Complete Nucleotide Sequence of Plasmid Rts1: Implications for Evolution of Large Plasmid Genomes

Abstract

Rts1, a large conjugative plasmid originally isolated from Proteus vulgaris, is a prototype for the IncT plasmids and exhibits pleiotropic thermosensitive phenotypes. Here we report the complete nucleotide sequence of Rts1. The genome is 217,182 bp in length and contains 300 potential open reading frames (ORFs). Among these, the products of 141 ORFs, including 9 previously identified genes, displayed significant sequence similarity to known proteins. The set of genes responsible for the conjugation function of Rts1 has been identified. A broad array of genes related to diverse processes of DNA metabolism were also identified. Of particular interest was the presence of tus-like genes that could be involved in replication termination. Inspection of the overall genome organization revealed that the Rts1 genome is composed of four large modules, providing an example of modular evolution of plasmid genomes.Rts1 is a low-copy-number kanamycin resistance plasmid originally isolated from a clinical strain of Proteus vulgaris (56). Its molecular mass was originally estimated to be about 140 MDa (29). This large plasmid is the prototype for the T incompatibility group (9) and expresses pleiotropic thermosensitive phenotypes in autonomous replication (13, 58), conjugative transfer (56), host cell growth (12, 57), and restriction of T-even phages (28,32,66).As in many large plasmids of gram-negative bacteria, Rts1 requires two elements for its autonomous replication, a replication initiation protein encoded by the plasmid (repA) and a short segment containing the replication origin, ori (30,34,59). Because the replication of this mini-Rts1 plasmid is stable at 37°C but is inhibited at 42°C in Escherichia coli (30), as observed for Rts1 (56, 58), the replication machinery of Rts1 itself is thermosensitive. A locus termed tdi (temperature-dependent instability) has also been identified as another locus responsible for the temperature sensitivity (48, 55), but its real physiological role in the replication (or maintenance) of Rts1 is unknown.As for the temperature-sensitive effect on host cell growth, Terawaki et al. (57) first reported that Rts1 inhibits its host cell growth at 42°C but not at 37°C. Since then, two loci responsible for this phenotype have been identified, tsg (43, 47) and hig (60-62). Since the tsg locus contains no open reading frame (ORF), the AT-rich DNA segment itself is thought to be responsible for the thermosensitivity of host cell growth. On the other hand, the hig locus encodes a system belonging to the proteic killer family, where the HigB and HigA proteins function as a toxin and an antitoxin, respectively. The temperaturesensitive host cell growth conferred by the hig system is mediated by selectively killing the host that has lost all copies of Rts1 at a nonpermissive temperature (postsegregation killing).Conjugation of Rts1 is also thermosensitive, but the temperature range for conjugation differs from that for replication, efficient at 25°C but not at 37°C (56). Thus, the thermosensitivity of ...

Functional and Mutational Analysis of Conjugative Transfer Region 2 (Tra2) from the IncHI1 Plasmid R27

“…AF030442) (41), pNL1 from Novosphingobium aromaticivorans (NC_002033) (44), the IncJ element R391 from Providencia rettgeri (AY090559) (5), the IncT plasmid Rts1 from Proteus vulgaris (NC_003905) (38), and the SXT element from Vibrio cholerae (AY055428) (reference 4 and data not shown).…”

F-Like Type IV Secretion Systems Encode Proteins with Thioredoxin Folds That Are Putative DsbC Homologues

“…Interestingly, many large F-and H-like conjugative plasmids encode at least one Dsb homologue containing the C-X-X-C motif. H-like transfer regions such as those of pNL1 (63), SXT (11), R391 (14), Rts1 (55), and pCAR1 (46) encode either DsbC H or HtdT H but not both, whereas R27 (40,41,65), R478 (26), and pHCM1 (57) have two transfer regions, each encoding either HtdT H or DsbC H . Both F-and H-like transfer regions encode TraF or TrhF; however, trbB is found only in plasmids encoding TraF lacking the C-X-X-C motif.…”

An inducible tellurite-resistance operon in Proteus mirabilis

“…Furthermore, the ter operon was not located on large IncJ or IncT plasmids found in some strains of P. vulgaris and Providencia spp. (Boltner et al, 2002;Murata et al, 2002). A phylogenetic analysis of Te r proteins suggests a rather recent dissemination among the enteric bacteria, most likely accelerated by transmissible plasmids (Taylor et al, 2002).…”