Synthesis Of Lipopolysaccharide Core Research Articles

Elucidation of a Novel Role of YebC in Surface Polysaccharides Regulation of Escherichia coli bipA-Deletion.

The BipA (BPI-inducible protein A) protein is ubiquitously conserved in various bacterial species and belongs to the translational GTPase family. Interestingly, the function of Escherichia coli BipA is not essential for cell growth under normal growth conditions. However, cultivation of bipA-deleted cells at 20°C leads to cold-sensitive growth defect and several phenotypic changes in ribosome assembly, capsule production, and motility, suggesting its global regulatory roles. Previously, our genomic library screening revealed that the overexpressed ribosomal protein (r-protein) L20 partially suppressed cold-sensitive growth defect by resolving the ribosomal abnormality in bipA-deleted cells at low temperature. Here, we explored another genomic library clone containing yebC, which encodes a predicted transcriptional factor that is not directly associated with ribosome biogenesis. Interestingly, overexpression of yebC in bipA-deleted cells diminished capsule synthesis and partially restored lipopolysaccharide (LPS) core maturation at a low temperature without resolving defects in ribosome assembly or motility, indicating that YebC may be specifically involved in the regulation of exopolysaccharide and LPS core synthesis. In this study, we collectively investigated the impacts of bipA-deletion on E. coli capsule, LPS, biofilm formation, and motility and revealed novel roles of YebC in extracellular polysaccharide production and LPS core synthesis at low temperature using this mutant strain. Furthermore, our findings suggest that ribosomal defects as well as increased capsule synthesis, and changes in LPS composition may contribute independently to the cold-sensitivity of bipA-deleted cells, implying multiple regulatory roles of BipA.

Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain.

Escherichia coli is a diverse bacterial species that comprises commensal nonpathogenic strains such as E. coli K-12 and pathogenic strains that cause a variety of diseases in different host species. Avian pathogenic E. coli strain chi7122 (O78:K80:H9) was used in a chicken infection model to identify bacterial genes that are expressed in infected tissues. By using the cDNA selection method of selective capture of transcribed sequences and enrichment for the isolation of pathogen-specific (non-E. coli K-12) transcripts, pathogen-specific cDNAs were identified. Pathogen-specific transcripts corresponded to putative adhesins, lipopolysaccharide core synthesis, iron-responsive, plasmid- and phage-encoded genes, and genes of unknown function. Specific deletion of the aerobactin siderophore system and E. coli iro locus, which were identified by selective capture of transcribed sequences, demonstrated that these pathogen-specific systems contribute to the virulence of strain chi7122. Consecutive blocking to enrich for selection of pathogen-specific genes did not completely eliminate the presence of transcripts that corresponded to sequences also present in E. coli K-12. These E. coli conserved genes are likely to be highly expressed in vivo and contribute to growth or virulence. Overall, the approach we have used simultaneously provided a means to identify novel pathogen-specific genes expressed in vivo and insight regarding the global gene expression and physiology of a pathogenic E. coli strain in a natural animal host during the infectious process.

Pseudomonas aeruginosa galU is required for a complete lipopolysaccharide core and repairs a secondary mutation in a PA103 (serogroup O11) wbpM mutant.

Insertional inactivation of wbpM in Pseudomonas aeruginosa serogroup O11 strain PA103 resulted in mutants exhibiting three distinct lipopolysaccharide (LPS) phenotypes. One mutant, PA103 wbpM-C, had a truncated LPS core and lacked O antigen. These defects were not complemented by the cloned wbpM gene, suggesting a secondary mutation was present. When the wild-type galU gene was introduced into strain PA103 wbpM-C containing the cloned wbpM gene, both LPS defects were corrected. Construction of galU mutants in P. aeruginosa serogroups O11, O5, O6 and O17 strains led to truncation of the LPS core, indicating the involvement of GalU in P. aeruginosa LPS core synthesis.

Pseudomonas aeruginosa galU is required for a complete lipopolysaccharide core and repairs a secondary mutation in a PA103 (serogroup O11) wbpM mutant

Insertional inactivation of wbpM in Pseudomonas aeruginosa serogroup O11 strain PA103 resulted in mutants exhibiting three distinct lipopolysaccharide (LPS) phenotypes. One mutant, PA103 wbpM- C, had a truncated LPS core and lacked O antigen. These defects were not complemented by the cloned wbpM gene, suggesting a secondary mutation was present. When the wild-type galU gene was introduced into strain PA103 wbpM- C containing the cloned wbpM gene, both LPS defects were corrected. Construction of galU mutants in P. aeruginosa serogroups O11, O5, O6 and O17 strains led to truncation of the LPS core, indicating the involvement of GalU in P. aeruginosa LPS core synthesis.

Genetics of lipopolysaccharide biosynthesis in enteric bacteria.

