Utilization of glycoprotein-derived N-acetylglucosamine-L-asparagine during Enterococcus faecalis infection depends on catabolic and transport enzymes of the glycosylasparaginase locus.

Enterococcus faecalis is a Gram-positive clinical pathogen causing severe infections. Its survival during infection depends on its ability to utilize host-derived metabolites, such as protein-deglycosylation products. We have identified in E. faecalis OG1RF a locus (ega) involved in the catabolism of the glycoamino acid N-acetylglucosamine-L-asparagine. This locus is separated into two transcription units, genes egaRP and egaGBCD1D2, respectively. RT-qPCR experiments revealed that the expression of the ega locus is regulated by the transcriptional repressor EgaR. Electromobility shift assays evidenced that N-acetylglucosamine-L-asparagine interacts directly with the EgaR protein, which leads to the transcription of the ega genes. Growth studies with egaG, egaB and egaC mutants confirmed that the encoded proteins are necessary for N-acetylglucosamine-L-asparagine catabolism. This glycoamino acid is transported and phosphorylated by a specific phosphotransferase system EIIABC components (OG1RF_10751, EgaB, EgaC) and subsequently hydrolyzed by the glycosylasparaginase EgaG, which generates aspartate and 6-P-N-acetyl-ß-d-glucosaminylamine. The latter can be used as a fermentable carbon source by E. faecalis. Moreover, Galleria mellonella larvae had a significantly higher survival rate when infected with ega mutants compared to the wild-type strain, suggesting that the loss of N-acetylglucosamine-L-asparagine utilization affects enterococcal virulence.

Enterococcus faecalis Maltodextrin Gene Regulation by Combined Action of Maltose Gene Regulator MalR and Pleiotropic Regulator CcpA.

Enterococci are Gram-positive bacteria present in the healthy human microbiota, but they are also a leading cause of nosocomial infections. Maltodextrin utilization by Enterococcus faecalis has been identified as an important factor for colonization of mammalians hosts. Here, we show that the LacI/GalR transcriptional regulator MalR, the maltose gene regulator, is also the main regulator of the operons encoding an ABC transporter (mdxEFG) and three metabolic enzymes (mmdH-gmdH-mmgT) required for the uptake and catabolism of maltotetraose and longer maltodextrins. The utilization of maltose and maltodextrins is consequently coordinated and induced by the disaccharide maltose, which binds to MalR. Carbon catabolite repression of the mdxEFG and mmdH-gmdH-mmgT operons is mediated by both P-Ser-HPr/MalR and P-Ser-HPr/CcpA. The latter complex exerts only moderate catabolite repression, which became visible when comparing maltodextrin operon expression levels of a malR- mutant (with a mutant allele for the malR gene) and a malR- ΔccpA double mutant grown in the presence of maltose, which is transported via a phosphotransferase system and, thus, favors the formation of P-Ser-HPr. Moreover, maltodextrin transport via MdxEFG slows rapidly when glucose is added, suggesting an additional regulation via inducer exclusion. This complex regulation of metabolic operons likely allows E. faecalis to fine-tune gene expression in response to changing environmental conditions.IMPORTANCEEnterococcus faecalis represents a leading cause of hospital-acquired infections worldwide. Several studies highlighted the importance of carbohydrate metabolism in the infection process of this bacterium. The genes required for maltodextrin metabolism are particularly induced during mouse infection and, therefore, should play an important role for pathogenesis. Since no data were hitherto available concerning the regulation of expression of the maltodextrin operons, we have conducted experiments to study the underlying mechanisms.

Enterococcus faecalis MalR acts as a repressor of the maltose operons and additionally mediates their catabolite repression via direct interaction with seryl-phosphorylated-HPr.

Enterococci are gram-positive pathogens and lead to cause hospital-acquired infections worldwide. Central carbon metabolism was shown as highly induced in Enterococcus faecalis during infection context. Metabolism of α-polysaccharides was previously described as an important factor for host colonisation and biofilm formation. A better characterisation of the adaptation of this bacterium to carbohydrate availabilities may lead to a better understanding of the link between carbohydrate metabolism and the infection process of E. faecalis. Here we show that MalR, a LacI/GalR transcriptional regulator, is the main factor in the regulation of the two divergent operons involved in maltose metabolism in this bacterium. The malR gene is transcribed from the malP promoter, but also from an internal promoter inside the gene located upstream of malR. In the absence of maltose, MalR acts as a repressor and in the presence of glucose, it exerts efficient CcpA-independent carbon catabolite repression. The central PTS protein P-Ser-HPr interacts directly with MalR and enhances its DNA binding capacity, which allows E. faecalis to adapt its metabolism to environmental conditions.

