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.

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.

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.

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.

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.

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.

Transcription regulators controlled by interaction with enzyme IIB components of the phosphoenolpyruvate: sugar phosphotransferase system.

Numerous bacteria possess transcription activators and antiterminators composed of regulatory domains phosphorylated by components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). These domains, called PTS regulation domains (PRDs), usually contain two conserved histidines as potential phosphorylation sites. While antiterminators possess two PRDs with four phosphorylation sites, transcription activators contain two PRDs plus two regulatory domains resembling PTS components (EIIA and EIIB). The activity of these transcription regulators is controlled by up to five phosphorylations catalyzed by PTS proteins. Phosphorylation by the general PTS components EI and HPr is usually essential for the activity of PRD-containing transcription regulators, whereas phosphorylation by the sugar-specific components EIIA or EIIB lowers their activity. For a specific regulator, for example the Bacillus subtilis mtl operon activator MtlR, the functional phosphorylation sites can be different in other bacteria and consequently the detailed mode of regulation varies. Some of these transcription regulators are also controlled by an interaction with a sugar-specific EIIB PTS component. The EIIBs are frequently fused to the membrane-spanning EIIC and EIIB-mediated membrane sequestration is sometimes crucial for the control of a transcription regulator. This is also true for the Escherichia coli repressor Mlc, which does not contain a PRD but nevertheless interacts with the EIIB domain of the glucose-specific PTS. In addition, some PRD-containing transcription activators interact with a distinct EIIB protein located in the cytoplasm. The phosphorylation state of the EIIB components, which changes in response to the presence or absence of the corresponding carbon source, affects their interaction with transcription regulators. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Membrane sequestration by the EIIB domain of the mannitol permease MtlA activates the Bacillus subtilis mtl operon regulator MtlR.

In most firmicutes expression of the mannitol operon is regulated by MtlR. This transcription activator is controlled via phosphorylation of its regulatory domains by components of the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS). We found that activation of Bacillus subtilis MtlR also requires an interaction with the EIIB(Mtl) domain of the mannitol permease MtlA (EIICB(Mtl) ). The constitutive expression of the mtlAFD operon in an mtlF mutant was prevented when entire mtlA or only its 3' part (EIIB(Mtl) ) were deleted. Yeast two-hybrid experiments revealed a direct interaction of the EIIB(Mtl) domain with the two C-terminal domains of MtlR. Complementation of the Δ3'-mtlA ΔmtlF or ΔmtlAFD mutants with mtlA restored constitutive MtlR activity, whereas complementation with only 3'-mtlA had no effect. Moreover, synthesis of EIIB(Mtl) in strains producing constitutively active MtlR caused MtlR inactivation. Interestingly, EIIB(Mtl) fused to the trans-membrane protein YwqC restored constitutive MtlR activity in the above mutants. Replacing the phosphorylatable Cys with Asp in MtlA or soluble EIIB(Mtl) lowered MtlR activation, indicating that MtlR does not interact with phosphorylatyed EIIB(Mtl) . Induction of the B. subtilis mtl operon therefore follows a novel regulation mechanism where the transcription activator needs to be sequestered to the membrane by unphosphorylated EIICB(Mtl) in order to be functional.

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose 6'-phosphate phosphatase (MapP).

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose-specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6-phospho-α-glucosidase, which in B. subtilis hydrolyses maltose 6'-P into glucose and glucose 6-P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6-P into glucose 1-P and glucose 6-P. However, purified MalP phosphorolyses maltose but not maltose 6'-P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6'-P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1-P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6'-P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6'-P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1-P. MapP therefore connects PTS-mediated maltose uptake to maltose phosphorylase-catalysed metabolism. Dephosphorylation assays with a wide variety of phospho-substrates revealed that MapP preferably dephosphorylates disaccharides containing an O-α-glycosyl linkage.

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.

Novel listerial glycerol dehydrogenase- and phosphoenolpyruvate-dependent dihydroxyacetone kinase system connected to the pentose phosphate pathway.

