A region of the IncHI2 plasmid R478, encoding the phenotypes of tellurite resistance (Te r ), phage inhibition (Phi), and colicin resistance (PacB), was cloned and sequenced. Analysis indicated seven open reading frames (ORFs), whose genes were designated terZ, -A, -B, -C, -D, -E, and -F. Five of these predicted ORFs (A to E) had extensive amino acid homology with the previously reported ORFs of the IncHI2 Te r operon from plasmid pMER610. There were domains of highly conserved amino acid residues within the group TerA, -D, -E, and -F and within TerD, -E, and -Z, but no consensus could be found among all five putative polypeptides. There were also regions of good identity and similarity between individual pairs of ORFs which was not reflected in the multiple alignments. The three phenotypes were expressed in Escherichia coli DH5␣ by an 8.4-kb EcoRI insert subcloned from a cosmid of R478. The latter insert was clonable only as a double insertion with a 4.5-kb fragment, and forced deletion of the smaller fragment was lethal to cells. This lethality was not dependent on the cloned orientation of either fragment, suggesting that there is a trans-acting element in the 4.5-kb fragment. Tn1000 mutagenesis of one of the double-insert clones, pDT2575, showed that the phenotypes, including multiple colicin resistance, were genetically linked. Transpositions into terD, terC, and terZ reduced or abolished all phenotypes, while inserts into terE and terF had no effect on the phenotypes. Insertions in terA reduced phage inhibition levels only. The presence of the terZ and terF ORFs in pMER610 was confirmed, and derivatives of this plasmid mediated Phi, PacB, and Te r .Enterobacterial IncHI2 plasmids mediate resistance to a wide range of antibiotics, heavy metals, and pore-forming colicins. Tellurite resistance (Te r

The IncHI2 plasmid R478 specifies resistance to potassium tellurite (Te r ), to some bacteriophages (Phi), and to pore-forming colicins (PacB). The genes encoding the three phenotypes are linked, and an 8.4-kb fragment of R478 DNA encoding them cannot be subcloned unless cocloned with a second section of the plasmid. Subclone pKFW4A contains a 5.9-kb BamHI-EcoRI fragment which caused some toxicity when present in Escherichia coli cells. Bacterial cells containing freshly transformed pKFW4A, examined by light microscopy and electron microscopy, had a filamentous morphology consistent with a block in septation. Insertion of transposon Tn1000 into terZ, -A, -B, and -C genes of pKFW4A resulted in the loss of the filamentation phenotype. Deletion of several regions of the clone confirmed that these latter components are involved in the filamentation phenotype. The region specifying protection from toxicity caused by the larger 8.4-kb fragment (encompassing this cluster and the entire 5.9-kb section of pKFW4A) was sequenced and analyzed by T7 polymerase expression and Tn1000 mutagenesis. Three open reading frames, terW, terY, and terX, were identified in a 2.6-kb region. Two polypeptides with approximate molecular masses of 18 and 28 kDa were expressed in CSRDE3 cells and were consistent with TerW (17.1 kDa; 155 amino acids [aa]) and TerY (26.9 kDa; 248 aa), whereas a protein of 213 aa deduced from terX was not observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The terX gene product shows strong identity with the previously identified TerE, TerD, and TerZ polypeptides, and there is a conserved motif of 13 residues, GDN(R/L)TG(E/A)GDGDDE, within this group of polypeptides. Complementation analysis indicated that terW, located approximately 6.0 kb upstream of terZ, brings about protection of cells from toxic effects of components of the Te r , Phi, and PacB cluster.R478 is a 272-kb self-transmissible plasmid of the incompatibility subgroup IncHI2 which mediates resistance to kanamycin, chloramphenicol, tetracycline, mercuric chloride, potassium tellurite (Te r ), and arsenic compounds, as well as resistance to some bacteriophages (Phi) and to pore-forming colicins (PacB) (18,23,30,32,33,42,43). During physical and genetic mapping of R478 (42), several plasmid subclones which expressed Te r but not PacB were obtained, whereas other subclones encoded Phi but not Te r . The Te r , Phi, and PacB phenotypes were linked by insertion mutagenesis to a 6.2-kb DNA fragment which contained seven genes, terZ, -A, -B, -C, -D, -E, and -F (43). Insertion mutagenesis within terZ, terC, or terD reduced or abolished resistance to phage T5, colicins A, B, and K, and potassium tellurite. The amino acid sequences deduced from terD, terE, and terZ were found to be related. Identity was also observed between the amino acid sequences of terA and terF; in addition, highly related amino acid domains were noted among various subsets of the five latter putative polypeptides (43).Subcloning and internal deletion within the Te r ...