Characterization of the gen locus involved in ß-1,6-oligosaccharide utilization by Enterococcus faecalis.

The bicistronic genBA operon (formerly named celBA) of the opportunistic pathogen Enterococcus faecalis, encodes a 6-phospho-ß-glucosidase (GenA) and a phosphotransferase system permease EIIC (GenB). It resembles the cel operon of Streptococcus pyogenes, which is implicated in the metabolism of cellobiose. However, genBA mutants grew normally on cellobiose, but not (genA) or only slowly (genB) on gentiobiose and amygdalin. The two glucosides were also found to be the main inducers of the operon, confirming that the encoded proteins are involved in the utilization of ß-1,6- rather than ß-1,4-linked oligosaccharides. Expression of the genBA operon is regulated by the transcriptional activator GenR, which is encoded by the gene upstream from genB. Thermal shift analysis showed that it binds gentiobiose-6'-P with a Kd of 0.04 mM and with lower affinity also other phospho-sugars. The GenR/gentiobiose-6'-P complex binds to the promoter region upstream from genB. The genBA promoter region contains a cre box and gel-shift experiments demonstrated that the operon is under negative control of the global carbon catabolite regulator CcpA. We also show that the orphan EIIC (GenB) protein needs the EIIA component of the putative OG1RF_10750-OG1RF_10755 operon situated elsewhere on the chromosome to form a functional PTS transporter.

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 (EIIACel1/EIIBCel1 and EIIACel2/EIIBCel2) and the permease EIICCel1 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∼EIIBCel1 or P∼EIIBCel2 at His823 inhibits it. Replacement of His823 with Ala or deletion of both celA or celB genes caused constitutive CelR regulon expression. Mutants lacking EIICCel1, CelR or both EIIACel 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/BCel pairs are similarly efficient as phosphoryl donors in EIICCel1-catalyzed cellobiose transport and CelR regulation. The permanent virulence gene repression in the celA double mutant further supports a role of PTSCel components in PrfA regulation.

Photoinactivation of the Staphylococcus aureus Lactose-Specific EIICB Phosphotransferase Component with p-azidophenyl-ß-D-Galactoside and Phosphorylation of the Covalently Bound Substrate.

BACKGROUND: The phosphoenolpyruvate (PEP):lactose phosphotransferase system of Staphylococcus aureus transports and phosphorylates lactose and various phenylgalactosides. Their phosphorylation is catalyzed by the Cys476-phosphorylated EIIB domain of the lactose-specific permease enzyme IICB (EIICBLac). Phosphorylation causes the release of galactosides bound to the EIIC domain into the cytoplasm by a mechanism not yet understood. RESULTS: Irradiation of a reaction mixture containing the photoactivatable p-azidophenyl-ß-D-galactopyranoside and EIICBLac with UV light caused a loss of EIICBLac activity. Nevertheless, photoinactivated EIICBLac could still be phosphorylated with [32P]PEP. Proteolysis of photoinactivated [32P]P-EIICBLac with subtilisin provided an 11-kDa radioactive peptide. Only the sequence of its first three amino acids (-H-G-P-, position 245-247) could be determined. They are part of the substrate binding pocket in EIICs of the lactose/cellobiose PTS family. Surprisingly, while acid treatment caused hydrolysis of the phosphoryl group in active [32P]P∼EIICBLac, photoinactivated [32P]P-EIICBLac remained strongly phosphorylated. CONCLUSION: Phosphorylation of the -OH group at C6 of p-nitrenephenyl-ß-D-galactopyranoside covalently bound to EIICLac by the histidyl-phosphorylated [32P]P∼EIIBLac domain is a likely explanation for the observed acid resistance. Placing p-nitrenephenyl-ß-D-galactopyranoside into the active site of modelled EIICLac suggested that the nitrene binds to the -NH- group of Ser248, which would explain why no sequence data beyond Pro247could be obtained.

