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Josef Deutscher

Publications and source records attributed to Josef Deutscher.

At least 19 recordsLinked to original sources
PubMed2025

Cellodextrin Metabolism and Phosphotransferase System-Catalyzed Uptake in Enterococcus faecalis.

Two PTS transporters involved in the uptake of cellobiose and short cellooligosaccharides were identified in Enterococcus faecalis. Genes coding for the different EII proteins are found in a locus composed of three operonic structures expressing two distinct EIIC (CelC1 and CelC2), two identical EIIB (CelB1 and CelB2) and a unique EIIA (CelA1). The EIIA plays a central role in β-glucoside uptake because it is required not only for β-homodiholosides but also for the diheteroside N-acetylglucosamine-L-asparagine. Depending on their size, cellooligosaccharides are preferably transported either by CelC1 (di-saccharides) or by CelC2 (4 glycosidic residues and more), with tri-saccharides being taken up by both EIIC transporters. Moreover, CelA1B2C2 require CelGHI to be functional, three small proteins, the function of which remains unknown. CelA1B1C1 is the main but not exclusive transporter of cellobiose and chitobiose. It is involved in the transport of other β-glucodisaccharides, such as laminaribiose and sophorose. This PTS can be complemented by other transporters highlighting the existence of a network for β-glucoside uptake. This locus is under the control of CelR, a LevR-like transcription activator.

Enterococcus faecalis↗
PubMed2007

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.

Amino Acid Sequence↗
PubMed2007

The phosphotransferase system of Lactobacillus casei: regulation of carbon metabolism and connection to cold shock response.

Genome sequencing of two different Lactobacillus casei strains (ATCC334 and BL23) is presently going on and preliminary data revealed that this lactic acid bacterium possesses numerous carbohydrate transport systems probably reflecting its capacity to proliferate under varying environmental conditions. Many carbohydrate transporters belong to the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but all different kinds of non-PTS transporters are present as well and their substrates are known in a few cases. In L. casei regulation of carbohydrate transport and carbon metabolism is mainly achieved by PTS proteins. Carbon catabolite repression (CCR) is mediated via several mechanisms, including the major P-Ser-HPr/catabolite control protein A (CcpA)-dependent mechanism. Catabolite response elements, the target sites for the P-Ser-HPr/CcpA complex, precede numerous genes and operons. PTS regulation domain-containing antiterminators and transcription activators are also present in both L. casei strains. Their activity is usually controlled by two PTS-mediated phosphorylation reactions exerting antagonistic effects on the transcription regulators: P~EIIB-dependent phosphorylation regulates induction of the corresponding genes and P~His-HPr-mediated phosphorylation plays a role in CCR. Carbohydrate transport of L. casei is also regulated via inducer exclusion and inducer expulsion. The presence of glucose, fructose, etc. leads to inhibition of the transport or metabolism of less favorable carbon sources (inducer exclusion) or to the export of accumulated non-metabolizable carbon sources (inducer expulsion). While P-Ser-HPr is essential for inducer exclusion of maltose, it is not necessary for the expulsion of accumulated thio-methyl-beta-D-galactopyranoside. Surprisingly, recent evidence suggests that the PTS of L. casei also plays a role in cold shock response.

Biological Transport↗
PubMed2006

Identification and characterization of a fructose phosphotransferase system in Bifidobacterium breve UCC2003.

In silico analysis of the Bifidobacterium breve UCC2003 genome allowed identification of four genetic loci, each of which specifies a putative enzyme II (EII) protein of a phosphoenolpyruvate:sugar phosphotransferase system. The EII encoded by fruA, a clear homologue of the unique EIIBCA enzyme encoded by the Bifidobacterium longum NCC2705 genome, was studied in more detail. The fruA gene is part of an operon which contains fruT, which is predicted to encode a homologue of the Bacillus subtilis antiterminator LicT. Transcriptional analysis showed that the fru operon is induced by fructose. The genetic structure, complementation studies, and the observed transcription pattern of the fru operon suggest that the EII encoded in B. breve is involved in fructose transport and that its expression is controlled by an antiterminator mechanism. Biochemical studies unequivocally demonstrated that FruA phosphorylates fructose at the C-6 position.

Amino Acid Sequence↗
PubMed2006

Molecular analysis of the glucose-specific phosphoenolpyruvate : sugar phosphotransferase system from Lactobacillus casei and its links with the control of sugar metabolism.

