Phage pilH alpha: a phage which adsorbs to IncHI and IncHII plasmid-coded pili

Phage pilH alpha was specific for bacterial strains, of various genera, harbouring plasmids of the HI and HII incompatibility groups. Plaque formation was temperature sensitive in that plaques formed at 26 degrees C but not at 37 degrees C. Plaques were fairly clear, irregular in outline and varied from pin point to about 2 mm in diameter on all hosts where plaques were detected. The phage had an isometric hexagonal outline with a diameter of 25 nm. It contained RNA but differed from all but one other plasmid-dependent RNA phage by being sensitive to chloroform. It adsorbed along the length of the shafts of IncHI and HII plasmid-coded pili.     

Phages I alpha and I2-2: IncI plasmid-dependent bacteriophages

Phage I alpha was isolated from sewage from Windhoek, South West Africa. It formed relatively clear plaques about 2 mm in diameter, on sensitive strains of Escherichia coli K12 and Salmonella typhimurium LT2. The phage had an hexagonal outline with a diameter of about 24 nm, contained RNA and was resistant to chloroform. Phage I alpha formed plaques or propagated only on organisms carrying I1 plasmids or the I gamma plasmid R621a. The efficiency of plating was higher on E. coli than on S. typhimurium hosts. The phage adsorbed along the length of shafts of I1 pili. Phage I2-2 was isolated from Pretoria sewage. It was a filamentous virus and individual virions varied considerably in length. Phage I2-2 formed turbid plaques which varied from pin point to about 1 mm in diameter on all hosts. It was resistant to RNAase and sensitive to chloroform. Phage I2-2 had a spectrum of activity limited to strains harbouring I2 plasmids but the adsorption site could not be demonstrated. The phage was not related serologically to phages Ifl or PR64FS.     

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.     

Phages C-2 and J: IncC and IncJ plasmid-dependent phages, respectively

Phages C-2 and J were isolated from sewage. Phage C-2 was filamentous and formed plaques on Salmonella typhimurium strains carrying various C plasmids. It also plated on Proteus mirabilis and Serratia marcescens strains carrying particular C plasmids, but failed to form plaques on lines of Escherichia coli K12 strains harbouring most of these plasmids, although in all cases, phage multiplication on the strains was demonstrated. No phage increase occurred in any strain which lacked a C plasmid or contained plasmids of other incompatibility groups. The phage was sensitive to chloroform and, unlike other filamentous bacterial viruses, adsorbed to shafts of conjugative pili. It had a disc-like structure at the end which attached to the pilus. Phage C-2 had a buoyant density of 1 . 30 g cm-3 and a single-stranded circular DNA genome of 3 . 0 MDal. Phage J had an hexagonal head with an inter-apical distance of 40 nm and a short noncontractile tail. It was resistant to chloroform and diethyl ether. The phage formed plaques or propagated on E. coli strains harbouring some IncC plasmids and all IncJ and IncD plasmids tested. The phage did not form plaques but propagated on P. mirabilis and Ser. marcescens strains carrying these plasmids. It did not plate or propagate on S. typhimurium strains harbouring the plasmids. The plaques were very hazy and variable in size. The phage attached sparsely, at a site which appeared to be located at the base of the tail, to sides of conjugative pili.     

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.     

Phage t: a group T plasmid-dependent bacteriophage

Phage t was isolated from sewage from Pretoria. It formed plaques only on Escherichia coli and Salmonella typhimurium strains that carried plasmids belonging to incompatibility group T. Five of six group T plasmids permitted visible lysis of R+ host strains. There was no visible lysis of E. coli J53-2 or S. typhimurium LT2trpA8 carrying the T plasmid Rts1 although the strains supported phage growth as indicated by at least a 10-fold increase in phage titre. The latter strains transferred the plasmid at high frequency to E. coli strain CSH2 and the resulting transconjugants plated the phage. Proteus mirabilis strain PM5006(R402) failed to support phage growth although it transferred the plasmid and concomitant phage sensitivity to E. coli J53-2. The phage was hexagonal in outline, RNA-containing, resistant to chloroform and adsorbed to the shafts of pili determined by T plasmids.     

Phage Folac: an Folac plasmid-dependent bacteriophage

By enriching sewage with Escherichia coli or Salmonella typhimurium strains harbouring the plasmid EDP208, a constitutive pilus-producing derivative of plasmid F olac, a phage was isolated which plated on these two organisms but not on isogenic strains without the plasmid. The phage was named F olac; it has a hexagonal outline with a diameter of 28 nm, contained RNA, was resistant to chloroform, and probably adsorbed preferentially to the sides of EDP208 pili very near the tip. Phage multiplication could be demonstrated on E. coli or S. typhimurium strains carrying the plasmid F olac, but an increase in titre did not occur on E. coli strains carrying plasmids of the F complex. Results of phage multiplication experiments on strains carrying the depressed pilus-producing plasmids R71 or TP224-Tc, which determine pili serologically related to those of EDP208, were inconclusive. Phage F olac was found to be serologically related to phage UA-6, another isolate specific for EDP208.     

