Biomedical subjects
Publications and source records attributed to J N Coetzee.
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.
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.
The T incompatibility group plasmid R394 can mobilize the chromosome of Proteus mirabilis strain PM5006. It transferred relatively large segments, corresponding to at least 20 min on the D plasmid chromosomal map of the organism. The frequency of recombination for a large number of selected markers was nearly constant at 5 X 10(-6) per donor cell and it is concluded that mobilization takes place from a number of chromosomal sites. All recombinants were R+ and displayed all properties of the plasmid. By analysing crosses for co-inheritance frequencies of unselected markers, a number of chromosomal loci were assembled in linear array. Linkage between markers at the ends of this linkage group was established to markers at the respective termini of the existing D plasmid linkage group. This established a composite circular linkage map of genes of the P. mirabilis strain PM5006 chromosome.
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.
R plasmids R40a, Rip69, R447b, R769 belonging to incompatibility groups A-C, M, N, V, respectively, were investigated for chromosomal mobilizing ability in Proteus mirabilis. Plasmids R40a, Rip69 and R447b mediated polarized transfer of markers in a clockwise direction from origins near tyr-1, metF and ser-2, respectively, on the linkage map. The recovery frequency per donor cell of proximal markers approached 1 x 10(-4) for these three plasmids and the efficiency of chromosomal transfer was higher than that of the previously studied plasmid D. The plasmid-guided chromosomal trajectories overlap and it was possible to complement results obtained with plasmid D to assemble a time-of-entry chromosomal map and directly establish the circularity of the linkage group. The map comprises a length of 93 min in terms of transfer time. Plasmid R769 had a different pattern of chromosome transfer. This plasmid produced recombinants for all markers at frequencies of about 4 x 10(-6) per donor. It effected multiple and more or less simultaneous entry of markers and produced recombination over lengths of chromosome rarely corresponding to more than 10 min on the linkage map.
Spheroplasts of Providence alcalifaciens strain P29 auxotrophs were prepared by combined treatment with glycine and lysozyme-EDTA. About 15% of spheroplasts had areas of cytoplasmic membrane exposed where cell wall was absent. The spheroplasts of different auxotrophs were mixed pairwise and fusion was attempted with polyethylene glycol or nascent calcium phosphate. After spheroplasts had regenerated to bacterial forms selection was made for recombinants. Recombinants arose at frequencies of 3.8 X 10(-6) to 1.7 X 10(-7) per spheroplast initially present, by both methods of fusion. The frequency was strongly dependent on the number of chromosomal loci used in selection. The possible order of five loci was determined and this corresponded to that on the closely related Proteus mirabilis chromosome. Control experiments excluded possibilities of auxotrophic reversion, conjugation, transformation, transfection or transduction as explanations of the results. Analysis of prototrophic clones yielded stable prototrophs or mixtures of stable prototrophs and stable recombinants. Parental types were not encountered. Unselected markers segregated among recombinants. It was concluded that the formation of recombinant bacteria was due to spheroplast fusion and that only stable products of the very temporary heteroploid state were haploid recombinants. The low frequency of recombination was ascribed to the limited number of spheroplasts with areas of exposed cytoplasmic membrane.
Mating procedures for detection of mobilization of the Proteus mirabilis chromosome were re-investigated. The chromosome was mobilized by plasmid D, the previously used hybrid between plasmids P-lac and R1drd19. About a 40-fold increase in recombinant recovery correlated with the absence of swarming during mating and a lower temperature of incubation. The modification introduced was that conjugation was allowed to proceed on a non-selective supplemented minimal medium at 30 degrees C before washing and plating on selective media. Final incubation was also at 30 degrees C. This technique enabled eight additional chromosomal markers to be mapped. Polarized transfer of the chromosome was shown by gradient of transmission experiments using a previously described marker as reference, by linkage analysis with reference to proximal and distal markers and (less successfully) by interrupted mating on solid medium. Markers of plasmid D transferred at high frequency to all recombinants. The plasmid was stable in recombinants and could transfer itself and chromosomal markers of the new hosts in further matings. Resulting recombination of markers occurred at usual frequencies. The marker order, his-1, ser-2, ura-2, pyrB1, trp-3, cysA1, ade-2, ilv-2, cysG1, gly-1, cysC1, argA2, metF2, nalA1, thr-1, leuB2, did not resemble the order of these markers in Escherichia coli.
