Selection pressure required for long-term persistence of blaCMY-2-positive IncA/C plasmids

Multidrug resistance blaCMY-2 plasmids that confer resistance to expanded-spectrum cephalosporins have been found in multiple bacterial species collected from different hosts worldwide. The widespread distribution of blaCMY-2 plasmids may be driven by antibiotic use that selects for the dissemination and persistence of these plasmids. Alternatively, these plasmids may persist and spread in bacterial populations in the absence of selection pressure if a balance exists among conjugative transfer, segregation loss during cell division, and fitness cost to the host. We conducted a series of experiments (both in vivo and in vitro) to study these mechanisms for three blaCMY-2 plasmids, peH4H, pAR060302, and pAM04528. Results of filter mating experiments showed that the conjugation efficiency of blaCMY-2 plasmids is variable, from <10(-7) for pAM04528 and peH4H to ～10(-3) for pAR060302. Neither peH4H nor pAM04528 was transferred from Escherichia coli strain DH10B, but peH4H was apparently mobilized by the coresident trimethoprim resistance-encoding plasmid pTmpR. Competition studies showed that carriage of blaCMY-2 plasmids imposed a measurable fitness cost on the host bacteria both in vitro (0.095 to 0.25) and in vivo (dairy calf model). Long-term passage experiments in the absence of antibiotics demonstrated that plasmids with limited antibiotic resistance phenotypes arose, but eventually drug-sensitive, plasmid-free clones dominated the populations. Given that plasmid decay or loss is inevitable, we infer that some level of selection is required for the long-term persistence of blaCMY-2 plasmids in bacterial populations.     

大肠埃希菌耐药性监测及耐药质粒的研究

为了解医院内感染大肠埃希菌的耐药情况,探讨其耐药发生、流行及传播机制,对从临床分离的32株大肠埃希菌进行了药敏试验、质粒图谱分析以及质粒接合、转化试验,结果表明大肠埃希菌对氨苄青霉素的耐药率＞88%,对庆大霉素的耐药率＞75%,其中28株大肠埃希菌检出质粒,均含有一条分子量为5.66 Mu的质粒带,是医院内感染大肠埃希菌的流行质粒.质粒的接合、转化试验证实了质粒具有横向传播的特点,是细菌产生耐药的主要原因.     

Conjugative plasmid transfer in gram-positive bacteria.

Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.     

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.     

Conjugative Plasmid Transfer in Gram-Positive Bacteria

Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.     

Conjugative plasmids in multi-resistant bacterial isolates from Indian soil

Aims:  Determination of heavy metal and antibiotic resistance and presence of conjugative plasmids in bacteria isolated from soil irrigated with wastewater.
Methods and Results:  Composite soil samples were collected from Ghaziabad, Uttar Pradesh, India. Forty different bacteria were selected from nutrient agar and characterized by morphological, cultural and biochemical tests. All the isolates were tested for their resistance to different heavy metals and antibiotics. The DNA derived from multiple metal and antibiotic-resistant bacterial isolates was PCR amplified and plasmid-specific sequences (IncP, IncN, IncW, IncQ and pMV158-type) were analysed by dot blot hybridization. All isolates gave PCR products with trfA2 and oriT primers of the IncP group. These PCR products also hybridized with the RP4-derived probes. However, the samples were negative for all the other investigated plasmids as proved by PCR and dot blots.
Conclusions:  The presence of conjugative/mobilizable IncP plasmids in the isolates indicates that these bacteria have gene-mobilizing capacity with implications for potential dissemination of introduced recombinant DNA.
Significance and Impact of the Study:  The detection of IncP plasmids in all the bacterial isolates is another proof for the prevalence of these plasmids. We propose that IncP plasmids are mainly responsible for the spread of multi-resistant bacteria in these soils.     

Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids

In this study, the prevalence and types of transferable antibiotic resistance plasmids in piggery manure were investigated. Samples from manure storage tanks of 15 farms in Germany were analysed, representing diverse sizes of herds, meat or piglet production. Antibiotic resistance plasmids from manure bacteria were captured in gfp-tagged rifampicin-resistant Escherichia coli and characterized. The occurrence of plasmid types was also detected in total community DNA by PCR and hybridization. A total of 228 transconjugants were captured from 15 manures using selective media supplemented with amoxicillin, sulfadiazine or tetracycline. The restriction patterns of 81 plasmids representing different antibiotic resistance patterns or different samples clustered into seven groups. Replicon probing revealed that 28 of the plasmids belonged to IncN, one to IncW, 13 to IncP-1 and 19 to the recently discovered pHHV216-like plasmids. The amoxicillin resistance gene bla-TEM was detected on 44 plasmids, and sulphonamide resistance genes sul1, sul2 and/or sul3 on 68 plasmids. Hybridization of replicon-specific sequences amplified from community DNA revealed that IncP-1 and pHHV216-like plasmids were detected in all manures, while IncN and IncW ones were less frequent. This study showed that 'field-scale' piggery manure is a reservoir of broad-host range plasmids conferring multiple antibiotic resistance genes.     

