Demonstration of a third incompatibility function on plasmids already incompatible with group P and group I plasmids

The addition of extra antibiotic resistance determinants to plasmids of a series previously shown to be incompatible in an atypical fashion with group I and group P plasmids, has enabled the existence of a third and major incompatibility function determined by these plasmids to be demonstrated. The in vitro construction of a plasmid consisting of the replication region of one of the plasmids, linked to the genes of the galactose operon, facilitated the identification of this third incompatibility function as similar to IncB plasmids.     

Plasmids for naphthalene biodegradation incompatible with IncP-2 and IncP-7 group plasmids

Fourteen conjugative naphthalene degradative plasmids have been classified by incompatibility. It is shown that the plasmids of IncP-9 group are characterized by the minor entry exclusion, with respect to the R plasmids belonging to IncP-2 or IncP-7 groups. On the other hand, the naphthalene degradative plasmids of incompatibility group P-7 exhibit a markedly pronounced entry exclusion, with respect to the R plasmids of the same incompatibility groups. Two naphthalene degradative plasmids reveal incompatibility with the reference plasmids of two Inc groups (P-2 and P-7). These plasmids control also resistance of bacterial cells to potassium tellurite, which is characteristic of the IncP-2 plasmids. Two other naphthalene degradative plasmids are capable of stable coexistence with the IncP-2, P-7 or P-9 reference plasmids.     

Enterococcus faecalis hemolysin-bacteriocin plasmids belong to the same incompatibility group

Plasmid pair coexistence was studied both among nine Enterococcus faecalis hemolysin-bacteriocin (Hly-Bcn) plasmids, including pJH2, pAD1, pAM gamma 1, and pIP964, and between pIP964 and five R plasmids. Some of the Hly-Bcn plasmids used were derivatives encoding resistance to erythromycin or tetracycline. The Hly-Bcn plasmids were incompatible with each other; 40 to 100% displacement was observed bilaterally for eight pairs and unilaterally for one pair. In contrast, pIP964 stably coexisted with each of the R plasmids. Entry exclusion was associated with incompatibility for most of the Hly-Bcn plasmids. The nine Hly-Bcn plasmids harbored by E. faecalis form a distinct incompatibility (Inc) group, designated IncHly.     

Incompatibility group P-7 plasmids responsible for biodegradation of naphthalene and salicylate in fluorescent pseudomonads

Analysis of seven plasmids (77 to 135 kbp in size) of the P-7 incompatibility group that are responsible for the biodegradation of naphthalene and salicylate has shown that the main natural host of IncP-7 plasmids is the species Pseudomonas fluorescens. The IncP-7 plasmids are structurally diverse and do not form groups, as is evident from their cluster analysis. The naphthalene catabolism genes of six of the IncP-7 plasmids are conservative and homologous to the catabolic genes of NAH7 and pDTG1 plasmids. The pAK5 plasmid contains the classical nahA gene, which codes for naphthalene dioxygenase, and the salicylate 5-hydroxylase gene (nagG) sequence, which makes the conversion of salicylate to gentisate possible.     

Enterococcus faecalis hemolysin-bacteriocin plasmids belong to the same incompatibility group.

Plasmid pair coexistence was studied both among nine Enterococcus faecalis hemolysin-bacteriocin (Hly-Bcn) plasmids, including pJH2, pAD1, pAM gamma 1, and pIP964, and between pIP964 and five R plasmids. Some of the Hly-Bcn plasmids used were derivatives encoding resistance to erythromycin or tetracycline. The Hly-Bcn plasmids were incompatible with each other; 40 to 100% displacement was observed bilaterally for eight pairs and unilaterally for one pair. In contrast, pIP964 stably coexisted with each of the R plasmids. Entry exclusion was associated with incompatibility for most of the Hly-Bcn plasmids. The nine Hly-Bcn plasmids harbored by E. faecalis form a distinct incompatibility (Inc) group, designated IncHly.     

Conjugative R plasmids in group C and G streptococci.