Manual and expert annotation of the nearly complete genome sequence of Staphylococcus sciuri strain ATCC 29059: A reference for the oxidase-positive staphylococci that supports the atypical phenotypic features of the species group.

Staphylococcus sciuri is considered to be one of the most ancestral species in the natural history of the Staphylococcus genus that consists of 48 validly described species. It belongs to the basal group of oxidase-positive and novobiocin-resistant staphylococci that diverged from macrococci approximately 250 million years ago. Contrary to other groups, the S. sciuri species group has not developed host-specific colonization strategies. Genome analysis of S. sciuri ATCC 29059 provides here the first genetic basis for atypical traits that would support the switch between the free-living style and the infective state in animals and humans. From among the most remarkable features, it was noticed in this extensive study that there were a number of phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS), almost twice as many as any other staphylococci, and the co-occurrence of mevalonate and non-mevalonate pathways for isoprenoid synthesis. The sequenced strain was devoid of the main virulence factors present in Staphylococcus aureus, although it exhibited numerous heme and iron acquisition systems, as well as crt and aldH genes necessary for gold pigment synthesis. The sensing and signaling networks, exemplified by a large and typical repertoire of two-component regulatory systems and a complete panel of master regulators, such as agr, rex, mgrA, rot, sarA and sarR genes, depict the background in which S. aureus virulence genes were later acquired. An additional sigma factor, a distinct set of electron transducer elements and many gene operons similar to those found in Bacillus spp. would constitute the most visible remnant links with Bacillaceae organisms.

Enzymes Required for Maltodextrin Catabolism in Enterococcus faecalis Exhibit Novel Activities.

Maltose and maltodextrins are formed during the degradation of starch or glycogen. Maltodextrins are composed of a mixture of maltooligosaccharides formed by α-1,4- but also some α-1,6-linked glucosyl residues. The α-1,6-linked glucosyl residues are derived from branching points in the polysaccharides. In Enterococcus faecalis, maltotriose is mainly transported and phosphorylated by a phosphoenolpyruvate:carbohydrate phosphotransferase system. The formed maltotriose-6″-phosphate is intracellularly dephosphorylated by a specific phosphatase, MapP. In contrast, maltotetraose and longer maltooligosaccharides up to maltoheptaose are taken up without phosphorylation via the ATP binding cassette transporter MdxEFG-MsmX. We show that the maltose-producing maltodextrin hydrolase MmdH (GenBank accession no. EFT41964) in strain JH2-2 catalyzes the first catabolic step of α-1,4-linked maltooligosaccharides. The purified enzyme converts even-numbered α-1,4-linked maltooligosaccharides (maltotetraose, etc.) into maltose and odd-numbered (maltotriose, etc.) into maltose and glucose. Inactivation of mmdH therefore prevents the growth of E. faecalis on maltooligosaccharides ranging from maltotriose to maltoheptaose. Surprisingly, MmdH also functions as a maltogenic α-1,6-glucosidase, because it converts the maltotriose isomer isopanose into maltose and glucose. In addition, E. faecalis contains a glucose-producing α-1,6-specific maltodextrin hydrolase (GenBank accession no. EFT41963, renamed GmdH). This enzyme converts panose, another maltotriose isomer, into glucose and maltose. A gmdH mutant had therefore lost the capacity to grow on panose. The genes mmdH and gmdH are organized in an operon together with GenBank accession no. EFT41962 (renamed mmgT). Purified MmgT transfers glucosyl residues from one α-1,4-linked maltooligosaccharide molecule to another. For example, it catalyzes the disproportionation of maltotriose by transferring a glucosyl residue to another maltotriose molecule, thereby forming maltotetraose and maltose together with a small amount of maltopentaose.IMPORTANCE The utilization of maltodextrins by Enterococcus faecalis has been shown to increase the virulence of this nosocomial pathogen. However, little is known about how this organism catabolizes maltodextrins. We identified two enzymes involved in the metabolism of various α-1,4- and α-1,6-linked maltooligosaccharides. We found that one of them functions as a maltose-producing α-glucosidase with relaxed linkage specificity (α-1,4 and α-1,6) and exo- and endoglucosidase activities. A third enzyme, which resembles amylomaltase, exclusively transfers glucosyl residues from one maltooligosaccharide molecule to another. Similar enzymes are present in numerous other Firmicutes, such as streptococci and lactobacilli, suggesting that these organisms follow the same maltose degradation pathway as E. faecalis.

