Woolwine S C, Wozniak D J
Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1064, USA.
J Bacteriol. 1999 Jan;181(1):107-16. doi: 10.1128/JB.181.1.107-116.1999.
Strains of Pseudomonas aeruginosa isolated from the respiratory tracts of patients with cystic fibrosis often display a mucoid morphology due to high levels of expression of the exopolysaccharide alginate. The response regulator AlgB is required for full transcription of the alginate biosynthetic operon. Repeated attempts to demonstrate a direct interaction between AlgB and the promoter region of algD, the first gene in the alginate operon, have thus far been unsuccessful. The possibility that AlgB exerts its effect on algD indirectly exists. To identify putative genes under the control of AlgB which affect algD transcription, transposon mutagenesis of nonmucoid algB derivatives of the mucoid strain FRD1 was employed. Of approximately 3,000 transposon mutants screened, 6 were found to display phenotypes which were mucoid relative to the phenotype of the parental algB strain. The phenotypes of these mutants ranged from being only slightly mucoid to being indistinguishable from that of the original FRD1 strain. One of the particularly mucoid transposon mutants was chosen for further study. This strain was found to be disrupted in a previously uncharacterized open reading frame with 56% amino acid identity to PepA of Escherichia coli. PepA is classified as a leucine aminopeptidase, and homologs have been detected in a number of bacterial, plant, and animal species. This novel gene has been designated phpA (P. aeruginosa homolog of pepA). The insertional inactivation of phpA was found to correlate with the mucoid phenotype and an increase in algD transcription in the algB strain. Expression of phpA from an ectopic chromosomal locus compensated for the transposon insertion in the native phpA gene, restoring algD transcription to levels similar to those observed in the parental algB strain. While phpA expression did not appear to be under the control of AlgB at the transcriptional level, this study demonstrates that loss of phpA in an algB genetic background had a positive effect on alginate expression and, more specifically, on transcription of the alginate biosynthetic operon.
从囊性纤维化患者呼吸道分离出的铜绿假单胞菌菌株，由于胞外多糖藻酸盐的高表达，常常呈现出黏液样形态。应答调节因子AlgB是藻酸盐生物合成操纵子进行完全转录所必需的。迄今为止，多次尝试证明AlgB与藻酸盐操纵子中第一个基因algD的启动子区域之间存在直接相互作用均未成功。AlgB间接影响algD的可能性是存在的。为了鉴定受AlgB控制且影响algD转录的假定基因，对黏液样菌株FRD1的非黏液样algB衍生物进行了转座子诱变。在大约3000个筛选的转座子突变体中，发现有6个呈现出相对于亲本algB菌株表型而言为黏液样的表型。这些突变体的表型范围从仅轻微黏液样到与原始FRD1菌株的表型无法区分。选择了其中一个特别黏液样的转座子突变体进行进一步研究。发现该菌株在一个先前未表征的开放阅读框中发生了破坏，该开放阅读框与大肠杆菌的PepA具有56%的氨基酸同一性。PepA被归类为亮氨酸氨肽酶，并且在许多细菌、植物和动物物种中都检测到了其同源物。这个新基因被命名为phpA（铜绿假单胞菌中pepA的同源物）。发现phpA的插入失活与黏液样表型以及algB菌株中algD转录的增加相关。从异位染色体位点表达phpA弥补了天然phpA基因中的转座子插入，将algD转录恢复到与亲本algB菌株中观察到的水平相似。虽然phpA的表达在转录水平上似乎不受AlgB的控制，但这项研究表明，在algB遗传背景中phpA的缺失对藻酸盐表达，更具体地说，对藻酸盐生物合成操纵子的转录有积极影响。
