses to oxidative stress by controlling the expression of important metabolic pathways.

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.

Functional and structural diversification of incomplete phosphotransferase system in cellulose-degrading clostridia.

Carbohydrate utilization is critical to microbial survival. The phosphotransferase system (PTS) is a well-documented microbial system with a prominent role in carbohydrate metabolism, which can transport carbohydrates through forming a phosphorylation cascade and regulate metabolism by protein phosphorylation or interactions in model strains. However, those PTS-mediated regulated mechanisms have been underexplored in non-model prokaryotes. Here, we performed massive genome mining for PTS components in nearly 15,000 prokaryotic genomes from 4,293 species and revealed a high prevalence of incomplete PTSs in prokaryotes with no association to microbial phylogeny. Among these incomplete PTS carriers, a group of lignocellulose degrading clostridia was identified to have lost PTS sugar transporters and carry a substitution of the conserved histidine residue in the core PTS component, HPr (histidine-phosphorylatable phosphocarrier). Ruminiclostridium cellulolyticum was then selected as a representative to interrogate the function of incomplete PTS components in carbohydrate metabolism. Inactivation of the HPr homolog reduced rather than increased carbohydrate utilization as previously indicated. In addition to regulating distinct transcriptional profiles, PTS associated CcpA (Catabolite Control Protein A) homologs diverged from previously described CcpA with varied metabolic relevance and distinct DNA binding motifs. Furthermore, the DNA binding of CcpA homologs is independent of HPr homolog, which is determined by structural changes at the interface of CcpA homologs, rather than in HPr homolog. These data concordantly support functional and structural diversification of PTS components in metabolic regulation and bring novel understanding of regulatory mechanisms of incomplete PTSs in cellulose-degrading clostridia.

Engineering D-glucose utilization in Azospirillum brasilense Sp7 promotes rice root colonization.

Bacteria of the genus Azospirillum include several plant associated bacteria which often promote the growth of their host plants. Although the host range of Azospirillum brasilense Sp7 is much wider than its close relative Azospirillum lipoferum 4B, it lacks the ability to efficiently utilize D-glucose for its growth. By comparing the genomes of both the species, the genes of A. lipoferum 4B responsible for conferring D-glucose utilization ability in A. brasilese Sp7 were identified by cloning individual or a combination of genes in a broad host range expression vector, mobilizing them in A. brasilense Sp7 and examining the ability of exconjugants to use D-glucose as sole carbon source for growth. These genes also included the homologs of genes involved in N-acetyl glucosamine utilization in Pseudomonas aeruginosa PAO1. A transcriptional fusion of the 5 genes encoding glucose-6-phosphate dehydrogenase and 4 components of glucose phosphotransferase system were able to improve D-glucose utilization ability in A. brasilense Sp7. The A. brasilense Sp7 strain engineered with D-glucose utilization ability showed significantly improved root colonization of rice seedling. The improvement in the ability of A. brasilense Sp7 to colonize rice roots is expected to bring benefits to rice by promoting its growth. KEY POINTS: • Genes required for glucose utilization in Azospirillum lipoferum were identified. • A gene cassette encoding glucose utilization was constructed. • Transfer of gene cassette in A. brasilense improves glucose utilization and rice root colonization..

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.

Glucose Transport through N-Acetylgalactosamine Phosphotransferase System in Escherichia coli C Strain.

When ptsG, a glucose-specific phosphotransferase system (PTS) component, is deleted in Escherichia coli, growth can be severely poor because of the lack of efficient glucose transport. We discovered a new PTS transport system that could transport glucose through the growth-coupled experimental evolution of ptsG-deficient E. coli C strain under anaerobic conditions. Genome sequencing revealed mutations in agaR, which encodes a repressor of N-acetylgalactosamine (Aga) PTS expression in evolved progeny strains. RT-qPCR analysis showed that the expression of Aga PTS gene increased because of the loss-of-function of agaR. We confirmed the efficient Aga PTS-mediated glucose uptake by genetic complementation and anaerobic fermentation. We discussed the discovery of new glucose transporter in terms of different genetic backgrounds of E. coli strains, and the relationship between the pattern of mixed-acids fermentation and glucose transport rate.

