Lightfoot J, Lam J S
Department of Microbiology, University of Guelph, Ontario, Canada.
Mol Microbiol. 1993 May;8(4):771-82. doi: 10.1111/j.1365-2958.1993.tb01620.x.
Pseudomonas aeruginosa can express two distinct forms of lipopolysaccharide (LPS), called A-band and B-band. As an attempt to understand the molecular biology of the synthesis and regulation of these LPS antigens, a recombinant plasmid, pFV3, containing genes for A-band expression was isolated previously. In the present study, P. aeruginosa strain PAO1 was mutagenized with transposon Tn5-751 and yielded a B-band-deficient mutant, called ge6. This mutant was mated with a PAO1 genomic library, and transconjugants were screened for complementation of B-band using B-band-specific monoclonal antibody MF15-4. Recombinant plasmid pFV100 was subsequently isolated by its ability to complement B-band expression in ge6. SDS-PAGE analysis of LPS from ge6 and ge6(pFV100) revealed that ge6 was deficient in expression of B-band, while ge6(pFV100) had an LPS profile similar to that of the parent strain PAO1. With A-band and B-band genes cloned in separate plasmids, pFV3 and pFV100 respectively, we were able to determine the map location of these LPS genes on the P. aeruginosa PAO1 chromosome using pulsed-field gel electrophoresis. A-band genes mapped at 5.75 to 5.89 Mbp (SpeI fragment SpK; DpnI fragment DpF2), while genes involved with expression of B-band LPS mapped at 1.9 Mbp (SpeI fragments SpC, SpI and SpAI; DpnI fragment DpD) on the 5.9 Mbp chromosome. We also performed initial characterization of a gene involved with synthesis of A-band present on pFV3. We previously reported that recombinant plasmid pFV3 and subcloned plasmid pFV36 complemented A-band synthesis in rd7513, an A- mutant derived from A+ strain AK1401. pFV36 was mutagenized with transposon Tn1000 to reveal a one-kilobase region capable of complementing the expression of A-band in the A- strain rd7513. This region was subcloned as a 1.6 kb KpnI fragment into plasmid vector pAK1900 and the resulting clone named pFV39. Labelling of proteins encoded by pAK1900 and pFV39 in Escherichia coli maxicells revealed a single unique polypeptide of approximately 37 kDa expressed by pFV39. Supernatants from disrupted cells of rd7513(pFV39) and AK1401 converted 14C-labelled-guanosine diphospho (GDP)-D-mannose to GDP-rhamnose, while supernatants from rd7513 did not show synthesis of GDP-rhamnose. The data therefore suggest that conversion of GDP-D-mannose to GDP-rhamnose is required for synthesis of A-band LPS, and that a 37 kDa protein is involved in this conversion.
铜绿假单胞菌可表达两种不同形式的脂多糖（LPS），称为A带和B带。为了试图了解这些LPS抗原合成与调控的分子生物学，先前已分离出一种含有A带表达基因的重组质粒pFV3。在本研究中，用转座子Tn5 - 751对铜绿假单胞菌PAO1菌株进行诱变，得到一个B带缺陷型突变体，称为ge6。将该突变体与PAO1基因组文库进行接合，利用B带特异性单克隆抗体MF15 - 4筛选转接合子的B带互补情况。随后通过其在ge6中互补B带表达的能力分离出重组质粒pFV100。对ge6和ge6(pFV100)的LPS进行SDS - PAGE分析表明，ge6缺乏B带表达，而ge6(pFV100)的LPS图谱与亲本菌株PAO1相似。由于A带和B带基因分别克隆在不同质粒pFV3和pFV100中，我们能够利用脉冲场凝胶电泳确定这些LPS基因在铜绿假单胞菌PAO1染色体上的图谱位置。A带基因定位于5.75至5.89 Mbp（SpeI片段SpK；DpnI片段DpF2），而与B带LPS表达相关的基因定位于5.9 Mbp染色体上的1.9 Mbp处（SpeI片段SpC、SpI和SpAI；DpnI片段DpD）。我们还对pFV3上存在的与A带合成相关的基因进行了初步表征。我们先前报道重组质粒pFV3和亚克隆质粒pFV36可互补rd7513中的A带合成，rd7513是源自A + 菌株AK1401的A - 突变体。用转座子Tn1000对pFV36进行诱变，以揭示一个能够互补A - 菌株rd7513中A带表达的1千碱基区域。该区域作为1.6 kb的KpnI片段亚克隆到质粒载体pAK1900中，得到的克隆命名为pFV39。在大肠杆菌大细胞中对pAK1900和pFV39编码的蛋白质进行标记，结果显示pFV39表达一种约37 kDa的独特单一多肽。rd7513(pFV39)和AK1401破碎细胞上清液可将14C标记的鸟苷二磷酸（GDP） - D - 甘露糖转化为GDP - 鼠李糖，而rd7513上清液未显示GDP - 鼠李糖的合成。因此，数据表明GDP - D - 甘露糖向GDP - 鼠李糖的转化是A带LPS合成所必需的，并且一种37 kDa的蛋白质参与了这种转化。
