SummaryNatural genetic transformation in Streptococcus pneumoniae is controlled in part by a quorum-sensing system mediated by a peptide pheromone called competence-stimulating peptide (CSP), which acts to coordinate transient activation of genes required for competence. To characterize the transcriptional response and regulatory events occurring when cells are exposed to competence pheromone, we constructed DNA microarrays and analysed the temporal expression profiles of 1817 among the 2129 unique predicted open reading frames present in the S. pneumoniae TIGR4 genome (84%). After CSP stimulation, responsive genes exhibited four temporally distinct expression profiles: early, late and delayed gene induction, and gene repression. At least eight early genes participate in competence regulation including comX , which encodes an alternative sigma factor. Late genes were dependent on ComX for CSPinduced expression, many playing important roles in transformation. Genes in the delayed class (third temporal wave) appear to be stress related. Genes repressed during the CSP response include ribosomal protein loci and other genes involved in protein synthesis. This study increased the number of identified CSP-responsive genes from approximately 40 to 188. Given the relatively large number of induced genes (6% of the genome), it was of interest to determine which genes provide functions essential to transformation. Many of the induced loci were subjected to gene disruption mutagenesis, allowing us to establish that among 124 CSP-inducible genes, 67 were individually dispensable for transformation, whereas 23 were required for transformation.

Natural genetic transformation offers a direct route by which synthetic gene constructs can be placed into the single circular chromosome of Streptococcus pneumoniae. However, the lack of a general negative-selection marker has hampered the introduction of constructs that do not confer a selectable phenotype. A 1.3-kb cassette was constructed comprising a kanamycin (Kn) resistance marker (kan) and a counterselectable rpsL ؉ marker. The cassette conferred dominant streptomycin (Sm) sensitivity in an Sm-resistant background in S. pneumoniae. It was demonstrated that it could be used in a two-step transformation procedure to place DNA of arbitrary sequence at a chosen target site. The first transformation into an Sm-resistant strain used the cassette to tag a target gene on the chromosome by homologous recombination while conferring Kn resistance but Sm sensitivity on the recombinant. Replacement of the cassette by an arbitrary segment of DNA during a second transformation restored Sm resistance (and Kn sensitivity), allowing construction of silent mutations and deletions or other gene replacements which lack a selectable phenotype. It was also shown that gene conversion occurred between the two rpsL alleles in a process that depended on recA and that was susceptible to correction by mismatch repair.

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MicroRNAs are small regulatory RNAs that post-transcriptionally regulate gene expression and can be encoded by viral as well as cellular genomes. The functions of most viral miRNAs are unknown and few have been studied in an in vivo context. Here we show that the murine polyomavirus (PyV) encodes a precursor microRNA that is processed into two mature microRNAs, both of which are active at directing the cleavage of the early PyV mRNAs. Furthermore, we identify a deletion mutant of polyomavirus that is defective in encoding the microRNAs. This mutant replicates normally and transforms cultured cells with efficiencies comparable to wildtype PyV. The miRNA mutant is competent to establish a transient infection of mice following parenteral inoculation, and is cleared post infection at approximately the same rate as the wildtype virus. In addition, under these laboratory conditions, we observe no differences in anti-viral CD8 T cell responses. These results indicate that PyV miRNA expression is not essential for infection of cultured cells or experimentally inoculated mice, and raise the possibility that its role in natural infection might involve aspects of acquisition or spread that are not recapitulated by experimental inoculation.

Natural genetic transformation in Streptococcus pneumoniae is controlled by a quorum-sensing system, which acts through the competence-stimulating peptide (CSP) for transient activation of genes required for competence. More than 100 genes have been identified as CSP regulated by use of DNA microarray analysis. One of the CSP-induced genes required for genetic competence is comW. As the expression of this gene depended on the regulator ComE, but not on the competence sigma factor ComX ( X ), and as expression of several genes required for DNA processing was affected in a comW mutant, comW appears to be a new regulatory gene. Immunoblotting analysis showed that the amount of the X protein is dependent on ComW, suggesting that ComW may be directly or indirectly involved in the accumulation of X . As X is stabilized in clpP mutants, a comW mutation was introduced into the clpP background to ask whether the synthesis of X depends on ComW. The clpP comW double mutant accumulated an amount of X higher (threefold) than that seen in the wild type but was not transformable, suggesting that while comW is not needed for X synthesis, it acts both in stabilization of X and in its activation. Modification of ComW with a histidine tag at its C or N terminus revealed that both amino and carboxyl termini are important for increasing the stability of X , but only the N terminus is important for stimulating its activity.