Inducer exclusion in Firmicutes: insights into the regulation of a carbohydrate ATP binding cassette transporter from Lactobacillus casei BL23 by the signal transducing protein P-Ser46-HPr.

Catabolite repression is a mechanism that enables bacteria to control carbon utilization. As part of this global regulatory network, components of the phosphoenolpyruvate:carbohydrate phosphotransferase system inhibit the uptake of less favorable sugars when a preferred carbon source such as glucose is available. This process is termed inducer exclusion. In bacteria belonging to the phylum Firmicutes, HPr, phosphorylated at serine 46 (P-Ser46-HPr) is the key player but its mode of action is elusive. To address this question at the level of purified protein components, we have chosen a homolog of the Escherichia coli maltose/maltodextrin ATP-binding cassette transporter from Lactobacillus casei (MalE1-MalF1G1K12 ) as a model system. We show that the solute binding protein, MalE1, binds linear and cyclic maltodextrins but not maltose. Crystal structures of MalE1 complexed with these sugars provide a clue why maltose is not a substrate. P-Ser46-HPr inhibited MalE1/maltotetraose-stimulated ATPase activity of the transporter incorporated in proteoliposomes. Furthermore, cross-linking experiments revealed that P-Ser46-HPr contacts the nucleotide-binding subunit, MalK1, in proximity to the Walker A motif. However, P-Ser46-HPr did not block binding of ATP to MalK1. Together, our findings provide first biochemical evidence that P-Ser-HPr arrests the transport cycle by preventing ATP hydrolysis at the MalK1 subunits of the transporter.

Enterococcus faecalis Uses a Phosphotransferase System Permease and a Host Colonization-Related ABC Transporter for Maltodextrin Uptake.

Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6″-P in the cell. MapP, which dephosphorylates maltose-6'-P, also releases Pi from maltotriose-6″-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection.IMPORTANCE Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics, and new strategies are urgently needed. A largely unexplored aspect is how these pathogens proliferate and which substrates they use in order to grow inside infected hosts. The use of maltodextrins as a source of carbon and energy was studied in Enterococcus faecalis and linked to its virulence. Our results demonstrate that E. faecalis can efficiently use glycogen degradation products. We show here that depending on the length of the maltodextrins, one of two different transporters is used: the maltose-PTS transporter MalT, or the MdxEFG-MsmX ABC transporter. MdxEFG-MsmX takes up longer maltodextrins as well as complex molecules, such as panose and isopanose.

Sophisticated Regulation of Transcriptional Factors by the Bacterial Phosphoenolpyruvate: Sugar Phosphotransferase System.

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a carbohydrate transport and phosphorylation system present in bacteria of all different phyla and in archaea. It is usually composed of three proteins or protein complexes, enzyme I, HPr, and enzyme II, which are phosphorylated at histidine or cysteine residues. However, in many bacteria, HPr can also be phosphorylated at a serine residue. The PTS not only functions as a carbohydrate transporter but also regulates numerous cellular processes either by phosphorylating its target proteins or by interacting with them in a phosphorylation-dependent manner. The target proteins can be catabolic enzymes, transporters, and signal transduction proteins but are most frequently transcriptional regulators. In this review, we will describe how PTS components interact with or phosphorylate proteins to regulate directly or indirectly the activity of transcriptional repressors, activators, or antiterminators. We will briefly summarize the well-studied mechanism of carbon catabolite repression in firmicutes, where the transcriptional regulator catabolite control protein A needs to interact with seryl-phosphorylated HPr in order to be functional. We will present new results related to transcriptional activators and antiterminators containing specific PTS regulation domains, which are the phosphorylation targets for three different types of PTS components. Moreover, we will discuss how the phosphorylation level of the PTS components precisely regulates the activity of target transcriptional regulators or antiterminators, with or without PTS regulation domain, and how the availability of PTS substrates and thus the metabolic status of the cell are connected with various cellular processes, such as biofilm formation or virulence of certain pathogens.

The Phosphocarrier Protein HPr Contributes to Meningococcal Survival during Infection.

