Microarray Analysis of Transposon Insertion Mutations inBacillus anthracis: Global Identification of Genes Required for Sporulation and Germination

Abstract

A transposon site hybridization (TraSH) assay was developed for functional analysis of the Bacillus anthracis genome using a mini-Tn10 transposon which permitted analysis of 82% of this pathogen's genes. The system, used to identify genes required for generation of infectious anthrax spores, spore germination, and optimal growth on rich medium, was predictive of the contributions of two conserved hypothetical genes for the phenotypes examined.The goal of obtaining a detailed understanding of Bacillus anthracis physiology has not been realized due to limited information concerning factors required for growth, sporulation, and virulence. For example, few factors or processes required for generation of the anthrax spore, which develops to enable long-term survival outside the host, have been described (28). Such information would be very valuable as the spore is the infectious form of the pathogen and has been the focus of several virulence and vaccine studies (1,5,15,16). Recently, a functional genomics assay termed the transposon site hybridization (TraSH) assay was described, and this assay has the potential to significantly increase our understanding of microbial physiology by directly identifying genes required for growth and survival in specific environments (18). The TraSH assay uses microarray technology to globally determine the locations of transposon insertions in a population of mutants and to compare the effects of each insertion on the representation of clones before and after exposure to an environmental stress ( Fig. 1) (19,20). Comprehensive TraSH screens depend on thorough distribution of transposon insertion mutations in the genome. Several investigators have noted that transposons commonly used in gram-positive bacteria (Tn916 and Tn917) appear to target preferentially one or both of the B. anthracis virulence plasmids and that few insertions are observed in the chromosome (9,11,24,26). Recently, a mariner-based transposon mutagenesis system which shows promise was described for B. anthracis; however, the utility of this system in global mutagenesis has not been demonstrated (23). Thus, such mutagenesis systems may not permit global genome-wide analysis of B. anthracis physiological systems in strains harboring one or both of the virulence plasmids. This presents a significant hurdle since strains that lack both virulence plasmids are completely attenuated and of limited use in genetic screens for virulence traits in animal models of disease (26). In this study we sought to adapt TraSH to functional genomic analyses of the B. anthracis genome using a mini-Tn10 transposon and whole-genome B. anthracis microarrays.Mini-Tn10 permits global mutagenesis of the B. anthracis genome. Plasmid pIC333, a mini-Tn10 vector encoding spectinomycin resistance and containing a tnpA allele with relaxed target specificity to increase the randomness of transposition (21), was used to mutagenize two B. anthracis strains, B. anthracis ⌬Ames harboring the capsule plasmid (pXO1 Ϫ pXO2 ϩ ) and a derivative of this strai...

A Novel Spore Protein, ExsM, Regulates Formation of the Exosporium in Bacillus cereus and Bacillus anthracis and Affects Spore Size and Shape

“…The amplicon was then digested with KpnI and ligated into the plasmid pASD2, an E. coli-B. cereus shuttle vector with a thermosensitive origin of replication in B. cereus (14). The resulting plasmid, pMFB4 (pASD2::exsM 200bp ), was passaged through E. coli INV110 (dam dcm mutant) to obtain unmethylated DNA and then introduced into B. cereus ATCC 4342 by electroporation, following a previously described protocol (62).…”

Dual effects of single-walled carbon nanotubes coupled with near-infrared radiation on Bacillus anthracis spores: inactivates spores and stimulates the germination of surviving spores

“…Spore germination has been studied intensively. Recently, 31 genes required for B. anthracis sporulation and germination were identified [34]. A few studies have been reported on how the expression of sporulation and germination related genes in B. anthracis spores are affected under different physical intervention techniques, such as heat treatment, pasteurization, and microfiltration [35].…”

“…In order to examine the effect of SWCNTs-NIR treatment on B. anthracis spores at the molecular level, real-time PCR was used to test the expression levels of six germination-related genes identified by the transposon site hybridization (TraSH) assay [34], seven virulence-related genes and three regulatory genes. Table  1 shows the fold changes in the expression levels of all the detected genes in B. anthracis spores upon SWCNTs-NIR treatment in comparison to the untreated spores.…”

Genetic Manipulation Methods inBacillus anthracis

“…These transposons, both Mariner and mini -Tn 10 -based systems, do not appear to preferentially insert into the virulence plasmids, and transposon insertions have been isolated in a wide variety of chromosomal locations (see the references cited in Table 4.1 ). Indeed, the mini -Tn 10 system described by Day et al (2007) associated with greater than 80% of the genes in the B. anthracis strain tested. This improved chromosome coverage allowed for TraSH analysis of genes important in growth, sporulation, and germination in laboratory growth media.…”

“…This improved chromosome coverage allowed for TraSH analysis of genes important in growth, sporulation, and germination in laboratory growth media. All of these studies were performed with non -pathogenic strains of B. anthracis , but Cote et al (2008) used the miniTn 10 system of Day et al (2007) to identify a protein recognized by anti -spore antibodies from a fully virulent strain.…”