David E. Bradley

Morphological and serological relationships of conjugative pili.

It is now known that conjugative pili are determined by representative plasmids for all incompatibility groups in Escherichia coli K-12. They fall into three basic morphological groups, which are described: thin flexible, thick flexible, and rigid filaments or rods. The main thrust of this study, however, has been the use of immune electron microscopy to survey pili of all established incompatibility groups for serological cross-reactions. Morphologically identical thin flexible pili were determined by plasmids of the I complex, as well as IncB and IncK. Immune electron microscopy revealed two unrelated serotypes typified by Ia and I2 pili; K and B pili belonged to the first serotype. Thick flexible pili were determined by plasmids of Inc groups C, D, the F complex, H1, H2, J, T, V, X, com9, the single plasmid F0 lac, and the unclassified plasmid R687. Serological tests showed that C pili were related to J pili, H1 pili to H2 pili, com9 pili to F0 lac pili, and R687 pili to D pili, the remainder being unrelated. Rigid pili were determined by plasmids of Inc groups M, N, P, W, and by the unclassified plasmids R775, RA3, and pAr-32. The only relationship detected was between RA3 and pAr-32 pili. No cross reactions were found between pili of the three different morphological groups.

Characteristics and Function of Thick and Thin Conjugative Pili Determined by Transfer-derepressed Plasmids of Incompatibility Groups I1, I2, I5, B, K and Z

Eleven transfer-derepressed plasmids from incompatibility groups I1, I5, B, K and Z were constructed using the dnaG3 mutant Escherichia coli strain BW86. All were found to determine thin flexible and thick rigid pili constitutively. Immune electron microscopy was used to relate thick and thin pilus serotypes with incompatibility grouping. Mutant plasmids that determined only thick pili constitutively transferred efficiently on an agar surface but not in a liquid, whereas plasmids with both kinds of pili transferred equally well in both environments. A mutant of the IncI2 plasmid R721 determined thin pili constitutively, and thick pili at a repressed level, as indicated by electron microscopy. Experiments with this indicated that thin pili were apparently not involved directly in conjugation but were only used to stabilize mating aggregates.

Morphology of pili determined by the N incompatibility group plasmid N3 and interaction with bacteriophages PR4 and IKe

Abstract The IncN plasmid N3 was transferred to bald strains of Salmonella typhimurium LT2 and Escherichia coli K-12. In both cases, transconjugants were found to carry short pili, which were designated N pili. They were very easily detached from cells and readily broken into short pieces. N pili were found to be straight inflexible rods 9.5 nm thick and sharply pointed at the distal end. The N-specific filamentous bacteriophage IKe and the lipid-containing phage PR4 both adsorbed to the pointed tips when mixed with cell-free suspensions of N pili.

R plasmids of a new incompatibility group determine constitutive production of H pili

Abstract R plasmids of a new incompatibility group, IncHII, determined the constitutive production of H pili, had high molecular weights, and determined tellurite resistance. They were designated IncHII because, during incompatibility tests, they sometimes eliminated or were eliminated by, previously described IncH plasmids, which they resembled in several respects. Nevertheless, stable and separate coexistence, i.e., compatibility, with plasmids of IncH1, IncH2, and IncH3 was demonstrated. The latter subgroups, members of which are all incompatible with one another, were distinguished on the basis of DNA-DNA hybridization experiments ( N. D. F. Grindley, G. O. Humphreys, and E. S. Anderson, 1973 , J. Bacteriol. 115, 387–398; A. F. Roussel, and Y. A. Chabbert, 1978 , J. Gen. Microbiol. 104, 269–276.); it is proposed that they be called IncHI, the subgroups being HI1, HI2, and HI3.

Detection of Tellurite-resistance Determinants in IncP Plasmids

Six IncP plasmids were tested for their ability to generate tellurite-resistant variants by plating bacterial strains harbouring them on medium containing potassium tellurite. Four plasmids, three of subgroup IncP alpha and one not allocated, formed variants that could transfer tellurite-resistance at the same frequency as plasmid-determined drug resistance. This property was not shared by two examples of subgroup IncP beta.

The TOL Plasmid is Naturally Derepressed for Transfer

Pseudomonas putida mt-2, formerly known as Pseudomonas arvilla mt-2, which carries the wild-type TOL plasmid, and P. putida strain AC37 carrying TOL, were completely lysed by the pilus-adsorbing plasmid-specific bacteriophages PR4 and PRD1. Pseudomonas putida strain PpS388, also harbouring the plasmid, was not lysed. In a P. putida mt-2 host, TOL transferred 18-fold better on a surface (2.5 X 10(-1) transconjugants per donor h-1) than in liquid; when P. putida PpS388 was the host, however, a frequency of only 2.3 X 10(-4) transconjugants per donor h-1 was obtained. Thus, TOL was derepressed for transfer in P. putida mt-2 and P. putida AC37, but not in P. putida PpS388. Electron microscopy revealed that TOL determined thick (8.5-10 nm diameter) flexible pili in large numbers, suggesting constitutive expression in its derepressed state.

Specification of the conjugative pili and surface mating systems of Pseudomonas plasmids.

Conjugative pili were identified for representative Pseudomonas plasmids of incompatibility groups P-2, P-3, P-5, P-7, P-8, P-10, P-11, and P-13, pili for groups P-1 and P-9 having already been described in detail. FP5 pili (unclassified) were also found. In most cases pili could be characterized by electron microscopy as rigid or flexible. The majority of Pseudomonas plasmids transferred significantly better on a surface than in a liquid. Examples of all incompatibility groups were tested.

Escherichia coli tolQ mutants are resistant to filamentous bacteriophages that adsorb to the tips, not the shafts, of conjugative pili.

The tolQ (previously fii) mutation in Escherichia coli K12 inhibits infection by filamentous bacteriophages f1 and IKe but not by RNA-containing phage f2. This work extends these observations to other plasmid-specific bacteriophages including various filamentous. RNA-containing, and lipid-containing isolates. Only tip-adsorbing filamentous phages were affected by tolQ 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 tolQ 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.

Organization and regulation of the conjugation genes of IncI1 plasmid ColIb-P9

The IncI1 plasmid ColIb-P9 is among a group of related plasmids that encode the I1 type of conjugation system. The I1 system is known to include two morphologically distinct types of pilus, a DNA primase gene (sog) and an exclusion determinant (exc). Transposon mutagenesis and analysis of cloned fragments of ColIb were used to identify the location of these determinants with respect to an EcoRI restriction map. Also identified were the location of the origin of transfer (oriT) and a gene determining an EDTA-resistant nuclease, which is coordinately regulated with the transfer genes. The results indicate that the ColIb tra genes are separated into at least three Tra regions. The pleiotropic nature of transposon insertion mutations in two of these regions suggests that two positive regulators are required for expression of the transfer genes and evidence is also found for a trans-acting repressor. It is suggested that the I1 conjugation system may have evolved following fusion of two distinct types of conjugative plasmid.

Characteristics of RP4 Tellurite-resistance Transposon Tn521

A restriction map of the tellurite-resistance (Ter) transposon Tn521 (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 Tn521 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).