Bacteriophages F0lac h, SR, SF: phages which adsorb to pili encoded by plasmids of the S-complex

Phage F0lac is an RNA-containing phage which plates only on strains carrying the plasmid EDP208, a pilus derepressed derivative of the unique incompatibility plasmid F0lac. A host range mutant, phage F0lac h, was selected which plated on strains carrying the ungrouped plasmid pPLS::Tn5 and lysed strains carrying another ungrouped plasmid TP224::Tn10 or the Com9 plasmid R71. An RNA-containing phage, SR, was isolated from sewage on bacteria harbouring plasmid pPLS::Tn5. It was antigenically distinct from the above two phages but had the same host range as phage F0lac h. Phages F0lac h and SR adsorbed unevenly to the shafts of the conjugative pili. Another phage, SF, was filamentous and plated or propagated on strains carrying any of the above plasmids as well as on strains harbouring IncD or F-complex plasmids. Plasmids TP224::Tn10 and pPLS::Tn5 were compatible with representative plasmids of all Inc groups also encoding thick flexible pili. The four plasmids EDP208, R71, TP224::Tn10 and pPLS::Tn5 were compatible with one another except for the reaction of TP224::Tn10 in the presence of pPLS::Tn5 which was slightly ambiguous. The host ranges of the bacteriophages, together with the serological relatedness of the thick flexible pili determined by these four compatible plasmids, suggested that they constitute a new complex, here designated S.     

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.     

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.     

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.     

Specification of surface mating systems among conjugative drug resistance plasmids in Escherichia coli K-12

Representative plasmids for most incompatibility groups in Escherichia coli K-12 were transferred to a "bald" strain to compare transfer frequencies for liquid and solid media. Standard broth matings were used for a liquid environment, but for solid surface mating, conjugation was allowed to take place on nutrient plates before washing off the cells for transconjugant selection on plates containing appropriate drugs. Plasmids that determine rigid pili transferred at least 2,000x better on plates than in broth. Some plasmids that determine thick flexible pili transferred 45 to 470x better, whereas others transferred equally well in both environments, as did plasmids of the I complex, which determine thin flexible pili. These results clearly distinguished a number of surface mating systems where most plasmids were derepressed for transfer and determined conjugative pili constitutively. The temperature-independent IncH2 plasmid R831b transferred best on plates, but other IncH plasmids transferred equally well in broth. This inconsistency led to the reclassification of R831b as IncM.     

Incompatibility of the Cad plasmid from Yersinia pestis with plasmids of the IncFI group

The relation of Yersinia pestis calcium dependence plasmid (pCad) to known Inc FI (F'lac, R386, pOX38) and IncFV (F0lac) plasmids has been studied. Evidence that plasmid pCad of Yersinia pestis belongs to FI incompatibility group is presented.     

Characterization of pili determined by drug resistance plasmids R711b and R778b.

The bacterial drug resistance plasmids R711b and R778b, at present classified in the X incompatibility group, determine pili (designated 711) that resemble F pili morphologically. Like F pili, 711 pili adsorb F-specific filamentous bacteriophages to their tips, though more often in pairs, than singly. However, F-specific RNA-containing bacteriophages are not adsorbed to their sides, and strains carrying the plasmids are resistant to these phages. Pili determined by the only IncFV plasmid Folac are similar to 711 pili in their phage adsorption properties, but they are serologically different, as are F pili. It is concluded that F, Folac and 711 pili have basic differences in spite of a morphological resemblance.     

Phage X: a plasmid-dependent, broad host range, filamentous bacterial virus

Phage X was isolated from sewage as plating on Escherichia coli or Salmonella typhimurium strains harbouring the incompatibility group X plasmid R6K. It also plated on a strain of Serratia marcescens carrying this plasmid. It failed to form plaques on Proteus mirabilis, P. morganii or Providencia alcalifaciens harbouring R6K, but did multiply on them. No phage increase occurred with homologous R- strains. Phage X also plated or registered an increase in titre on E. coli or S. typhimurium strains carrying various plasmids of incompatibility groups M, N, P-1, U or W as well as the unassigned plasmid R775. It adsorbed to pili determined by a group P-10 plasmid in a Pseudomonas aeruginosa strain but did not multiply on this organism. The phage was filamentous and curly, resistant to ribonuclease and diethyl ether and sensitive to chloroform. It adsorbed to the tips of pili.     