Two streptococcal isolates of groups C and G harbored conjugative R plasmids with molecular weights of 17 X 10(6) (pIP646) and 20 X 10(6) (pIP920). These plasmids carried genetic markers for resistance to macrolides and related drugs, as well as to chloramphenicol (pIP920), and have very similar HindIII restriction enzyme patterns.     

Sequence homology between Inc N group plasmids

DNA-DNA hybridization combined with "Southern blotting" was used to analyse the genetic organization and the nucleotide sequence homology between different regions of a previously characterized Inc N group plasmid pCUI and nine other Inc N group plasmids. The following conclusions could be reached: (1) N plasmids isolated from different parts of the world share substantial DNA sequence homology and also some similarity of overall genetic organization, (2) the majority of the N plasmids used in this study showed conservation of distribution of BglII and KpnI cleavage sites. Often, restriction endonuclease fragments of similar electrophoretic mobility encoded the same genetic function, (3) in one case, the N-specific properties appear to be integrated into the bacterial chromosome. (4) the plasmid DNA in strains carrying two Inc N plasmids, R199 and R113 were each composed of two molecular species only one of which constituted an N group plasmid.     

Comparative genetic organization of incompatibility group P degradative plasmids.

Plasmids that encode genes for the degradation of recalcitrant compounds are often examined only for characteristics of the degradative pathways and ignore regions that are necessary for plasmid replication, incompatibility, and conjugation. If these characteristics were known, then the mobility of the catabolic genes between species could be predicted and different catabolic pathways might be combined to alter substrate range. Two catabolic plasmids, pSS50 and pSS60, isolated from chlorobiphenyl-degrading strains and a 3-chlorobenzoate-degrading plasmid, pBR60, were compared with the previously described IncP group (Pseudomonas group P-1) plasmids pJP4 and R751. All three of the former plasmids were also members of the IncP group, although pBR60 is apparently more distantly related. DNA probes specific for known genetic loci were used to determine the order of homologous loci on the plasmids. In all of these plasmids the order is invariant, demonstrating the conservation of this "backbone" region. In addition, all five plasmids display at least some homology with the mercury resistance transposon, Tn501, which has been suggested to be characteristic of the beta subgroup of the IncP plasmids. Plasmids pSS50 and pSS60 have been mapped in detail, and repeat sequences that surround the suspected degradation genes are described.     

Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5.

Large plasmids from Agrobacterium tumefaciens, Salmonella typhimurium, Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa were routinely and consistently isolated using a procedure which does not require ultracentrifugation but includes steps designed to separate large-plasmid DNA from the bacterial folded chromosome. It also selectively removes fragments of broken chromosome. A variety of large plasmids was readily visualized with agarose gel electorphoresis, including five between 70 and 85 megadaltons (Mdal) in size, six between 90 and 143 Mdal, one that was larger than 200 Mdal, and one that was larger than 300 Mdal. This isolation procedure allowed initial estimation of the molecular sizes of the two IncP2 plasmids, pMG1 and pMG5, which were 312 and 280 Mdal, respectively. A standard curve for size determination by gel electrophoresis including plasmids between 23 and 143 Mdal in size did not extrapolate linearly for plasmids of the 300-Mdal size range. Unique response of different plasmids to the isolation procedure included sensitivity of IncP1 plasmids to high pH and the co-isolation of a 20-Mdal "cryptic" plasmid in conjunction.     

Protoplast transformation of group N streptococci with cryptic plasmids

Abstract By using an extension to group N streptococci of a contransformation procedure we have introduced 4 different-sized cryptic plasmids for Streptococcus lactis into the plasmid-free S. lactis IL1403. A mixture of 4 cryptic plasmids with an indicator plasmid (pHV1301) conferring erythromycin resistance was used for IL1403 protoplast transformation. Under such conditions, 41.5% of the erythromycin-resistant transformants were contransformed with one of the cryptic plasmids in addition to pHV1301. Indicator plasmid pHV1301 was later spontaneously segregated from doubly transformed cells. This protocol should be very useful for constructing lactic streptococcal strains bearing any phenotypically cryptic plasmid.     