Lactobacillus casei transports glucose preferentially by a mannose-class phosphoenolpyruvate : sugar phosphotransferase system (PTS). The genomic analysis of L. casei allowed the authors to find a gene cluster (manLMNO) encoding the IIAB (manL), IIC (manM) and IID (manN) proteins of a mannose-class PTS, and a putative 121 aa protein of unknown function (encoded by manO), homologues of which are also present in man clusters that encode glucose/mannose transporters in other Gram-positive bacteria. The L. casei man operon is constitutively expressed into a manLMNO messenger, but an additional manO transcript was also detected. Upstream of the man operon, two genes (upsR and upsA) were found which encode proteins resembling a transcriptional regulator and a membrane protein, respectively. Disruption of either upsR or upsA did not affect manLMNO transcription, and had no effect on glucose uptake. Cells carrying a manO deletion transported glucose at a rate similar to that of the wild-type strain. By contrast, a manM disruption resulted in cells unable to transport glucose by the PTS, thus confirming the functional role of the man genes. In addition, the manM mutant exhibited neither inducer exclusion of maltose nor glucose repression. This result confirms the need for glucose transport through the PTS to trigger these regulatory processes in L. casei.

Amino Acid Sequence↗
PubMed2006

Cloning and characterization of a gene encoding a cold-shock protein in Lactobacillus casei.

One csp-like gene, called cspA, from the lactic acid bacterium Lactobacillus casei was identified by an inverse polymerase chain reaction approach based on degenerate primers. cspA encodes a protein of 66 amino acid residues, which displays at least 74% identity with Csp proteins of Lactobacillus genera. Northern blot analysis revealed that cspA is transcribed monocistronically and that its expression is induced after a temperature downshift from 37 degrees C to 20 degrees C. The transcriptional start site has been determined and is situated 98 bp upstream of the initiation codon. A cspA mutant strain was constructed and it showed reduced growth rate compared with the wild type at both optimal and low temperatures, demonstrating that CspA plays an important role in the physiology of L. casei.

Amino Acid Sequence↗
PubMed2006

How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria.

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

Bacteria↗
PubMed2005

The glycolytic genes pfk and pyk from Lactobacillus casei are induced by sugars transported by the phosphoenolpyruvate:sugar phosphotransferase system and repressed by CcpA.

In Lactobacillus casei BL23, phosphofructokinase activity was higher in cells utilizing sugars transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphofructokinase gene (pfk) was cloned from L. casei and shown to be clustered with the gene encoding pyruvate kinase (pyk). pfk and pyk genes are cotranscribed and induced upon growth on sugars transported by the PTS. Contrarily to the model proposed for Lactococcus lactis, where the global catabolite regulator protein (CcpA) is involved in PTS-induced transcription of pfk and pyk, a ccpA mutation resulted in a slight increase in pfk-pyk expression in L. casei. This weak regulation was evidenced by CcpA binding to a region of the pfk-pyk promoter which contained two cre sequences significantly deviated from the consensus. The PTS induction of pfk-pyk seems to be counteracted by the CcpA-mediated repression. Our results suggest that the need to accommodate the levels of pfk-pyk mRNA to the availability of sugars is fulfilled in L. casei by a PTS/CcpA-mediated signal transduction different from L. lactis.

Amino Acid Sequence↗
PubMed2005

P-Ser-HPr--a link between carbon metabolism and the virulence of some pathogenic bacteria.

HPr kinase/phosphorylase phosphorylates HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system, at serine-46. P-Ser-HPr is the central regulator of carbon metabolism in Gram-positive bacteria, but also plays a role in virulence development of certain pathogens. In Listeria monocytogenes, several virulence genes, which depend on the transcription activator PrfA, are repressed by glucose, fructose, etc., in a catabolite repressor (CcpA)-independent mechanism. However, the catabolite co-repressor P-Ser-HPr was found to inhibit the activity of PrfA. In an hprKV267F mutant, in which most of the HPr is transformed into P-Ser-HPr, PrfA was barely active. The ptsH1 mutation (Ser-46 of HPr replaced with an alanine) prevented the inhibitory effect of the hprKV267F mutation. Interestingly, disruption of ccpA also inhibited PrfA activity. This effect is probably also mediated via P-Ser-HPr, since ccpA disruption leads to elevated amounts of P-Ser-HPr. Indeed, a ccpA ptsH1 double mutant exhibited normal PrfA activity. In S. pyogenes, the expression of several virulence genes depends on the transcription activator Mga. Interestingly, the mga promoter is preceded by an operator site, which serves as target for the CcpA/P-Ser-HPr complex. Numerous Gram-negative pathogens also contain hprK, which is often organised in an operon with transcription regulators necessary for the development of virulence, indicating that in these organisms P-Ser-HPr also plays a role in pathogenesis. Indeed, inactivation of Neisseria meningitidis hprK strongly diminished cell adhesion of this pathogen.