Comparative Biochemical Studies on F and EDP208 Conjugative Pili

EDP208 pili are encoded by a derepressed derivative of a naturally occurring lac plasmid, F(0)lac (incompatibility group FV), originally isolated from Salmonella typhi. EDP208 pili are serologically unrelated to F pili and do not promote infection by F-specific ribonucleic acid bacteriophages. However, they do confer sensitivity to the F-specific filamentous deoxyribonucleic acid phages. EDP208-containing cells are multi-piliated and contain approximately 20 pili per cell. These pili contain a single polypeptide subunit of 11,500 daltons. EDP208-specific RNA phages were readily isolated from local sewage. These phages were somewhat smaller in diameter than the F-specific ribonucleic acid phages and absorbed relatively weakly to EDP208 pili. Comparing EDP208 pilin to F, it was found that both contain the equivalent of two to three hexose units per subunit as well as blocked N-termini. EDP208 pilin contains one covalently linked phosphate residue per subunit, whereas the F pilin subunit contains two such residues. Although notable differences were found in the case of three or four amino acids, the overall amino acid compositions of F and EDP208 were very similar. Moreover, the tryptic peptide maps of the two proteins contained seven peptides with similar mobilities, suggesting considerable homology in their amino acid sequences. Substantial similarities were also noted in the secondary structures of F and EDP208 pilin on the basis of circular dichroism studies. The alpha-helix content of both proteins was calculated to be 65 to 70%. X-ray fiber diffraction studies have indicated that the arrangements of subunits in F and EDP208 pili are also similar. It was concluded that F and EDP208 pili are closely related structures.     

Plasmid-Associated Functions of a Stable Flac

Several functions associated with the stable plasmid, FlacS, have been examined. Our results indicate that the sex pili synthesized by Escherichia coli strains carrying FlacS are altered in some manner as evidenced by a very inefficient adsorption of male-specific phages. On the other hand, FlacS-mediated entry exclusion of related plasmids and plasmid incompatibility function(s) appear normal. The presence of covalently closed circular deoxyribonucleic acid in E. coli strains harboring FlacS indicates that it is an autonomously replicating plasmid. Based on beta-galactosidase levels and the percentage of covalently closed circular deoxyribonucleic acid, it appears that the stability of the FlacS is not the result of multiple copies of this plasmid. FlacS appears larger than its precursor, F(ts114)lac, in sedimentation through alkaline sucrose gradients.     

Distribution of plasmid maintenance regions among IncFIV plasmids

The distribution of the IncFI basic replicons among IncFIV plasmids was assessed by DNA hybridization. In addition these and 20 other plasmids from 16 incompatibility groups were screened for the presence of IncIV, an incompatibility determinant recently found on the IncFIV plasmid R124. The IncIV determinant was found commonly but not universally among the IncFIV plasmids. It was also detected on the IncFI reference plasmid R386 and plasmids from IncB, IncI alpha and IncI gamma. The frequency and distribution of IncFI replicons among the IncFIV plasmids is similar to that observed in other F groups. The similarity of the IncFIV plasmids to plasmids of the other IncF groups and the failure to find replicons unique to IncFIV plasmids indicates that their division into a separate incompatibility group is not justified.     

Use of a known plasmid and transposon as tracers for the incompatibility classification of a cryptic plasmid

Summary We have developed a novel genetic technique by which an unknown plasmid can be classified by the use of a plasmid of known incompatibility group. In phenocopy state, cells harboring plasmids of known incompatibility group behave as normal recipients and receive plasmids belonging to the same incompatibility group at a high frequency. This work provides additional evidence that plasmids in phenocopy hosts do not replicate and therefore fail to demonstrate their incompatibility barrier to the incoming plasmids. A cryptic plasmid, now called pWS7, has been identified by this method in a pili, fla female strain of E. coli K12. Genetic analysis shows the plasmid pWS7 is in fact, a sex-factor which is curable with acridine orange. It belongs to the Inc F1 group. Physical analysis confirms its size to be 124 Kb. The plasmid has been labelled genetically with a transposon Tn903 in a recA host and further characterized by heteroduplex analysis. A DNA sequence homology between pWS7 and F'lac plasmid extends only in F-regions, 2.8F-94.5F. The pili, fla host strain of pWS7 shows a high frequency of transformation for recombinant DNA and rapid propagation for a male-specific RNA phage, R17.     

Molecular cloning and functional analysis of DNA regions of plasmid RP4 determining the incompatibility properties

Sau3A-generated DNA fragments determining incompatibility functions of the plasmid RP4 were cloned on the vectors pTK16 and pBR322. Inc+ recombinant plasmids were divided into two types: 1) expressing incompatibility only towards the homologous RP4 replicon, 2) expressing incompatibility - both towards the homologous RP4 replicon and towards the heterologous replicons of plasmids R906 and R751. For one member of the first type plasmids it was shown that the cloned Inc+-specific insertion derived from the region of location of the EcoRI restriction site. The majority of the Inc+ recombinant plasmids showed asymmetric expression of incompatibility, predominantly eliminating the resident IncP plasmid.     

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.     

Properties of R plasmid R772 and the corresponding pilus-specific phage PR772.