Detection of large plasmids from the Bacillus cereus group

The members of the Bacillus cereus group, Bacillus anthracis, Bacillus thuringiensis, and B. cereus senso stricto, are largely defined by their content of large plasmids, which encode major virulence factors. Here we offer an easy, fast, and reliable protocol for the isolation and detection of large plasmids up to the size of at least 350kb. Furthermore, using this method, we report that Bacillus mycoides contain large plasmids.     

Survey of the plasmid content of group A streptococci

Abstract Examination of 70 M-prototype group A streptococci showed small plasmids (2.0–2.5 MDa) to be present in strains representative of M-types 28, 57, 61, 63, 64 and 69. Identical results were obtained from M _r and restriction endonuclease analyses of a 2.2-MDa plasmid (pDN691) found in the M-type 69 strain and similar plasmids in the M57 prototype and two other independently isolated M-type 57 strains. In all four strains the presence of plasmid correlated with the production of bacteriocin-like inhibitory activity identifiable as P type 614. Similar analysis revealed a possible relationship between a 2.5-MDa plasmid in the prototype M61 and M64 strains and the production of P-type 216 inhibitory activity. A survey of 56 group A streptococci recovered in association with either rheumatic fever or nephritis failed to demonstrate plasmid DNA with the exception of 2.2-MDa plasmids in four nephritis-associated M-type 57 isolates.     

Analysis of the Inc P-1 group plasmids R906 and R751 and their relationship to RP1

Three Inc-W group plasmids R388, Sa, and R7K have been studied by a variety of physical and genetic techniques. The data presented here permit the mapping of function onto the restriction enzyme maps of all three plasmids. When the physical and functional maps of these three plasmids are then compared they show a high degree of sequence conservation in the regions encoding replication and transfer functions but large differences in the regions which code for antibiotic resistance. In all three plasmids the DNA in and around the antibiotic resistance genes contains the majority of the restriction enzyme sites. The evolution of these three plasmids is discussed.     

Characterization of incompatibility group HI1 plasmids from Salmonella typhi by restriction endonuclease digestion and hybridization of DNA probes for Tn3, Tn9, and Tn10

Chloramphenicol resistance in Salmonella typhi is medicated by plasmids of the incompatibility group H, subgroup 1 (IncHI1). Eight IncHI1 plasmids from S. typhi strains originating in Mexico, Vietnam, Thailand, and India were examined by restriction enzyme digestion. The restriction enzymes, Apal, Xbal, and PstI were found to be most useful for comparison of plasmid DNAs. Four plasmids from S. typhi isolated in Mexico, Vietnam, and Thailand between 1972 and 1974 had identical restriction patterns with all three enzymes. The other IncHI1 plasmids showed only minor differences. However, some significant differences were noted between these IncHI1 plasmids and the prototype IncHI1 plasmid R27, which was isolated from S. typhimurium in 1961 and for which a restriction map has been constructed. Southern transfer hybridization with a nick-translated HI1 plasmid as a probe confirmed that there is a great deal of sequence homology among the IncHI1 plasmids. DNA probes were used to locate DNA sequences for ampicillin resistance (Tn3), chloramphenicol resistance (Tn9), tetracycline resistance (Tn10), and the one-way incompatibility between IncHI1 plasmids and the F factor, a characteristic property of IncHI1 plasmids. The results demonstrate that IncHI1 plasmids isolated from S. typhi from widely different geographic sources are very similar. Comparisons between the S. typhi plasmids and R27 indicated that conserved regions of DNA were those involved in conjugative transfer.     

Conjugal transfer and characterization of bacteriocin plasmids in group N (lactic acid) streptococci.