Several bacteria use glycerol dehydrogenase to transform glycerol into dihydroxyacetone (Dha). Dha is subsequently converted into Dha phosphate (Dha-P) by an ATP- or phosphoenolpyruvate (PEP)-dependent Dha kinase. Listeria innocua possesses two potential PEP-dependent Dha kinases. One is encoded by 3 of the 11 genes forming the glycerol (gol) operon. This operon also contains golD (lin0362), which codes for a new type of Dha-forming NAD(+)-dependent glycerol dehydrogenase. The subsequent metabolism of Dha requires its phosphorylation via the PEP:sugar phosphotransferase system components enzyme I, HPr, and EIIA(Dha)-2 (Lin0369). P∼EIIA(Dha)-2 transfers its phosphoryl group to DhaL-2, which phosphorylates Dha bound to DhaK-2. The resulting Dha-P is probably metabolized mainly via the pentose phosphate pathway, because two genes of the gol operon encode proteins resembling transketolases and transaldolases. In addition, purified Lin0363 and Lin0364 exhibit ribose-5-P isomerase (RipB) and triosephosphate isomerase activities, respectively. The latter enzyme converts part of the Dha-P into glyceraldehyde-3-P, which, together with Dha-P, is metabolized via gluconeogenesis to form fructose-6-P. Together with another glyceraldehyde-3-P molecule, the transketolase transforms fructose-6-P into intermediates of the pentose phosphate pathway. The gol operon is preceded by golR, transcribed in the opposite orientation and encoding a DeoR-type repressor. Its inactivation causes the constitutive but glucose-repressible expression of the entire gol operon, including the last gene, encoding a pediocin immunity-like (PedB-like) protein. Its elevated level of synthesis in the golR mutant causes slightly increased immunity against pediocin PA-1 compared to the wild-type strain or a pedB-like deletion mutant.

Mutational analysis of glucose transport regulation and glucose-mediated virulence gene repression in Listeria monocytogenes.

Listeria monocytogenes transports glucose/mannose via non-PTS permeases and phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS). Two mannose class PTS are encoded by the constitutively expressed mpoABCD and the inducible manLMN operons. The man operon encodes the main glucose transporter because manL or manM deletion significantly slows glucose utilization, whereas mpoA deletion has no effect. The PTS(Mpo) mainly functions as a constitutively synthesized glucose sensor controlling man operon expression by phosphorylating and interacting with ManR, a LevR-like transcription activator. EIIB(Mpo) plays a dual role in ManR regulation: P~EIIB(Mpo) prevailing in the absence of glucose phosphorylates and thereby inhibits ManR activity, whereas unphosphorylated EIIB(Mpo) prevailing during glucose uptake is needed to render ManR active. In contrast to mpoA, deletion of mpoB therefore strongly inhibits man operon expression and glucose consumption. A ΔptsI (EI) mutant consumes glucose at an even slower rate probably via GlcU-like non-PTS transporters. Interestingly, deletion of ptsI, manL, manM or mpoB causes elevated PrfA-mediated virulence gene expression. The PTS(Man) is the major player in glucose-mediated PrfA inhibition because the ΔmpoA mutant showed normal PrfA activity. The four mutants showing PrfA derepression contain no or only little unphosphorylated EIIAB(Man) (ManL), which probably plays a central role in glucose-mediated PrfA regulation.

A cell-cell communication system regulates protease production during sporulation in bacteria of the Bacillus cereus group.

In sporulating Bacillus, major processes like virulence gene expression and sporulation are regulated by communication systems involving signalling peptides and regulators of the RNPP family. We investigated the role of one such regulator, NprR, in bacteria of the Bacillus cereus group. We show that NprR is a transcriptional regulator whose activity depends on the NprX signalling peptide. In association with NprX, NprR activates the transcription of an extracellular protease gene (nprA) during the first stage of the sporulation process. The transcription start site of the nprA gene has been identified and the minimal region necessary for full activation has been characterized by promoter mutagenesis. We demonstrate that the NprX peptide is secreted, processed and then reimported within the bacterial cell. Once inside the cell, the mature form of NprX, presumably the SKPDIVG heptapeptide, directly binds to NprR allowing nprA transcription. Alignment of available NprR sequences from different species of the B. cereus group defines seven NprR clusters associated with seven NprX heptapeptide classes. This cell-cell communication system was found to be strain-specific with a possible cross-talk between some pherotypes. The phylogenic relationship between NprR and NprX suggests a coevolution of the regulatory protein and its signalling peptide.

Aerobic glycerol dissimilation via the Enterococcus faecalis DhaK pathway depends on NADH oxidase and a phosphotransfer reaction from PEP to DhaK via EIIADha.

