Transfer systems of K88 and K99 plasmids

The conjugation systems of three K88-mobilizing plasmids were characterized for the morphology of their pili and type of mating system (surface only or surface + liquid). pREI had a typical IncI1 transfer system with both thick and thin pili. pVIDO determined aggregating thick flexible pili and pPLS nonaggregating thick flexible pili. All three transferred equally well in broth and on plates. pPLS alone was naturally transfer-depressed. pREI and pVIDO were tested for K88 mobilization efficiency, which was greater from their wild-type host strains to Escherichia coli K-12 than between E. coli K-12 strains. The K99 conjugative plasmid from strain B41 was repressed for transfer and determined thick flexible pili that were receptors for the filamentous phage fd.     

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.     

Significance of filter mating in integrative incompatibility test for plasmid classification

For classification of plasmids in epidemiological studies, an integrative incompatibility test using liquid mating was developed by Sasakawa et al (Plasmid 3: 116-127, 1980). This test was designed to compare the relative mating frequency of a donor carrying a test plasmid with that of recA recipients carrying various integrated plasmids. To improve the accuracy of this method by increasing transfer frequency of a test plasmid, filter mating was introduced. A transfer frequency 10 to 30,000 times higher than that achieved by liquid mating was attained by filter mating. The degree of increase varied among the incompatibility groups and the majority of members belonging to the same incompatibility group exhibited a similar degree of increase. Standard plasmids were classified correctly with the integrative incompatibility test using filter mating. Out of 26 naturally occurring plasmids of poor transferability in liquid mating, all of domestic animal origin, 25 were correctly classified as IncH with the integrative incompatibility test using filter mating. Moreover, the method is capable of subdividing IncH plasmids directly into IncH1 and IncH2 , because IncH2 , but not IncH1 , plasmids showed incompatibility with the integrated plasmid, R478 , of the IncH2 group.     

pHH502, a plasmid with IncP and IncI alpha characters, loses the latter by a specific recA-independent deletion event

Plasmid pHH502, of molecular weight 70 X 10(6), determined resistance to tetracycline, chloramphenicol, trimethoprim, sulphonamides and mercuric chloride and was incompatible with members of IncP and IncI alpha. It resembled other plasmids of IncI alpha in the following properties: it determined pili that were morphologically and serologically I alpha pili, whose production was repressed in established plasmid-carrying (R+) cultures; its transfer was equally efficient in liquid or on solid medium; it exerted surface exclusion against other IncI alpha plasmids; it was non-transferable to Proteus. In a reproducible, recA-independent event, pHH502 gave rise to pHH502-1, a plasmid of molecular weight 40 X 10(6), lacking determinants for resistance to tetracycline and chloramphenicol and all detectable IncI alpha characteristics. pHH502-1 was incompatible only with IncP plasmids and resembled other IncP plasmids in determining constitutive production of rigid pili, in its surface exclusion, in transferring at greater frequency on solid than in liquid medium and in being transmissible to Proteus mirabilis. It differed from other IncP plasmids in the morphology and serological type of its pili and in failing to transfer to Pseudomonas aeruginosa or Acinetobacter calcoaceticus. Small numbers of pHH502-1 rigid pili were present on bacteria carrying pHH502. Possible mechanisms for the generation of pHH502 and pHH502-1 are discussed.     

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.     

Capacity of aquatic bacteria to act as recipients of plasmid DNA.

A total of 68 gram-negative freshwater bacterial isolates were screened for their ability to receive and express plasmids from Pseudomonas aeruginosa donors. The plate mating technique identified 26 of the isolates as recipient active for the self-transmissible wide-host-range plasmid R68; 10 were recipient active by R68 mobilization for the wide-host-range plasmid cloning vector R1162. Frequencies of transfer were compared by using three conjugal transfer procedures: broth, plate, and filter mating. For every recipient tested, a solid environment was superior to a liquid environment for transfer. The broth mating technique failed to demonstrate R68 transfer in 63% of the recipient-active isolates. Filter mating, in general, yielded the highest transfer frequencies. The more-rapid plate mating procedure, however, was just as sensitive for testing the capacity of natural isolates to participate in conjugal plasmid transfer.     