From a historical perspective, the study of both the biochemistry and the genetics of lipopolysaccharide (LPS) synthesis began with the enteric bacteria. These organisms have again come to the forefront as the blocks of genes involved in LPS synthesis have been sequenced and analyzed. A number of new and unanticipated genes were found in these clusters, indicating a complexity of the biochemical pathways which was not predicted from the older studies. One of the most dramatic areas of LPS research has been the elucidation of the lipid A biosynthetic pathway. Four of the genes in this pathway have now been identified and sequenced, and three of them are located in a complex operon which also contains genes involved in DNA and phospholipid synthesis. The rfa gene cluster, which contains many of the genes for LPS core synthesis, includes at least 17 genes. One of the remarkable findings in this cluster is a group of several genes which appear to be involved in the synthesis of alternate rough core species which are modified so that they cannot be acceptors for O-specific polysaccharides. The rfb gene clusters which encode O-antigen synthesis have been sequenced from a number of serotypes and exhibit the genetic polymorphism anticipated on the basis of the chemical complexity of the O antigens. These clusters appear to have originated by the exchange of blocks of genes among ancestral organisms. Among the large number of LPS genes which have now been sequenced from these rfa and rfb clusters, there are none which encode proteins that appear to be secreted across the cytoplasmic membrane and surprisingly few which encode integral membrane proteins or proteins with extensive hydrophobic domains. These data, together with sequence comparison and complementation experiments across strain and species lines, suggest that the LPS biosynthetic enzymes may be organized into clusters on the inner surface of the cytoplasmic membrane which are organized around a few key membrane proteins.

Genetics of lipopolysaccharide biosynthesis in enteric bacteria.

From a historical perspective, the study of both the biochemistry and the genetics of lipopolysaccharide (LPS) synthesis began with the enteric bacteria. These organisms have again come to the forefront as the blocks of genes involved in LPS synthesis have been sequenced and analyzed. A number of new and unanticipated genes were found in these clusters, indicating a complexity of the biochemical pathways which was not predicted from the older studies. One of the most dramatic areas of LPS research has been the elucidation of the lipid A biosynthetic pathway. Four of the genes in this pathway have now been identified and sequenced, and three of them are located in a complex operon which also contains genes involved in DNA and phospholipid synthesis. The rfa gene cluster, which contains many of the genes for LPS core synthesis, includes at least 17 genes. One of the remarkable findings in this cluster is a group of several genes which appear to be involved in the synthesis of alternate rough core species which are modified so that they cannot be acceptors for O-specific polysaccharides. The rfb gene clusters which encode O-antigen synthesis have been sequenced from a number of serotypes and exhibit the genetic polymorphism anticipated on the basis of the chemical complexity of the O antigens. These clusters appear to have originated by the exchange of blocks of genes among ancestral organisms. Among the large number of LPS genes which have now been sequenced from these rfa and rfb clusters, there are none which encode proteins that appear to be secreted across the cytoplasmic membrane and surprisingly few which encode integral membrane proteins or proteins with extensive hydrophobic domains. These data, together with sequence comparison and complementation experiments across strain and species lines, suggest that the LPS biosynthetic enzymes may be organized into clusters on the inner surface of the cytoplasmic membrane which are organized around a few key membrane proteins.

Role of outer membrane components in the nonspecific binding ofEscherichia coli to cellulose

The involvement of lipopolysaccharide and outer membrane proteins in the binding ofEscherichia coli to cellulose was investigated. Cellulose binding was assayed in defined strains with or without O-antigenic polysaccharide and in mutants with defects in lipopolysaccharide core synthesis. Binding was also tested in strains lacking major outer membrane proteins. Optimal cellulose binding was exhibited by rough strains and was reduced to various extents in the presence of different O-antigens. Core defects also reduced but did not abolish binding to cellulose. Reduced binding was also found in mutants lacking OmpC protein, but OmpC/OmpA double mutants orompB mutants lacking OmpC and OmpF were not affected. Mutants with reduced cellulose binding were also isolated directly through selection of nonbinding populations after chromatography on cellulose columns. Each of the independent isolates derived fromE. coli K12 with reduced cellulose binding had multiple mutations, with additional phenotypic changes such as phage resistance, increased sensitivity to bile salts, or altered patterns of outer membrane proteins. These results suggest that no single receptor that could be altered by mutation was responsible for the binding ofE. coli to cellulose. Rather, the nonspecific binding of cellulose was more likely to be due to interaction with, or the combined activity of, several integral outer membrane components that could be masked by O-antigen.

Mutations in Salmonella typhimurium conferring resistance to Felix O phage without loss of smooth character.

Several mutants obtained from smooth Salmonella typhimurium strains by selection for resistance to Felix O (FO) phage [whose receptor site includes the N-acetylglucosamine branch of the lipopolysaccharide (LPS) core] were smooth in cultural properties, antigenic character and phage sensitivity pattern (except for their FO resistance). However, the affected genes of several such 'FOR' (FO-resistant) mutants were shown by transduction of map in the short cysE-pyrE segment, which includes nearly all known rfa genes responsible for synthesis of LPS core. All of seven FOR mutants differed from their parents, and resembled rfa mutants with defects in the deeper part of the LPS core, by increased sensitivity to various antibiotics. One FOR mutant was non-virulent (LD50 greater than 10-7, compared with smaller than 100 for its parent); LT7 derivatives given this FOR gene by co-transduction with cysE+ were likewise non-virulent. It is inferred that FOR mutations affect the assembly of the inner part of the LPS core, perhaps causing incomplete blocks in glycosyl transferase reactions.