Thirteen bacteriocin-producing strains of group N (lactic acid) streptococci were screened for their potential to transfer this property by conjugation to Streptococcus lactis subsp. diacetylactis Bu2-60. Bacteriocin production in three strains was plasmid encoded as shown by conjugal transfer and by analysis of cured, bacteriocin-negative derivatives of the donor strains and the transconjugants. With Streptococcus cremoris strains 9B4 and 4G6 and S. lactis subsp. diacetylactis 6F7 as donors, bacteriocin-producing transconjugants were isolated with frequencies ranging from ca. 2 X 10(-2) to 2 X 10(-1) per recipient cell. Bacteriocin-producing transconjugants had acquired a 39.6-megadalton plasmid from the donor strains 9B4 and 4G6, and a 75-megadalton plasmid from the donor strain 6F7. As shown by restriction endonuclease analysis, the plasmids from strains 9B4 and 4G6 were almost identical. The plasmid from strain 6F7 yielded some additional fragments not present in the two other plasmids. In hybridization experiments any of the three plasmids strongly hybridized with each other and with some other bacteriocin but nontransmissible plasmids from other S. cremoris strains. Homology was also detected to a variety of cryptic plasmids in lactic acid streptococci.     

Incompatibility group K plasmids in bacteria isolated from a urinary tract infection

Citrobacter freundii and Klebsiella pneumoniae were concurrently isolated from a patient with a urinary tract infection. Transferable drug resistant plasmids were isolated from both strains, pMS434 and pMS435. These plasmids belonged to incompatibility group K and both carried genes governing resistance to various aminoglycoside antibiotics, i.e., kanamycin, gentamicin C complex, streptomycin, and 3',4'-dideoxykanamycin B, in addition to those governing resistance to sulfanilamide and ampicillin. They inactivated kanamycin, gentamicin C complex and 3',4'-dideoxykanamycin by adenylylation and kanamycin by phosphorylation. Electron microscopic observations disclosed that the molecular weights of the plasmids were about 67.8 megadaltons. These results indicated the similarity in genetic constitution of the two plasmids. This was the second isolation of incompatibility group K plasmids, following that reported by Hedges and Datta (Nature 234: 220-221, 1971).     

Genetic and physical studies of recombinant plasmids formed between an R plasmid of compatibility group FI and sex factor F of HfrH.

Recombinant plasmids between an R plasmid of the FI group (R162/3) and the sex factor F or HfrH were produced after the conjugal transfer of this R plasmid into HfrH. Three types of recombinant plasmids were identified after the mating of HfrH (R162/3) with recA and rec+ recipients. One specimen of each type (pIP218, pIP222, pIP226) was studied in this report. All three recombinant plasmids carry the same genetic information for resistance to antibiotics (CSSuT) retained from R162/3. pIP218 retained all the other properties from F of HfrH: derepression for pilus synthesis, mobilization of the chromosome for the proximally transferred HfrH genes (thr, leu, proA), interference with T7 propagation, and ability to be cured by acridine orange. pIP222 retained from F of HfrH the derepression for pilus synthesis and the same polarity of chromosome transfer (thr, leu, proA), while pIP226 retained the interference with T7 propagation and acridine orange curing. Physical studies revealed that replication control and/or recovery of F and pIP218 as covalent circles of deoxyribonucleic acid are similar, and are different from R162/3. The new plasmids are more likely the result of a substitutive recombination event than a fusion. We propose genetic maps of these recombinant plasmids, showing the unequal participation of the parental plasmids in their formation.     

Distribution of plasmids in cyanobacteria of the LPP group

Abstract The distribution of plasmid DNA has been studied in 23 strains and variants of non-heterocystous filamentous cyanobacteria that are susceptible to infection by the LPP-1 cyanophage (archetype). 21 have identical plasmid profiles and contain 3 plasmids of M _rs 0.9, 10 and approx. 12 · 10⁶ respectively. In one strain, Plectonema FS180, the plasmids have been designated pMP1, pMP2, and pMP3, respectively. pMP1 shows sequence homology with pMP2 and pMP3 but not with DNA from an LPP-type cyanophage. Plectonema UTEX 598 lacks the small plasmid only, while Plectonema UTEX 1541 is distinct amongst all these strains with 3 plasmids of M _rs 3, 10, and > 30 · 10⁶, respectively. The findings support the view that the majority of these strains may be independent isolates of a single species.