Neisseria meningitidis is an exclusively human pathogen frequently carried asymptomatically in the nasopharynx but it can also provoke invasive infections such as meningitis and septicemia. N. meningitidis uses a limited range of carbon sources during infection, such as glucose, that is usually transported into bacteria via the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), in which the phosphocarrier protein HPr (encoded by the ptsH gene) plays a central role. Although N. meningitidis possesses an incomplete PTS, HPr was found to be required for its virulence. We explored the role of HPr using bioluminescent wild-type and ΔptsH strains in experimental infection in transgenic mice expressing the human transferrin. The wild-type MC58 strain was recovered at higher levels from the peritoneal cavity and particularly from blood compared to the ΔptsH strain. The ΔptsH strain provoked lower levels of septicemia in mice and was more susceptible to complement-mediated killing than the wild-type strain. We tested whether meningococcal structures impacted complement resistance and observed that only the capsule level was decreased in the ΔptsH mutant. We therefore compared the transcriptomic profiles of wild-type and ΔptsH strains and identified 49 differentially expressed genes. The HPr regulon contains mainly hypothetical proteins (43%) and several membrane-associated proteins that could play a role during host interaction. Some other genes of the HPr regulon are involved in stress response. Indeed, the ΔptsH strain showed increased susceptibility to environmental stress conditions. Our data suggest that HPr plays a pleiotropic role in host-bacteria interactions most likely through the innate immune response that may be responsible for the enhanced clearance of the ΔptsH strain from blood.

Transport and Catabolism of Pentitols by Listeria monocytogenes.

Transposon insertion into Listeria monocytogenes lmo2665, which encodes an EIIC of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), was found to prevent D-arabitol utilization. We confirm this result with a deletion mutant and show that Lmo2665 is also required for D-xylitol utilization. We therefore called this protein EIICAxl. Both pentitols are probably catabolized via the pentose phosphate pathway (PPP) because lmo2665 belongs to an operon, which encodes the three PTSAxl components, two sugar-P dehydrogenases, and most PPP enzymes. The two dehydrogenases oxidize the pentitol-phosphates produced during PTS-catalyzed transport to the PPP intermediate xylulose-5-P. L. monocytogenes contains another PTS, which exhibits significant sequence identity to PTSAxl. Its genes are also part of an operon encoding PPP enzymes. Deletion of the EIIC-encoding gene (lmo0508) affected neither D-arabitol nor D-xylitol utilization, although D-arabitol induces the expression of this operon. Both operons are controlled by MtlR/LicR-type transcription activators (Lmo2668 and Lmo0501, respectively). Phosphorylation of Lmo0501 by the soluble PTSAxl components probably explains why D-arabitol also induces the second pentitol operon. Listerial virulence genes are submitted to strong repression by PTS sugars, such as glucose. However, D-arabitol inhibited virulence gene expression only at high concentrations, probably owing to its less efficient utilization compared to glucose.

Transport and Catabolism of Carbohydrates by Neisseria meningitidis.

We identified the genes encoding the proteins for the transport of glucose and maltose in Neisseria meningitidis strain 2C4-3. A mutant deleted for NMV_1892(glcP) no longer grew on glucose and deletion of NMV_0424(malY) prevented the utilization of maltose. We also purified and characterized glucokinase and α-phosphoglucomutase, which catalyze early catabolic steps of the two carbohydrates. N. meningitidis catabolizes the two carbohydrates either via the Entner-Doudoroff (ED) pathway or the pentose phosphate pathway, thereby forming glyceraldehyde-3-P and either pyruvate or fructose-6-P, respectively. We purified and characterized several key enzymes of the two pathways. The genes required for the transformation of glucose into gluconate-6-P and its further catabolism via the ED pathway are organized in two adjacent operons. N. meningitidis also contains genes encoding proteins which exhibit similarity to the gluconate transporter (NMV_2230) and gluconate kinase (NMV_2231) of Enterobacteriaceae and Firmicutes. However, gluconate might not be the real substrate of NMV_2230 because N. meningitidis was not able to grow on gluconate as the sole carbon source. Surprisingly, deletion of NMV_2230 stimulated growth in minimal medium in the presence and absence of glucose and drastically slowed the clearance of N. meningitidis cells from transgenic mice after intraperitoneal challenge.

