Characterization of the Batl (Bacteroides aerotolerance) operon in Bacteroides fragilis: isolation of a B-fragilis mutant with reduced aerotolerance and impaired growth in in vivo model systems

YT135.2.8, a Tn4400 ' insertion mutant of Bacteroides fragilis strain TM400 0, grows poorly when used to infect Monika or Chinese hamster ovary (CHO) c ell 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 mo noculture and is completely outcompeted by the wild-type strain in a mixed infection. In addition, this mutant shows defects in a new model system con sisting of CHO suspension cell columns. All of these defects may be explain ed by the finding that YT135.2.8 shows decreased tolerance to exposure to a tmospheric oxygen (less aerotolerant). The monolayer growth defect (MGD) of YT135.2.8 can be influenced significantly by the presence of sulphur-conta ining reducing agents (cysteine, dithiothreitol, thiodiglycol) or the non-s ulphur reducing agent Tris-(2-carboxylethyl)phosphine (TCEP). The defects i n YT135.2.8 can be complemented by a 6.6 kb fragment of the B. fragilis chr omosome. DNA sequencing of this fragment and of the regions flanking the Tn 4400 ' insertion in the B, fragilis chromosome revealed the presence of fiv e open reading frames, corresponding to genes bat(Bacteroides aerotolerance ) A, B, C, D, E, which form the Batl operon; Tn4400 ' inserted within batD. All of the hypothetical proteins possess one or more membrane-spanning dom ains. BatA and Bats show high similarity to each other but, like BatD, they show no match to sequences of known function in the databases. BatC and Ba ts contain 2-4 repeated sequences similar to the tetra tricopeptide 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 Batl complex may be involved in the generation or export of reducing power equivalents to the periplasm of the B. fragili s cell.