Guenzi E, Gasc A M, Sicard M A, Hakenbeck R
Max-Planck Institut für molekulare Genetik, Berlin, Germany.
Mol Microbiol. 1994 May;12(3):505-15. doi: 10.1111/j.1365-2958.1994.tb01038.x.
Penicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin-binding protein (PBP) variants with decreased affinities for beta-lactam antibiotics. Cefotaxime-resistant laboratory mutants, selected after several steps on increasing concentrations of this beta-lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, ciaR and ciaH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal-transducing proteins). Two hydrophobic peptide regions divided the histidine kinase CiaH into two putative domains: an N-terminal extracellular sensor part, and an intracellular C-terminal domain with the conserved His-226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime-resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime-resistant mutants were each located in the C-terminal half of CiaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated ciaH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal-transducing system cia is involved in early steps of competence regulation.
肺炎链球菌对青霉素的耐药性至今被认为是由于产生了对β-内酰胺抗生素亲和力降低的青霉素结合蛋白（PBP）变体。在逐步增加这种β-内酰胺浓度的情况下筛选出的耐头孢噻肟实验室突变体，也变得缺乏转化能力。从突变体C306中分离并克隆出一个赋予头孢噻肟抗性和转化缺陷的DNA片段。与该抗性决定簇相关的头孢噻肟抗性并未伴随着PBP特性的明显变化，并且它定位在与已知抗性决定簇（编码PBP2x、PBP1a或PBP2b的基因）不同的染色体上。对该抗性决定簇的2265 bp DNA序列的测定揭示了两个开放阅读框，ciaR和ciaH，其推导的氨基酸序列分别将相应的蛋白质鉴定为应答调节因子和组氨酸激酶受体（细菌信号转导蛋白的两个家族的成员）。两个疏水肽区域将组氨酸激酶CiaH分为两个假定结构域：一个N端细胞外传感部分，以及一个带有保守His-226残基（推测的磷酸化位点）的细胞内C端结构域。导致C306以及另外两个独立分离的耐头孢噻肟突变体的头孢噻肟抗性和转化缺陷的单点突变均位于CiaH的C端一半区域。一种小的细胞外蛋白，即感受态因子，是诱导感受态所必需的。C306和用突变的ciaH基因获得的转化体均不产生感受态因子，并且外源性感受态因子不能弥补转化缺陷，这表明信号转导系统cia参与感受态调节的早期步骤。
