A genomics‐based approach was used to identify the entire gene complement of putative two‐component signal transduction systems (TCSTSs) in Streptococcus pneumoniae. A total of 14 open reading frames (ORFs) were identified as putative response regulators, 13 of which were adjacent to genes encoding probable histidine kinases. Both the histidine kinase and response regulator proteins were categorized into subfamilies on the basis of phylogeny. Through a systematic programme of mutagenesis, the importance of each novel TCSTS was determined with respect to viability and pathogenicity. One TCSTS was identified that was essential for the growth of S. pneumoniaeThis locus was highly homologous to the yycFG gene pair encoding the essential response regulator/histidine kinase proteins identified in Bacillus subtilis and Staphylococcus aureus. Separate deletions of eight other loci led in each case to a dramatic attenuation of growth in a mouse respiratory tract infection model, suggesting that these signal transduction systems are important for the in vivo adaptation and pathogenesis of S. pneumoniae. The identification of conserved TCSTSs important for both pathogenicity and viability in a Gram‐positive pathogen highlights the potential of two‐component signal transduction as a multicomponent target for antibacterial drug discovery.

SummaryStreptococcus pneumoniae remains a serious cause of morbidity and mortality in humans, but relatively little is known about the molecular basis of its pathogenesis. We used signature-tagged mutagenesis together with an analysis of S. pneumoniae genome sequence to identify and characterize genes required for pathogenesis. A library of signaturetagged mutants was created by insertion±duplication mutagenesis, and 1786 strains were analysed for their inability to survive and replicate in murine models of pneumonia and bacteraemia. One hundred and eightysix mutant strains were identified as attenuated, and 56 were selected for further genetic characterization based on their ability to excise the integrated plasmid spontaneously. The genomic DNA inserts of the plasmids were cloned in Escherichia coli and sequenced. These sequences were subjected to database searches, including the S. pneumoniae genome sequence, which allowed us to examine the chromosomal regions flanking these genes. Most of the insertions were in probable operons, but no pathogenicity islands were found. Forty-two novel virulence loci were identified. Five strains mutated in genes involved in gene regulation, cation transport or stress tolerance were shown to be highly attenuated when tested individually in a murine respiratory tract infection model. Additional experiments also suggest that induction of competence for genetic transformation has a role in virulence.

The legionnaires' disease bacterium, LegioneUa pneumophila, is a facultative intracellular parasite. Its interaction with phagocytes has characteristics in common with several other intracellular parasites. Critical aspects of L. pneumophila intracellular multiplication are evasion of lysosomal host cell defenses and the presence of a nutritionally appropriate environment. Following phagocytosis, wild-type

Differential fluorescence induction technology was used to identify promoters of Streptococcus pneumoniae genes that are expressed during lung infection of the mouse. Among the promoter clones that were identified multiple times was the psa promoter, which drives expression of the psaBCA operon. These genes have been identified previously and shown to encode a manganese permease system as well as play a role in the virulence of this organism. Mutations in psaB, psaC or psaA result in growth limitation in low manganese. The expression of the psa operon was examined in vivo and the virulence of deletion mutants of psaB, psaC, psaA and psaBCA was assessed in four different animal models of infection. The psa promoter was induced more than ten-fold in vivo using an intraperitoneal chamber implant model. The psaB, psaC and psaA mutants were completely attenuated in systemic, respiratory tract and otitis media infections. In addition, these mutants were unable to grow in an implanted peritoneal chamber, but growth was restored by the addition of manganese to the chambers.

The Yersinia pseudotuberculosis invasin protein mediates bacterial entry into mammalian cells by binding multiple beta 1‐chain integrins. Invasin binding to purified alpha 5 beta 1 integrin is inhibited by Arg‐Gly‐Asp (RGD)‐containing peptides, although invasin contains no RGD sequence. Fifteen mutations that diminished binding and bacterial entry were isolated after mutagenesis of the entire inv gene. All of the mutations altered residues within the C‐terminal 192 amino acids of invasin, previously delineated as the integrin binding domain, and 10 of the mutations fell within an 11 residue region. This small region was subjected to site‐directed mutagenesis and almost half of the 35 mutations generated decreased invasin‐mediated entry. D911 within this region was the most critical residue, as even a conservative glutamate substitution abolished bacterial penetration. Purified invasin derivatives altered at this residue were defective in promoting cell attachment and this defect was reflected in a 10‐fold or greater increase in IC50 for integrin binding. D911 may have a function similar to that of the aspartate residue in RGD‐containing sequences.