GENETIC AND NUCLEOTIDE-SEQUENCE ANALYSIS OF THE GENE HTDA, WHICH REGULATES CONJUGAL TRANSFER OF INCHI PLASMIDS

IncHI plasmids are naturally repressed for conjugative transfer and do not allow efficient propagation of the IncH pilus-specific phage Hgal . Transposons Tn7, Tn5, and TnlacZ were inserted into IncHI plasmids R 478, R477-1, and R27, respectively, leading to the isolation of severa l plasmid mutants which exhibited increased levels of transfer and als o permitted good lysis with phage Hgal. A 4.3-kb HindIII fragment from R478 reversed both phenotypic effects of derepression for the R477-1: :Tn5 and the R478::Tn7 derivatives, pKFW99 and pKFW100, respectively. Exonuclease III deletions of this fragment and nucleotide sequence ana lysis indicated that the gene responsible for transfer repression, nam ed here htdA, encoded a polypeptide of 150 amino acids. Cloning and se quence analysis of pDT2454 (R27::TnlacZ) revealed that the transposon had inserted into an open reading frame (ORF) which had an 83% amino a cid identity with the R478 htdA gene. Maxicell analysis showed both th e R27 and R478 HtdA products had molecular masses of 19.9 kDa. Conjuga tion experiments showed that the cloned htdA determinants caused a sig nificant reduction of the transfer frequencies of wild-type R478 and R 27 plasmids. Examination of both R378 derepressed mutants, pKFW100 and pKFW101, indicated that both transposon insertions occurred upstream of the htdA ORF. The results suggest that HtdA is a regulatory compone nt of IncH plasmid transfer and also show that the region upstream of the htdA ORF is involved in transfer repression. The locations of the htdA determinants were identified on the plasmid maps of R27 and R478.