Martin D W, Schurr M J, Yu H, Deretic V
Department of Microbiology, University of Texas Health Science Center at San Antonio, 78284-7758.
J Bacteriol. 1994 Nov;176(21):6688-96. doi: 10.1128/jb.176.21.6688-6696.1994.
Alginate overproducition by mucoid Pseudomonas aeruginosa is a critical pathogenic determinant expressed by this organism during chronic infections in cystic fibrosis. Conversion to mucoidy and a subsequent loss of mucoid character can occur via different mutations in the algU mucA mucB gene cluster. The algU gene encodes a 22.2-kDa putative alternative sigma factor required for expression of the critical alginate biosynthetic gene algD. In this work, algU transcription was studied by S1 nuclease protection analysis. Transcription from the promoter proximal to the algU coding region was found to be dependent on AlgU. The -35 and -10 sequences of this newly mapped promoter showed strong similarity ot the promoters of two other critical alg genes: algD and algR. The proximal promoter of algR was also shown to depend on algU. Interestingly, the putative -35 and -10 regions of all three promoters displayed striking similarity to the consensus sequence of the sigma E-dependent promoters in Escherichia coli and Salmonella typhimurium. This 24-kDa sigma factor, controlling genes participating in resistance to high temperatures and oxidative stress, has been previously biochemically characterized, but the gene for sigma E remained unidentified. To examine whether AlgU is related to sigma E, the effect of algU inactivation on the sensitivity of P. aeruginosa to killing by heat and reactive oxygen intermediates was tested. Two isogenic pairs of algU+ and algU mutant strains were compared. The algU mutants, irrespective of the mucoid status of the parental strains, displayed increased sensitivity to killing by paraquat, known to generate intracellular superoxide radicals, and heat. Further lgobal homology searches revealed the presence of a previously unrecognized E. coli gene with the predicted gene product showing a striking 66% identity to AlgU. The corresponding gene from S. typhimurium was cloned and sequenced, and it is displayed one amino acid substitution relative to its E. coli equivalent. AlgU and its close homologs in E. coli and S. typhimurium may be functionally related.
黏液型铜绿假单胞菌过量产生藻酸盐是该菌在囊性纤维化慢性感染过程中表达的一个关键致病决定因素。通过藻酸操纵子基因簇（algU、mucA、mucB）中的不同突变，可发生向黏液型的转变以及随后黏液型特征的丧失。algU基因编码一个22.2 kDa的假定替代σ因子，它是关键的藻酸盐生物合成基因algD表达所必需的。在本研究中，通过S1核酸酶保护分析对algU转录进行了研究。发现从靠近algU编码区的启动子转录依赖于AlgU。这个新定位的启动子的-35和-10序列与另外两个关键藻酸基因algD和algR的启动子有很强的相似性。algR的近端启动子也显示依赖于algU。有趣的是，所有这三个启动子的假定-35和-10区域与大肠杆菌和鼠伤寒沙门氏菌中σE依赖型启动子的共有序列有显著相似性。这个24 kDa的σ因子控制参与高温和氧化应激抗性的基因，此前已进行了生化特性鉴定，但σE的基因仍未确定。为了检验AlgU是否与σE相关，测试了algU失活对铜绿假单胞菌对热和活性氧中间体杀伤敏感性的影响。比较了两对同源的algU+和algU突变株。algU突变体，无论亲本菌株的黏液型状态如何，对百草枯（已知可产生细胞内超氧自由基）和热杀伤的敏感性都增加。进一步的全局同源性搜索发现存在一个以前未被识别的大肠杆菌基因，其预测的基因产物与AlgU有惊人的66%同一性。克隆并测序了鼠伤寒沙门氏菌的相应基因，它与其大肠杆菌对应基因相比有一个氨基酸替换。AlgU及其在大肠杆菌和鼠伤寒沙门氏菌中的密切同源物可能在功能上相关。
