HI plasmids are distinguished by their thermosensitive mode of conjugation (transfer efficiency is optimal at 22-30 degrees C) and their capacity to encode multiple antibiotic resistance. These traits have implicated HI plasmids as potential vectors in the dissemination of antibiotic resistance among pathogenic and indigenous bacterial species in water and soil environments. We compared the transfer efficiency of HI plasmids with that of plasmids from 13 other incompatibility groups at 37, 24, and 14 degrees C in intragenic conjugations between laboratory strains of Escherichia coli K-12 under in vitro conditions. Only the HI plasmids and a representative plasmid from incompatibility groups M, N, P alpha, T, and W were observed to be transmissible at 14 degrees C. These plasmids, along with HI plasmids and the related HII representative, were tested for their host range and transfer proficiency to Enterobacteria species and some other Gram-negative organisms of environmental significance at 24 and 14 degrees C. Notable differences in the host range of HI plasmids compared with plasmid representatives from the other enterobacterial groups were not evident at 24 degrees C. At 14 degrees C, R478 (HI2) displayed the broadest host range and transfer proficiency among the test plasmids. The ability of several plasmid groups, including HI, to transfer at 14-24 degrees C to Vibrio cholerae non 01, Salmonella typhi, and the fish pathogens Aeromonas salmonicida, Vibrio anguillarum, and Yersinia ruckeri needs to be corroborated by in situ studies.

The kinetics of pilus outgrowth were examined for Escherichia coli containing pDT1942, a TnkacZ insertion derivative of the IncHIl plasmid R27, which was derepressed for transfer. IncHI1 plasmids are thermosensitive for transfer. The pili specified by pDT1942 were examined by transmission electron microscopy after the pilus had been labeled with the H-pilus-specific bacteriophage Hgal, which had been inactivated with RNase A. H pili were extended by extrusion from the cell surface and not by the addition of pilin subunits to the pilus tip. After pili were removed by vortexing, the outgrowth of full-length pili (2 ;Lm long) required 20 min. H pili expressed at the transfer optimal temperature (270C) remained stable after incubation at the transfer inhibitory temperature (370C), but the formation of mating aggregates was inhibited at 370C. Within 1 min of exposure of the host cell to a heat stimulus of 50'C, pili vanished. Pili were observed in straight and flexible forms with a field emission scanning electron microscope, which may indicate a dynamic role for the pilus in conjugation.H pili are conjugation appendages specified by incompatibility group HI and HII plasmids of the family Enterobacteriaceae (for reviews of H plasmids, see references 15 and 27). With the exception of the rigid pilus specified by the HI3 plasmid MIP233 (7), all plasmids belonging to the HIl, HI2, and HII groups specify thick, flexible pili (3). Thick, flexible pili are also specified by plasmids of the incompatibility groups FI, FII, FV, C, D, J, T, V, and X (3, 5, 6). Members of these plasmid incompatibility groups transfer to recipient cells with equal proficiency by both liquid and surface mating (9). HI group plasmids differ from those in the other groups in being thermosensitive for conjugation, i.e., transferring optimally below 30'C and negligibly (<10-8) at 370C (30). The basis for thermosensitive conjugation is not understood. H-pilus biochemistry is also unknown, and the basic pilin subunit(s) has not been characterized.Plasmid pDT1942, also known as R27::TnlacZ (16) Two single-stranded RNA bacteriophage, pilHa and Hgal (10, 16, 21), adsorb uniformly along the H-pilus shaft. The goal of this study was to exploit phage adsorption, constitutive pilus production, and the thermosensitive conjugation system of R27::TnlacZ to examine several aspects of H-pilus assembly, stability, and morphology by transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM). * Corresponding author. MATERIALS AND METHODSBacterial strains, plasmid, and bacteriophage. The donor strain, Escherichia coli DT1944, is a rifampin-resistant mutant of JE2571 (leu thr str fla pit) (4) harboring plasmid pDT1942 (R27::TnlacZ) (16). This derivative of the tetracycline-resistant HIU plasmid R27 (28) mediates constitutive pilus production and kanamycin resistance. The recipient strain was E. coli K-12 strain J53-1 (pro met F-Nal) (2). Bacteriophage Hgal (21) is an H-pilus-specific RNA phage, 18.7 nm in size (16), which adsorbs to ...

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.

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