Phage tf-1: a filamentous bacteriophage specific for bacteria harbouring the IncT plasmid pIN25

Phage tf-1 is a filamentous phage which is about 800 nm in length, 10 nm in width and has slightly tapered ends. The phage was isolated from sewage and formed plaques or propagated only on Escherichia coli, Salmonella typhimurium and Klebsiella oxytoca strains harbouring the IncT plasmid pIN25 at 30 degrees C. It adsorbed in large numbers to pIN25-encoded long thick flexible conjugative pili formed at 30 degrees C and also to the short form of these pili synthesized at 37 degrees C. The reason for the failure to form plaques at 37 degrees C is not known. The adsorption site is a short length of the pilus shaft extending 100-200 nm back from the distal tip. Efficient phage tf-1 adsorption to the same site was found for pili determined by other IncT plasmids in spite of the fact that phage tf-1 did not plate or propagate on strains harbouring them. However, areas of specific partial clearing on lawns of these plasmid-containing bacteria were produced by phage in high concentrations. Lack of plaque-formation could be due to inefficient intracellular assembly coupled to avid adsorption of any liberated phage to pili. The phage differs from all but one other filamentous phage by being sensitive to diethyl ether.     

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.     

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.     

IncI2 plasmids specify sensitivity to filamentous bacteriophage IKe.

Bacterial strains carrying the derepressed incompatibility group IncI2 plasmids TP114drp-l or R721pilc were lysed by the filamentous bacteriophages IKe, I2-2, and X. Phage I2-2 was serologically related to IKe, but phage X was not. Phage IKe adsorbed to the tips of thick pili determined by the IncI2 plasmids, but not to the well-known thin I2 pili.     

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.     

Effects of high temperature on Escherichia coli F pili.

The effects of high temperatures (46 to 50 degrees C) on the production of F pili by Escherichia coli were studied by electron microscopy. Attached F pili rapidly disappeared at 48 and 50 degrees C but not at 46 degrees C. Free pili were not denatured at these temperatures. The pili that disappeared from the cells at 50 degrees C did not appear as free pili in the culture supernatant fluid, indicating that the pili had retracted to the cell surface or into the cell. The adsorption of either R17 phage or F pili antibody to the sides of pili prevented retraction. The disappearance of pili was accompanied by a loss in the ability to adsorb R17 phage but not M13 phage, suggesting that the tip of a pilus remains exposed after retraction.     

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.     

Growth of Male-Specific Bacteriophage in Pasteurella Harboring F-Genotes Derived from Escherichia coli

When either the F' lac or the F'Cm plasmid was transferred from Escherichia coli into Pasteurella pseudotuberculosis, the P. pseudotuberculosis (F') strains isolated formed plaques with both ribonucleic acid (RNA)-containing and deoxyribonucleic acid-containing male-specific phages. In contrast, strains of P. pestis harboring E. coli (F') plasmids did not form plaques with male-specific phages, although such strains permitted limited multiplication of phage MS2. The adsorption and burst size of MS2 were approximately the same in both species of Pasteurella, but the per cent of adsorbed MS2 that produced infective centers was much lower in P. pestis than it was in P. pseudotuberculosis. By use of a sib-selection technique of P. pestis (F') cells, we isolated a single clone that could form MS2 plaques. (32)P-labeled MS2 adsorbed equally to and its RNA penetrated equally into both the typical MS2-nonpermissive P. pestis cells and the MS2-permissive P. pestis cells. No host modification occurred after growth of MS2 in Pasteurella. Our data suggest that typical strains of P. pestis inhibit the intracellular development of phage MS2.     

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.     

Inhibition Phenomenon of Staphylococci by Bacteriophages

In the study of the relationship between bacteriophage and strains of staphylococci showing inhibition, slight differences were observed in the ability to adsorb phage between staphylococci of full phage sensitivity and those showing inhibition by phage. Only a few plaques were produced by inhibitory phages adsorbed on strains showing inhibition, whereas almost all of the phages adsorbed on corresponding phage-propagating strains produced plaques. Some strains showing inhibition were converted to full sensitivity to certain phages by heat shock or trypaflavine treatment. Treated strains adsorbed inhibitory phages to almost the same degree as nontreated strains, but most of the phages adsorbed on treated strains produced plaques. Killing was not always observed in cells adsorbing inhibitory phages. These results suggest that inhibition is not due to low adsorption rates, but rather to plaque formation by a small number of the sensitive fraction of the population and overgrowth by nonlysed cells.     

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.     

Transduction of Myxococcus virescens by coliphage P1CM: generation of plasmids containing both phage and Myxococcus genes.

Chloramphenicol-resistant Myxococcus virescens were obtained by infecting myxococci with Escherichia coli specialized transducing phage P1CM. The drug-resistant myxococci were phenotypically unstable. They contained more than one type of plasmid; these plasmids were not found in the parent strain. Chloramphenicol-resistant E. coli were obtained by transformation with either a fraction of myxococcal DNA containing the plasmids or with P1CM prophage DNA. These transformants contained plasmids. Escherichia coli transformed by DNA from the myxococci contained both P1CM and myxococcal genes. Individual transformant clones differed in the genetic make-up of their plasmids. Among the myxococcal genes expressed in these plasmid-harbouring E. coli strains were a capacity for self-transmissibility and a pattern of phage sensitivity characteristic of R factor incompatibility group W. Escherichia coli transformed with P1CM prophage contained incomplete P1CM genomes; none of the chloramphenicol-resistant transformants produced P1CM phage particles. The significance of these findings for an understanding of mechanisms for the generation of R factors is discussed.     

Growth of Bacteriophage H on Male and Female Strains of Escherichia coli

Phage H propagated on Yersinia pestis was reported by Molnar and Lawton to be rapidly adsorbed to female but not to male strains of Escherichia coli. In contrast, we find phage H adsorbs to all E. coli strains tested (both male and female) and forms plaques on a wide variety of male strains. Phage H appears to be related to the T3-T7 group of coliphages.