The phosphocarrier protein HPr of Neisseria meningitidis interacts with the transcription regulator CrgA and its deletion affects capsule production, cell adhesion, and virulence.

The bacterial phosphotransferase system (PTS) transports and phosphorylates sugars, but also carries out numerous regulatory functions. The ß-proteobacterium Neisseria meningitidis possesses an incomplete PTS unable to transport carbon sources because it lacks a membrane component. Nevertheless, the residual phosphorylation cascade is functional and the meningococcal PTS was therefore expected to carry out regulatory roles. Interestingly, a ΔptsH mutant (lacks the PTS protein HPr) exhibited reduced virulence in mice and after intraperitoneal challenge it was rapidly cleared from the bloodstream of BALB/c mice. The rapid clearance correlates with lower capsular polysaccharide production by the ΔptsH mutant, which is probably also responsible for its increased adhesion to Hec-1-B epithelial cells. In addition, compared to the wild-type strain more apoptotic cells were detected when Hec-1-B cells were infected with the ΔptsH strain. Coimmunoprecipitation revealed an interaction of HPr and P-Ser-HPr with the LysR type transcription regulator CrgA, which among others controls its own expression. Moreover, ptsH deletion caused increased expression of a ΦcrgA-lacZ fusion. Finally, the presence of HPr or phospho-HPr's during electrophoretic mobility shift assays enhanced the affinity of CrgA for its target sites preceding crgA and pilE, but HPr did not promote CrgA binding to the sia and pilC1 promoter regions.

PTS-Mediated Regulation of the Transcription Activator MtlR from Different Species: Surprising Differences despite Strong Sequence Conservation.

The hexitol D-mannitol is transported by many bacteria via a phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). In most Firmicutes, the transcription activator MtlR controls the expression of the genes encoding the D-mannitol-specific PTS components and D-mannitol-1-P dehydrogenase. MtlR contains an N-terminal helix-turn-helix motif followed by an Mga-like domain, two PTS regulation domains (PRDs), an EIIB(Gat)- and an EIIA(Mtl)-like domain. The four regulatory domains are the target of phosphorylation by PTS components. Despite strong sequence conservation, the mechanisms controlling the activity of MtlR from Lactobacillus casei, Bacillus subtilis and Geobacillus stearothermophilus are quite different. Owing to the presence of a tyrosine in place of the second conserved histidine (His) in PRD2, L. casei MtlR is not phosphorylated by Enzyme I (EI) and HPr. When the corresponding His in PRD2 of MtlR from B. subtilis and G. stearothermophilus was replaced with alanine, the transcription regulator was no longer phosphorylated and remained inactive. Surprisingly, L. casei MtlR functions without phosphorylation in PRD2 because in a ptsI (EI) mutant MtlR is constitutively active. EI inactivation prevents not only phosphorylation of HPr, but also of the PTS(Mtl) components, which inactivate MtlR by phosphorylating its EIIB(Gat)- or EIIA(Mtl)-like domain. This explains the constitutive phenotype of the ptsI mutant. The absence of EIIB(Mtl)-mediated phosphorylation leads to induction of the L. caseimtl operon. This mechanism resembles mtlARFD induction in G. stearothermophilus, but differs from EIIA(Mtl)-mediated induction in B. subtilis. In contrast to B. subtilis MtlR, L. casei MtlR activation does not require sequestration to the membrane via the unphosphorylated EIIB(Mtl) domain.

Protein-tyrosine phosphorylation in Bacillus subtilis: a 10-year retrospective.

The discovery of tyrosine-phosphorylated proteins in Bacillus subtilis in the year 2003 was followed by a decade of intensive research activity. Here we provide an overview of the lessons learned in that period. While the number of characterized kinases and phosphatases involved in reversible protein-tyrosine phosphorylation in B. subtilis has remained essentially unchanged, the number of proteins known to be targeted by this post-translational modification has increased dramatically. This is mainly due to phosphoproteomics and interactomics studies, which were instrumental in identifying new tyrosine-phosphorylated proteins. Despite their structural similarity, the two B. subtilis protein-tyrosine kinases (BY-kinases), PtkA and PtkB (EpsB), seem to accomplish different functions in the cell. The PtkB is encoded by a large operon involved in exopolysaccharide production, and its main role appears to be the control of this process. The PtkA seems to have a more complex role; it phosphorylates and regulates a large number of proteins involved in the DNA, fatty acid and carbon metabolism and engages in physical interaction with other types of kinases (Ser/Thr kinases), leading to mutual phosphorylation. PtkA also seems to respond to several activator proteins, which direct its activity toward different substrates. In that respect PtkA seems to function as a highly connected signal integration device.

