Rocchetta H L, Pacan J C, Lam J S
Department of Microbiology and Canadian Bacterial Diseases Network, University of Guelph, ON.
Mol Microbiol. 1998 Sep;29(6):1419-34. doi: 10.1046/j.1365-2958.1998.01024.x.
Pseudomonas aeruginosa is capable of producing various cell-surface polysaccharides including alginate, A-band and B-band lipopolysaccharides (LPS). The D-mannuronic acid residues of alginate and the D-rhamnose (D-Rha) residues of A-band polysaccharide are both derived from the common sugar nucleotide precursor GDP-D-mannose (D-Man). Three genes, rmd, gmd and wbpW, which encode proteins involved in the synthesis of GDP-D-Rha, have been localized to the 5' end of the A-band gene cluster. In this study, WbpW was found to be homologous to phosphomannose isomerases (PMIs) and GDP-mannose pyrophosphorylases (GMPs) involved in GDP-D-Man biosynthesis. To confirm the enzymatic activity of WbpW, Escherichia coli PMI and GMP mutants deficient in the K30 capsule were complemented with wbpW, and restoration of K30 capsule production was observed. This indicates that WbpW, like AlgA, is a bifunctional enzyme that possesses both PMI and GMP activities for the synthesis of GDP-D-Man. No gene encoding a phosphomannose mutase (PMM) enzyme could be identified within the A-band gene cluster. This suggests that the PMM activity of AlgC may be essential for synthesis of the precursor pool of GDP-D-Man, which is converted to GDP-D-Rha for A-band synthesis. Gmd, a previously reported A-band enzyme, and Rmd are predicted to perform the two-step conversion of GDP-D-Man to GDP-D-Rha. Chromosomal mutants were generated in both rmd and wbpW. The Rmd mutants do not produce A-band LPS, while the WbpW mutants synthesize very low amounts of A band after 18 h of growth. The latter observation was thought to result from the presence of the functional homologue AlgA, which may compensate for the WbpW deficiency in these mutants. Thus, WbpW AlgA double mutants were constructed. These mutants also produced low levels of A-band LPS. A search of the PAO1 genome sequence identified a second AlgA homologue, designated ORF488, which may be responsible for the synthesis of GDP-D-Man in the absence of WbpW and AlgA. Polymerase chain reaction (PCR) amplification and sequence analysis of this region reveals three open reading frames (ORFs), orf477, orf488 and orf303, arranged as an operon. ORF477 is homologous to initiating enzymes that transfer glucose 1-phosphate onto undecaprenol phosphate (Und-P), while ORF303 is homologous to L-rhamnosyltransferases involved in polysaccharide assembly. Chromosomal mapping using pulsed field gel electrophoresis (PFGE) and Southern hybridization places orf477, orf488 and orf303 between 0.3 and 0.9 min on the 75 min map of PAO1, giving it a map location distinct from that of previously described polysaccharide genes. This region may represent a unique locus within P. aeruginosa responsible for the synthesis of another polysaccharide molecule.
铜绿假单胞菌能够产生多种细胞表面多糖，包括藻酸盐、A带和B带脂多糖（LPS）。藻酸盐的D-甘露糖醛酸残基和A带多糖的D-鼠李糖（D-Rha）残基均源自共同的糖核苷酸前体GDP-D-甘露糖（D-Man）。三个基因，rmd、gmd和wbpW，编码参与GDP-D-Rha合成的蛋白质，已定位到A带基因簇的5'端。在本研究中，发现WbpW与参与GDP-D-Man生物合成的磷酸甘露糖异构酶（PMI）和GDP-甘露糖焦磷酸化酶（GMP）同源。为了确认WbpW的酶活性，用wbpW对缺乏K30荚膜的大肠杆菌PMI和GMP突变体进行互补，并观察到K30荚膜产生的恢复。这表明WbpW与AlgA一样，是一种双功能酶，具有用于合成GDP-D-Man的PMI和GMP活性。在A带基因簇中未鉴定到编码磷酸甘露糖变位酶（PMM）酶的基因。这表明AlgC的PMM活性对于GDP-D-Man前体池的合成可能是必不可少的，该前体池被转化为GDP-D-Rha用于A带合成。Gmd是先前报道的A带酶，Rmd预计执行GDP-D-Man到GDP-D-Rha的两步转化。在rmd和wbpW中均产生了染色体突变体。Rmd突变体不产生A带LPS，而WbpW突变体在生长18小时后合成极少量的A带。后一观察结果被认为是由于功能性同源物AlgA的存在，其可能补偿了这些突变体中WbpW的缺陷。因此，构建了WbpW AlgA双突变体。这些突变体也产生低水平的A带LPS。对PAO1基因组序列的搜索鉴定出第二个AlgA同源物，命名为ORF488，其可能在没有WbpW和AlgA的情况下负责GDP-D-Man的合成。该区域的聚合酶链反应（PCR）扩增和序列分析揭示了三个开放阅读框（ORF），orf477、orf488和orf303，它们作为一个操纵子排列。ORF477与将葡萄糖1-磷酸转移到十一碳烯醇磷酸（Und-P）上的起始酶同源，而ORF303与参与多糖组装的L-鼠李糖基转移酶同源。使用脉冲场凝胶电泳（PFGE）和Southern杂交进行的染色体定位将orf477、orf488和orf303定位在PAO1的75分钟图谱上0.3至0.9分钟之间，使其图谱位置与先前描述的多糖基因不同。该区域可能代表铜绿假单胞菌内负责另一种多糖分子合成的独特位点。