Bacterial Proteins↗
PubMed2005

Horizontal gene transfer in the molecular evolution of mannose PTS transporters.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) represents hitherto the only example of group translocation transport systems. PTS transporters are exclusively found in bacteria and can be grouped on the basis of sequence and structure into six classes. We have analyzed the evolution of mannose-class PTS transporters. These transporters have a limited distribution among bacteria being mostly harbored by species associated to animals. The results obtained indicate that these genes have undergone a complex evolutionary history, including extensive horizontal gene transfer events, duplications, and nonorthologous displacements. The phylogenetic analysis revealed an early diversification to specialize in different transport capabilities, but these events have also occurred relatively recently. In addition, these transporters can be further divided into seven groups and this division correlates with their transport capabilities. Finally, the consideration of the genomic context allowed us to propose putative functional roles for some uncharacterized PTS transporters. The functional role and distribution of mannose-class PTS transporters suggest that their expansion may have played a significant role in the establishment of symbiotic relationships between animals and some bacteria.

Bacteria↗
PubMed2005

Elaborate transcription regulation of the Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids through global regulators of CcpA, CodY and TnrA.

The Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids is under negative regulation mediated by TnrA and CodY, which recognize and bind to their respective cis-elements located upstream of the ilv-leu promoter. This operon is known to be under CcpA-dependent positive regulation. We have currently identified a catabolite-responsive element (cre) for this positive regulation (bases -96 to -82; +1 is the ilv-leu transcription initiation base) by means of DNase I-footprinting in vitro, and deletion and base-substitution analyses of cre. Under nitrogen-rich growth conditions in glucose-minimal medium supplemented with glutamine and amino acids, CcpA and CodY exerted positive and negative regulation of ilv-leu, respectively, but TnrA did not function. Moreover, CcpA and CodY were able to function without their counteracting regulation of each other, although the CcpA-dependent positive regulation did not overcome the CodY-dependent negative regulation. Furthermore, under nitrogen-limited conditions in glucose-minimal medium with glutamate as the sole nitrogen source, CcpA and TnrA exerted positive and negative regulation, respectively, but CodY did not function. This CcpA-dependent positive regulation occurred without the TnrA-dependent negative regulation. However, the TnrA-dependent negative regulation did not occur without the CcpA-dependent positive regulation, raising the possibility that this negative regulation might decrease the CcpA-dependent positive regulation. The physiological role of this elaborate transcription regulation of the B. subtilis ilv-leu operon in overall metabolic regulation in this organism is discussed.

Amino Acids, Branched-Chain↗
PubMed2005

In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE.

Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains one such CpsB-like PTP, YwqE, in addition to two class II Cys-based PTPs, YwlE and YfkJ. The substrates for both YwlE and YfkJ are presently unknown, while YwqE was shown to dephosphorylate two phosphotyrosine-containing proteins implicated in UDP-glucuronate biosynthesis, YwqD and YwqF. In this study, we characterize YwqE, compare the activities of the three B. subtilis PTPs (YwqE, YwlE, and YfkJ), and demonstrate that the two B. subtilis class II PTPs do not dephosphorylate the physiological substrates of YwqE.

Amino Acid Sequence↗
PubMed2005

Ser/Thr/Tyr protein phosphorylation in bacteria - for long time neglected, now well established.

The first clearly established example of Ser/Thr/Tyr phosphorylation of a bacterial protein was isocitrate dehydrogenase. In 1979, 25 years after the discovery of protein phosphorylation in eukaryotes, this enzyme was reported to become phosphorylated on a serine residue. In subsequent years, numerous other bacterial proteins phosphorylated on Ser, Thr or Tyr were discovered and the corresponding protein kinases and P-protein phosphatases were identified. These protein modifications regulate all kinds of physiological processes. Ser/Thr/Tyr phosphorylation in bacteria therefore seems to play a similar important role as in eukaryotes. Surprisingly, many bacterial protein kinases do not exhibit any similarity to eukaryotic protein kinases, but rather resemble nucleotide-binding proteins or kinases phosphorylating diverse low-molecular-weight substrates.

Bacteria↗
PubMed2005

Protein-tyrosine phosphorylation in Bacillus subtilis.

In recent years bacterial protein-tyrosine kinases have been found to phosphorylate a growing number of protein substrates, including RNA polymerase sigma factors, UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. The activity of these protein substrates was affected by tyrosine phosphorylation, indicating that this post-translational modification could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this field was done in Bacillus subtilis, and we here present the current state of knowledge on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network of protein-tyrosine phosphorylation. We discuss the approaches currently used to chart this network: ranging from studies of substrate specificity and the physiological role of tyrosine phosphorylation of individual enzymes to the global approaches at the level of systems biology.

Adaptation, Physiological↗
PubMed2005

How seryl-phosphorylated HPr inhibits PrfA, a transcription activator of Listeria monocytogenes virulence genes.

