YT135.2.8, a Tn4400′ insertion mutant of Bacteroides fragilis strain TM4000, grows poorly when used to infect Monika or Chinese hamster ovary (CHO) cell monolayers and is outcompeted by wild‐type strains in mixed infections. YT135.2.8 also shows defects in the rat granuloma pouch model system in monoculture and is completely outcompeted by the wild‐type strain in a mixed infection. In addition, this mutant shows defects in a new model system consisting of CHO suspension cell columns. All of these defects may be explained by the finding that YT135.2.8 shows decreased tolerance to exposure to atmospheric oxygen (less aerotolerant). The monolayer growth defect (MGD) of YT135.2.8 can be influenced significantly by the presence of sulphur‐containing reducing agents (cysteine, dithiothreitol, thiodiglycol) or the non‐sulphur reducing agent Tris‐(2‐carboxylethyl)phosphine (TCEP). The defects in YT135.2.8 can be complemented by a 6.6 kb fragment of the B. fragilis chromosome. DNA sequencing of this fragment and of the regions flanking the Tn4400′ insertion in the B. fragilis chromosome revealed the presence of five open reading frames, corresponding to genes bat (Bacteroides aerotolerance) A, B, C, D, E, which form the BatI operon; Tn4400′ inserted within batD. All of the hypothetical proteins possess one or more membrane‐spanning domains. BatA and BatB show high similarity to each other but, like BatD, they show no match to sequences of known function in the databases. BatC and BatE contain 2–4 repeated sequences similar to the tetratricopeptide repeats (TPRs) seen in many eukaryotic proteins. The function of TPR sequences in protein interactions in other systems leads to the suggestion that the Bat proteins form a complex. The BatI complex may be involved in the generation or export of reducing power equivalents to the periplasm of the B. fragilis cell.

A modified version of the Bacteroides fragilis transposon Tn4400, designated Tn4400, enabling rapid isolation and analysis of B. fragilis mutants has been constructed. To identify potential virulence factors, Tn4400-generated mutants were screened by a new method; this resulted in the isolation of 21 mutant strains with impaired growth characteristics on tissue culture monolayers but normal growth in rich medium anaerobically. Tn4400 mutagenesis. Bacteroides fragilis is the most frequently isolated anaerobic species from human intraperitoneal and intra-abdominal infections involving anaerobes (2, 3, 9). Compound transposon Tn4400 was isolated from B. fragilis plasmid pBFTM10 by Robillard et al. (7). A similar transposon, Tn4351, studied in the laboratories of Smith (11) and Salyers (5, 10), was used to generate B. fragilis and Bacteroides thetaiotaomicron mutants. Here we describe further modifications of Tn4400 which have increased its transposition frequency as well as facilitated the rapid isolation of B. fragilis chromosomal fragments abutting the inserted transposon.The entire transposon Tn4400, including a clindamycin-resistant (Cln r ) determinant active in B. fragilis, was cloned as a single BglII fragment from a pBFTM10-FЈ lac fusion plasmid, pOX446R1 (7), into a plasmid based on pDG5, which contains the pBR322 replicon, the bla gene for ampicillin resistance (Amp r ) in Escherichia coli, and the origin of transfer (oriT) from the IncP plasmid RK2 (4). The resulting plasmid, pNJR609, apparently underwent an oriT-mediated rearrangement upon prolonged storage and consequently lost the oriT region. This plasmid was named pNJR609⌬ (Table 1).To restore the ability of the plasmid to be mobilized by RK2, the oriT of RP4 was isolated as a blunt-ended 760-bp HaeII fragment from pJST51 (14) and cloned into the HincII site within the bla gene of pNJR609⌬. The resulting plasmids, pYT644A and pYT644B, were able to generate Cln r colonies by transposition upon transfer into B. fragilis. A tetQ gene cassette, which confers tetracycline resistance in B. fragilis but not E. coli (6), was cloned as a SacI fragment into a pSP72 vector plasmid, generating pRG23. A BglII-BamHI fragment from pRG23 containing the tetQ gene was cloned into the BglII sites of pYT644A and pYT644B. The resulting plasmids, pYT645A and pYT645B (Fig. 1A), were able to generate both Cln r and tetracycline-resistant (Tet r ) B. fragilis strains. For conjugal transfer of the pYT644 or pYT645 plasmid from E. coli to B. fragilis, mid-log-phase broth cultures of an E. coli donor strain, a second E. coli strain containing the mobilizer RK231, and a B. fragilis recipient were mixed (2 ml-2 ml-5 ml) and concentrated by centrifugation, and the mixture was placed on a sterile filter (type HAWP 047; Millipore Corp., Bedford, Mass.) on the surface of a plate containing solidified brain heart infusion broth supplemented with 0.5% yeast extract and 5 g of hemin per ml (BHIS). After overnight, aerobic incubation at 37°C, the bacteria were plated on BHIS plates cont...

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...