Genetic and nucleotide sequence analysis of the gene htdA, which regulates conjugal transfer of IncHI plasmids

Abstract

IncHI plasmids are naturally repressed for conjugative transfer and do not allow efficient propagation of the IncH pilus-specific phage Hgal. Transposons Tn7, TnS, and TnlacZ were inserted into IncHI plasmids R478, R477-1, and R27, respectively, leading to the isolation of several plasmid mutants which exhibited increased levels of transfer and also permitted good lysis with phage Hgal. A 4.3-kb HindlIl fragment from R478 reversed both phenotypic effects of derepression for the R477-1::TnS and the R478::Tn7 derivatives, pKFW99 and pKFWIOO, respectively. Exonuclease III deletions of this fragment and nucleotide sequence analysis indicated that the gene responsible for transfer repression, named here htdAl, encoded a polypeptide of 150 amino acids.Cloning and sequence analysis of pDT2454 (R27::TnlacZ) revealed that the transposon had inserted into an open reading frame (ORF) which had an 83% amino acid identity with the R478 htd4 gene. Maxicell analysis showed both the R27 and R478 HtdA products had molecular masses of 19.9 kDa. Coijugation experiments showed that the cloned htdA determinants caused a significant reduction of the transfer frequencies of wild-type R478 and R27 plasmids. Examination of both R478 derepressed mutants, pKFW100 and pKFW101, indicated that both transposon insertions occurred upstream of the htdA ORF. The results suggest that HtdA is a regulatory component of IncH plasmid transfer and also show that the region upstream of the htd4 ORF is involved in transfer repression. The locations of the htdA4 determinants were identified on the plasmid maps of R27 and R478.

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

“…Since mutations were made in drR27 (wild-type R27 with TnlacZ inserted into htdA), we inserted a CAT cassette into htdA within wild-type R27 to see if a different disruption of this gene (CAT [1 kb] versus TnlacZ [8.7 kb]) had the same effect on transfer. An insertional disruption of htdA by CAT increased the transfer frequency of R27 by 6,000-fold, as did the insertion by TnlacZ (52). These data, combined with those from the random transposon mutagenesis experiments, indicate that 11 genes within the Tra2 region are essential for conjugative transfer.…”

“…E. coli strains containing drR27 with mutations in transfer genes trhU and trhN were capable of forming plaques, although they were notably smaller and not as clear as drR27 plaques. Disruption of htdA in wild-type R27 resulted in plaques which were larger than those produced by wild-type R27; this is attributed to the increase in H-pilus production by donors (52). E. coli cells containing an insertional disruption of orf030, orf028, orf027, trhO, orf017, orf016, htdF, htdK, orf009, orf004, or trhI were all capable of forming plaques, suggesting that these genes are not essential for H-pilus biosynthesis.…”

“…DNA manipulations. R27 DNA was isolated using either ultracentrifugation in a CsCl-ethidium bromide gradient (52) or the large-construct kit (Qiagen Inc., Mississauga, Ontario, Canada). Standard recombinant DNA methods were performed as described by Sambrook and Russell (46).…”

“…We have previously characterized the Tra1 region of derepressed R27 (drR27) and found that it contains the origin of transfer and genes encoding the relaxosome, coupling protein, and three Mpf proteins (26). drR27 is the prototypical IncHI1 plasmid that has an elevated transfer frequency compared to the wild type due to an insertion into htdA (48,52). In addition, we have performed a preliminary analysis of the Tra2 region (45).…”

Functional and Mutational Analysis of Conjugative Transfer Region 1 (Tra1) from the IncHI1 Plasmid R27

“…DNA manipulations. R27 DNA was isolated using either ultracentrifugation in a CsCl-ethidium bromide gradient (35) or with a Qiagen (Mississauga, Ont.) Large-Construct Kit.…”