The complete DNA sequence and analysis of R27, a large IncHI plasmid from Salmonella typhi that is temperature sensitive for transfer

Abstract

Salmonella typhi, the causative agent of typhoid fever, annually infects 16 million people and kills 600 000 world wide. Plasmid-encoded multiple drug resistance in S. typhi is always encoded by plasmids of incompatibility group H (IncH). The complete DNA sequence of the large temperature-sensitive conjugative plasmid R27, the prototype for the IncHI1 family of plasmids, has been compiled and analyzed. This 180 kb plasmid contains 210 open reading frames (ORFs), of which 14 have been previously identified and 56 exhibit similarity to other plasmid and prokaryotic ORFs. A number of insertion elements were found, including the full Tn 10 transposon, which carries tetracycline resistance genes. Two transfer regions, Tra1 and Tra2, are present, which are separated by a minimum of 64 kb. Homologs of the DNA-binding proteins TlpA and H-NS that act as temperature-regulated repressors in other systems have been located in R27. Sequence analysis of transfer and replication regions supports a mosaic-like structure for R27. The genes responsible for conjugation and plasmid maintenance have been identified and mechanisms responsible for thermosensitive transfer are discussed.

Figure 1

Linear representation of R27, showing all ORFs, their orientation and with color coding by functional groups. The upper rectangles are ORFs transcribed from right to left and the lower rectangles are transcribed left to right. Color coding of functional groups is as follows: yellow, Tn and IS elements; black, replication and stability; red, drug resistance; blue, citrate utilization; magenta, conjugal transfer. The GenBank entry of ORFs were assigned unique identifiers in the form Rxxxx. This figure was created using GeneScene (DNASTAR).

Figure 2

G+C profile of the complete DNA sequence of R27. This figure was plotted using data generated from the GCG program Window.

Figure 3

Alignment of TraI (ORF120) with nickase/relaxase proteins from other conjugative plasmids (IncW R388, IncP RP4, IncI R64, Ti and IncHI1 R27). Amino acid residues that follow the guidelines of the nickase/relaxase distinguishing motifs, as set out in the discussion, are highlighted.

Figure 4

PILEUP comparison of the TraF homologs identifying a conserved motif between the proposed TrhF and other proteins related to the TraF protein of the IncPα plasmid RP4 (30,31). The illustrated sequences are from R27 (TraF_R27), the IncPα plasmid RP4 (TraF_PAlph), the IncPβ plasmid R751 (TraF_PBeta), the A.tumefaciens octopine-type Ti plasmid (TraF_OctTi), the A.tumefaciens nopaline-type Ti plasmid (TraF_NopTi) and ORF2 from the Vir2 region of the Ti plasmid (Orf2_TiVir). Catalytic residues described in the text are denoted above the alignment with an asterisk. Catalytic domains I, II and III as described by Haase and Lanka (37) are denoted above the alignment with a line.

Figure 5

(A) Amino acid alignment of the TlpA DNA-binding domain (45), underlined, with surrounding sequences, and the proposed equivalent in ORF158. (B) The COILS2 output for ORF158 and TlpA indicating the probability (P) of distinct regions of each protein having a coiled-coil conformation. ORF158 and TlpA also have similar outputs using the PARCOIL program (21) (data not shown).