Brian J. Akerley

A genome-scale analysis for identification of genes required for growth or survival of Haemophilus influenzae

A high-density transposon mutagenesis strategy was applied to the Haemophilus influenzae genome to identify genes required for growth or viability. This analysis detected putative essential roles for the products of 259 ORFs of unknown function. Comparisons between complete genomes defined a subset of these proteins in H. influenzae having homologs in Mycobacterium tuberculosis that are absent in Saccharomyces cerevisiae, a distribution pattern that favors their use in development of antimicrobial therapeutics. Three genes within this set are essential for viability in other bacteria. Interfacing the set of essential gene products in H. influenzae with the distribution of homologs in other microorganisms can detect components of unrecognized cellular pathways essential in diverse bacteria. This genome-scale phenotypic analysis identifies potential roles for a large set of genes of unknown function.

Analysis of gene function in bacterial pathogens by GAMBIT.

Publisher Summary This chapter describes practical guide to the use of the GAMBIT (genome analysis and mapping by in vitro transposition) approach for analysis of genetic function on a genomic scale or to study individual genomic regions of interest. As part of this discussion, versatile components of this method are described including transposase purification, in vitro transposon mutagenesis with mariner-derived transposons, and genetic footprinting. The chapter focuses on studies in naturally transformable bacteria; however, elements of the GAMBIT system have been adapted to genetic analysis of organisms. GAMBIT owes its versatility to its simplicity. The method creates a bank of transposon mutations spanning the entire genome or within user-defined subgenomic regions and uses an analytical PCR-based approach, genetic footprinting, to locate the positions of genes essential for growth or viability under any specific experimental condition. Transposition is conducted using purified components in vitro and therefore does not require complex genetic arrangements such as conditional replicons, organism-specific transposase expression systems, or “suicide” DNA delivery systems. The only requirement for conducting this procedure in any naturally transformable organism is the presence of a gene within the transposon conferring antibiotic resistance or other selectable traits.

Understanding signal transduction during bacterial infection

Many known or suspected bacterial virulence factors require environmentally responsive control factors for expression. In Bordetella species, the BvgAS system represses and activates sets of genes, and mediates a biphasic phenotypic transition. Studies using mutants with altered signaling pathways and reversed regulatory connections have provided insights into the role of BvgAS and this phenotypic transition during the Bordetella-host interaction.

Position-Based Scanning for Comparative Genomics and Identification of Genetic Islands in Haemophilus influenzae Type b

ABSTRACT Bacteria exhibit extensive genetic heterogeneity within species. In many cases, these differences account for virulence properties unique to specific strains. Several such loci have been discovered in the genome of the type b serotype of Haemophilus influenzae, a human pathogen able to cause meningitis, pneumonia, and septicemia. Here we report application of a PCR-based scanning procedure to compare the genome of a virulent type b (Hib) strain with that of the laboratory-passaged Rd KW20 strain for which a complete genome sequence is available. We have identified seven DNA segments or H. influenzae genetic islands (HiGIs) present in the type b genome and absent from the Rd genome. These segments vary in size and content and show signs of horizontal gene transfer in that their percent G+C content differs from that of the rest of the H. influenzae genome, they contain genes similar to those found on phages or other mobile elements, or they are flanked by DNA repeats. Several of these loci represent potential pathogenicity islands, because they contain genes likely to mediate interactions with the host. These newly identified genetic islands provide areas of investigation into both the evolution and pathogenesis of H. influenzae. In addition, the genome scanning approach developed to identify these islands provides a rapid means to compare the genomes of phenotypically diverse bacterial strains once the genome sequence of one representative strain has been determined.