PynA is a pyrimidine 5'-nucleotidase that functions as an antimutator protein in Streptococcus pneumoniae.

Streptococcus pneumoniae is a Gram-positive bacterium that is a major agent of community-acquired bacterial pneumonia, meningitis and sepsis. Although the mismatch repair function of S. pneumoniae has been assigned to the hexA-hexB gene products, an enzyme capable of the direct elimination of noncanonical nucleotides from the cytoplasm has not been described for this bacterium. Our results show that Spr1057, a protein with previously unknown function, is involved in the inactivation of mutagenic pyrimidine nucleotides and was accordingly designated PynA (pyrimidine nucleotidase A). Biochemical assays confirmed the phosphatase activity of the recombinant enzyme and revealed its metal ion dependence for optimal enzyme activity. We demonstrated that PynA forms a homodimer with higher in vitro activity towards noncanonical 5-fluoro-2'-deoxyuridine monophosphate than towards canonical thymidine monophosphate. Furthermore, we showed via in vivo assays that PynA protects cells against noncanonical pyrimidine derivatives such as 5-fluoro-2'-deoxyuridine and prevents the incorporation of the potentially mutagenic 5-bromo-2'-deoxyuridine (5-BrdU) into DNA. Fluctuation analysis performed under S. pneumoniae exposure to 5-BrdU revealed that the pynA null strain accumulates random mutations with high frequency, resulting in a 30-fold increase in the mutation rate. The data support a model in which PynA, a protein conserved in other Gram-positive bacteria, functions as a house-cleaning enzyme by selectively eliminating noncanonical nucleotides and maintaining the purity of dNTP pools, similar to the YjjG protein described for Escherichia coli.

Characterization of pneumococcal Ser/Thr protein phosphatase phpP mutant and identification of a novel PhpP substrate, putative RNA binding protein Jag.

BACKGROUND: Reversible protein phosphorylation catalyzed by protein kinases and phosphatases is the primary mechanism for signal transduction in all living organisms. Streptococcus pneumoniae encodes a single Ser/Thr protein kinase, StkP, which plays a role in virulence, stress resistance and the regulation of cell wall synthesis and cell division. However, the role of its cognate phosphatase, PhpP, is not well defined. RESULTS: Here, we report the successful construction of a ΔphpP mutant in the unencapsulated S. pneumoniae Rx1 strain and the characterization of its phenotype. We demonstrate that PhpP negatively controls the level of protein phosphorylation in S. pneumoniae both by direct dephosphorylation of target proteins and by dephosphorylation of its cognate kinase, StkP. Catalytic inactivation or absence of PhpP resulted in the hyperphosphorylation of StkP substrates and specific phenotypic changes, including sensitivity to environmental stresses and competence deficiency. The morphology of the ΔphpP cells resembled the StkP overexpression phenotype and conversely, overexpression of PhpP resulted in cell elongation mimicking the stkP null phenotype. Proteomic analysis of the phpP knock-out strain permitted identification of a novel StkP/PhpP substrate, Spr1851, a putative RNA-binding protein homologous to Jag. Here, we show that pneumococcal Jag is phosphorylated on Thr89. Inactivation of jag confers a phenotype similar to the phpP mutant strain. CONCLUSIONS: Our results suggest that PhpP and StkP cooperatively regulate cell division of S. pneumoniae and phosphorylate putative RNA binding protein Jag.

The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor.

The genome of Streptomyces coelicolor encodes six potential WD-40 genes. Two of them, the wdpB (SCO5953) and the wdpC (SCO4422) genes, were studied to determine their function. Deletion of the wdpB gene resulted in a considerable decrease of aerial hyphae formation, leading to a conditionally bald phenotype, and reduced undecylprodigiosin production. In addition, the aerial hyphae of the ΔwdpB mutant strain were unusually branched and showed the signs of irregular septation and precocious lysis. Disruption of wdpC resulted in the reduction of undecylprodigiosin and delayed actinorhodin production. The ΔwdpC mutant strain showed precocious lysis of hyphae and delayed sporulation without typical curling of aerial hyphae in the early sporulation stage. The whole-genome transcriptome analysis revealed that deletion of wdpB affects the expression of genes involved in aerial hyphae differentiation, sporulation and secondary metabolites production. Deletion of wdpC caused downregulation of several gene clusters encoding secondary metabolites. Both the wdp genes seem to possess transcriptional autoregulatory function. Overexpression and genetic complementation studies confirmed the observed phenotype of both mutants. The results obtained suggest that both genes studied have a pleiotropic effect on physiological and morphological differentiation.

A eukaryotic-type signalling system of Pseudomonas aeruginosa contributes to oxidative stress resistance, intracellular survival and virulence.

BACKGROUND: The genome of Pseudomonas aeruginosa contains at least three genes encoding eukaryotic-type Ser/Thr protein kinases, one of which, ppkA, has been implicated in P. aeruginosa virulence. Together with the adjacent pppA phosphatase gene, they belong to the type VI secretion system (H1-T6SS) locus, which is important for bacterial pathogenesis. To determine the biological function of this protein pair, we prepared a pppA-ppkA double mutant and characterised its phenotype and transcriptomic profiles. RESULTS: Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pyoverdine. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the plant model of infection. Whole-genome transcriptome analysis revealed that pppA-ppkA deletion affects the expression of oxidative stress-responsive genes, stationary phase σ-factor RpoS-regulated genes, and quorum-sensing regulons. The transcriptome of the pppA-ppkA mutant was also analysed under conditions of oxidative stress and showed an impaired response to the stress, manifested by a weaker induction of stress adaptation genes as well as the genes of the SOS regulon. In addition, expression of either RpoS-regulated genes or quorum-sensing-dependent genes was also affected. Complementation analysis confirmed that the transcription levels of the differentially expressed genes were specifically restored when the pppA and ppkA genes were expressed ectopically. CONCLUSIONS: Our results suggest that in addition to its crucial role in controlling the activity of P. aeruginosa H1-T6SS at the post-translational level, the PppA-PpkA pair also affects the transcription of stress-responsive genes. Based on these data, it is likely that the reduced virulence of the mutant strain results from an impaired ability to survive in the host due to the limited response to stress conditions.