Characterization of R-plasmids in environmental isolates ofsalmonella: Host range and stability

Four environmental isolates ofSalmonella, resistant to several drugs, were examined for plasmid carriage with four different plasmid DNA isolation procedures. The method of Birnboim and Doly gave the best results. Three of the strains possessed a single plasmid with molecular weights of 60 (kanamycin resistant), 44.5 (kanamcin resistant), and 23.4 Md (ampicillin and amoxicillin resistant); the other strain (resistant to tetracycline) harbored two plasmids of 69.8 and 2.2 Md. The 69.8 Md was the one responsible for resistance. All plasmids were fi^–, and the 44.5 Md Kc^r plasmid synthesized a sex pilus type F. Some properties related to the dissemination of R-plasmids, such as host range, transfer frequencies, and in vitro stability, were studied. Plasmids generally showed a wide host range and high stability in the transconjugants tested. It could be concluded that these plasmids may be widely disseminated in the environment studied.     

Molecular Nature of Two Nonconjugative Plasmids Carrying Drug Resistance Genes

Two nonconjugative R-plasmids, N-SuSm and N-Tc, have been characterized. Both were of relatively small size (5 x 10(6) to 6 x 10(6) daltons) and present in multiple copies within their respective bacterial hosts. N-SuSm possessed a guanine plus cytosine content of 55%, whereas N-Tc was 49% guanine plus cytosine. Although these plasmids were inherently nontransmissible they could be mobilized by a large variety of transfer agents including Ent, Hly, and K88. The fi(-) transfer factors tested were far more likely (about 200x) to mobilize these nonconjugative plasmids than were the fi(+) transfer factors tested. Although the mobilization phenomenon was not found to be associated with a detectable level of direct stable recombinational union between N-SuSm or N-Tc with a transfer factor, we were able to demonstrate a low level of recombination between these replicons and a transfer factor by P1-mediated transduction. The isolation of recombinants between transfer factors and nonconjugative plasmids presumably represents one means by which unitary molecular types of R-plasmids arise and by which existing R-plasmids may acquire new resistance determinants.     

Incidence and transfer of R-plasmids at a hospital in Saudi Arabia

One hundred and fifty Gram-negative bacteria isolated from patient specimens at King Faisal Specialist Hospital were examined for their ability to transfer antibiotic resistance plasmids to a sensitive Escherichia coli recipient in conjugation and transformation experiments. Agarose gel electrophoresis was used to enumerate and size the R-plasmids found, and Southern DNA hybridization was used to assess similarities between antibiotic resistance plasmids from different bacteria and sources. Of the bacterial isolates tested 65% contained plasmids, 70% of these transferred antibiotic resistance to E. coli, and 40% transferred multiple, linked resistances on R-plasmids. DNA hybridization of these R-plasmids demonstrated widespread similarities between plasmids from different bacterial genera and from different hospital locations. In particular, a gene encoding ampicillin resistance appeared especially widespread, indicating that a transposon may be mediating transmission of this resistance.     

Antibiotic resistance and R-plasmids in food chain Salmonella: evidence of plasmid relatedness.

A large number of strains (1,783) belonging to 15 Salmonella serovars isolated, in Canada, from the three major links of the human food chain were screened for multiple antibiotic resistance and the presence of R-plasmids. Multiresistant strains occurred among animal feed, livestock, and human isolates at frequencies of 4, 22, and 14%, respectively. Conjugation analysis revealed that 58% of the isolates from feeds, 87% of those from livestock, and 89% of the human strains carried all or part of their resistance determinants extrachromosomally on R-plasmids. Conjugative plasmids representing nine different incompatibility groups were detected, with the Inc I alpha group being predominant. Within the limits of the parameters measured, certain of these plasmids show a degree of relatedness suggestive of a common ancestry.     

Characterization of Pseudomonas aeruginosa derepressed R-plasmids.

A genetic study of conjugal transmissibility of two R-plasmids was undertaken in Pseudomonas aeruginosa. Conjugally derepressed mutants of the R-plasmids were isolated, and examination of 11 independent mutants revealed that 10 were recessive to the wild-type transfer repressor, whereas 1 mutant was cis dominant. Cross-repression was observed between the two R-plasmids, suggesting that they have functionally equivalent systems for regulating the expression of tra loci. The derepressed R-plasmid mutants exhibited several characteristics, in addition to derepressed transfer, that were not expressed by the parental plasmids. These included sensitivity to certain donor-specific phages, inhibition of multiplication of a transducing phage, and, in the one case examined, a high degree of entry exclusion. The coexpression of these different functions suggests that their respective genetic loci are controlled by the same regulatory system as that of tra, or else that they are part of the tra complex.     

