John P. Throup

A genomic analysis of two‐component signal transduction in Streptococcus pneumoniae

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.

A novel strategy for the isolation of luxl homologues: evidence for the widespread distribution of a LuxR:Luxl superfamily in enteric bacteria

The pheromone N‐(3‐oxohexanoyl)‐L‐homoserine lactone (OHHL) regulates expression of bioluminescence in the marine bacterium Vibrio fischeri, the production of carbapenem antibiotic in Erwinia carotovora and exoenzymes in both E. carotovora and Pseudomonas aeruginosa. A characteristic feature of this regulatory mechanism in V. fischeri is that it is cell density‐dependent, reflecting the need to accumulate sufficient pheromone to trigger the induction of gene expression. Using a lux plasmid‐based bioluminescent sensor for OHHL, pheromone production by E. carotovora, Enterobacter agglomerans, Hafnia alvei, Rahnella aquatilis and Serratia marcescens has been demonstrated and shown also to be cell density‐dependent. Production of OHHL implies the presence in these bacteria of a gene equivalent to luxl. Chromosomal banks from all five enteric bacteria have yielded clones capable of eliciting OHHL production when expressed in Escherichia coli. The luxl homologue from both E. carotovora (carl) and E. agglomerans (eagl) were characterized at the DNA sequence level and the deduced protein sequences have only 25% identity with the V. fischeri Luxl. Despite this, carl, eagl and luxl are shown to be biologically equivalent. An insertion mutant of eagl demonstrates that this gene is essential for OHHL production in E. agglomerans.

A hierarchical quorum‐sensing system in Yersinia pseudotuberculosis is involved in the regulation of motility and clumping

In cell‐free Yersinia pseudotuberculosis culture supernatants, we have chemically characterized three N‐acyl homoserine lactone (AHL) molecules, N‐octanoyl homoserine lactone (C8‐HSL), N‐(3‐oxohexanoyl)homoserine lactone (3‐oxo‐C6‐HSL) and N‐hexanoyl homoserine lactone (C6‐HSL). We have identified, cloned and sequenced two pairs of LuxR/I homologues termed YpsR/I and YtbR/I. In Escherichia coli at 37°C, YpsI and YtbI both synthesize C6‐HSL, although YpsI is responsible for 3‐oxo‐C6‐HSL and YtbI for C8‐HSL synthesis respectively. However, in a Y. pseudotuberculosis ypsI‐negative background, YtbI appears capable of adjusting the AHL profile from all three AHLs at 37°C and 22°C to the absence of 3‐oxo‐C6‐HSL at 28°C. Insertion deletion mutagenesis of ypsR leads to the loss of C8‐HSL at 22°C, which suggests that at this temperature the YpsR protein is involved in the hierarchical regulation of the ytbR/I locus. When compared with the parent strain, the ypsR and ypsI mutants exhibit a number of phenotypes, including clumping (ypsR mutant), overexpression of a major flagellin subunit (ypsR mutant) and increased motility (both ypsR and ypsI mutants). The clumping and motility phenotypes are both temperature dependent. These data are consistent with a hierarchical quorum‐sensing cascade in Y. pseudotuberculosis that is involved in the regulation of clumping and motility.

Characterisation of the yenI/yenR locus from Yersinia enterocolitica mediating the synthesis of two N‐acylhomoserine lactone signal molecules

Yersinia enterocolitica produces compounds capable of transcriptionally activating the Photobacterium fischeri bioluminescence (lux) operon. Using high‐performance liquid chromatography, high resolution tandem mass spectrometry in conjunction with chemical synthesis, two signal molecules were identified and shown to be N‐hexanoyl‐l‐homoserine lactone (HHL) and N‐(3‐oxohexanoyl)‐l‐homoserine lactone (OHHL). A gene (yenI) was isolated from Y. enterocolitica and demonstrated to direct the synthesis of both HHL and OHHL. DNA sequence analysis revealed an open reading frame (ORF) of 642 bp encoding a protein (YenI) of 24.6 kDa with ≈20% identity to the LuxI family of proteins. Northern blot analysis of yenI expression indicated yenI is transcribed as a single gene and 5′ transcript mapping of yenI identified a transcriptional start site 89 bp upstream of the ORF. DNA sequence analysis of the region downstream of yenI located a second ORF, termed yenR, with significant homology to the LuxR family of transcriptional activators. An insertion mutation of yenI abolishes HHL and OHHL production, indicating its central role in N‐acylhomo‐serine lactone synthesis in Y. enterocolitica. Transcriptional analysis using a chromosomal yenI::luxAB fusion has demonstrated that yenI is not subject to autoinduction but is expressed constitutively. Whilst production of the Yop proteins in the wild type and in yenI mutants is indistinguishable, two‐dimensional SDS‐PAGE analysis of total cell proteins indicated that a number of proteins lack the yenI mutant.

Plants genetically modified to produce N-acylhomoserine lactones communicate with bacteria.

N-acylhomoserine lactones (AHLs) play a critical role in plant/microbe interactions. The AHL, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), induces exoenzymes that degrade the plant cell wall by the pathogenic bacterium Erwinia carotovora. Conversely, the antifungal activity of the biocontrol bacterium Pseudomonas aureofaciens 30–84 is due (at least in part) to phenazine antibiotics whose synthesis is regulated by N-hexanoylhomoserine lactone (HHL). Targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in plants of the cognate AHL signaling molecules (OHHL and HHL). The AHLs produced by the transgenic plants were sufficient to induce target gene expression in several recombinant bacterial AHL biosensors and to restore biocontrol activity to an HHL-deficient P. aureofaciens strain. In addition, pathogenicity was restored to an E. carotovora strain rendered avirulent as a consequence of a mutation in the OHHL synthase gene, carI. The ability to generate bacterial quorum-sensing signaling molecules in the plant offers novel opportunities for disease control and for manipulating plant/microbe interactions.

The Contribution of Genomics to the Discovery of New Antibiotics

The emergence of common bacterial pathogens that are resistant to multiple antibiotics, coupled with the failure of traditional methods to yield new anti-infective agents, threatens current paradigms of therapeutic intervention (Omura, 1992; Shlaes et al., 1991; Tenover and Hughes, 1996). The current focus has been on improving existing antibiotic classes while little progress has been made in discovering chemically novel anti-infective agents. This unmet medical need may now be addressed if we can successfully exploit the new wealth of genomic sequence data to devise novel strategies for drug discovery (Moir et al., 1999). Whole genome comparative sequence analysis now allows the identification of genes/gene products that are common to many or all pathogenic bacteria of clinical importance. bioinformatics-based gene homology and motif analyses allow rapid phylogenetic comparisons to be made and, in addition, predict functional information critical to target selection. Putative targets can then be assessed rapidly using gene essentiality testing methods which allow analyses both in vitro and in models of the infection state. Sensitive and direct in vivo expression analyses confirm that the expression of the target gene is relevant to the establishment and maintenance of infection. Ultimately, the gene products must be screened against novel chemical libraries and natural product banks derived from a wide bio-diversity in order to identify lead compounds with potential antibiotic activities that will be developed to provide the next generation of therapeutic agents. Those companies which can adapt and implement these genomic-based technologies, derive significant competitive advantage in creating product portfolios that will address the unmet clinical need.

Quorum Sensing: Bacterial Cell-Cell Signalling from Bioluminescence to Pathogenicity

The integration of signals from the bacterial environment, through a network of cellular transduction mechanisms, determines the profile of genes expressed and thereby the bacterial phenotype. Quorum sensing transmits one such signal, i.e. population density, by relying on the accumulation of a small extracellular signal molecule to modulate transcription of target operons.