Bifunctional alcohol/aldehyde dehydrogenase AdhE controls phospho-transferase system sugar utilization and virulence gene expression by interacting PtsH in Edwardsiella piscicida

The bifunctional alcohol/aldehyde dehydrogenase (AdhE), one of the key enzymes in the bacterial ethanol anaerobic fermentation pathway, is critical for appropriate expression of the genes for the utilization of carbon sources. Knowledge about its global roles in modulating gene expression and metabolomics remains limited. Edwardsiella bacteria includes several important zoonotic pathogenic species including Edwardsiella piscicida, a leading fish pathogen that causes severe economic losses in the aquaculture industry. It is well known to utilize few sugars. In this study, we showed that AdhE is involved in various processes including sugar utilization, bacteria growth, intracellular pH homeostasis, type III/VI secretion system (T3/T6SS) production, and survival in fish. Moreover, our unbiased metabolomics approaches revealed that AdhE modulates a large quantity of metabolic pathways, including amino acids, tricarboxylic acid (TCA) intermediates, sugar and fatty acids. Pull-down and Co-immunoprecipitation (IP) analysis revealed that AdhE interacts with the phospho-transferase system component PtsH that supports the transform of its PTS sugars including mannose to mannose-6P, the established metabolic ligand modulating EvrA activity to control T3/T6SS expression. Collectively, AdhE appears to play important roles in bacterial adapting to the internal environment changes by regulating sugar metabolic pathways and bacterial virulence expression. These observations support a model in which AdhE acts a macromolecule hub accommodating proteins to modulate the PTS and other signaling cascades related to pathogenesis and environmental adaptation in bacterial pathogens, which may provide new perspectives for attempts to attenuate bacterial virulence.

Coordinate regulation of carbohydrate metabolism and virulence by PtsH in pathogen Edwardsiella piscicida.

Carbohydrate metabolism of bacterial pathogens conducts crucial roles in regulating pathogenesis but the molecular mechanisms by which metabolisms and virulence are been modulated and coordinated remain to be illuminated. Here, we investigated in this regard Edwardsiella piscicida, a notorious zoonotic pathogen previously named E. tarda that could ferment very few PTS sugars including glucose, fructose, mannose, N-acetylglucosamine, and N-acetylgalactosamine. We systematically characterized the roles of each of the predicted 23 components of phosphotransferase system (PTS) with the respective in-frame deletion mutants and defined medium containing specific PTS sugar. In addition, PtsH was identified as the crucial PTS component potentiating the utilization of all the tested PTS sugars. Intriguingly, we also found that PtsH while not Fpr was involved in T3SS gene expression and was essential for the pathogenesis of E. piscicida. To corroborate this, His15 and Ser46, the two established PtsH residues involved in phosphorylation cascade, showed redundant roles in regulating T3SS yields. Moreover, PtsH was shown to facilitate mannose uptake and transform it into mannose-6-phosphate, an allosteric substrate established to activate EvrA to augment bacterial virulence. Collectively, our observations provide new insights into the roles of PTS reciprocally regulating carbohydrate metabolism and virulence gene expression.Key points • PTS components’ roles for sugar uptake are systematically determined in Edwardsiella piscicida. • PtsH is involved in saccharides uptake and in the regulation of E. piscicida’s T3SS expression. • PtsH phosphorylation at His15 and Ser46 is essential for the T3SS expression and virulence.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-022-11848-8.

Enzyme systems involved in glucosinolate metabolism in Companilactobacillus farciminis KB1089

Cruciferous vegetables are rich sources of glucosinolates (GSLs). GSLs are degraded into isothiocyanates, which are potent anticarcinogens, by human gut bacteria. However, the mechanisms and enzymes involved in gut bacteria-mediated GSL metabolism are currently unclear. This study aimed to elucidate the enzymes involved in GSL metabolism in lactic acid bacteria, a type of gut bacteria. Companilactobacillus farciminis KB1089 was selected as a lactic acid bacteria strain model that metabolizes sinigrin, which is a GSL, into allylisothiocyanate. The sinigrin-metabolizing activity of this strain is induced under glucose-absent and sinigrin-present conditions. A quantitative comparative proteomic analysis was conducted and a total of 20 proteins that were specifically expressed in the induced cells were identified. Three candidate proteins, β-glucoside-specific IIB, IIC, IIA phosphotransferase system (PTS) components (CfPttS), 6-phospho-β-glucosidase (CfPbgS) and a hypothetical protein (CfNukS), were suspected to be involved in sinigrin-metabolism and were thus investigated further. We hypothesize a pathway for sinigrin degradation, wherein sinigrin is taken up and phosphorylated by CfPttS, and subsequently, the phosphorylated entity is degraded by CfPbgS. As expression of both pttS and pbgS genes clearly gave Escherichia coli host strain sinigrin converting activity, these genes were suggested to be responsible for sinigrin degradation. Furthermore, heterologous expression analysis using Lactococcus lactis suggested that CfPttS was important for sinigrin degradation and CfPbgS degraded phosphorylated sinigrin.