The P-1 incompatibility group plasmid R772 can mobilize the chromosome of Proteus mirabilis strain PM5006. The decreasing gradient of recombinant recovery frequencies found for markers which were increasingly distal to 0 min with plasmid D donors was not found with R772. Instead, it produced recombinants for all markers at frequencies of about 5 X 10(-5) per donor. This is about 10-fold lower than the plasmid transfer frequency. Recombinants were stable and recombination was only detected over short segments of the chromosome which corresponded to about 10 min on the D plasmid map of the chromosome. All recombinants had inherited R772 and expressed all properties of the plasmid. Attempts to isolate variant plasmids with increased frequencies of recombinant formation were unsuccessful.
Infective complications were analysed in 74 patients who underwent renal transplantation. Bacterial infections were seen in 69%, viral infections in 48% and fungal infections in 17% of these patients. The urinary tract was involved in 56% of patients, the lungs in 24% and the upper respiratory tract in 17%. Less common were cellulitis (7%), CNS infections (4%), and other sites of infection.
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Plasmids have been constructed by insertion of DNA from Rhizobium leguminosarum or Proteus mirabilis into RP4 (an R factor of group P). Such recombinant plasmids retain the wide host range of the parental plasmid, being as efficiently transmissible as the unmodified RP4 and are stably maintained in rapidly growing cultures. The recombinant plasmids, even though each contained a DNA sequence absolutely identical with that of the host strain, are no more efficient at mobilizing the transfer of chromosomal genetic information from that host strain than was unmodified RP4. We therefore conclude that an unknown factor must be essential in the process of chromosome mobilization and rate limiting for that process.
High frequency transducing (HFT) phages 5006MHFTk and 5006MHFTak for kanamycin or ampicillin plus kanamycin resistance, derived from Proteus mirabilis strains PM5006(R394) and PM5006(R394) respectively, transduced (at low multiplicities of infection, m.o.i.) antibiotic resistance and prototrophy to PM5006 leu-I at high frequency. Simultaneous transduction of these markers occurred at very much lower frequencies. The latter result was correlated with the proportion of multiply-infected bacteria which, due to the great transducing potential of the phage, could register as transductants. Each HFT lysate was thus heterogenous with regard to high frequency transducing phage. Apart from the additional antibiotic resistance marker carried by one phage, no other difference between the two lysates was detected. High segregation frequencies of antibiotic-resistant or prototrophic transductants indicated transduction by lysogenization. Although antibiotic-sensitive segregants of antibiotic-resistant prototrophic transductants occurred at high frequency, no auxotrophic segregants of these transductants were found. This suggests transduction by a double cross-over event in the leucine region. Most transductants, even at low m.o.i., were lysogenically converted to homologous phage non-adsorption as a result of interaction between the transducing phage genome and the resident cryptic prophage. They could, however, be retransduced by appropriate phage lysates; thus, lysogenic conversion to non-adsorption was not absolute. Some prototrophic transductants were non-lysogenic although their segregants liberated low-titre phage. The latter anomaly, and the fact that the leucine marker and antibiotic resistance were not cotransduced, are explained by the mode of integration of the phage into the host chromosome in relation to the resident cryptic prophage and the leucine region.
