Abstract
BackgroundPorphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with periodontal disease onset and progression. Genetic tools for the manipulation of bacterial genomes allow for in-depth mechanistic studies of metabolism, physiology, interspecies and host-pathogen interactions. Analysis of the essential genes, protein-coding sequences necessary for survival of P. gingivalis by transposon mutagenesis has not previously been attempted due to the limitations of available transposon systems for the organism. We adapted a Mariner transposon system for mutagenesis of P. gingivalis and created an insertion mutant library. By analyzing the location of insertions using massively-parallel sequencing technology we used this mutant library to define genes essential for P. gingivalis survival under in vitro conditions.ResultsIn mutagenesis experiments we identified 463 genes in P. gingivalis strain ATCC 33277 that are putatively essential for viability in vitro. Comparing the 463 P. gingivalis essential genes with previous essential gene studies, 364 of the 463 are homologues to essential genes in other species; 339 are shared with more than one other species. Twenty-five genes are known to be essential in P. gingivalis and B. thetaiotaomicron only. Significant enrichment of essential genes within Cluster of Orthologous Groups ‘D’ (cell division), ‘I’ (lipid transport and metabolism) and ‘J’ (translation/ribosome) were identified. Previously, the P. gingivalis core genome was shown to encode 1,476 proteins out of a possible 1,909; 434 of 463 essential genes are contained within the core genome. Thus, for the species P. gingivalis twenty-two, seventy-seven and twenty-three percent of the genome respectively are devoted to essential, core and accessory functions.ConclusionsA Mariner transposon system can be adapted to create mutant libraries in P. gingivalis amenable to analysis by next-generation sequencing technologies. In silico analysis of genes essential for in vitro growth demonstrates that although the majority are homologous across bacterial species as a whole, species and strain-specific subsets are apparent. Understanding the putative essential genes of P. gingivalis will provide insights into metabolic pathways and niche adaptations as well as clinical therapeutic strategies.
Highlights
Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with periodontal disease onset and progression
Generation of the mutant library We generated transposon insertion libraries in P. gingivalis using a Himar 1 Mariner mini-transposon system created for use in Bacteroides thetaiotaomicron [30]
‘Incorrect’ transpositions can include portions of the vector backbone inserting with the transposon, the vector being stably maintained within the bacterium extrachromosomally or multiple insertions within the same
Summary
Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with periodontal disease onset and progression. Genetic tools for the manipulation of bacterial genomes allow for in-depth mechanistic studies of metabolism, physiology, interspecies and host-pathogen interactions. We adapted a Mariner transposon system for mutagenesis of P. gingivalis and created an insertion mutant library. Porphyromonas gingivalis is an oral Gram-negative, anaerobic, asaccharolytic and black-pigmented bacterium that is highly correlated with the development and progression of periodontal diseases and systemic comorbidities [1,2,3,4,5]. While the use of the mutant libraries generated with these systems led to important insights into P. gingivalis pathogenesis, both elements inserted preferentially into ‘hot-spots’ in the genome limiting the distribution of interrupted genes and were limited to which strains could be mutagenized. The lack of insertion saturation with these transposons into P. gingivalis genes resulted in libraries that were not suitable for the genome-wide identification of essential genes
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