R plasmid R772 was isolated from a strain of Proteus mirabilis and is a self-transmissible P-1 incompatibility group plasmid having a molecular weight of about 27 x 10(6). It renders bacterial hosts resistant to kanamycin. Phage PR772 was isolated as a phage dependent on the presence of R772 in bacterial hosts. It is hexagonal-shaped with a diameter of 53 nm, has a thick inner membrane and no tail. Vaguely defined appendages are sometimes apparent at some vertices and the phage possesses double-stranded DNA. The DNA has a guanine plus cytosine molar content of 48%. The phage is sensitive to chloroform and has a buoyant density of 1.26 g cm(-3). These observations suggested that the inner membrane of the phage could contain lipid. Phage PR772 differs in morphology from the double-stranded DNA plasmid-specific phages PR4 and PRR1 which adsorb to tips and sides, respectively, of sex pili coded for by P-1 incompatibility group plasmids. Phage PR772 formed clear plaques which varied in diameter. Serologically, phages PR772 and PR4 are possibly related though very distantly, but the two phages have identical host ranges. Phage PR772 adsorbed by one of its apices to tips of sex pili coded for by plasmid R772 in Escherichia coli. It also formed plaques on Salmonella typhimurium Proteus morganii and Providence strains harbouring this plasmid as well as strains of E. coli carrying plasmids of incompatibility groups N or W. The phage produced areas of partial clearing on lawns of P. mirabilis PM5006 harbouring plasmid R772, the P-1 incompatibility group plasmid RP4, the W group plasmid RSa or the N group plasmid N3, and on lawns of Providence strain P29 carrying plasmid RP4.     

Bacteriophage D: an IncD group plasmid-specific phage

The existence of the plasmid incompatibility group D was reaffirmed as a result of compatibility experiments done on plasmids R687, R711b, R778b and R840 which were previously tentatively accepted as constituting the group. The group was further delineated by the isolation of a phage, phage D, which adsorbed specifically to IncD plasmid-encoded pili produced by Escherichia coli K12 strains and strains of Salmonella typhimurium, Proteus morganii and Klebsiella oxytoca harbouring one of these plasmids. Plaque formation, like that of phage pilH alpha, was temperature sensitive in that plaques formed at 26 degrees C but not at 37 degrees C. Plaques were fairly clear, regular in outline and varied from pinpoint to about 1.5 mm in diameter on E. coli hosts where plaques were detected, but on the other hosts the plaques were more turbid and often irregular in outline. The phage did not plate (or propagate) on IncD plasmid-carrying strains of Providencia alcalifaciens, Providencia stuartii or Serratia marcescens. The phage had an isometric hexagonal outline with a diameter of about 27 nm. It contained RNA and resembled two other RNA-containing phages, M and pilH alpha, by being sensitive to chloroform. It adsorbed to the sides of the very distal ends of the shafts of IncD plasmid-coded pili.     

Genetic and molecular characterization of an epidemic plasmid coding for multidrug resistance in Salmonella typhimurium of human origin

All 201 multidrug resistant Salmonella typhimurium strains isolated from epidemics in India contained nonconjugative (157 strains) or conjugative (44 strains) Inc F1me multiresistance plasmids. Two small R-plasmids of 7 MDa which coded for resistance to either ampicillin or streptomycin and sulfamethoxazole were also detected along with other plasmids. The small plasmids were members of group 1 and group 2 incompatibility groups. Restriction endonuclease analysis of conjugative (96 MDa) and nonconjugative (88 MDa) Inc F1me plasmids showed considerable similarity except for the presence of unique fragments among both the groups and the loss of fragments corresponding to the smaller size of the nonconjugative plasmid. A single Inc F1me plasmid appears responsible for various outbreaks of multiresistant S. typhimurium in different parts of India.     

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.     

The molecular relatedness among alpha-hemolytic plasmids from various incompatibility groups

Deoxyribonucleic acid (DNA) reassociation studies among α-hemolytic (Hly) plasmids from FVI and FIII–IV incompatibility groups showed a close similarity between the nucleotide sequences of plasmids from the same group. With respect to R plasmids from the F overgroup, they have 20–26 Mdal in common, an amount of DNA close to the amount involved in the traF operon. No more extensive sequence homology was found between pSU316 (IncFIII–IV) and the incompatible plasmids ColB-K98 (IncFIII) or R124 (IncFIV). The IncIα I2 plasmid pSU5 has only the α-hemolytic region (5 Mdal) in common with plasmid pSU316 but it is much more closely related to IncFVI plasmids where the DNA in common amounts to 22 Mdal. Finally, the genetically unrelated plasmid pSU233 shares 66% of its nucleotide sequences (40 Mdal) with the IncFVI plasmids and has 16–23 Mdal in common with various F-like plasmids.     

Distribution of basic replicons having homology with RepFIA, RepFIB, and RepFIC among IncF group plasmids

Plasmids encoding F-like pili have been divided into groups on the basis of their incompatibility behavior. Three basic replicons have been recognized previously in the IncFI plasmid group and we have now examined their distribution in representative plasmids from 22 of the currently recognized incompatibility groups. The occurrence of these basic replicons was found to be rare outside of the IncF group, and significant hybridization was shown only for RepFIA to IncH1 and I group plasmids. Homology to the RepFIC basic replicon was found in all but one of the IncF group plasmids examined but RepFIA and RepFIB have a more restricted distribution. It appears likely that some plasmids carry vestiges of replicons which still express incompatibility but are incapable of replication. We suggest that evolutionary divergence among the plasmids of the IncF group has resulted from various genetic rearrangements among these basic replicons.