Capacity of aquatic bacteria to act as recipients of plasmid DNA

A total of 68 gram-negative freshwater bacterial isolates were screened for their ability to receive and express plasmids from Pseudomonas aeruginosa donors. The plate mating technique identified 26 of the isolates as recipient active for the self-transmissible wide-host-range plasmid R68; 10 were recipient active by R68 mobilization for the wide-host-range plasmid cloning vector R1162. Frequencies of transfer were compared by using three conjugal transfer procedures: broth, plate, and filter mating. For every recipient tested, a solid environment was superior to a liquid environment for transfer. The broth mating technique failed to demonstrate R68 transfer in 63% of the recipient-active isolates. Filter mating, in general, yielded the highest transfer frequencies. The more-rapid plate mating procedure, however, was just as sensitive for testing the capacity of natural isolates to participate in conjugal plasmid transfer.     

Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities

The transferability of plasmids pCAR1, pB10, R388, and NAH7 was compared using the same donor-recipient system at different cell density combinations in liquid or on a solid surface. pCAR1 was efficiently transferred in liquid, whereas the other plasmids were preferentially transferred on a solid surface. Difference of liquid or solid affected the transfer frequency especially at lower cell densities.     

H-pilus assembly kinetics determined by electron microscopy.

The kinetics of pilus outgrowth were examined for Escherichia coli containing pDT1942, a TnlacZ insertion derivative of the IncHI1 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 microns long) required 20 min. H pili expressed at the transfer optimal temperature (27 degrees C) remained stable after incubation at the transfer inhibitory temperature (37 degrees C), but the formation of mating aggregates was inhibited at 37 degrees C. Within 1 min of exposure of the host cell to a heat stimulus of 50 degrees 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.     

Changes in the nature of the sensitivity of an E. coli M strain carrying Inc-group R plasmids, to coliphages T3 and T7

After the transfer of prototype plasmids R6K (IncX), R387 (IncK), R27 (IncH1) and T (IncN) to E. coli M nalr the appearance of histidine-dependent mutants (R27, T), histidine-leucine-dependent mutants (R6K), methionine-proline-dependent mutants (R387) was observed among the resulting transconjugates. The mutations of E. coli M nalr R+ cells induced by the introduction of the plasmids were accompanied by the transformation of the cells from the S-form into the R-form. In contrast to the prototrophs E. coli M nalr, the auxotrophs carrying plasmids R6K, R27, T acquired sensitivity to phage T7, and the methionine-proline-dependent mutant became sensitive to phages T and T7. The above-mentioned plasmids rendered E. coli M cells capable of synthetizing the donor pili. But the adsorption of phages T3 and T7 on the auxotrophic cells, both with and without plasmids, occurred due to their interaction with the cell-wall receptors.     

Horizontal transfer of CS1 pilin genes of enterotoxigenic Escherichia coli

CS1 is one of a limited number of serologically distinct pili found in enterotoxigenic Escherichia coli (ETEC) strains associated with disease in people. The genes for the CS1 pilus are on a large plasmid, pCoo. We show that pCoo is not self-transmissible, although our sequence determination for part of pCoo shows regions almost identical to those in the conjugative drug resistance plasmid R64. When we introduced R64 into a strain containing pCoo, we found that pCoo was transferred to a recipient strain in mating. Most of the transconjugant pCoo plasmids result from recombination with R64, leading to acquisition of functional copies of all of the R64 transfer genes. Temporary coresidence of the drug resistance plasmid R64 with pCoo leads to a permanent change in pCoo so that it is now self-transmissible. We conclude that when R64-like plasmids are transmitted to an ETEC strain containing pCoo, their recombination may allow for spread of the pCoo plasmid to other enteric bacteria.     