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.

Deciphering the Regulation of the Mannitol Operon Paves the Way for Efficient Production of Mannitol in Lactococcus lactis.

Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positive bacteria, mtlF and mtlD, encoding the enzyme IIA component (EIIAmtl) of the mannitol phosphotransferase system (PTS) and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA, encoding the enzyme IIBC component (EIIBCmtl) of the mannitol PTS, mtlR, encoding a transcriptional activator, and mtlF, as well as a separately expressed mtlD gene. The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive element (cre) genes. The presence of carbon catabolite repression was demonstrated and was shown to be relieved in stationary-phase cells. The transcriptional activator MtlR (mtlR), in some Gram-positive bacteria, is repressed by phosphorylation by EIIAmtl, and when we knocked out mtlF, we indeed observed enhanced expression from the two promoters, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary-phase cells as biocatalysts, we attained 10.1 g/liter mannitol with a 55% yield, which, to the best of our knowledge, is the highest titer ever reported for L. lactis. Summing up, the results of our study should be useful for improving the mannitol-producing capacity of this important industrial organism. IMPORTANCE Lactococcus lactis is the most studied species of the lactic acid bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis to produce mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated and demonstrate how this impacts mannitol production capability. We harnessed this information and managed to establish efficient mannitol production without introducing foreign genes.

Streptococcal phosphotransferase system imports unsaturated hyaluronan disaccharide derived from host extracellular matrices.

Certain bacterial species target the polysaccharide glycosaminoglycans (GAGs) of animal extracellular matrices for colonization and/or infection. GAGs such as hyaluronan and chondroitin sulfate consist of repeating disaccharide units of uronate and amino sugar residues, and are depolymerized to unsaturated disaccharides by bacterial extracellular or cell-surface polysaccharide lyase. The disaccharides are degraded and metabolized by cytoplasmic enzymes such as unsaturated glucuronyl hydrolase, isomerase, and reductase. The genes encoding these enzymes are assembled to form a GAG genetic cluster. Here, we demonstrate the Streptococcus agalactiae phosphotransferase system (PTS) for import of unsaturated hyaluronan disaccharide. S. agalactiae NEM316 was found to depolymerize and assimilate hyaluronan, whereas its mutant with a disruption in the PTS genes included in the GAG cluster was unable to grow on hyaluronan, while retaining the ability to depolymerize hyaluronan. Using toluene-treated wild-type cells, the PTS activity for import of unsaturated hyaluronan disaccharide was significantly higher than that observed in the absence of the substrate. In contrast, the PTS mutant was unable to import unsaturated hyaluronan disaccharide, indicating that the corresponding PTS is the only importer of fragmented hyaluronan, which is suitable for PTS to phosphorylate the substrate at the C-6 position. This is distinct from Streptobacillus moniliformis ATP-binding cassette transporter for import of sulfated and non-sulfated fragmented GAGs without substrate modification. The three-dimensional structure of streptococcal EIIA, one of the PTS components, was found to contain a Rossman-fold motif by X-ray crystallization. Docking of EIIA with another component EIIB by modeling provided structural insights into the phosphate transfer mechanism. This study is the first to identify the substrate (unsaturated hyaluronan disaccharide) recognized and imported by the streptococcal PTS. The PTS and ABC transporter for import of GAGs shed light on bacterial clever colonization/infection system targeting various animal polysaccharides.

Regulation of Iron Uptake by Fine-Tuning the Iron Responsiveness of the Iron Sensor Fur.

Iron is one of most abundant environmental metal ions but is highly limited in organisms. It is an important metal ion as it facilitates various biological processes, including catalysis of metabolic enzymes and DNA biogenesis. In bacteria, the ferric uptake regulator (Fur) protein controls iron uptake by regulating genes coding for iron transporters in response to iron concentration. This iron response is ascribed to Fur's intrinsic affinity for iron because its binding to iron dictates its regulatory function. However, we now report that the pathogen Salmonella achieves a proper response of Fur to changes in environmental iron concentrations via EIIANtr (a nitrogen metabolic phosphotransferase system component). We establish that EIIANtr increases expression of iron transporter-coding genes under low-iron conditions (i.e., nanomolar ranges) in a Fur-dependent manner, which promotes Salmonella growth under such conditions. EIIANtr directly hampers Fur binding to DNA, thereby inducing expression of those genes. This regulation allows Salmonella to express Fur-regulated genes under low-iron conditions. Our findings reveal a potentially widespread control mechanism of bacterial iron uptake systems operating in response to iron availability.IMPORTANCE Iron is a fundamental metal ion for living organisms as it facilitates various biological processes. The ferric uptake regulator (Fur) protein controls iron homeostasis in various bacterial species. It is believed that Fur's iron-dependent regulatory action is sufficient for it to function as an iron sensor. However, we now establish that the bacterial pathogen Salmonella enables Fur to properly reflect changes in surrounding iron availability by fine-tuning its responsiveness to iron. This process requires a protein that hampers Fur DNA binding at low iron concentrations. In this way, Salmonella broadens the range of iron concentrations that Fur responds to. Our findings reveal a potentially widespread control mechanism of bacterial iron homeostasis.

