ABSTRACTDesulfitobacterium hafniensestrain TCE1 is capable of metabolically reducing tetra- and trichloroethenes by organohalide respiration. A previous study revealed that thepcegene cluster responsible for this process is located on an active composite transposon, Tn-Dha1. In the present work, we investigated the effects on the stability of the transposon during successive subcultivations of strain TCE1 in a medium depleted of tetrachloroethene. At the physiological level, an increased fitness of the population was observed after 9 successive transfers and was correlated with a decrease in the level of production of the PceA enzyme. The latter observation was a result of the gradual loss of thepcegenes in the population of strain TCE1 and not of a regulation mechanism, as was postulated previously for a similar phenomenon described forSulfurospirillum multivorans. A detailed molecular analysis of genetic rearrangements occurring around Tn-Dha1showed two independent but concomitant events, namely, the transposition of the first insertion sequence, ISDha1-a, and homologous recombination across identical copies of ISDha1flanking the transposon. A new model is proposed for the genetic heterogeneity around Tn-Dha1inD. hafniensestrain TCE1, along with some considerations for the cleavage mechanism mediated by the transposase TnpA1 encoded by ISDha1.

The strategic adaptation of prokaryotes in polluted niches involves the efficient regulation of their metabolism. The obligate anaerobe and metabolically versatile Desulfitobacterium hafniense reductively dechlorinates halogenated organic compounds (socalled organohalides). Some D. hafniense strains carry out organohalide respiration (OHR), a process which requires the use of corrinoid as a cofactor in reductive dehalogenases, the key enzymes in OHR. We report here the diversity of the cobalamin riboswitches that possibly regulate the corrinoid metabolism for D. hafniense. The analysis of available D. hafniense genomes indicates the presence of 18 cobalamin riboswitches located upstream of genes whose products are mainly involved in corrinoid biosynthesis and transport. To obtain insight into their function, the secondary structures of three of these RNA elements were predicted by Mfold, as well as analyzed by in-line probing. These RNA elements both display diversity in their structural elements and exhibit various affinities toward adenosylcobalamin that possibly relates to their role in the regulation of corrinoid metabolism. Furthermore, adenosylcobalamin-induced in vivo repression of RNA synthesis of the downstream located genes indicates that the corrinoid transporters and biosynthetic enzymes in D. hafniense strain TCE1 are regulated at the transcriptional level. Taken together, the riboswitch-mediated regulation of the complex corrinoid metabolism in D. hafniense could be of crucial significance in environments polluted with organohalides both to monitor their intracellular corrinoid level and to coexist with corrinoid-auxotroph OHR bacteria.