Alain Mazé

Transmembrane modulator-dependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases

Protein‐tyrosine kinases regulating bacterial exopolysaccharide synthesis autophosphorylate on tyrosines located in a conserved C‐terminal region. So far no other substrates have been identified for these kinases. Here we demonstrate that Bacillus subtilis YwqD not only autophosphorylates at Tyr‐228, but that it also phosphorylates the two UDP‐glucose dehydrogenases (UDP‐glucose DHs) YwqF and TuaD at a tyrosine residue. However, phosphorylation of YwqF and TuaD occurs only in the presence of the transmembrane protein YwqC. The presumed intracellular C‐terminal part of YwqC (last 50 amino acids) seems to interact with the tyrosine‐kinase and to allow YwqD‐catalysed phosphorylation of the two UDP‐glucose DHs, which are key enzymes for the synthesis of acidic polysaccharides. However, only when phosphorylated by YwqD do the two enzymes exhibit detectable UDP‐glucose DH activity. Dephosphorylation of P‐Tyr‐YwqF and P‐Tyr‐TuaD by the P‐Tyr‐protein phosphatase YwqE switched off their UDP‐glucose DH activity. YwqE, which is encoded by the fourth gene of the B.subtilis ywqCDEF operon, also dephosphorylates P‐Tyr‐YwqD.

Complete Genome Sequence of the Probiotic Lactobacillus casei Strain BL23

The entire genome of Lactobacillus casei BL23, a strain with probiotic properties, has been sequenced. The genomes of BL23 and the industrially used probiotic strain Shirota YIT 9029 (Yakult) seem to be very similar.

Identification and Characterization of a Fructose Phosphotransferase System in Bifidobacterium breve UCC2003

ABSTRACT 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.

Transcription Regulators Potentially Controlled by HPr Kinase/Phosphorylase in Gram-Negative Bacteria

Phosphorylation and dephosphorylation at Ser-46 in HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is controlled by the bifunctional HPr kinase/phosphorylase (HprK/P). In Gram-positive bacteria, P-Ser-HPr controls (1) sugar uptake via the PTS; (2) catabolite control protein A (CcpA)-mediated carbon catabolite repression, and (3) inducer exclusion. Genome sequencing revealed that HprK/P is absent from Gram-negative enteric bacteria, but present in many other proteobacteria. These organisms also possess (1) HPr, the substrate for HprK/P; (2) enzyme I, which phosphorylates HPr at His-15, and (3) one or several enzymes IIA, which receive the phosphoryl group from P∼His-HPr. The genes encoding the PTS proteins are often organized in an operon with hprK. However, most of these organisms miss CcpA and a functional PTS, as enzymes IIB and membrane-integrated enzymes IIC seem to be absent. HprK/P and the rudimentary PTS phosphorylation cascade in Gram-negative bacteria must therefore carry out functions different from those in Gram-positive organisms. The gene organization in many HprK/P-containing Gram-negative bacteria as well as some preliminary experiments suggest that HprK/P might control transcription regulators implicated in cell adhesion and virulence. In α-proteobacteria, hprK is located downstream of genes encoding a two-component system of the EnvZ/OmpR family. In several other proteobacteria, hprK is organized in an operon together with genes from the rpoN region of Escherichia coli (rpoN encodes a σ54). We propose that HprK/P might control the phosphorylation state of HPr and EIIAs, which in turn could control the transcription regulators.

The Cold Shock Response of Lactobacillus casei: Relation between HPr Phosphorylation and Resistance to Freeze/Thaw Cycles

When carrying out a proteome analysis with a ptsH3 mutant of Lactobacillus casei, we found that the cold shock protein CspA was significantly overproduced compared to the wild-type strain. We also noticed that CspA and CspB of L. casei and CSPs from other organisms exhibit significant sequence similarity to the C-terminal part of EIIAGlc, a glucose-specific component of the phosphoenolpyruvate:sugar phosphotransferase system. This similarity suggested a direct interaction of HPr with CSPs, as histidyl-phosphorylated HPr has been shown to phosphorylate EIIAGlc in its C-terminal part. We therefore compared the cold shock response of several carbon catabolite repression mutants to that of the wild-type strain. Following a shift from 37°C to lower temperatures (20, 15 or 10°C), all mutants showed significantly reduced growth rates. Moreover, glucose-grown mutants unable to form P-Ser-HPr (ptsH1, hprK) exhibited drastically increased sensitivity to freeze/thaw cycles. However, when the same mutants were grown on ribose or maltose, they were similarly resistant to freezing and thawing as the wild-type strain. Although subsequent biochemical and genetic studies did not allow to identify the form of HPr implicated in the resistance to cold and freezing conditions, they strongly suggested a direct interaction of HPr or one of its phospho-derivatives with CspA and/or another, hitherto undetected cold shock protein in L. casei.

Utilization of d-Ribitol by Lactobacillus casei BL23 Requires a Mannose-Type Phosphotransferase System and Three Catabolic Enzymes

Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment D-ribitol (also called D-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates D-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in D-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented D-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a D-ribitol-5-phosphate (D-ribitol-5-P) 2-dehydrogenase, a D-ribulose-5-P 3-epimerase, a D-ribose-5-P isomerase, and a D-xylulose-5-P phosphoketolase. In the first catabolic step, the protein D-ribitol-5-P 2-dehydrogenase uses NAD(+) to oxidize D-ribitol-5-P formed during PTS-catalyzed transport to D-ribulose-5-P, which, in turn, is converted to D-xylulose-5-P by the enzyme D-ribulose-5-P 3-epimerase. Finally, the resulting D-xylulose-5-P is split by D-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate D-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as D-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation.

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.