The conjugational transfer of plasmids to Legionella strains

In experiments on conjugation the transfer of a number of R-plasmids having a wide range of hosts, such as plasmids RP1, R68.45, RP4, N3, RK2, S-a, those having a narrow range of hosts, such as plasmid R64, to strains of different Legionella species was shown. The frequency of transfer varied from 3.1 X 10(-3) to 9.4 X 10(-7). The fact that the conjugation transfer was confirmed by the reverse transfer of plasmids from Legionella transconjugates to Escherichia coli strain K12, as well as by the detection of the DNA of the transferred plasmid by means of electrophoresis in agar gel. Plasmid RP1 showed different behavior in transconjugates of various Legionella species after several passages in a medium free of antibiotics. In the Legionella strain under study the unstable preservation of plasmid R64 was observed.     

Incompatibility Reactions of R Plasmids Isolated from Escherichia coli of Animal Origin

The incompatibility reactions of a group of 90 R plasmids, isolated from the fecal Escherichia coli of calves, pigs, and chickens, have been determined against reference plasmids of the incompatibility groups F(11), I, N, P, and W. Twenty plasmids belonged to incompatibility groups F(11), I, N, or P. Forty-nine plasmids were compatible with all of the five reference plasmids. Eight plasmids exhibited marked incompatibility with representatives of two or more of the above groups, whereas two strains showed incompatibility reactions consistent with the coexistence of two compatible plasmids. In addition, 19 plasmids remain untyped because they showed intermediate reactions with one or more reference plasmids.     

Worldwide distribution of the conjugative Clostridium perfringens tetracycline resistance plasmid, pCW3

The aim of this study was to test the hypothesis that all conjugative R-plasmids of Clostridium perfringens are closely related to the previously characterized tetracycline resistance plasmid, pCW3. Fourteen conjugative R-plasmids derived from 11 C. perfringens strains isolated in Australia, the United States, France, Belgium, and Japan were analyzed. Eleven of the plasmids encoded tetracycline resistance while three carried both tetracycline and chloramphenicol resistance. Each of these plasmids was compared, by restriction analysis, to the reference plasmid, pCW3. Seven of the tetracycline resistance plasmids had EcoRI, XbaI, and ClaI restriction profiles that were identical to those of the corresponding pCW3 digests. The seven remaining R-plasmids were different from pCW3. Comparison of partial restriction maps of these plasmids with a complete map of pCW3 indicated that they contained at least 17 kb of DNA that also was present in pCW3. Hybridization analysis confirmed that these plasmids shared substantial homology with pCW3. The three tetracycline and chloramphenicol resistance plasmids frequently lost a 6-kb chloramphenicol resistance segment during conjugation. Cloning experiments showed that the chloramphenicol resistance determinant was expressed in Escherichia coli and that the chloramphenicol resistance gene of one of these plasmids, pIP401, was contained within a 1.5-kb region of the 6-kb deletion segment. Hybridization analysis indicated that the deletion segment of pIP401 was related to those of the other two chloramphenicol resistance plasmids. During the course of this study, conjugative R-plasmids which appear to be identical to pCW3 or closely related to pCW3 were identified from C. perfringens strains from human, animal and environmental sources in five countries. It is concluded that C. perfringens strains in humans and animals throughout the world have overlapping gene pools and that all the conjugative C. perfringens R-plasmids examined probably evolved from a pCW3-like element.     

Plasmids encoding trimethoprim resistance in bacterial isolates from man and pigs

Trimethoprim (Tp) resistant Gram negative bacteria were isolated from humans and pigs. The bacterial hosts were characterized by their resistance pattern and biotype. The presence of transferable Tp plasmids was demonstrated in 86% of 59 porcine isolates and 37% of 49 human isolates. The Tp R-plasmids carried a diversity of resistance determinants such as Tc, Cm, Sp, Sm and Su. Incompatibility tests distinguished two major groups, Inc FIV and Inc N. Thirty of 99 Tp R-plasmids isolated from pigs were grouped as Inc FIV and three as Inc N. Eleven of 26 Tp R-plasmids isolated from humans were grouped as Inc FIV and eight as Inc N. The results of molecular weight determination of Tp R-plasmids performed by agarose gel electrophoresis were consistent with the existence of two groups—larger R-plasmids (76 to 104 Md) belonging to Inc FIV and lower molecular weight R-plasmids (25 to 36 Md) belonging to Inc N. Results from this study indicate that the Tp R-plasmids isolated in Perth have evolved independently from those described in Europe and the United Kingdom. There is also evidence for their local spread between Escherichia, Klebsiella, Enterobacter, Citrobacter and Acinetobacter from man and animals.     