The properties of three additional Proteus mirabilis hosts for the high frequency transducing (HET) phages 5006MHFTkappa and 5006MHFTak are described. The phages transduce resistance to kanamycin and to ampicillin plus kanamycin, respectively, and were produced by ultraviolet induction of derivatives of P. mirabilis strain PM5006. Strain PM804 could not be shown to adsorb the phages although it yielded a few transductants. All transductants, even those produced at low multiplicities of phage input were lysogenic and segregated markers at high frequency. Ultraviolet induced phage lysates of these transductants transduced PM804 at higher frequencies and PM5006 at lower frequencies than the original phages. Strain PM804 or its derivatives did not adsorb phage from these lysates. Tranmission experiments through PM5006 of phage in the transductant lysates confirmed that PM804 had a host-controlled modification system which modified HFT phage from PM5006. That PM804 also possessed a restriction system was inferred from the greater numbers of transductants obtained with page which bore a compatible modification pattern. Strain N and a restrictionless mutant of it named NHI, adsorbed the HET phages avidly and failed to modify their host range. Transduction frequencies of the phage markers were about 10(-4)/p.f.u. adsorbed to strain NHI and only about tenfold lower to strain N which did not plate the phages. Tranductants also had the features of heterogenotes and those obtained at low multiplicites of infection were non-lysogenic. The latter transductants adsorbed homolgous phage while NHI transductants also plated the HFT lysates. Strain NHI, lysogenized by the parent phage 5006M, did not adsorb the HFT phages. These findings suggest that the HFT phages were most likely defective for lysogenic conversion to homologous phage non-adsorption. The postulated restriction enzymes of PM804 and strain N could not be shown to be thermolabile.
Properties of two transducing systems with phages capable of high frequency transduction (HFT) of streptomycin and sulphonamide resistance markers of the V group plasmid R905, and of these markers plus the kanamycin resistance marker derived from a previously described HFT phage 5006MHFTak, are described. Transducing particles of the former phage, named 5006MHFTsus, were detected using the replica-plate technique in an ultraviolet-induced lysate of Proteus mirabilis strain PM5006 transduced to streptomycin and sulphonamide resistance by phage 5006M grown on PM5006 carrying R905. Phage 5006MHFTsusk was also detected by the replica-plate technique in ultraviolet-induced lysates of of phage 5006MHFTsus transductants retransduced to ampicillin and kanamycin resistance by phage 5006MHFTak. Both phages were serologically identical to the parent phage 5006M. Ultraviolet-induced lysates transduced their markers to PM5006 at frequencies of about 5 X 10(-2)/plaque-forming unit adsorbed for both the phages. With phage 5006MHFTsusk, this frequency was increased about 10-fold by simultaneous infection of recipients with homologous non-transducing phage, while phage 5006MHFTsus transductions only underwent a twofold increase. Transductants took about 60 min to express complete resistance to 50 mug streptomycin/ml, and resistance to 1600 mug sulphadiazine/ml was complete within 120 min after phage adsorption. Phage 5006MHFTsusk was slightly more resistant to ultraviolet inaction of its transducing potential and reasons are given for the belief that transductants of both phages are heterogenote-like. Both phage lysates were also capable of generalized transduction and, like previously described HFT phages, lysates transduced the leucine marker at increased frequencies. Using previously described extra- and intra-species phages hosts, it was found that the phages could transduce in single infection and were defective in the lysogenic conversion function as well as in a maturation step. Possible modes of formation of the HFT particles are discussed9
Recombinant plasmids have been produced both by transduction of genetic material from FIIR factors into Proteus mirabilis strains carrying plasmids of group J and by insertion of a transposon conferring streptomycin and trimethoprim resistances into a J group R factor. The transposon-carrying derivative and one of the transductants were shown to be members of group J whereas another transductant was shown to be compatible with members of this group. This recombinant plasmid was able to eliminate but not to be eliminable by R factors of group FII. A model for the origin of this anomalous compatibility characteristic is presented based on the assumption that the recombinant plasmid carries part but not all of a complex of binding sites for the repressor of replication [Uhlin and Nordström (1975)].
A 5 Megadalton segment of DNA carrying a gene for kanamycin resistance from R447 b (a plasmid of group N of molecular weight 33 Megadaltons) has been inserted into Plac (a plasmid of the A--C complex of molecular weight 101 Megadaltons) to produce the recombinant plasmid Plac-R447 b (Coetzee, 1974). The recombinant plasmid is a typical member of the A--C complex except that entry of an N group plasmid into a Plac-R447 b+ recipient frequently leads to the loss of 5 Megadaltons of DNA (including the kanamycin resistance determinant) from the resident plasmid. In those transcipients from which kanamycin resistance is not eliminated, both plasmids are stably inherited.