Iwase Tadayuki, Matsuo Takashi, Nishioka Saiko, Tajima Akiko, Mizunoe Yoshimitsu
Department of Bacteriology, The Jikei University School of MedicineTokyo, Japan.
Graduate School of Materials Science, Nara Institute of Science and TechnologyNara, Japan.
Front Microbiol. 2017 Apr 26;8:656. doi: 10.3389/fmicb.2017.00656. eCollection 2017.
RpoS is a key stress-inducible sigma factor that regulates stress resistance genes in , such as the gene encoding catalase HPII and the genes encoding glycogen synthesis proteins. Monitoring RpoS activity can provide information on the stress sensitivity of isolates in clinical settings because the RpoS in these isolates is often mutated. In the present study, we found a novel, missense point mutation at RpoS residue 128 in a clinical Shiga toxin-producing (STEC) isolate. This mutation caused RpoS dysfunction and increased stress sensitivity. A mutant was cloned from a clinical STEC that is vulnerable to cold temperature and oxidative stresses. Mutant RpoS protein expression was detected in the clinical isolate, and this RpoS was non-functional according to HPII activity and glycogen levels, which are positively regulated by RpoS and thus are used as indicators for RpoS function. A reporter assay with β-galactosidase indicated that the dysfunction occurred at the transcriptional level of genes regulated by RpoS. Furthermore, substitution analysis indicated that the hydrophobicity of the amino acid at residue 128 was critical for RpoS activity; the simulation analysis indicated that the amino acids of RNA polymerase (RNAP) that interact with RpoS residue 128 are hydrophobic, suggesting that this hydrophobic interaction is critical for RpoS activity. In addition, substitution of Ile128 to Pro128 abolished RpoS activity, possibly as a result of disruption of the secondsary structure around residue 128, indicating that the structure is also a crucial factor for RpoS activity. These results indicate that only one point mutation at a hydrophobic residue of the complex formed during transcription leads to a critical change in RpoS regulation. Moreover, we found that Ile128 is widely conserved among various bacteria: several bacterial strains have Met128 or Leu128, which are hydrophobic residues, and these strains had similar or higher RpoS activity than that observed with Ile128 in this study. These data indicate that the hydrophobicity of the amino acid at residue 128 is critical for RpoS activity and is consequently important for bacterial survival. Taken together, these findings may contribute to a deeper understanding of protein functional mechanisms and bacterial stress responses.
RpoS是一种关键的应激诱导型σ因子，可调节大肠杆菌中的应激抗性基因，例如编码过氧化氢酶HPII的基因和编码糖原合成蛋白的基因。监测RpoS活性可以提供临床环境中大肠杆菌分离株应激敏感性的信息，因为这些分离株中的RpoS经常发生突变。在本研究中，我们在一株临床产志贺毒素大肠杆菌（STEC）分离株的RpoS第128位残基处发现了一个新的错义点突变。该突变导致RpoS功能障碍并增加了应激敏感性。从一株对低温和氧化应激敏感的临床STEC中克隆出一个突变体。在临床分离株中检测到突变型RpoS蛋白表达，根据HPII活性和糖原水平判断，这种RpoS无功能，HPII活性和糖原水平受RpoS正调控，因此用作RpoS功能的指标。用β-半乳糖苷酶进行的报告基因检测表明，功能障碍发生在RpoS调控基因的转录水平。此外，替代分析表明，第128位残基处氨基酸的疏水性对RpoS活性至关重要；模拟分析表明，与RpoS第128位残基相互作用的RNA聚合酶（RNAP）氨基酸是疏水性的，这表明这种疏水相互作用对RpoS活性至关重要。此外，将Ile128替换为Pro128消除了RpoS活性，这可能是由于第128位残基周围二级结构的破坏，表明该结构也是RpoS活性的关键因素。这些结果表明，转录过程中形成的复合物中一个疏水性残基处的单点突变会导致RpoS调控的关键变化。此外，我们发现Ile128在各种细菌中广泛保守：几种细菌菌株具有Met128或Leu128，它们都是疏水性残基，这些菌株的RpoS活性与本研究中Ile128的活性相似或更高。这些数据表明，第128位残基处氨基酸的疏水性对RpoS活性至关重要，因此对细菌生存很重要。综上所述，这些发现可能有助于更深入地了解蛋白质功能机制和细菌应激反应。