Interaction with enzyme IIBMpo (EIIBMpo) and phosphorylation by phosphorylated EIIBMpo exert antagonistic effects on the transcriptional activator ManR of Listeria monocytogenes.

UNLABELLED: Listeriae take up glucose and mannose predominantly through a mannose class phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS(Man)), whose three components are encoded by the manLMN genes. The expression of these genes is controlled by ManR, a LevR-type transcription activator containing two PTS regulation domains (PRDs) and two PTS-like domains (enzyme IIA(Man) [EIIA(Man)]- and EIIB(Gat)-like). We demonstrate here that in Listeria monocytogenes, ManR is activated via the phosphorylation of His585 in the EIIA(Man)-like domain by the general PTS components enzyme I and HPr. We also show that ManR is regulated by the PTS(Mpo) and that EIIB(Mpo) plays a dual role in ManR regulation. First, yeast two-hybrid experiments revealed that unphosphorylated EIIB(Mpo) interacts with the two C-terminal domains of ManR (EIIB(Gat)-like and PRD2) and that this interaction is required for ManR activity. Second, in the absence of glucose/mannose, phosphorylated EIIB(Mpo) (P∼EIIB(Mpo)) inhibits ManR activity by phosphorylating His871 in PRD2. The presence of glucose/mannose causes the dephosphorylation of P∼EIIB(Mpo) and P∼PRD2 of ManR, which together lead to the induction of the manLMN operon. Complementation of a ΔmanR mutant with various manR alleles confirmed the antagonistic effects of PTS-catalyzed phosphorylation at the two different histidine residues of ManR. Deletion of manR prevented not only the expression of the manLMN operon but also glucose-mediated repression of virulence gene expression; however, repression by other carbohydrates was unaffected. Interestingly, the expression of manLMN in Listeria innocua was reported to require not only ManR but also the Crp-like transcription activator Lin0142. Unlike Lin0142, the L. monocytogenes homologue, Lmo0095, is not required for manLMN expression; its absence rather stimulates man expression. IMPORTANCE: Listeria monocytogenes is a human pathogen causing the foodborne disease listeriosis. The expression of most virulence genes is controlled by the transcription activator PrfA. Its activity is strongly repressed by carbohydrates, including glucose, which is transported into L. monocytogenes mainly via a mannose/glucose-specific phosphotransferase system (PTS(Man)). Expression of the man operon is regulated by the transcription activator ManR, the activity of which is controlled by a second, low-efficiency PTS of the mannose family, which functions as glucose sensor. Here we demonstrate that the EIIB(Mpo) component plays a dual role in ManR regulation: it inactivates ManR by phosphorylating its His871 residue and stimulates ManR by interacting with its two C-terminal domains.

Quantitative proteome analyses identify PrfA-responsive proteins and phosphoproteins in Listeria monocytogenes.

Protein phosphorylation is a major mechanism of signal transduction in bacteria. Here, we analyzed the proteome and phosphoproteome of a wild-type strain of the food-borne pathogen Listeria monocytogenes that was grown in either chemically defined medium or rich medium containing glucose. We then compared these results with those obtained from an isogenic prfA* mutant that produced a constitutively active form of PrfA, the main transcriptional activator of virulence genes. In the prfA* mutant grown in rich medium, we identified 256 peptides that were phosphorylated on serine (S), threonine (T), or tyrosine (Y) residues, with a S/T/Y ratio of 155:75:12. Strikingly, we detected five novel phosphosites on the virulence protein ActA. This protein was known to be phosphorylated by a cellular kinase in the infected host, but phosphorylation by a listerial kinase had not previously been reported. Unexpectedly, SILAC experiments with the prfA* mutant grown in chemically defined medium revealed that, in addition to previously described PrfA-regulated proteins, several other proteins were significantly overproduced, among them were several proteins involved in purine biosynthesis. This work provides new information for our understanding of the correlation among protein phosphorylation, virulence mechanisms, and carbon metabolism.

The bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions.

The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.