Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis

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.

Post-translational regulation of a Porphyromonas gingivalis regulator

“…In addition, acetyl-CoA can also be generated from acetate via the reversible activities of acetate kinase (PGN_1081) and phosphotransacetylase (PGN_1082). Both the latter were identified as essential genes in strain ATCC 33277 [31] and interestingly are located four genes upstream from pat (PGN_1185) and rprY (PGN_1186), raising the possibility that they are or once were part of an acetylation unit.…”

Porphyromonas gingivalis infection in gestational diabetes mellitus and survival in tobacco smokers.

“…Further, we compared the generated library with the existing library (Klein et al, 2012) in order to identify the common inherently essential genes. We entered the identified inherently essential genes in DEG database, which has only 36 bacterial essential genes apart from P. gingivalis.…”

“…The genome of ATCC 33277 comprised a single circular chromosome of 2,354,886 bp with an average G + C content of 48.4% [147]. The ATCC 33277 genome contained 2155 genes, 2090 protein coding sequences (CDSs) with a mean size of 970 bp, covering 86.1% of the whole chromosome sequence, approximately 117,000 TA sites, and no plasmids [32,147,148]. It contains 4 RNA operons and 53 tRNA genes that provide specificity for amino acids.…”

“…ATCC 33277 contained a variety of mobile genetic elements [147]. Only four NCBI annotated genes coding hypothetical proteins lack TA sites [148]. A total of 93 insertion sequence elements and 48 miniature inverted-repeat transposable elements were found in ATCC 33277.…”

“…The genes essential for survival in GAM broth were compared with the previously published list of essential genes for survival in supplemented brain-heart infusion broth [148]. The function and interrelationships of the 281 common inherently essential genes for planktonic growth of P. gingivalis in complex media were investigated in silico by …”