A restriction map of the tellurite-resistance (Te') transposon Tn52I (parent plasmid RP4Ter) was prepared. Five sites from RP4Ter, including the EcoRI origin, were found in pIN25 : : Tn521. Tn521 was inserted into a transferable 27-5 kb vector (pCU109) to make three different insertion mutants, in which the size of Tn521 was measured accurately at 4.5 kb. Unlike the Ter of IncHI2 plasmids, that of Tn52I in RP4Ter was non-inducible. Ter was expressed in five widely differing bacterial species to which RP4Ter was transferred from Escherichia coli. Electron micrographs of bacteria expressing the Ter of RP4Ter, H complex plasmids, and chromosomal mutants, all revealed similar tellurium metal crystallites when the bacteria were grown in potassium tellurite medium. No other Ter determinants were found amongst 54 plasmids representing most incompatibility groups (excluding the H complex).
2665Four IncT plasmids were compared for various chatacters, in particular pilus synthesis and function at different temperatures. The prototype Rtsl differed in some respects from the others (R402, R394, pIN25). At 37 "C, the supposedly temperature-sensitive conjugation systems of the plasmids could still function efficiently on a surface, but not in a liquid. Long conjugative pili were synthesized at 30 "C, but only short ones (approx. 200 nm) were produced at 37 "C. The long pili converted two surface-obligatory conjugation systems to surface + liquid ones at 30 "C. I N T R O D U C T I O NThe prototype IncT plasmid Rtsl (Terawaki et al. 1967; Terawaki & Rownd, 1972) has temperature-sensitive replication and transfer systems. Other members of this incompatibility group (R394, R401, R402) have been described by Coetzee et al. (1972), with more being added recently (Matthew & Hedges, 1976; Odakura et a/., 1977; Levy et a/., 1985). R394 alone is not temperature-sensitive (Coetzee et ul., 1972).Although it is the prototype IncT plasmid, Rtsl appears to be significantly different from the others. (For a more detailed description of IncT plasmids, see Bradley et al., 1981.) Because of their temperature-sensitivity, the repressed nature of Rtsl, and the availability of a pilusspecific phage (Bradley et ul., 1981), the IncT plasmids were chosen for investigating some aspects of the function of thick flexible pili in conjugation. The temperature-sensitivities of four IncT plasmid transfer systems were compared to find out if they were due to failure of pilus function or synthesis at elevated temperatures. M E T H O D SBncteriu, pIa.st?iids uiid bacteriophages. The host organism for plasmids was Eschurichiu coli strain JE257 1 (feu riir .str,Pu pil), together with its nalidixic acid-and rifampicin-resistant derivatives (suffices -1 and -2 respectively). Srrlmoncllu tl~hit?iurrutti LTZ strain SQ1 139 (purCproA ilr str.flo pip) was also used in one experiment. Plasmids and resistances determined were : R402 (ampicillin, carbenicillin, streptomycin), Rts 1 (kanamycin), R394 (ampicillin. carbenicillin, kanamycin), pI N?S (ampicillin, carbenicillin, kanamycin). All save pIN25 were supplied by Dr N. Datta (Datta, 1977;Jacob et d . , 1977). pIN25 was supplied by D r S. B. Levy (Levy et uf.. 1985). The IncHIl plasmid R27 : :Tn7, a derepressed mutant of R27, was supplied by Dr D. E. Taylor (Taylor, 1983). The T pilus-specific R N A bacteriophage t u a s described by Bradley et ul. (1981).Media. niatings und huctc~riophqq~ rcdiniqut~s. These, including drug concentrations, were as described by Bradley (1983) and Bradley t't uf. (1981). For comparative plate and broth matings, an overnight static broth culture, incubated at the optimum transfer temperature of the test plasmid (RP4 and Sa, 37 ' T i lncT plasmids, 30 "C), was used to inoculate a shake culture. which was grown at 1-2 x 10'' bacteria ml-' at the mating temperature. A sample (2.5 nil) ofthis was mixed with a similar amount of an identical culture of recipient strain ...
~~ ~~~The tole (previously f i i ) mutation in Escherichia coli K 12 inhibits infection by filamentous bacteriophages fl and IKe but not by RNA-containing phage f 2 . This work extends these observations to other plasmid-specific bacteriophages including various filamentous, RNAcontaining, and lipid-containing isolates. Only tip-adsorbing filamentous phages were affected by toIQ and not shaft-adsorbing ones. Electron microscopy showed that RP4-specific filamentous phage Pf3 was one of the latter kind. Several tip-adsorbing filamentous phages inhibited conjugation between to@ strains carrying their specific plasmids, implicating the phage receptors (conjugative pili) as mating organelles. tolQ mutant strains were as proficient as their parents in conjugation mediated by a wide range of plasmids.
An Enterobacter cloacae strain isolated from the faeces of a child with diarrhoea in Indonesia contained a transferable 216 MDa plasmid, pIN32, exhibiting IncHI2 phenotypic characters, including temperature sensitivity of transfer and the expression of H serotype pili at a repressed level. A derivative plasmid (pIN32-1), which had lost the IncHI2 phenotype, and contained only 60 MDa of the original replicon, was obtained after mating at 37 degrees C. It was IncFII, showed regions of homology with plasmid R100, determined IncFII serotype conjugative pili constitutively and was transfer-derepressed. After overnight growth at 37 degrees C in non-selective medium, pIN32 gave rise to another derivative, pIN32-2 (size 184.3 MDa), which retained the IncHI2 phenotype and several other pIN32 characters.
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