Constitutive competence for genetic transformation in Streptococcus pneumoniae caused by mutation of a transmembrane histidine kinase

Competence for DNA uptake and genetic transformation in Streptococcus pneum oniae is regulated by a quorum-sensing system. A competence-stimulating pol ypeptide (CSP) is secreted by the bacteria and acts back on the cells via a transmembrane histidine kinase. This enzyme phosphorylates a response regu lator that activates synthesis of a SigH-like protein. The new sigma factor enables expression of a set of proteins transcribed from a novel promoter. A mutation called trt had been found that circumvented this regulation. Th e mutant cells are constitutively competent; that is, they can be transform ed at low cell densities, in the presence of proteases that attack CSP, or during growth at low pH. In this work, cells containing trt were shown to b e competent even in the presence of a comAB mutation that blocks secretion of CSP. The trt mutation was localized to comD, the gene encoding the trans membrane histidine kinase. A DNA segment of the trt mutant corresponding to comCDE was cloned, and it was shown to contain the trt mutation by its abi lity to confer constitutive competence. A two-step assay, which was based o n transfer of trt to a wild strain and screening for transformability in th e presence of trypsin, served to locate the trt mutation precisely. It corr esponds to a GC-->AT transition, which changes Asp(299) in the histidine ki nase to Asn. This alteration in the carboxyl terminal half of the protein, which is cytoplasmically located and contains the phosphorylase activity, p resumably alters the enzyme conformation so that it is permanently activate d, independent of signals from the transmembrane domain. These results may help illuminate the mechanism by which external signals affect kinase actio n in two-component regulatory systems, and they may be of practical value i n facilitating genetic studies by rendering pneumococcal strains permanentl y competent.