Characterization of Mutants Deficient in the L,D-Carboxypeptidase (DacB) and WalRK (VicRK) Regulon, Involved in Peptidoglycan Maturation of Streptococcus pneumoniae Serotype 2 Strain D39
Characterization of Mutants Deficient in the L,D-Carboxypeptidase (DacB) and WalRK (VicRK) Regulon, Involved in Peptidoglycan Maturation of Streptococcus pneumoniae Serotype 2 Strain D39
复制标题
DOI：
10.1128/jb.01555-10
复制
发表时间：
2011-05-01
影响因子：
3.2
通讯作者：
Winkler, Malcolm E.
中科院分区：
文献类型：
--
作者：
Barendt, Skye M.;Sham, Lok-To;Winkler, Malcolm E.
肽聚糖（PG）水解酶在细菌分裂过程中细胞壁的重塑中起着关键作用。PG水解酶已经在大肠杆菌和枯草芽孢杆菌等几种芽孢杆菌中得到了广泛的研究，但在卵球菌中，如肺炎链球菌（肺炎球菌），PG水解酶的特征仍然相对不明确。在这项工作中，我们在肺炎链球菌菌株D39基因组中鉴定了编码具有假定PG水解结构域的蛋白质的基因。构建这些基因的敲除突变，并将产生的突变体与亲本菌株在生长、细胞形态、PG肽掺入以及某些情况下PG肽组成方面进行比较。此外，我们还发现了WalRKSpn双组分系统调控中功能未知的非必需基因的缺失突变，该调控还包含pcsB细胞分裂的必需基因。一些突变体没有表现出明显的表型，这可能是冗余的指示。相比之下，两个新的突变体在PG生物合成方面表现出明显的缺陷。其中一个突变发生在一个名为dacB （spd_0549）的基因上，我们发现该基因编码一种参与PG成熟的L， d -羧基肽酶。值得注意的是，与dacA （D，D-羧基肽酶）突变体类似，dacB突变体在细胞形状和分隔上表现出缺陷，这与PG肽前体的可用性对PG的适当生物合成很重要的观点一致。上位分析表明，DacA在D-Ala去除中先于DacB发挥作用，免疫荧光显微镜显示，DacA和DacB位于肺炎球菌细胞的整个表面。另一个突变是WalRKSpn调节基因spd_0703，该基因编码一种被认为可能作为一种保守的链球菌形状、延伸、分裂和产孢（SEDS）蛋白的膜蛋白。
Peptidoglycan (PG) hydrolases play critical roles in the remodeling of bacterial cell walls during division. PG hydrolases have been studied extensively in several bacillus species, such as Escherichia coli and Bacillus subtilis, but remain relatively uncharacterized in ovococcus species, such as Streptococcus pneumoniae (pneumococcus). In this work, we identified genes that encode proteins with putative PG hydrolytic domains in the genome of S. pneumoniae strain D39. Knockout mutations in these genes were constructed, and the resulting mutants were characterized in comparison with the parent strain for growth, cell morphology, PG peptide incorporation, and in some cases, PG peptide composition. In addition, we characterized deletion mutations in nonessential genes of unknown function in the WalRKSpn two-component system regulon, which also contains the essential pcsB cell division gene. Several mutants did not show overt phenotypes, which is perhaps indicative of redundancy. In contrast, two new mutants showed distinct defects in PG biosynthesis. One mutation was in a gene designated dacB (spd_0549), which we showed encodes an L, D-carboxypeptidase involved in PG maturation. Notably, dacB mutants, similar to dacA (D,D-carboxypeptidase) mutants, exhibited defects in cell shape and septation, consistent with the idea that the availability of PG peptide precursors is important for proper PG biosynthesis. Epistasis analysis indicated that DacA functions before DacB in D-Ala removal, and immunofluorescence microscopy showed that DacA and DacB are located over the entire surface of pneumococcal cells. The other mutation was in WalRKSpn regulon gene spd_0703, which encodes a putative membrane protein that may function as a type of conserved streptococcal shape, elongation, division, and sporulation (SEDS) protein.