Bacteriophage X-2: a filamentous phage lysing IncX-plasmid-harbouring bacterial strains.

Phage X-2, a filamentous rod about 950 nm in length, was isolated from sewage as plating on strains of Escherichia coli, Salmonella typhimurium or Serratia marcescens carrying either the IncX plasmid R6K, or the unique plasmid R775. Phage X-2 differs morphologically from a previously described very broad host range filamentous phage X which also lyses plasmid R6K-carrying strains and the phages differ in their resistance to inactivation by diethyl ether. Phage X-2 is serologically unrelated to phage X and the X-like phages IKe and I2-2. The adsorption site of the phage on the plasmid-bearing strains could not be determined but evidence implicating conjugative pili is presented.

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.

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.

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.

Characterization of phages derived from strains of Rhodococcus australis and R. equii.

Rhodococcus australis strain CSIR-A201 and R. equii strain CSIR-A655 spontaneously liberated phages A and B, respectively. The phages have different host ranges, but both infect R. rubropertinctus ATCC 14352. phage infection of strains resulted in stable lysogeny. The phages have similar morphologies and belong to the family Styloviridae. The DNA restriction enzyme patterns of the phages differ and do not correspond to those of actinophage phi EC. These temperate phages did not transduce prototrophic or antibiotic resistance markers to appropriate hosts.

Bacteriophage M: an incompatibility group M plasmid-specific phage.

Phage M was specific for bacterial strains, of various genera, harbouring plasmids of the M incompatibility group. It formed turbid plaques which varied from pin point to more than 2 mm in diameter on all hosts where plaques were detected. The phage had an hexagonal outline with a diameter of 27 nm. It contained RNA but differed from other plasmid-dependent RNA phages in being sensitive to chloroform. It adsorbed along the length of shafts of M pili.

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.

Plasmid R394 is a cointegrate.

Plasmids R394a and R394b which cointegrate to form R394 are described. They have molecular masses of 102 +/- 4 and 11.0 +/- 0.4 MDal, belong to the T and N incompatibility groups and confer resistance to kanamycin and ampicillin, respectively. R394a is self transmissible and mobilizes R394b, which is non-self transmissible. These findings clarify anomalies in the behaviour of R394 and support suggestions based on the properties of variant phages derived from R394.

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.

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.

In vitro-constructed RP4-prime plasmids mediate orientated mobilization of the Proteus morganii chromosome.

RP4-prime plasmids containing inserts of Proteus morganii strain 2815 chromosomal DNA (0.5-13 megadaltons in size) were constructed in vitro. When introduced into the latter organism, the hybrid plasmids promoted transfer of chromosomal markers in an orientated manner, with recombinant frequencies from 10(-4) to 10(-6) per donor. Transconjugants expressed the tetracycline-resistant phenotype of the plasmids. Plasmid-guided trajectories overlapped and a circular map of chromosome markers was constructed. Correspondence between size of inserted chromosomal DNA in hybrid plasmids and numbers of markers transferred suggested that homology played a role in chromosome mobilization by these plasmids.

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.

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 C-1: an IncC group; plasmid-specific phage.

A phage was isolated from sewage and shown to form plaques on Salmonella typhimurium strains carrying C plasmids. It failed to multiply on strains lacking plasmids of this group. It also plated on Proteus mirabilis and Serratia marcescens strains carrying various IncC plasmids but failed to form plaques on Escherichia coli strains harbouring most of these plasmids, although in all cases, phage multiplication on the strains was demonstrated. No phage increase occurred in any of the strains which lacked an IncC plasmid or contained plasmids of other incompatibility groups. The phage is small, hexagonal in outline, contains RNA, is resistant to chloroform and adsorbs to the shafts of pili coded for by IncC plasmids.

Genetic recombination between Proteus mirabilis and Providencia alcalifaciens.

Chromosome transfer occurred in plate matings between Proteus mirabilis strain PM5006 and Providencia alcalifaciens strain P29 in either direction with the use of plasmid D or R772 as sex factor. Auxotrophic chromosomal markers of recipients were converted to prototrophy and the galactose fermentation marker of donor PM5006 could also be selected. Recombination frequencies for a group of selected markers in PM5006(D) x P29 matings varied between 3 x 10(-5) (trp+) and 1.2 x 10(-7) (lys+) per donor. In the reverse cross, plasmid D mobilized markers on the P29 chromosome randomly with a recombination frequency of about 1.7 x 10(-7) per donor for all selected P29 markers. R772 produced random mobilization of markers on both chromosomes yielding recombinants at a frequency of about 1.8 x 10(-7) per donor. Unselected markers separated by no more than about 10 min from selected markers on the PM5006 chromosome were cotransferred from P29 by both plasmids. Despite the low degree of DNA homology existing between the two species, all hybrids behaved as stable haploids. Progeny from P29(D or R772) x PM5006 auxotroph matings displayed similar sets of naturally occurring P29 unselected markers irrespective of the selected prototroph allele. In reverse crosses, a similar range of PM5006 naturally occurring unselected markers registered in P29 recipients, although differences existed in the sets of markers mediated by the two plasmids. Weak linkage was detected between PM5006 gal+ allele(s) and some P29 auxotroph markers. Adsorption of donor-specific phages 5006M or PL25 to hybrids could not be demonstrated and many recombinants failed to express some or all of the plasmid markers.

Mobilization of the Proteus morganii chromosome by R plasmids.

R plasmids R702, R711b, R1, D, Rip69, R447b, R471 and R394, belonging to different incompatibility groups, mobilized the Proteus morganii 2815 chromosome. Matings employing plasmids R711b or R702 as sex factors with doubly auxotrophic recipients produced recombinants characterized by the obligatory inheritance of ser-1+, irrespective of the selected marker.

Properties of a filamentous phage which adsorbs to pili coded by plasmids of the IncI complex.

A filamentous phage, PR64FS, which adsorbs to tips of I pili was isolated. PR64FS is shorter than the I pilus-adsorbing phage If1 and differs from it in plaque morphology. Phages PR64FS and Ifl are serologically related and, like the latter, PR64FS adsorbs to pili coded by all Inc groups of the I plasmid complex.