Mapping of transfer and H pilus coding regions of the IncHII plasmid pHH1508a

The IncHII plasmid pHH1508a (208 kilobases) encodes resistance to potassium tellurite, trimethoprim, and streptomycin. Conjugative pili encoded by pHH1508a were isolated, purified, and used for preparation of anti-H pilus antiserum. Immuno-gold labelling experiments using H pilus specific antiserum showed that antigenic determinants were located along the entire length of the H pilus. Immuno-gold labelling and lysis studies using pilH alpha, a bacteriophage specific for H pili, were used to investigate transfer-deficient mutants of pHH1508a obtained by Tn5 mutagenesis and an in vitro constructed derivative of 96 kilobases, pDT1178, which also conferred resistance to potassium tellurite, trimethoprim, and streptomycin. The transfer-deficient mutants did not specify H pili, whereas pDT1178, which transferred at low frequency (1 x 10(-4) transconjugants per recipient), specified a small number of H pili. A naturally occurring plasmid, pMG110, was found to encode the production of H pili, but was completely transfer deficient (less than 1 x 10(-7) transconjugants per recipient). This study suggests that genes required for H pilus production and assembly as well as low level transfer are located separately within the 96-kilobase fragment of pDT1178 and that other genes, located outside this region, are essential for the regulation and full expression of conjugative transfer.