Oligotropha carboxidovorans harbors the low-copy-number, circular, 133,058-bp DNA megaplasmid pHCG3, which is essential in the chemolithoautotrophic utilization of CO (carboxidotrophy), H(2) (hydrogenotrophy) and CO(2) under aerobic conditions. The complete nucleotide sequence of pHCG3 revealed 125 open reading frames. Of these, 95 were identified as putative structural genes. The plasmid carries the four gene clusters cox (14.54 kb, 12 genes), cbb (13.33 kb, 13 genes), hox (23.35 kb, 19 genes plus one ORF) and tra/trb (25.01 kb, 22 genes plus 2 ORFs), which assemble the functions required for the utilization of CO, CO(2) or H(2), and the conjugal transfer of the plasmid, respectively. The gene clusters cox, cbb and hox form a 51.2-kb chemolithoautotrophy module. The tra/trb cluster on the plasmid pHCG3 of O. carboxidovorans has a similar architecture as the Ti-plasmid of Agrobacterium tumefaciens. The tra/trb cluster is separated from the chemolithoautotrophy module by two regions (25.2 and 29.6 kb) with miscellaneous or mostly unknown functions. These regions carry a number of single genes coding for replication and stabilization of pHCG3 as well as the components of a putative system of global regulation of plasmid replication in O. carboxidovorans. An oriV encodes the replication proteins RepABC. Sequence comparisons of pHCG3-encoded genes suggest that major genetic exchange between O. carboxidovorans and the proteobacteria has occurred.