Dataset Information

Mutational analysis of the carboxy-terminal (YGX)4 repeat domain of CpsD, an autophosphorylating tyrosine kinase required for capsule biosynthesis in Streptococcus pneumoniae.

ABSTRACT: In Streptococcus pneumoniae, CpsB, CpsC, and CpsD are essential for encapsulation, and mutants containing deletions of cpsB, cpsC, or cpsD exhibit rough colony morphologies. CpsD is an autophosphorylating protein-tyrosine kinase, CpsC is required for CpsD tyrosine phosphorylation, and CpsB is a phosphotyrosine-protein phosphatase. We have previously shown that autophosphorylation of CpsD at tyrosine attenuates its activity and consequently reduces the level of encapsulation and negatively regulates CPS production. In this study, we further investigated the role of the carboxy-terminal (YGX)(4) repeat domain of CpsD in encapsulation. A CpsD truncation mutant in which the entire (YGX)(4) repeat domain was removed was indistinguishable from a strain in which the entire cpsD gene had been deleted, indicating that the carboxy-terminal (YGX)(4) tail is required for CpsD activity in capsular polysaccharide production. Double mutants having a single tyrosine residue at position 2, 3, or 4 in the (YGX)(4) repeat domain and lacking CpsB exhibited a rough colony morphology, indicating that in the absence of an active protein-tyrosine phosphatase, phosphorylation of just one of the tyrosine residues in the (YGX)(4) repeat was sufficient to inactivate CpsD. When various mutants in which CpsD had either one or combinations of two or three tyrosine residues in the (YGX)(4) repeat domain were examined, only those with three tyrosine residues in the (YGX)(4) repeat domain were indistinguishable from the wild-type strain. The mutants with either one or two tyrosine residues exhibited mucoid colony morphologies. Further analysis of the mucoid strains indicated that the mucoid phenotype was not due to overproduction of capsular polysaccharide, as these strains actually produced less capsular polysaccharide than the wild-type strain. Thus, the tyrosine residues in the (YGX)(4) repeat domain are essential for normal functioning of CpsD.

SUBMITTER: Morona JK

PROVIDER: S-EPMC154072 | biostudies-literature | 2003 May

REPOSITORIES: biostudies-literature

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Publications

Mutational analysis of the carboxy-terminal (YGX)4 repeat domain of CpsD, an autophosphorylating tyrosine kinase required for capsule biosynthesis in Streptococcus pneumoniae.

Morona Judy K JK Morona Renato R Miller David C DC Paton James C JC

Journal of bacteriology 20030501 10

In Streptococcus pneumoniae, CpsB, CpsC, and CpsD are essential for encapsulation, and mutants containing deletions of cpsB, cpsC, or cpsD exhibit rough colony morphologies. CpsD is an autophosphorylating protein-tyrosine kinase, CpsC is required for CpsD tyrosine phosphorylation, and CpsB is a phosphotyrosine-protein phosphatase. We have previously shown that autophosphorylation of CpsD at tyrosine attenuates its activity and consequently reduces the level of encapsulation and negatively regula ...[more]

PMID: 12730159

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Project description:Bacterial capsular polysaccharides (CPS) are produced by a multi-protein membrane complex, in which a particular type of tyrosine-autokinases named BY-kinases, regulate their polymerization and export. However, our understanding of the role of BY-kinases in these processes remains incomplete. In the human pathogen Streptococcus pneumoniae, the BY-kinase CpsD localizes at the division site and participates in the proper assembly of the capsule. In this study, we show that the cytoplasmic C-terminal end of the transmembrane protein CpsC is required for CpsD autophosphorylation and localization at mid-cell. Importantly, we demonstrate that the CpsC/CpsD complex captures the polysaccharide polymerase CpsH at the division site. Together with the finding that capsule is not produced at the division site in cpsD and cpsC mutants, these data show that CPS production occurs exclusively at mid-cell and is tightly dependent on CpsD interaction with CpsC. Next, we have analyzed the impact of CpsD phosphorylation on CPS production. We show that dephosphorylation of CpsD induces defective capsule production at the septum together with aberrant cell elongation and nucleoid defects. We observe that the cell division protein FtsZ assembles and localizes properly although cell constriction is impaired. DAPI staining together with localization of the histone-like protein HlpA further show that chromosome replication and/or segregation is defective suggesting that CpsD autophosphorylation interferes with these processes thus resulting in cell constriction defects and cell elongation. We show that CpsD shares structural homology with ParA-like ATPases and that it interacts with the chromosome partitioning protein ParB. Total internal reflection fluorescence microscopy imaging demonstrates that CpsD phosphorylation modulates the mobility of ParB. These data support a model in which phosphorylation of CpsD acts as a signaling system coordinating CPS synthesis with chromosome segregation to ensure that daughter cells are properly wrapped in CPS.

Project description:The polysaccharide capsule of Streptococcus pneumoniae defines over ninety serotypes, which differ in their carriage prevalence and invasiveness for poorly understood reasons. Recently, an inverse correlation between carriage prevalence and oligosaccharide structure of a given capsule has been described. Our previous work suggested a link between serotype and growth in vitro. Here we investigate whether capsule production interferes with growth in vitro and whether this predicts carriage prevalence in vivo. Eighty-one capsule switch mutants were constructed representing nine different serotypes, five of low (4, 7F, 14, 15, 18C) and four of high carriage prevalence (6B, 9V, 19F, 23F). Growth (length of lag phase, maximum optical density) of wildtype strains, nontypeable mutants and capsule switch mutants was studied in nutrient-restricted Lacks medium (MLM) and in rich undefined brain heart infusion broth supplemented with 5% foetal calf serum (BHI+FCS). In MLM growth phenotype depended on, and was transferred with, capsule operon type. Colonization efficiency of mouse nasopharynx also depended on, and was transferred with, capsule operon type. Capsule production interfered with growth, which correlated inversely with serotype-specific carriage prevalence. Serotypes with better growth and higher carriage prevalence produced thicker capsules (by electron microscopy, FITC-dextran exclusion assays and HPLC) than serotypes with delayed growth and low carriage prevalence. However, expression of cpsA, the first capsule gene, (by quantitative RT-PCR) correlated inversely with capsule thickness. Energy spent for capsule production (incorporation of H3-glucose) relative to amount of capsule produced was higher for serotypes with low carriage prevalence. Experiments in BHI+FCS showed overall better bacterial growth and more capsule production than growth in MLM and differences between serotypes were no longer apparent. Production of polysaccharide capsule in S. pneumoniae interferes with growth in nutrient-limiting conditions probably by competition for energy against the central metabolism. Serotype-specific nasopharyngeal carriage prevalence in vivo is predicted by the growth phenotype.

Dataset Information

Mutational analysis of the carboxy-terminal (YGX)4 repeat domain of CpsD, an autophosphorylating tyrosine kinase required for capsule biosynthesis in Streptococcus pneumoniae.

Publications

Mutational analysis of the carboxy-terminal (YGX)4 repeat domain of CpsD, an autophosphorylating tyrosine kinase required for capsule biosynthesis in Streptococcus pneumoniae.

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