Abstract
TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of approximately 650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.
Highlights
Bacterial conjugation is the primary mechanism by which many plasmids and conjugative transposons are spread throughout a bacterial population
It has become clear that transmissible plasmids encode a conjugative DNA transfer (CDT) initiator protein that plays a key role in initiating DNA strand transfer
Based on the above description, known conjugative transesterases may be grouped into two classes: (i) those lacking an intrinsic helicase activity (e.g. RP4 plasmid TraI) [15] and (ii) those in which transesterase and helicase activities have been shown to reside within a single protein, as is the case only for the R388 plasmid TrwC protein and the F plasmid TraI protein [8, 10, 12]
Summary
Bacterial conjugation is the primary mechanism by which many plasmids and conjugative transposons are spread throughout a bacterial population. The process begins with the formation of a stable mating pair involving a donor cell that contains a conjugative plasmid (or transposon) and a recipient cell that lacks the plasmid This establishes the close cell-cell contact required for physical transfer of single-stranded DNA (ssDNA) from the donor to the recipient. It has become clear that transmissible plasmids encode a conjugative DNA transfer (CDT) initiator protein that plays a key role in initiating DNA strand transfer. Regardless of class, the CDT transesterases exhibit structural motifs and functional similarities that reflect conservation of the catalytic mechanism [4, 16, 17] The majority of these do not have helicase motifs. F oriT nic site 30-mer 5Ј-PCR primer for 348C intein fusion construct 5Ј-PCR primer for 309C intein fusion construct 5Ј-PCR primer for 226C intein fusion construct 3Ј-PCR primer for traI helicase-intein fusions 5Ј-PCR primer for unique XbaI site upstream of traI Shine-Dalgarno sequence 3Ј-PCR primer for N306 construct 3Ј-PCR primer for N235 construct 3Ј-PCR primer for N200 construct 5Ј-PCR primer for TraI⌬252 construct 3Ј-PCR primer for TraI⌬252 construct
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