Heteroduplex analysis of P-plasmid evolution: the role of insertion and deletion of transposable elements

DNA homology of thirteen R-plasmids of group P was examined by heteroduplex analysis and Southern blotting. Ten of these plasmids showed homology for extensive regions including all genes reported as necessary for replication and conjugational transfer. The differences between these plasmids could be explained by gain or loss of DNA sequences, many of which have been shown to be transposons. Of the other three plasmids, two showed unambiguous homology with the typical P-plasmids but this homology was imperfect, implying that these plasmids are products of lines which have evolved separately for long periods. One plasmid failed to produce heteroduplexes with the reference P plasmid.     

Plasmids encoding trimethoprim resistance in bacterial isolates from man and pigs

Trimethoprim (Tp) resistant Gram negative bacteria were isolated from humans and pigs. The bacterial hosts were characterized by their resistance pattern and biotype. The presence of transferable Tp plasmids was demonstrated in 86% of 59 porcine isolates and 37% of 49 human isolates. The Tp R-plasmids carried a diversity of resistance determinants such as Tc, Cm, Sp, Sm and Su. Incompatibility tests distinguished two major groups, Inc FIV and Inc N. Thirty of 99 Tp R-plasmids isolated from humans were grouped as Inc FIV and eight as Inc N. The results of molecular weight determination of Tp R-plasmids performed by agarose gel electrophoresis were consistent with the existence of two groups--larger R-plasmids (76 to 104 Md) belonging to Inc FIV and lower molecular weight R-plasmids (25 to 35 Md) belonging to Inc N. Results from this study indicate that the Tp R-plasmids isolated in Perth have evolved independently from those described in Europe and the United Kingdom. There is also evidence for their local spread between Escherichia, Klebsiella, Enterobacter, Citrobacter and Acinetobacter from man and animals.     

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.     

Plasmid analysis of bacteria of Bacillus genus used in the development of probiotics

Presence of plasmid-contained strains in probiotics prepared on the basis of Bacillus bacteria was assessed. Plasmid analysis was performed by the method based on neutralization of bacterial suspension after alkaline treatment with lithium chloride. Presence of 4-6 plasmids with molecular weight from 1 to 75 MDa was revealed in 4 commercially available probiotics. Since plasmids can facilitate transfer of genes of the antimicrobial resistance to pathogenic microorganisms it was recommended to perform thorough search of probiotic microorganisms without plasmids or ensure the elimination of R-plasmids in start cultures for the most promising biopreparations.     

Deletion and rearrangement of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules.

When E. coli was transformed with linearized pBR322 DNA, many transformants contained recircularized plasmids bearing deletions and other rearrangements. Most aberrant molecules were less than monomeric length and had lost the restriction site used for linearization, with the deleted region extending mono- (type Ia) or bi-directionally (type Ib). Type II deletants were greater than monomeric but less than dimeric and contained the pBR322 sequence in direct repeat with deletion at one or both junctions (type IIa) or in inverted repeat with loss of sequence at both junctions (type IIb). Type III deletants were greater than dimeric but less than trimeric, consisting of pBR322 sequences in both direct and inverse repeat with deletions at two or more junctions. Transformation frequencies for linear DNA were drastically reduced in xth-1- bacteria with type IIb deletants predominating in transformants. This indicates that exonuclease III is important for perfect recyclization of plasmids and the generation of type I deletants. In vivo recyclization of in vitro ligation products explains many of the aberrant DNA molecules that are encountered during gene cloning.     

Formation of delta tra F' plasmids: specific recombination at oriT

Delta tra F' plasmids can be isolated from matings between Hfr donors and recA- recipients, with selection for transfer of proximal chromosomal genes. Previous experiments indicate that F DNA from the neighborhood of the transfer origin up to the proximal junction with the chromosomal DNA is present on these plasmids, together with chromosomal segments, some of which belong to distinct size classes. We have sequenced across the novel joints contained in five delta tra FproA+ plasmids and in five delta tra FpurE+ plasmids, and we have compared these with the F sequence near oriT and with a chromosomal site near purE. The previously reported specificity in formation of some of these classes is confirmed at the nucleotide sequence level. The F DNA in nine of these novel joints extended beyond the nicking sites identified by others in lambda oriT+ bacteriophages up to a position between two sequenced oriT- mutations. Small plasmids containing these novel joints are mobilized in trans by pOX38 at frequencies less than 5 X 10(-7) times the mobilization frequencies for similar plasmids that contain oriT. The relations of these findings to the location of the nicking site at oriT are discussed.     

