Identification and Cloning of Genes from Porphyromonas gingivalis after Mutagenesis with a Modified Tn 4400 Transposon from Bacteroides fragilis

Abstract

Porphyromonas gingivalis is a gram-negative, black-pigmented, oral anaerobe strongly associated with adult periodontitis. Previous transposon mutagenesis studies with this organism were based on the Bacteroides transposon Tn4351. Characterization of Tn4351-disrupted genes by cloning has not been an efficient way to analyze large numbers of mutants and is further complicated by the high rate of cointegration of the suicide delivery vector containing Tn4351. In this study, we mutagenized P. gingivalis with a modified version of the Bacteroides fragilis transposon Tn4400. Plasmid pYT646B carrying the transposon was mobilized from Escherichia coli to P. gingivalis ATCC 33277 by conjugation. Both normal and inverse transposition frequencies were similar (3 ؋ 10 ؊8 ). However, the inverse transposon (Tn4400) contains a pBR322 replicon and a ␤-lactamase gene; thus, the cloning of disrupted genomic DNAs from inverse transposition mutants was easily accomplished after ligation of genomic fragments and transformation into E. coli. Thousands of transconjugants could be obtained in a single mating experiment, and inverse transposition was random as demonstrated by Southern hybridization. By this procedure the disrupted genes from P. gingivalis pleiotropic mutants were quickly cloned, sequenced, and identified. The ability to isolate mutations in bacterial virulence genes is a powerful technique for defining their role in pathogenesis.Transposon mutagenesis is the method of choice because mutations are ideally random and mutants can be readily selected and identified. Plasmid vectors and transposons developed for colonic Bacteroides spp. have proved invaluable for genetic studies with Porphyromonas gingivalis, a gram-negative, blackpigmented, oral anaerobe. Tn4351, the first transposon developed for mutagenesis of P. gingivalis, has been used to generate mutants of several strains (2, 12), but its use has limitations. For example, in a separate study with strain ATCC 33277 (1), only 20% of mutations were simple transposon insertions, the rest being cointegrates containing the delivery vector and the transposon. While mutant phenotypes appeared stable, the cointegration of 53 kb of extra plasmid DNA complicated the molecular characterization of mutant genes.In this report we describe a new mutagenesis system for P. gingivalis based on Tn4400Ј, a modified version of the Bacteroides transposon Tn4400 (10). The transposon delivery vector, pYT646B (11), shown in Fig. 1A, carries on its Escherichia coli vector backbone a conjugational transfer origin, oriT, from the broad-host-range IncP plasmid RK2 (3) and the following tetracycline resistance genes: tetX, which is expressed in aerobically grown E. coli, and tetQ, which is expressed in P. gingivalis (6). The vector pYT646B does not contain a P. gingivalis replicon; therefore, upon conjugal transfer, tetracycline-resistant (Tc r ) transconjugants can be obtained only from either inverse transposition of Tn4400 (Fig. 1B) or cointegration of pYT646B (see below). Because pYT646B cont...

Porphyromonas gingivalis Resistance to Polymyxin B Is Determined by the Lipid A 4′‐Phosphatase, PGN_0524

“…We performed a Tn4400'-based mutagenesis in P. gingivalis (ATCC 33277) according to an established procedure to obtain approximately 2,700 independent colonies containing random genomic insertions of the Tn4400' transposon (Chen et al, 2000;Tang et al, 2000). The colonies were screened by replica plating on media plates with or without PMB (50 µg⋅mL -1 ).…”

“…Subsequent sequencing analyses of the resulting PCR fragments were performed using the primers that interact with the 5'-end (primer SJ149) or the 3'-end (primer SJ150) of the coding sequence to determine the site of transposon integration. The sequences of the primers used in this study are in the PGN_0524 locus was determined by PCR analyses using primers derived from the upstream (primer SJ163) and downstream (primer SC337) flanking regions of the PGN_0524 gene in combination with primers derived from the left (primer a) and right (primer c) ends of the insertion sequences of Tn4400' (grey shaded arrows) (Chen et al, 2000). Sequencing of the PCR fragments derived from these PCR analyses using primers that interact with the 5'-end of the coding sequence (primer SJ149) and the 3'-end of the coding sequence (primer SJ150) revealed that Tn4400' was integrated at base-pair (bp) position 404 bp of the 717 bp PGN_0524 coding sequence (vertical arrow).…”

“…A conjugation method based upon a previously described procedure was used to generate random Tn4400'-mediated insertion mutations in the genome of P. gingivalis ATCC 33277 (Chen et al, 2000;Tang et al, 2000). For a typical tri-parental mating, a HB101 strain of E. coli containing pRK231 (plasmid encoding mobilization element) and a DH10B strain of E. coli containing pYT-646B (plasmid encoding the Tn4400 transposon) were grown in Luria broth to an OD 600 ~ 0.8.…”

“…Polymerase chain reactions (PCR) using clone 8-1 genomic DNA were performed using primers that are specific for the 5'-and 3'-flanking regions of PGN_0524 ( SJ163 and SC337 respectively) in combination with primers that are specific for the right hand side ('a') or left hand side ('c') of the Tn4400' insertion element (Chen et al, 2000) to determine the orientation of the Tn4400' transposon within the PGN_0524 locus ( Figure 1). Subsequent sequencing analyses of the resulting PCR fragments were performed using the primers that interact with the 5'-end (primer SJ149) or the 3'-end (primer SJ150) of the coding sequence to determine the site of transposon integration.…”

“…We employed a previously developed transposon-mediated mutagenesis system that utilizes Tn4400' to generate a library of random mutants in P. gingivalis ATCC 33277 (Chen et al, 2000;Tang et al, 2000). The resulting mutants (approximately 2,700 independent clones) were subjected to a simple screen to identify PMB-sensitive mutants.…”