Studies of the Listeria monocytogenes Cellobiose Transport Components and Their Impact on Virulence Gene Repression

Background: Many bacteria transport cellobiose via a phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). In Listeria monocytogenes, two pairs of soluble PTS components (EIIA^Cel1/EIIB^Cel1 and EIIA^Cel2/EIIB^Cel2) and the permease EIIC^Cel1 were suggested to contribute to cellobiose uptake. Interestingly, utilization of several carbohydrates, including cellobiose, strongly represses virulence gene expression by inhibiting PrfA, the virulence gene activator. Results: The LevR-like transcription regulator CelR activates expression of the cellobiose-induced PTS operons celB1-celC1-celA1, celB2-celA2, and the EIIC-encoding monocistronic celC2. Phosphorylation by P∼His-HPr at His550 activates CelR, whereas phosphorylation by P∼EIIB^Cel1 or P∼EIIB^Cel2 at His823 inhibits it. Replacement of His823 with Ala or deletion of both celA or celB genes caused constitutive CelR regulon expression. Mutants lacking EIIC^Cel1, CelR or both EIIA^Cel exhibitedslow cellobiose consumption. Deletion of celC1 or celR prevented virulence gene repression by the disaccharide, but not by glucose and fructose. Surprisingly, deletion of both celA genes caused virulence gene repression even during growth on non-repressing carbohydrates. No cellobiose-related phenotype was found for the celC2 mutant. Conclusion: The two EIIA/B^Cel pairs are similarly efficient as phosphoryl donors in EIIC^Cel1-catalyzed cellobiose transport and CelR regulation. The permanent virulence gene repression in the celA double mutant further supports a role of PTS^Cel components in PrfA regulation.

Effective mucosal live attenuated Salmonella vaccine by deleting phosphotransferase system component genes ptsI and crr.

Salmonella enterica is a major human pathogen that causes invasive non-typhoidal Salmonellosis (iNTS), resulting in significant morbidity and mortality. Although a number of pre-clinical and clinical studies have reported on the feasibility of developing a safe and effective vaccine against iNTS, there have been no licensed Salmonella vaccines available to protect against NTS strains. Vaccine formulations of highest priority for NTS are live attenuated vaccines, which can elicit effective induction of intestinal mucosal and intracellular bacteria-specific cell mediated immune responses. Since glucose is crucial for intracellular survival and replication in host cells, we constructed strains with mutations in components of the glucose uptake system, called the phosphotransferase system (PTS), and compared the relative virulence and immune responses in mice. In this study, we found that the strain with mutations in both ptsI and crr (KST0556) was the most attenuated strain among the tested strains, and proved to be highly effective in inducing a mucosal immune response that can protect against NTS infections in mice. Thus, we suggest here that KST0556 (ΔptsIΔcrr) is a potential live vaccine candidate for NTS, and may also be a candidate for a live delivery vector for heterologous antigens. Moreover, since PTS is a well-conserved glucose transporter system in both Gramnegative and Gram-positive bacteria, the ptsI and crr genes may be potential targets for creating live bacterial vectors or vaccine strains.

Characterisation of the action mechanism of a Lactococcus-specific bacteriocin, lactococcin Z

Lactococcin Z is a novel Lactococcus-specific bacteriocin produced by Lactococcus lactis QU 7 that shares 55.6% identity with lactococcin A. To identify the receptor targeted by lactococcin Z, several lactococcin Z-resistant mutants were generated from the sensitive strain, L.lactis IL1403. The resistant mutants showed difficulties in utilising mannose and glucose as sole carbon sources, contrary to their pattern of growth in the presence of galactose as a sole carbon source. Mutations were found in the ptnC and ptnD genes of lactococcin Z-resistant mutants, which encode the mannose phosphotransferase system (Man-PTS) components, IIC and IID, respectively; therefore, IIC and IID are proposed as potential receptors employed by lactococcin Z and are the same receptors targeted by lactococcin A. Both lactococcins A and Z share a high percentage identity in their N-termini regions in contrast to their C-termini that show less similarity; this may explain the difference in their action mechanisms as well as the lack of cross-immunity between them. Although lactococcin Z showed bactericidal activity, it neither dissipated membrane potential nor formed pores on the membranes of sensitive cells, in sharp contrast to the pore-forming lactococcin A.