Two pathways for glycerol dissimilation are present in Enterococcus faecalis. Either glycerol is first phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase with molecular oxygen as the electron acceptor (GlpO/GlpK pathway), or it is first oxidized by glycerol dehydrogenase with NAD(+) as the acceptor of the reduction equivalents and then phosphorylated by dihydroxyacetone kinase (GldA/DhaK pathway). The final end product in both cases is dihydroxyacetone phosphate (DHAP). The genes of the GldA/DhaK pathway are present in a four-gene operon structure encoding GldA, a small hypothetical protein (EF1359), and two subunits of dihydroxyacetone kinase (DhaK and DhaL). We demonstrate in this study that protein EF1359 is part of a phosphorylation cascade which phosphorylates dihydroxyacetone in a phosphoenolpyruvate (PEP)-dependent reaction via EI, HPr, EF1359 and DhaLK. Furthermore we show that aerobic dissimilation of glycerol via the GldA/DhaK pathway is dependent on active NADH oxidase to regenerate NADH in Ent. faecalis. A refined model of the aerobic metabolism of glycerol via the GldA/DhaK pathway is presented.

Fine-tuned transcriptional regulation of malate operons in Enterococcus faecalis.

In Enterococcus faecalis, the mae locus is constituted by two putative divergent operons, maePE and maeKR. The first operon encodes a putative H(+)/malate symporter (MaeP) and a malic enzyme (MaeE) previously shown to be essential for malate utilization in this bacterium. The maeKR operon encodes two putative proteins with significant similarity to two-component systems involved in sensing malate and activating its assimilation in bacteria. Our transcriptional and genetic assays showed that maePE and maeKR are induced in response to malate by the response regulator MaeR. In addition, we observed that both operons were partially repressed in the presence of glucose. Accordingly, the cometabolism of this sugar and malate was detected. The binding of the complex formed by CcpA and its corepressor P-Ser-HPr to a cre site located in the mae region was demonstrated in vitro and explains the carbon catabolite repression (CCR) observed for the maePE operon. However, our results also provide evidence for a CcpA-independent CCR mechanism regulating the expression of both operons. Finally, a biomass increment of 40 or 75% was observed compared to the biomass of cells grown only on glucose or malate, respectively. Cells cometabolizing both carbon sources exhibit a higher rate of glucose consumption and a lower rate of malate utilization. The growth improvement achieved by E. faecalis during glucose-malate cometabolism might explain why this microorganism employs different regulatory systems to tightly control the assimilation of both carbon sources.

Tyrosine phosphorylation: an emerging regulatory device of bacterial physiology.

Tyrosine phosphorylation is a key device in numerous cellular functions in eukaryotes, but in bacteria this protein modification was largely ignored until the mid-1990s. The first conclusive evidence of bacterial tyrosine phosphorylation came only a decade ago. Since then, several tyrosine kinases exhibiting unexpected features have been identified in a variety of bacteria. These enzymes use homologues of Walker motifs of nucleotide-binding proteins for their catalytic mechanism, thus defining an idiosyncratic type of bacterial tyrosine kinases. Recently, bacterial tyrosine kinases have been found to phosphorylate an increasing list of endogenous protein substrates. This discovery contributes to the emerging picture that bacterial tyrosine phosphorylation is an important regulatory arsenal of bacterial physiology in addition to the classical serine/threonine kinases, and the 'two-component' and phosphotransferase systems.

Analysis of the serine/threonine/tyrosine phosphoproteome of the pathogenic bacterium Listeria monocytogenes reveals phosphorylated proteins related to virulence.

Phosphorylation is the most common and widely studied post-translational protein modification in bacteria. It plays an important role in all kinds of cellular processes and controls key regulatory mechanisms, including virulence in certain pathogens. To gain insight into the role of protein phosphorylation in the pathogen Listeria monocytogenes, the serine (Ser), threonine (Thr) and tyrosine (Tyr) phosphoproteome of this bacterium was determined. We used the "gel free" proteomic approach with high accuracy mass spectrometry after enrichment of phosphopeptides. A total of 143 sites of phosphorylation were clearly identified, on 155 unique peptides of 112 phosphoproteins. The Ser/Thr/Tyr phosphorylation site distribution was 93:43:7. All identified phosphopeptides are monophosphorylated, except one and many identified phosphoproteins are related to virulence, translation, phosphoenolpyruvate:sugar phosphotransferase system, glycolysis and stress response. A description of these phosphoproteins is provided together with a comparison of the phosphosites in the L. monocytogenes proteins and in their homologues of other bacteria for which the phosphoproteome has been determined. Compared with the previous studies, we noticed a more extended conservation of the phosphorylation sites in glycolytic enzymes as well as ribosomal proteins.