Using Tn-seq To Identify Pigmentation-Related Genes of Porphyromonas gingivalis: Characterization of the Role of a Putative Glycosyltransferase

Abstract

Cellular pigmentation is an important virulence factor of the oral pathogen Porphyromonas gingivalis. Pigmentation has been associated with many bacterial functions, including but not limited to colonization, maintaining a local anaerobic environment by binding oxygen molecules, and defense against reactive oxygen species (ROS) produced by immune cells. Pigmentation-associated loci identified to date have involved lipopolysaccharide, fimbriae, and heme acquisition and processing. We utilized a transposon mutant library of P. gingivalis strain ATCC 33277 and screened for pigmentation-defective colonies using massively parallel sequencing of the transposon junctions (Tn-seq) to identify genes involved in pigmentation. Transposon insertions at 235 separate sites, located in 67 genes and 15 intergenic regions, resulted in altered pigmentation: 7 of the genes had previously been shown to be involved in pigmentation, while 75 genes and intergenic regions had not. To further confirm identification, we generated a smaller transposon mutant library in P. gingivalis strain W83 and identified pigment mutations in several of the same loci as those identified in the screen in ATCC 33277 but also eight that were not identified in the ATCC 33277 screen. PGN_0361/ PG_0264, a putative glycosyltransferase gene located between two tRNA synthetase genes and adjacent to a miniature inverted-repeat transposable element, was identified in the Tn-seq screen and then verified through targeted deletion and complementation. Deletion mutations in PGN_0361/PG_0264 glycosyltransferase abolish pigmentation, modulate gingipain protease activity, and alter lipopolysaccharide. The mechanisms of involvement in pigmentation for other loci identified in this study remain to be determined, but our screen provides the most complete survey of genes involved in pigmentation to date. IMPORTANCE P. gingivalis has been implicated in the onset and progression of periodontal disease. One important virulence factor is the bacterium's ability to produce pigment. Using a transposon library, we were able to identify both known and novel genes involved in pigmentation of P. gingivalis. We identified a glycosyltransferase, previously not associated with pigmentation, that is required for pigmentation and determined its mechanism of involvement. A better understanding of the genes involved in pigmentation may lead to new insights into the complex mechanisms involved in this important virulence characteristic and could facilitate development of novel therapeutics.

Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase

“…Next, we determined whether hydrogen peroxide was the major component of STYM1 inhibitory activity and whether we could isolate P. gingivalis-resistant mutants. To do this, we took advantage of a transposon library constructed in the W83 strain of P. gingivalis (30). We exposed the P. gingivalis transposon library to STYM1 extract and then plated for survivors.…”

Identification of genes encoding glycosyltransferases involved in lipopolysaccharide synthesis in Porphyromonas gingivalis

“…We found more than 20 proteins belonging to various glycosyltransferase families in the Pfam protein family database for the P. gingivalis strains W83 and ATCC 33277 (Table ) . The 7 genes encoding glycosyltransferase group 1 were as follows: PGN_0227, PGN_0242, PGN_0428, PGN_1134, PGN_1135, PGN_1240 and gtfB (PGN_1251).…”

“…O‐LPS was present in the gtfD (encoding the second glycosyltransferase for the conventional O‐polysaccharide) and wbaP (encoding the initial glycosyltransferase for the anionic O‐polysaccharide) double mutant, suggesting that GtfC may recognize the first sugar formed by wbaQ (encoding the initial glycosyltransferase for the conventional O‐polysaccharide) or other unknown glycosyltransferases. Very recently, it has been reported that GtfC is involved in the synthesis of A‐LPS …”

The bare necessities: Uncovering essential and condition‐critical genes with transposon sequencing

“…Black pigmentation is a virulence‐associated attribute of P gingivalis required for iron sequestration and protection against oxidative stress . Klein et al screened a transposon library for non‐pigmented colonies on agar plates, pooled non‐pigmented strains and then sequenced en masse using massively parallel sequencing. This screen, in P gingivalis ATCC 33277, identified 82 genes/intergenic regions that are required for correct pigmentation.…”