Conjugational plasmid transfer from A, B and H streptococci to N streptococci

Plasmid-mediated resistance to erythromycin and chloramphenicol was successfully transferred from group A, B and H streptococci to group N streptococci by a process akin to conjugation. The results showed that plasmids from streptococcal groups other than N were able to replicate in lactic streptococci as well. The transfer experiments were carried out by using a membrane filter mating technique. Four of the five plasmids used (pSM15346, pSM10419, pIP501, and pEL1) were transferred at frequencies ranging from 10(-1) to 10(-8) transconjugants per donor colony-forming unit. The highest transfer frequencies were obtained when S. pyogenes strain 15346 (pSM15346) served as the donor strain. The identy of transconjugants was verified by testing for the presence of unselected markers of the recipient strains, and both transduction and transformation were ruled out as the mechanisms of transfer.     

Expression of R-plasmid functions during anaerobic growth of an Escherichia coli K-12 host.

The normal habitat of enteric bacteria is largely anaerobic. Expression of the three characteristic properties of R-plasmids, drug resistance, vegetative replication, and fertility, was therefore studied in Escherichia coli K-12 during anaerobiosis. Replication and drug resistance functions were not altered in the 45 R-plasmids studies, whereas the expression of fertility varied considerably among different R-plasmids during anaerobiosis. The R-plasmids could be divided into three groups, one showing a strong, the second a moderate, and the third little or no reduction of fertility by anaerobiosis. Plasmid-determined sensitivity to F-, N-, and I-specific phage, respectively, was well, although not absolutely, correlated with each of three groups mentioned. Anaerobiosis-aerobiosis appears to change the fertility of type F R-plasmids by influencing the degree of repression of their fertility functions such as the formation of sex pili. Although the minimum inhibitory concentrations of ampicillin, chloramphenicol, streptomycin, and tetracycline were unaltered by anaerobiosis, sulfonamide was found to be four- to eightfold less active under this condition in both resistant and sensitive strains. A surprisingly high frequency and uniformity of minimal inhibitory concentrations was observed for R-plasmid-mediated resistance to streptomycin and chloramphenicol.     

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.     

F factor mobilization of non-conjugative chimeric plasmids in Escherichia coli: General mechanisms and a role for site-specific recA-independent recombination at oriV1

Plasmid pSC101 is neither self-transmissible nor efficiently mobilized (made to transfer) by the Escherichia coli F factor. When fragments of F factor DNA were inserted into pSC101 the resulting chimeric plasmids were mobilized by the F factor at enhanced frequencies. These chimeric plasmids, which were not self-transmissible, fell into three classes according to their relative ability to be mobilized by an autonomous or integrated F factor: (1) class I pSC101-F chimeric plasmids contain the origin of transfer of the F factor (oriT) and were mobilized in trans at an efficiency nearly equal to that of F factor transfer; (2) class II pSC101-F chimeric plasmids lacked both oriT and the origin of vegetative F replication (oriV1), and were mobilized in cis via fusion with the F factor in a recA-dependent recombination to yield a transferable co-integrated plasmid; (3) class III pSC101-F chimeric plasmids lacked oriT but contained oriV1 and were mobilized in cis via co-integration with the F factor probably at oriV1 in a recA-independent recombination. A fourth class of mobilization event, not exhibited by pSC101-F chimeric plasmids, was also observed. Mobilization of pBR322 and pSC101 occurred in cis via transposon-mediated recA-independent fusion with F. On the basis of these results we present a general classification scheme of non-conjugative plasmids and also suggest mechanisms for their mobilization.     

Iron-sulfur clusters in type I reaction centers.

Type I reaction centers (RCs) are multisubunit chlorophyll-protein complexes that function in photosynthetic organisms to convert photons to Gibbs free energy. The unique feature of Type I RCs is the presence of iron-sulfur clusters as electron transfer cofactors. Photosystem I (PS I) of oxygenic phototrophs is the best-studied Type I RC. It is comprised of an interpolypeptide [4Fe-4S] cluster, F(X), that bridges the PsaA and PsaB subunits, and two terminal [4Fe-4S] clusters, F(A) and F(B), that are bound to the PsaC subunit. In this review, we provide an update on the structure and function of the bound iron-sulfur clusters in Type I RCs. The first new development in this area is the identification of F(A) as the cluster proximal to F(X) and the resolution of the electron transfer sequence as F(X)-->F(A)-->F(B)-->soluble ferredoxin. The second new development is the determination of the three-dimensional NMR solution structure of unbound PsaC and localization of the equal- and mixed-valence pairs in F(A)(-) and F(B)(-). We provide a survey of the EPR properties and spectra of the iron-sulfur clusters in Type I RCs of cyanobacteria, green sulfur bacteria, and heliobacteria, and we summarize new information about the kinetics of back-reactions involving the iron-sulfur clusters.