Abstract
The identification of genes essential for a bacterium’s growth reveals much about its basic physiology under different conditions. Bordetella pertussis , the causative agent of whooping cough, adopts both virulent and avirulent states through the activity of the two-component system, Bvg. The genes essential for B. pertussis growth in vitro were defined using transposon sequencing, for different Bvg-determined growth states. In addition, comparison of the insertion indices of each gene between Bvg phases identified those genes whose mutation exerted a significantly different fitness cost between phases. As expected, many of the genes identified as essential for growth in other bacteria were also essential for B. pertussis . However, the essentiality of some genes was dependent on Bvg. In particular, a number of key cell wall biosynthesis genes, including the entire mre/mrd locus, were essential for growth of the avirulent (Bvg minus) phase but not the virulent (Bvg plus) phase. In addition, cell wall biosynthesis was identified as a fundamental process that when disrupted produced greater fitness costs for the Bvg minus phase compared to the Bvg plus phase. Bvg minus phase growth was more susceptible than Bvg plus phase growth to the cell wall-disrupting antibiotic ampicillin, demonstrating the increased susceptibility of the Bvg minus phase to disruption of cell wall synthesis. This Bvg-dependent conditional essentiality was not due to Bvg-regulation of expression of cell wall biosynthesis genes; suggesting that this fundamental process differs between the Bvg phases in B. pertussis and is more susceptible to disruption in the Bvg minus phase. The ability of a bacterium to modify its cell wall synthesis is important when considering the action of antibiotics, particularly if developing novel drugs targeting cell wall synthesis.
Highlights
Essential genes Allelic replacement mutagenesis is a cornerstone of the study of bacterial pathogenicity
Plasmid ep1 cannot replicate in B. pertussis, but on delivery into these bacteria by conjugation, the transient expression of the transposase enables transposition of the kanamycin cassette onto the B. pertussis chromosome but subsequent loss of the plasmid
490 000 transposon insertion mutants were recovered for each Bvg phase condition
Summary
Essential genes Allelic replacement mutagenesis is a cornerstone of the study of bacterial pathogenicity. Comparison between the phenotypes of the WT and mutant reveals differences in behaviour that are ascribed to the loss of the targeted gene from the mutant. In this way, the genetic bases of numerous traits have been established and targeted mutagenesis has been used to perturb traits to understand them. Genes that are essential for the viability of a bacterium cannot be studied in this way because, by definition, the bacteria cannot withstand a knock-o ut mutation in these genes and remain viable. Often it is not easy to establish that a gene is essential, as the failure to obtain viable mutants is a
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