Complete nucleotide sequence and determination of the replication region of the sporulation inhibiting plasmid p576 from Bacillus pumilus NRS576

Large plasmids, presumably replicating via the theta mechanism, have been identified in numerous gram-positive bacteria. However, their characterization is rather poor and predominantly limited to those harbored by some (opportunistic) pathogenic bacteria. Here we determined the DNA sequence of the 43.3 kb plasmid p576 from Bacillus pumilus strain NRS576, the first B. pumilus theta-replicating plasmid sequenced. Plasmid p576 has a modular structure, but surprisingly, it does not seem to encode a Rep protein found on most theta-replicating plasmids. However, a ∼1 kb region was identified showing homology with the Rep-independent replication region of Bacillus subtilis plasmid pLS20, and we demonstrated that this region is sufficient for autonomous replication. The plasmid contains various large direct repeat sequences. A likely function could be attributed to at least 15 putative p576 genes. Some of these are predicted to be involved in stable maintenance of the plasmid; others are likely to encode proteins involved in conjugation. p576 also carries a rap-phr cassette whose possible function is discussed.