SummaryLipopolysaccharides (LPSs) are complex glycolipids found in the outer membrane of Gram-negative bacteria. The lipid A-core component of the LPS molecule provides a versatile anchor to which a surface polymer:lipid A-core ligase enzyme can attach one or more structurally distinct surface polymers in a single bacterial strain. In some cases the same polymer can be found on the cell surface in both lipid A-core-linked and -unlinked forms. Analysis by SDS-PAGE of populations of LPS molecules extracted from bacterial cells indicates that there is extensive heterogeneity in their size distribution. Much of the heterogeneity results from complex modal distributions in the chain length of the polymers which are attached to lipid A-core. This is the result of preferential ligation of polymers with specific degrees of polymerization during the assembly of the LPS molecule. The surface architecture of the Gram-negative bacterial cell is therefore profoundly affected by the activities of the surface polymer:lipid A-core ligase and by molecular determinants of polymer chain length. Because of the involvement of cell-surface polymers in interactions between pathogenic bacteria and their hosts, these enzymatic activities also have an important impact on virulence. In this review, the organization of LPSs and related surface polymers will be described and the current understanding of the molecular mechanisms involved in surface diversity will be discussed. Emphasis is placed on the Enterobacteriaceae, but similarities to other bacteria suggest that aspects of the enterobacterial system will have broader significance.
Unlike Glf, RfbD KPO1 showed a requirement for NADH or NADPH, which could not be replaced by NAD or NADP. RfbD KPO1 was used to synthesize milligram quantities of UDP-Galf, allowing this compound to be purified and fully characterized in an intact form for the first time. The structure of UDP-Galf was proven by NMR spectroscopy.
The nucleotide sequence of a 3.4-kb EcoRI-PstI DNA fragment of Xanthomonas campestris pv. campestris revealed two open reading frames, which were designated xanA and xanB. The genes xanA and xanB encode proteins of 448 amino acids (molecular weight of 48,919) and 466 amino acids (molecular weight of 50,873), respectively. These genes were identified by analyzing insertion mutants which were known to be involved in xanthan production. Specific tests for the activities of enzymes involved in the biosynthesis of UDP-glucose and GDP-mannose indicated that the xanA gene product was involved in the biosynthesis of both glucose 1-phosphate and mannose 1-phosphate. The deduced amino acid sequence of xanB showed a significant degree of homology (59%) to the phosphomannose isomerase of Pseudomonas aeruginosa, a key enzyme in the biosynthesis of alginate. Moreover, biochemical analysis and complementation experiments with the Escherichia coli manA fragment revealed that xanB encoded a bifunctional enzyme, phosphomannose isomerase-GDP-mannose pyrophosphorylase.
A 2.6 kb ClaI-BamHI DNA fragment of megaplasmid 2 of Rhizobium meliloti 2011 was found to carry genes involved in exopolysaccharide synthesis and infection of alfalfa nodules. The analysis of the nucleotide sequence of this DNA fragment revealed the existence of two open reading frames (ORFs) running in opposite directions. Plasmid integration mutagenesis showed that these ORFs are organized as two monocistronic transcription units. One of the ORFs represents a new exo gene designated exoZ, which is involved in, but not essential for, the production of acidic exopolysaccharide. However, exoZ is not necessary for nodule formation with alfalfa. The ExoZ protein was found to show homology (23.3%) to the NodX protein of the R. leguminosarum biovar viciae strain TOM, known to be essential for nodulating the primitive Afghanistan pea. The second identified ORF corresponds to the exoB locus. The deduced amino acid sequence of the ExoB protein is homologous (39.6%) to that of the Escherichia coli GalE protein. In R. meliloti, exoB codes for a UDP-glucose 4-epimerase. A deficiency in the activity of this enzyme fully accounts for all the multiple carbohydrate defects that have been observed in exoB mutants.
