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A study was conducted to identify a gene cluster enabling Lactobacillus casei BL23 to utilize myo-inositol (MI). The results reveal that Lactobacillus casei BL23 utilizes Mi carbon source and iolk gene codes for a malonate semialdehyde decraboxylate.

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.

In silico analysis of the Bifidobacterium breve UCC2003 genome has helped in identifying the four genetic loci, each of which has specified a putative enzyme II (EII) protein of a phosphoenolpyruvate:sugar phosphotransferase system. Biochemical studies have shown that FruA has phosphorylated fructose at the C-6 position.

The mechanisms that guide the carbohydrate phosphotransferse system (PTS), which controls carbohydrate metabolism by catalyzing the transport of several polysaccharides, polyols and other sugar derivatives, are discussed. Findings have shown that PTS regulation network, apart from controlling the metabolism, also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

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 manLMNQ messenger, but an additional manQ 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.

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 YflJ. The substrates for both YwlE and YfkJ are presently unknown, while YwqE was shown to dephosphorylate two phosphotyrosine-containiug 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.

A proteome analysis of Lactobacillus casei mutants that are affected in carbon catabolite repression revealed that a 15-kDa protein was strongly overproduced in a ptsHI47T mutant. This protein was identified as EIIA of a mannose class phosphotransferase system (PTS). A 7.1-kb DNA fragment containing the EIIA-encoding open reading frame and five other genes was sequenced. The first gene encodes a protein resembling the RpoN ([[sigma].sup.54])-dependent Bacillus subtilis transcription activator LevR. The following pentacistronic operon is oriented in the opposite direction and encodes four proteins with strong similarity to the proteins of the B. subtilis Lev-PTS and one protein of unknown function. The genes present on the 7.1-kb DNA fragment were therefore called levR and levABCDX. The levABCDX operon was induced by fructose and mannose. No '-12, -24' promoter typical of RpoN-dependent genes precedes the L. casei lev operon, and its expression was therefore RpoN independent but required LevR. Phosphorylation of LevR by P~His-HPr stimulates its activity, while phosphorylation by P~EII[B.sup.Lev] inhibits it. Disruption of the EII[B.sup.Lev]-encoding levB gene therefore led to strong constitutive expression of the lev operon, which was weaker in a strain carrying a ptsI mutation preventing phosphorylation by both P~EII[B.sup.Lev] and P~His-HPr. Expression of the L. casei lev operon is also subject to P-Ser-HPr-mediated catabolite repression. The observed slow phosphoenolpyruvate- and ATP-dependent phosphorylation of HPrI47T as well as the slow phosphoryl group transfer from the mutant P~His-HPr to EII[A.sup.Lev] are assumed to be responsible for the elevated expression of the lev operon in the ptsHI47T mutant.

Research has been conducted on Bacillus subtilis antiterminator LicT. The effect of gene mutations on LicT activity, inducibility and antitermination-mediated carbon catabolite repression has been investigated and the results are reported.

Results show that the serine-phosphorylated HPr of Lactococcus lactis mediates carbon catabolite repression of operons encoding enzymes and excluding non-phosphotransferase system carbohydrates, such as maltose and ribose. Data further indicate that the serine-phosphorylated Hpr enzyme is not involved in inducer exclusion.

Results reveal that HPr kinase/P-Ser-HPr phosphatase of gram-positive bacteria is a regulator protein and controls catabolic expression. Data also point out that the bifunctional enzyme may not be involved in the inducer expulsion regulatory process.

Research was conducted to demonstrate the mechanism of glycerol transport and phosphorylation of glycerol kinase in Thermus flavus. Results show glucose represses glycerol transport and metabolism, and phosphoenol-pyruvate dependent phosphorylate glycerol kinase.

Research was conducted to examine any changes in the catalytic and regulatory functions of catabolite repression HPr (Crh) protein by substituting glutamine-15 to a histidine residue. Results show that the modification, although it enables Crh carry out phosphorylation, it disables sugar transport function to Crh.

Results demonstrate that pta gene is involved in the carbon excretion and encoding for phosphotransacetylase in Bacillus subtilis. The data also indicate that two proteins influence pta expression by catabolite activation.

The role of P-Ser-HPr and P-Ser Crh in carbon catabolite repression (CCR) of the Bacillus subtilis levanase operon was investigated by constructing a ptsH1 crh1 double mutant and examining the interaction of the CcpA/P-Ser-HPr and CcpA/P-Ser Crh complexes with the cre sequence of the operon. The involvement of HPr kinase in the regulation of the levanase operon was also examined. The expression of the levanase operon was found to be totally relieved from CCR in ptsH1 crh1 and hprK mutants. It was also observed that both P-Ser-HPr and P-Ser Crh induced the binding of CcpA to the cre sequence of the operon.

Genetic and biochemical proofs that LevD in Bacillus subtilis can be phosphorylated by phosphoenolpyruvate (PEP), enzyme I and histidine-containing phosphocarrier protein (HPr) and that phosphorylation of LevE needs LevD in addition to enzyme I, HPr and PEP, are shown. Also, the phosphorylation site of LevD and LevE were identified and the phosphorylatable amino acid in LevD was replaced by an alanyl residue and that in LevE with an aspartate, glutamate or alanyl residue. These mutations resulted in greatly reduced fructose uptake.

The (phosphoenolpyruvate) PEP-dependent phoshorylation site in the seryl-phosphorylated HPr of Bacillus subtilis affects the HPr-mediated carbon catabolite repression of the gnt operon. The P-ser Hpr is involved in both the phosphotransferase system (PTS) sugar transport and non-PTS sugra transport of the gram-positive bacteria. The His-15 site of PEP-dependent phosphorylation of Hpr is required for the metabolite-activated formation of the complex between P-Ser HPr and the catabolite control protein A.

A research effort focuses on how the phosphotransferase system (PTS) transports its sugar substrates, represses the synthesis of carbohydrate catabolic enzymes, inhibits PTS and non-PTS sugar uptake, and stimulates the efflux of accumulated sugars in a species-specific manner. Results show that the metabolite-activated protein kinase-mediated phosphorylation of Ser-46 in HPr has a significant role in catabolite repression and inducer level control in low-GC Gram-positive bacteria.