Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6.

We report the identification and characterization of the eps gene cluster of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis. This report is the first genetic work concerning EPS production in a food microorganism. The EPS secreted by this strain consists of the following tetrasaccharide repeating unit:-->3)-beta-D-Galp-(1-->3)-[alpha-D-Galp-(1-->6)]-beta-D- D-Galp-(1-->3)-alpha-D-Galp-D-GalpNAc-(1-->. The genetic locus The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants. Sequence analysis of the gene region, which was obtained from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames. EPS expression in the non-EPS-producing heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 genes epsA to epsM was capable of directing EPS synthesis and secretion in this host. Homology searches of the predicted proteins in the Swiss-Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococcus pneumoniae and Streptococcus agalactiae. Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for eps K and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and epsM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments.     

Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome

Background  

The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs.     

Exopolysaccharide biosynthesis genes required for social motility in Myxococcus xanthus

Social (S)-motility in Myxococcus xanthus is a flagellum-independent gliding motility system that allows bacteria to move in groups on solid surfaces. S-motility has been shown to require type IV pili (TFP), exopolysaccharide (EPS; a component of fibrils) and lipopolysaccharide (LPS). Previously, information concerning EPS biogenesis in M. xanthus was lacking. In this study, we screened 5000 randomly mutagenized colonies for defects in S-motility and EPS and identified two genetic regions essential for EPS biogenesis: the EPS synthesis (eps) region and the EPS-associated (eas) region. Mutants with insertions in the eps and eas regions were defective in S-motility and fruiting body formation. These mutants failed to bind the dye calcofluor white, indicating that they lacked EPS; however, they retained normal TFP and LPS. Analysis of the eps locus showed several open reading frames (ORFs) that encode homologues to glycosyltransferases, glucanases and EPS transporters as well as regulatory proteins; the eas locus contains two ORFs: one exhibits homology to hypothetical proteins with a conserved domain of unknown function and the other displays no apparent homology to other proteins in the database. Further genetic mutagenesis analysis indicates that the whole eps region is involved in the biosynthesis of fibrils and fibril EPS. The operon at the proximal end of the eps region was analysed by generating in-frame deletion mutations. These mutants showed varying degrees of defects in the bacterium's ability to produce EPS or perform EPS-related functions, confirming the involvement of these genes in M. xanthus EPS biogenesis.     

Structure of the high molecular weight exopolysaccharide produced by Bifidobacterium animalis subsp. lactis IPLA-R1 and sequence analysis of its putative eps cluster

The bile adapted strain Bifidobacterium animalis subsp. lactis IPLA-R1 secretes a high molecular weight exopolysaccharide (HMW-EPS) when grown on the surface of agar-MRSC. This EPS is composed of l-rhamnopyranosyl, d-glucopyranosyl, d-galactopyranosyl and d-galactofuranosyl residues in the ratio of 3:1:1:1. Linkage analysis and 1D and 2D NMR spectroscopy were used to show that the EPS has a hexasaccharide repeating unit with the following structure:Treatment of the EPS with mild acid cleanly removed the terminal d-galactofuranosyl residue. The eps cluster sequenced for strain IPLA-R1 showed high genetic homology with putative eps clusters annotated in the genomes of strains from the same species. It is of note that several genes coding for rhamnose-precursors are present in the eps cluster, which could be correlated with the high percentage of rhamnose detected in its EPS repeated unit.     

Gene fusions to the ptsM/pel locus of Escherichia coli

Summary We have constructed gene fusions between ptsM/pel and lacZ. These fusions affect both phenotypes assigned to the ptsM/pel locus (at 40 min), namely, no growth on mannose or glucosamine and inhibition of the penetration of bacteriophage DNA, as well as that of other lambdoid phages such as Hy-2. Since the lacZ gene fusions are insertion mutations that abolish target gene function by disrupting the linear contiguity of the gene, it would appear that ptsM and pel are either the same gene, or two genes within the same operon. Several size classes of these ptsM/pel-lacZ fusions have been isolated and the corresponding hybrid proteins are associated with the cytoplasmic membrane of Escherichia coli. This is consistent with the proposal that ptsM/pel codes for Enzyme II of the phosphotransferase transport system (PTS) specific for mannose, glucosamine, fructose and glucose. However, we have also identified Tn10 insertion mutations that confer a Man^- phenotype but have no effect on the Pel phenotype. Complementation analysis indicates that the Tn10 insertions and the lacZ gene fusions are in different genes. Both of these genes are involved in mannose uptake. This suggests that the locus at 40 min can be subdivided into two genes whose products are required for mannose uptake and that only one of these is involved in the penetration of DNA.     

Ralstonia solanacearum genes induced during growth in tomato: an inside view of bacterial wilt

The phytopathogen Ralstonia solanacearum has over 5000 genes, many of which probably facilitate bacterial wilt disease development. Using in vivo expression technology (IVET), we screened a library of 133 200 R. solanacearum strain K60 promoter fusions and isolated approximately 900 fusions expressed during bacterial growth in tomato plants. Sequence analysis of 307 fusions revealed 153 unique in planta-expressed (ipx) genes. These genes included seven previously identified virulence genes (pehR, vsrB, vsrD, rpoS, hrcC, pme and gspK) as well as seven additional putative virulence factors. A significant number of ipx genes may reflect adaptation to the host xylem environment; 19.6%ipx genes are predicted to encode proteins with metabolic and/or transport functions, and 9.8%ipx genes encode proteins possibly involved in stress responses. Many ipx genes (18%) encode putative transmembrane proteins. A majority of ipx genes isolated encode proteins of unknown function, and 13% were unique to R. solanacearum. The ipx genes were variably induced in planta; beta-glucuronidase reporter gene expression analysis of a subset of 44 ipx fusions revealed that in planta expression levels were between two- and 37-fold higher than in culture. The expression of many ipx genes was subject to known R. solanacearum virulence regulators. Of 32 fusions tested, 28 were affected by at least one virulence regulator; several fusions were controlled by multiple regulators. Two ipx fusion strains isolated in this screen were reduced in virulence on tomato, indicating that gene(s) important for bacterial wilt pathogenesis were interrupted by the IVET insertion; mutations in other ipx genes are necessary to determine their roles in virulence and in planta growth. Collectively, this profile of ipx genes suggests that in its host, R. solanacearum confronts and overcomes a stressful and nutrient-poor environment.     

Mutations in the csgD Promoter Associated with Variations in Curli Expression in Certain Strains of Escherichia coli O157:H7

Single-base-pair csgD promoter mutations in human outbreak Escherichia coli O157:H7 strains ATCC 43894 and ATCC 43895 coincided with differential Congo red dye binding from curli fiber expression. Red phenotype csgD::lacZ promoter fusions had fourfold-greater expression than white promoter fusions. Cloning the red variant csgDEFG operon into white variants induced the red phenotype. Substrate utilization differed between red and white variants.