The nucleotide sequence of a 3.6-kb HindIII-SmaI DNA fragment of Xanthomonas campestris pv. campestris revealed four open reading frames which, based on sequence homologies, were designated tonB, exbB, exbD1, and exbD2. Analysis of translational fusions to alkaline phosphatase and -galactosidase confirmed that the TonB, ExbB, ExbD1, and ExbD2 proteins are anchored in the cytoplasmic membrane. The TonB protein of X. campestris pv. campestris lacks the conserved (Glu-Pro) n and (Lys-Pro) m repeats but harbors a 13-fold repeat of proline residues. By mutational analysis, the tonB, exbB, and exbD1 genes were shown to be essential for ferric iron import in X. campestris pv. campestris. In contrast, the exbD2 gene is not involved in the uptake of ferric iron.
By mutational analysis it was found that a 3.9-kb SmaI-XhoII DNA fragment of Xanthomonas campestris pv. campestris is involved in lipopolysaccharide (LPS) biosynthesis. LPS samples isolated from different mutants carrying mutations in the 3.9-kb SmaI-XhoII DNA fragment exhibited banding patterns in silver-stained sodium dodecyl sulfate-polyacrylamide gels markedly different from that of the wild-type LPS. Moreover, comparison of the monosaccharide composition obtained by high-performance anion-exchange chromatography with pulsed amperometric detection of LPS purified from wild-type Xanthomonas campestris pv. campestris B100 and from mutants with mutations in the 3.9-kb SmaI-XhoII DNA fragment revealed a lack of rhamnose moieties in the mutant LPS. Sequence analysis of this DNA fragment revealed four open reading frames (ORFs), designated ORF302, ORF183, ORF295, and ORF351. The deduced amino acid sequences of these ORFs showed a high degree of homology to the deduced amino acid sequences of the rfbC, rfbD, rfbA, and rfbB genes of Salmonella typhimurium LT2, which have been shown to encode a set of enzymes responsible for conversion of glucose 1-phosphate to dTDP-rhamnose.
A 2.1-kb SmaI-EcoRI DNA fragment upstream of the xanA and xanB genes of Xanthomonas campestris pv. campestris carries two ORFs encoding putative proteins with sequence similarities to the alpha- and beta-subunits of 3-oxoacid-CoA transferases. The two ORFs were termed lpsI and lpsJ because strains carrying appropriate mutations showed an autoagglutination phenotype and because lipopolysaccharides of these mutant strains were altered according to silver-stained polyacrylamide gels. The monosaccharide composition of the exopolysaccharide xanthan produced by lpsI and lpsJ mutants remained unchanged.
The pyruvate dehydrogenase complex from the thermophilic bacterium Thermus aquuticus was purified by Triton X-100 extraction and chromatography on phenyl-Sepharose CL-4B and HPLC-hydroxyapatite. The electrophoretic pattern of the purified enzyme complex was similar to that of the enzyme complex from Bacillus subtifis, with four bands: the a-chain (Mr 39600) and @-chain (Mr 37500) of the pyruvate dehydrogenase component, the dihydrolipoamide acetyltransferase component ( Mr 58500) and the dihydrolipoamide dehydrogenase component (Mr 53900). Antibodies against the purified T. aquuticus pyruvate dehydrogenase complex cross-reacted with the enzyme complex from B. subtilis and, to a minor extentiwith that from bovine heart. No cross-reactivity could be observed with the enzyme complex from Escherkhiu coli. The T. uquuticus enzyme complex had a temperature maximum at 72 OC. 2-Oxobutyrate was a poor substrate and other 2-oxoacids were competitive inhibitors of the overall reaction. Long-chain 2-oxoacids showed a greater inhibitory effect, possibly caused by hydrophobic interactions. GTP inhibited the enzyme activity. Regulation of the pyruvate dehydrogenase complex from T. aquaticus by allosteric mechanisms or by reversible phosphorylation could not be demonstrated.
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