Serological characteristics of pili determined by the plasmids R711b and F0lac.

The plasmids R711b (at present IncX) and F0lac (IncFV) both determine pili morphologically like those of F (IncFI), and confer sensitivity to the F-specific filamentous bacteriophages, but not to the F-specific isometric RNA phages. Detailed serological studies show that the two pilus types are unrelated, and that neither is related to any of the previously defined F pilus serotypes. Adsorption of the isometric RNA phage MS2 to R711b pili occurs in the presence but not in the absence of formalin, which presumably prevents elution of reversibly adsorbed virions. No adsorption occurs with F0lac pili. MS2 multiplication, as measured by titre increase tests in liquid medium, is found with neither plasmid. The two plasmids are not incompatible. These observations indicate that R744b and F0lac are different both from one another and from the plasmids belonging to the incompatibility groups IncFI--IV.     

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

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.     

Monitoring the spread of broad host and narrow host range plasmids in soil microcosms

Abstract: Escherichia coli recipient and E. coli donor strains carrying streptothricin-resistance genes were inoculated together into different soil microcosms. These genes were localized on the narrow host range plasmids of incompatibility (Inc) groups FII, Il, and on the broad host range plasmids of IncP1, IncN, IncW3, and IncQ. The experiments were intended to study the transfer of these plasmids in sterile and non-sterile soil with and without antibiotic selective pressure and in planted soil microcosms. Transfer of all broad host range plasmids from the introduced E. coli donor into the recipient was observed in all microcosm experiments. These results indicate that broad host range plasmids encoding short and rigid pili might spread in soil environments by conjugative transfer. In contrast, transfer of the narrow host range plasmids of IncFII and IncI1, into E. coli recipients was not found in sterile or non-sterile soil. These plasmids encoded flexible pili or flexible and rigid pili, respectively. In all experiments highest numbers of transconjugants were detected for the IncP1-plasmid (pTH16). There was evidence with plasmids belonging to IncP group transferred by conjugation into a variety of indigenous soil bacteria at detectable frequencies. Significantly higher numbers of indigenous transconjugants were obtained for the IncP-plasmid under antibiotic selection pressure, and a greater diversity of transconjugants was detected. Availability of nutrients and rhizosphere exudates stimulated transfer in soil. Furthermore, transfer of the IncN-plasmid (pIE1037) into indigenous bacteria of the rhizosphere community could be detected. The transconjugants were determined by BIOLOG as Serratia liquefaciens . Despite the known broad host range of IncW3 and IncQ-plasmids, transfer into indigenous soil bacteria could not be detected.     

Purification and Characterization of Thin Pili of IncI1 Plasmids ColIb-P9 and R64: Formation of PilV-Specific Cell Aggregates by Type IV Pili

Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which Escherichia coli cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the pilS gene. Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa protein by the function of the pilU product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of the pilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.     

Determination of pili by conjugative bacterial drug resistance plasmids of incompatibility groups B, C, H, J, K, M, V, and X.

Representative plasmids from incompability groups B, C, H, J, K, M, V, and X were transferred to "bald" strains of Escherichia coli or Salmonella typhimurium. By using a new technique, pili were detected by electron microscopy for each incompatibility group. Morphology varied but was similar for plasmids within a group. These findings suggest that all conjugative plasmids in the Enterobacteriaceae may determine pili.     

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.     

Conjugation systems of IncT plasmids

Four IncT plasmids were compared for various characters, in particular pilus synthesis and function at different temperatures. The prototype Rts1 differed in some respects from the others (R402, R394, pIN25). At 37 degrees 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 degrees C, but only short ones (approx. 200 nm) were produced at 37 degrees C. The long pili converted two surface-obligatory conjugation systems to surface + liquid ones at 30 degrees C.