A restriction map of the 272-kb IncHI2 plasmid R478 was constructed by using the enzymes ApaI, XbaI, Sail, and XhoI. The map was derived from cloned restriction fragments from R478 inserted into cosmid and plasmid vectors as well as from double-digestion analysis of R478 and R478 miniplasmids. All previously known resistance determinants were cloned from R478, and their positions were located on the restriction map. A region involved in incompatibility was cloned and mapped. The location of a previously unreported arsenite resistance gene was also determined. The genes encoding tellurite resistance, colicin B resistance, and phage inhibition were found to be associated with a 6.7-kb Sail fragment of R478.The first reported isolation of a plasmid of the H incompatibility group was from the chloramphenicol-resistant Salmonella typhi strain responsible for the 1972 Mexican typhoid epidemic (2). IncH plasmids have since been shown to encode for multiple drug and metal resistances in bacteria of the family Enterobacteriaceae (1,31,36 30°C (25, 36). IncHII plasmids differ from IncHI plasmids in that they are nonthermosensitive for transfer, are not repressed for pilus synthesis, and transfer at high frequencies (6).On the basis of DNA-DNA hybridization studies, the IncHI group of plasmids is further divided into three subgroups, IncHIl, IncHI2, and IncHI3 (42 (Table 1). In this paper, we describe the construction of the first restriction map of R478 and indicate locations for several of the genes on this plasmid. MATERIALS AND METHODSBacterial strains, plasmids, cosmids, and media. All strains, plasmids, and cosmids used in this study are listed in Table 1. E. coli JM109 was used for plasmid cloning by using the vector pUC13. Transformants were isolated on Luria-Bertani (LB) medium containing 20 ,ug of 5-bromo-4-chloro-3-indolyl-o-D-galactopyranoside per ml, 20 jig of isopropyl-P-D-thiogalactopyranoside (Sigma Chemical Co. Ltd.) per ml, and 100 jig of ampicillin per ml (38). E. coli DH1 was used for all other experiments, unless otherwise stated, and all plasmid clones and deletion plasmids were ultimately transformed into this strain. The plasmid R478 was maintained in E. coli J53-2. Antibacterial agents were used at the following concentrations: ampicillin, 100 jig/ml; kanamycin, 100 ,ug! ml; chloramphenicol, 30 ,ug/ml; nalidixic acid, 30 ,ug/ml; tetracycline, 20 ,ug/ml; mercuric chloride, 80 jig/ml; potassium tellurite, 30 ,ug/ml; and rifampin, 50 ,ugIml. All antibacterial plates were made from tryptone soya agar or brain heart infusion agar (Oxoid Ltd.), unless otherwise indicated.Resistance to arsenate was detected by using 3.5% (wt/ vol) sodium arsenate in LB agar plates. Resistance to arsenite was examined by using 0.5% sodium arsenite (12) in brain heart infusion agar plates. Resistance to both arsenic compounds was tested by examining colony formation on

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.

The pore-forming colicins N and A require the porin, OmpF, in order to translocate across the outer membrane of Escherichia coli. We investigated the hypothesis that in vivo, colicins N and A may traverse the outer membrane through the OmpF channel. In order to accommodate a polypeptide in the pore, the mid-channel constriction loop of OmpF, L3, would need to undergo a conformational change. We used five OmpF cystine mutants, which fix L3 in the conformation determined by X-ray crystallography, to investigate L3 movement during colicin activity in vivo. Sensitivity to colicins N and A of E. coli cells expressing these OmpF cystine mutants was determined using cell survival and in vivo potassium efflux and fluorescence assays. Results indicate that gross movement of L3 is not required for colicin N or A activity and that neither of these colicins crosses the outer membrane of E. coli through the lumen of the OmpF pore.z 1998 Federation of European Biochemical Societies.