The oligomerization state of bacterial enzyme I (EI) determines EI's allosteric stimulation or competitive inhibition by α-ketoglutarate

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by phosphorylation of enzyme I (EI) by phosphoenolpyruvate (PEP). The EI phosphorylation state determines the phosphorylation states of all other PTS components and is thought to play a central role in the regulation of several metabolic pathways and to control the biology of bacterial cells at multiple levels, for example, affecting virulence and biofilm formation. Given the pivotal role of EI in bacterial metabolism, an improved understanding of the mechanisms controlling its activity could inform future strategies for bioengineering and antimicrobial design. Here, we report an enzymatic assay, based on Selective Optimized Flip Angle Short Transient (SOFAST) NMR experiments, to investigate the effect of the small-molecule metabolite α-ketoglutarate (αKG) on the kinetics of the EI-catalyzed phosphoryl transfer reaction. We show that at experimental conditions favoring the monomeric form of EI, αKG promotes dimerization and acts as an allosteric stimulator of the enzyme. However, when the oligomerization state of EI is shifted toward the dimeric species, αKG functions as a competitive inhibitor of EI. We developed a kinetic model that fully accounted for the experimental data and indicated that bacterial cells might use the observed interplay between allosteric stimulation and competitive inhibition of EI by αKG to respond to physiological fluctuations in the intracellular environment. We expect that the mechanism for regulating EI activity revealed here is common to several other oligomeric enzymes.

Dissecting the genetic and metabolic mechanisms of adaptation to the knockout of a major metabolic enzyme in Escherichia coli

Unraveling the mechanisms of microbial adaptive evolution following genetic or environmental challenges is of fundamental interest in biological science and engineering. When the challenge is the loss of a metabolic enzyme, adaptive responses can also shed significant insight into metabolic robustness, regulation, and areas of kinetic limitation. In this study, whole-genome sequencing and high-resolution 13C-metabolic flux analysis were performed on 10 adaptively evolved pgi knockouts of Escherichia coliPgi catalyzes the first reaction in glycolysis, and its loss results in major physiological and carbon catabolism pathway changes, including an 80% reduction in growth rate. Following adaptive laboratory evolution (ALE), the knockouts increase their growth rate by up to 3.6-fold. Through combined genomic-fluxomic analysis, we characterized the mutations and resulting metabolic fluxes that enabled this fitness recovery. Large increases in pyridine cofactor transhydrogenase flux, correcting imbalanced production of NADPH and NADH, were enabled by direct mutations to the transhydrogenase genes sthA and pntAB The phosphotransferase system component crr was also found to be frequently mutated, which corresponded to elevated flux from pyruvate to phosphoenolpyruvate. The overall energy metabolism was found to be strikingly robust, and what have been previously described as latently activated Entner-Doudoroff and glyoxylate shunt pathways are shown here to represent no real increases in absolute flux relative to the wild type. These results indicate that the dominant mechanism of adaptation was to relieve the rate-limiting steps in cofactor metabolism and substrate uptake and to modulate global transcriptional regulation from stress response to catabolism.

The general PTS component HPr determines the preference for glucose over mannitol

Preferential sugar utilization is a widespread phenomenon in biological systems. Glucose is usually the most preferred carbon source in various organisms, especially in bacteria where it is taken up via the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The currently proposed model for glucose preference over non-PTS sugars in enteric bacteria including E. coli is strictly dependent on the phosphorylation state of the glucose-specific PTS component, enzyme IIAGlc (EIIAGlc). However, the mechanism of the preference among PTS sugars is largely unknown in Gram-negative bacteria. Here, we show that glucose preference over another PTS sugar, mannitol, is absolutely dependent on the general PTS component HPr, but not on EIIAGlc, in E. coli. Dephosphorylated HPr accumulates during the transport of glucose and interacts with the mannitol operon regulator, MtlR, to augment its repressor activity. This interaction blocks the inductive effect of mannitol on the mannitol operon expression and results in the inhibition of mannitol utilization.