Sweetman Wendy A, Moxon E Richard, Bayliss Christopher D
Paediatric Molecular Infectious Diseases Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
Microbiology (Reading). 2005 Aug;151(Pt 8):2751-2763. doi: 10.1099/mic.0.27996-0.
Haemophilus influenzae has microsatellite repeat tracts in 5' coding regions or promoters of several genes that are important for commensal and virulence behaviour. Changes in repeat number lead to switches in expression of these genes, a process referred to as phase variation. Hence, the virulence behaviour of this organism may be influenced by factors that alter the frequency of mutations in these repeat tracts. In Escherichia coli, induction of the SOS response destabilizes dinucleotide repeat tracts. H. influenzae encodes a homologue of the E. coli SOS repressor, LexA. The H. influenzae genome sequence was screened for the presence of the minimal consensus LexA-binding sequence from E. coli, CTG(N)(10)CAG, in order to identify genes with the potential to be SOS regulated. Twenty-five genes were identified that had LexA-binding sequences within 200 bp of the start codon. An H. influenzae non-inducible LexA mutant (lexA(NI)) was generated by site-directed mutagenesis. This mutant showed increased sensitivity, compared with wild-type (WT) cells, to both UV irradiation and mitomycin C (mitC) treatment. Semi-quantitative RT-PCR studies confirmed that H. influenzae mounts a LexA-regulated SOS response following DNA assault. Transcript levels of lexA, recA, recN, recX, ruvA and impA were increased in WT cells following DNA damage but not in lexA(NI) cells. Induction of the H. influenzae SOS response by UV irradiation or mitC treatment did not lead to any observable SOS-dependent changes in phase variation rates at either dinucleotide or tetranucleotide repeat tracts. Treatment with mitC caused a small increase in phase variation rates in both repeat tracts, independently of an SOS response. We suggest that the difference between H. influenzae and E. coli with regard to the effect of the SOS response on dinucleotide phase variation rates is due to the absence of any of the known trans-lesion synthesis DNA polymerases in H. influenzae.
流感嗜血杆菌在几个对共生和致病行为很重要的基因的5'编码区或启动子中具有微卫星重复序列。重复序列数量的变化会导致这些基因表达的切换，这一过程称为相变。因此，这种生物体的致病行为可能受到改变这些重复序列中突变频率的因素的影响。在大肠杆菌中，SOS反应的诱导会使二核苷酸重复序列不稳定。流感嗜血杆菌编码大肠杆菌SOS阻遏物LexA的同源物。为了鉴定具有SOS调控潜力的基因，对流感嗜血杆菌基因组序列进行筛选，寻找来自大肠杆菌的最小共有LexA结合序列CTG(N)(10)CAG。鉴定出25个在起始密码子200 bp内具有LexA结合序列的基因。通过定点诱变产生了一种流感嗜血杆菌非诱导型LexA突变体（lexA(NI)）。与野生型（WT）细胞相比，该突变体对紫外线照射和丝裂霉素C（mitC）处理均表现出更高的敏感性。半定量逆转录聚合酶链反应（RT-PCR）研究证实，流感嗜血杆菌在DNA攻击后会产生LexA调控的SOS反应。DNA损伤后，WT细胞中lexA、recA、recN、recX、ruvA和impA的转录水平升高，但lexA(NI)细胞中没有升高。紫外线照射或mitC处理诱导流感嗜血杆菌的SOS反应，在二核苷酸或四核苷酸重复序列处的相变率没有导致任何可观察到的SOS依赖性变化。mitC处理导致两个重复序列处的相变率略有增加，与SOS反应无关。我们认为，流感嗜血杆菌和大肠杆菌在SOS反应对二核苷酸相变率的影响方面存在差异，是由于流感嗜血杆菌中不存在任何已知的跨损伤合成DNA聚合酶。
