Ali A, Mahmud Z H, Morris J G, Sozhamannan S, Johnson J A
Departments of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Infect Immun. 2000 Dec;68(12):6857-64. doi: 10.1128/IAI.68.12.6857-6864.2000.
Vibrio cholerae can switch from a smooth to a wrinkled or rugose colony phenotype characterized by the secretion of a polysaccharide that enables the bacteria to survive harsh environmental conditions. In order to understand the genetic basis of rugosity, we isolated TnphoA-induced stable, smooth mutants of two O1 El Tor rugose strains and mapped the insertion sites in several of the mutants using a modified Y-adapter PCR technique. One of the TnphoA insertions was mapped to the first gene of the vps region that was previously shown to encode the rugose polysaccharide biosynthesis cluster. Three insertions were mapped to a previously unknown hlyA-like gene, also in the vps region. Five other insertions were found in loci unlinked to the vps region: (i) in the epsD gene (encodes the "secretin" of the extracellular protein secretion apparatus), (ii) in a hydG-like gene (encodes a sigma(54)-dependent transcriptional activator similar to HydG involved in labile hydrogenase production in Escherichia coli, (iii) in a gene encoding malic acid transport protein upstream of a gene similar to yeiE of E. coli (encodes a protein with similarities to LysR-type transcriptional activators), (iv) in dxr (encodes 1-deoxy-D-xylulose 5-phosphate reductoisomerase), and (v) in the intergenic region of lpd and odp (encode enzymes involved in the pyruvate dehydrogenase complex formation). These data suggest the involvement of a complex regulatory network in rugose polysaccharide production and highlight the general utility of the Y-adapter PCR technique described here for rapid mapping of transposon insertion sites.
霍乱弧菌能够从光滑菌落表型转变为褶皱或粗糙菌落表型，其特征是分泌一种多糖，这种多糖能使细菌在恶劣环境条件下存活。为了了解粗糙表型的遗传基础，我们分离了两株O1埃托型粗糙菌株经TnphoA诱导产生的稳定光滑突变体，并使用改良的Y-衔接子PCR技术定位了其中几个突变体的插入位点。其中一个TnphoA插入位点被定位到vps区域的第一个基因，该区域先前已被证明编码粗糙多糖生物合成簇。另外三个插入位点被定位到vps区域中一个以前未知的hlyA样基因。还在与vps区域不连锁的位点发现了另外五个插入位点：（i）在epsD基因中（编码细胞外蛋白质分泌装置的“分泌素”），（ii）在hydG样基因中（编码一种与HydG类似的σ54依赖性转录激活因子，HydG参与大肠杆菌中不稳定氢化酶的产生），（iii）在与大肠杆菌yeiE类似的基因上游的一个编码苹果酸转运蛋白的基因中（编码一种与LysR型转录激活因子相似的蛋白质），（iv）在dxr基因中（编码1-脱氧-D-木酮糖5-磷酸还原异构酶），以及（v）在lpd和odp的基因间区域（编码参与丙酮酸脱氢酶复合体形成的酶）。这些数据表明存在一个复杂的调控网络参与粗糙多糖的产生，并突出了本文所述的Y-衔接子PCR技术在快速定位转座子插入位点方面的普遍实用性。
