Identification of Genes Required by Bacillus thuringiensis for Survival in Soil by Transposon-Directed Insertion Site Sequencing
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Transposon-directed insertion site sequencing was used to identify genes required by Bacillus thuringiensis to survive in non-axenic plant/soil microcosms. A total of 516 genetic loci fulfilled the criteria as conferring survival characteristics. Of these, 127 (24.6 %) were associated with uptake and transport systems; 227 loci (44.0 %) coded for enzymatic properties; 49 (9.5 %) were gene regulation or sensory loci; 40 (7.8 %) were structural proteins found in the cell envelope or had enzymatic activities related to it and 24 (4.7 %) were involved in the production of antibiotics or resistance to them. Eighty-three (16.1 %) encoded hypothetical proteins or those of unknown function. The ability to form spores was a key survival characteristic in the microcosms: bacteria, inoculated in either spore or vegetative form, were able to multiply and colonise the soil, whereas a sporulation-deficient mutant was not. The presence of grass seedlings was critical to colonisation. Bacteria labelled with green fluorescent protein were observed to adhere to plant roots. The sporulation-specific promoter of spo0A, the key regulator of sporulation, was strongly activated in the rhizosphere. In contrast, the vegetative-specific promoters of spo0A and PlcR, a pleiotropic regulator of genes with diverse activities, were only very weakly activated.
KeywordsSupplementary Information Tryptone Soya Broth Peptidoglycan Synthesis Input Pool Fluorescent Bacterium
The authors are very grateful to The Defense Threat Reduction Agency (DTRA) for funding. AHB was also supported by funding from the Ministry of Defence, UK. We also acknowledge the help from Gill Hartley (Dstl) for confocal microscopy and Bry Lingard (Dstl) for assistance with sequence analysis. We thank Dr Sari Paikoff (DTRA) and Prof. Petra Oyston for support and advice.
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