Listeria monocytogenes PrfA, a transcription activator for several virulence genes, including the hemolysin-encoding hly, is inhibited by rapidly metabolizable carbon sources (glucose, fructose, etc.). This inhibition is not mediated via the major carbon catabolite repression mechanism of gram-positive bacteria, since inactivation of the catabolite control protein A (CcpA) did not prevent the repression of virulence genes by the above sugars. In order to test whether the catabolite co-repressor P-Ser-HPr might be involved in PrfA regulation, we used a Bacillus subtilis strain (BUG1199) containing L. monocytogenes prfA under control of pspac and the lacZ reporter gene fused to the PrfA-activated hly promoter. Formation of P-Ser-HPr requires the bifunctional HPr kinase/phosphorylase (HprK/P), which, depending on the concentration of certain metabolites, either phosphorylates HPr at Ser-46 or dephosphorylates P-Ser-HPr. The hprKV267F allele codes for an HprK/P leading to the accumulation of P-Ser-HPr, since it has normal kinase, but almost no phosphorylase activity. Interestingly, introducing hprKV267F into BUG1199 strongly inhibited transcription activation by PrfA. Preventing the accumulation of P-Ser-HPr in the hprKV267F mutant by replacing Ser-46 in HPr with an alanine restored PrfA activity, while ccpA inactivation had no effect. Interestingly, disruption of ccpA in the hprK wild-type strain BUG1199 also led to inhibition of PrfA. The lowered lacZ expression in the ccpA strain is probably also due to elevated amounts of P-Ser-HPr, since it disappeared when Ser-46 in HPr was replaced with an alanine. To carry out its catalytic function in sugar transport, HPr of the phosphotransferase system (PTS) is also phosphorylated by phosphoenolpyruvate and enzyme I at His-15. However, P-Ser-HPr is only very slowly phosphorylated by enzyme I, which probably accounts for PrfA inhibition. In agreement with this concept, disruption of the enzyme I- or HPr-encoding genes also strongly inhibited PrfA activity. PrfA activity therefore seems to depend on a fully functional PTS phosphorylation cascade.

Amino Acid Substitution↗
PubMed2004

Is 2-phosphoglycerate-dependent automodification of bacterial enolases implicated in their export?

We observed that in vivo and in vitro a small fraction of the glycolytic enzyme enolase became covalently modified by its substrate 2-phosphoglycerate (2-PG). In modified Escherichia coli enolase, 2-PG was bound to Lys341, which is located in the active site. An identical reversible modification was observed with other bacterial enolases, but also with enolase from Saccharomyces cerevisiae and rabbit muscle. An equivalent of Lys341, which plays an important role in catalysis, is present in enolase of all organisms. Covalent binding of 2-PG to this amino acid rendered the enzyme inactive. Replacement of Lys341 of E.coli enolase with other amino acids prevented the automodification and in most cases strongly reduced the activity. As reported for other bacteria, a significant fraction of E.coli enolase was found to be exported into the medium. Interestingly, all Lys341 substitutions prevented not only the automodification, but also the export of enolase. The K341E mutant enolase was almost as active as the wild-type enzyme and therefore allowed us to establish that the loss of enolase export correlates with the loss of modification and not the loss of glycolytic activity.

Animals↗
PubMed2004

HPr kinase/phosphorylase, a Walker motif A-containing bifunctional sensor enzyme controlling catabolite repression in Gram-positive bacteria.

Carbon catabolite repression (CCR) in Gram-positive bacteria is regulated by the bifunctional enzyme HPr kinase/phosphorylase (HprK/P). This enzyme catalyses the ATP- as well as the pyrophosphate-dependent phosphorylation of Ser-46 in HPr, a phosphocarrier protein of a sugar transport and phosphorylation system. HprK/P also catalyses the pyrophosphate-producing, inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr (P-Ser-HPr). P-Ser-HPr functions as catabolite co-repressor by interacting with the LacI/GalR-type repressor, catabolite control protein A (CcpA), and allowing it to bind to operator sites preceding catabolite-regulated transcription units. HprK/P thus indirectly controls the expression of about 10% of the genes of Gram-positive bacteria. The two antagonistic activities of HprK/P are regulated by intracellular metabolites, which change their concentration in response to the absence or presence of rapidly metabolisable carbon sources (glucose, fructose, etc.) in the growth medium. Biochemical and structural studies revealed that HprK/P exhibits no similarity to eukaryotic protein kinases and that it contains a Walker motif A (or P-loop) as nucleotide binding site. Interestingly, HprK/P has a structural fold resembling that in kinases phosphorylating certain low molecular weight substrates such as nucleosides, nucleotides or oxaloacetate. The structures of the complexes of HprK/P with HPr and P-Ser-HPr have also been determined, which allowed proposing a detailed mechanism for the kinase and phosphorylase functions of HprK/P.

Amino Acid Motifs↗