Deep Sequencing of Porphyromonas gingivalis and Comparative Transcriptome Analysis of a LuxS Mutant

Takanori Hirano,Donald R Demuth,Murray Hackett,Richard J Lamont,David A C Beck

doi:10.3389/fcimb.2012.00079

Abstract

Porphyromonas gingivalis is a major etiological agent in chronic and aggressive forms of periodontal disease. The organism is an asaccharolytic anaerobe and is a constituent of mixed species biofilms in a variety of microenvironments in the oral cavity. P. gingivalis expresses a range of virulence factors over which it exerts tight control. High-throughput sequencing technologies provide the opportunity to relate functional genomics to basic biology. In this study we report qualitative and quantitative RNA-Seq analysis of the transcriptome of P. gingivalis. We have also applied RNA-Seq to the transcriptome of a ΔluxS mutant of P. gingivalis deficient in AI-2-mediated bacterial communication. The transcriptome analysis confirmed the expression of all predicted ORFs for strain ATCC 33277, including 854 hypothetical proteins, and allowed the identification of hitherto unknown transcriptional units. Twelve non-coding RNAs were identified, including 11 small RNAs and one cobalamin riboswitch. Fifty-seven genes were differentially regulated in the LuxS mutant. Addition of exogenous synthetic 4,5-dihydroxy-2,3-pentanedione (DPD, AI-2 precursor) to the ΔluxS mutant culture complemented expression of a subset of genes, indicating that LuxS is involved in both AI-2 signaling and non-signaling dependent systems in P. gingivalis. This work provides an important dataset for future study of P. gingivalis pathophysiology and further defines the LuxS regulon in this oral pathogen.

Highlights

Porphyromonas gingivalis, a Gram-negative anaerobe, is a major pathogen in periodontal disease, one of the most common bacterial infections of humans in developed countries (Sabbah et al, 2010)
GINGIVALIS TRANSCRIPTOME In order to characterize the P. gingivalis transcriptome by SOLiD based RNA-Seq, RNA was prepared from three biological replicate cultures of ATCC 33277 and its ∆luxS mutant
We previously showed that AI-2 synthesis in P. gingivalis peaks in early exponential phase (James et al, 2006), cultures were grown to early exponential phase (OD600 0.5)

Summary

Introduction

Porphyromonas gingivalis, a Gram-negative anaerobe, is a major pathogen in periodontal disease, one of the most common bacterial infections of humans in developed countries (Sabbah et al, 2010). A major ecological niche of P. gingivalis is the subgingival crevice or periodontal pocket (Lamont and Jenkinson, 1998), and this region experiences fluctuation in oxygen levels, temperature, pH, and nutrient availability. In P. gingivalis, LuxS regulates the expression of a number of genes involved in the uptake of hemin and inorganic iron, including hmuR, fetB, feoB1, and ftn, which are negatively regulated, and tlr and kgp which are positively regulated (James et al, 2006). Expression of the luxS gene is controlled by a phospho-dependent signaling pathway that converges on the transcriptional regulator CdhR (Maeda et al, 2008). A microarray analysis of P. gingivalis has shown that LuxS is involved in the control of resistance to stress (Yuan et al, 2005); little else is known regarding the broader LuxS regulon

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