Design and synthesis of a quintessential self-transmissible IncX1 plasmid, pX1.0

DNA exchange in bacteria via conjugative plasmids is believed to be among the most important contributing factors to the rapid evolution- and diversification rates observed in bacterial species. The IncX1 plasmids are particularly interesting in relation to enteric bacteria, and typically carry genetic loads like antibiotic resistance genes and virulence factors. So far, however, a "pure" version of these molecular parasites, without genetic loads, has yet to be isolated from the environment. Here we report the construction of pX1.0, a fully synthesized IncX1 plasmid capable of horizontal transfer between different enteric bacteria. The designed pX1.0 sequence was derived from the consensus gene content of five IncX1 plasmids and three other, more divergent, members of the same phylogenetic group. The pX1.0 plasmid was shown to replicate stably in E. coli with a plasmid DNA per total DNA ratio corresponding to approximately 3-9 plasmids per chromosome depending on the growth phase of the host. Through conjugation, pX1.0 was able to self-transfer horizontally into an isogenic strain of E. coli as well as into two additional species belonging to the family Enterobacteriaceae. Our results demonstrate the immediate applicability of recent advances made within the field of synthetic biology for designing and constructing DNA systems, previously existing only in silica.     

Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from Gram-positive organisms

A 2.6 kb plasmid, named pBBR1, was isolated from Bordetella bronchiseptica S87. After insertion of an antibiotic resistance marker, this plasmid could be transferred into Escherichia coli, Bordetella pertussis, B. bronchiseptica, Vibrio cholerae, Rhizobium meliloti, and Pseudomonas putida by transformation or conjugation. Conjugation was possible only when the IncP group transfer functions were provided in trans. As shown by incompatibility testing, pBBR1 does not belong to the broad-host-range IncP, IncQ or IncW groups. DNA sequence analysis revealed two open reading frames: one was called Rep, involved in replication of the plasmid, and the other, called Mob, was involved in mobilization. Both the amino-terminal region of Mob and its promoter region show sequence similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. In spite of these sequence similarities, pBBR1 does not replicate via the rolling-circle mechanism commonly used by small Gram-positive plasmids. We therefore speculate that pBBR1 may combine a mobilization mechanism of Gram-positive organisms with a replication mechanism of Gram-negative organisms. Determination of the plasmid copy number in E. coli and B. pertussis indicated that pBBR1 has a rather high copy number, which, in conjunction with its small size and broad host range, renders it particularly interesting for studies of broad-host-range replicons and for the development of new cloning vectors for a wide range of Gram-negative bacteria.     

Characteristics of new pKMR-plasmids detected in natural strains of Enterobacteriaceae

pKMR-plasmids controlling the antibiotic resistance and adhesive properties were isolated from clinical strains of E. coli O26 and O124, and Sh. sonnei. Two of them, i.e. pKMR 207 and pKMR 208 were conjugative. On conjugation they jointly transferred the features of the antibiotic resistance and capacity for production of the colonization antigen. The studies on transformation of E. coli K 12 802 with the plasmid DNA of E. coli O124 showed that the antibiotic resistance and colonization properties in E. coli O124 were controlled by the nonconjugative plasmid pKMR 209. It was found that plasmids pKMR 207 and pKMR 208 had the fi(-)-phenotype. None of the plasmids allotted the host cells sensitivity to the donor specific phages of the incompatibility groups F, N, P, W, and I. Probably, the plasmids did not belong to these incompatibility groups. When the cells of E. coli K 12 802 were transformed with the plasmid DNA of the wild strain to the hemolytic strain of S. typhimurium with multiple antibiotic resistance, 3 pKMR 210 plasmids with different markers of the antibiotic resistance were detected in the transformants. One of the plasmids controlled both the drug resistance and the capacity for production of hemolysin. The ability of the detected pKMR plasmids to inhibit fertility and relation to the donor specific phages was studied.     

Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-like plasmids

Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors in Escherichia coli CV601 and Pseudomonas putida UWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ oriV probe in several P. putida UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ repB or IncQ oriT probes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different EcoRV restriction pattern, were selected for a more thorough plasmid characterization after transfer into E. coli K-12 strain DH5alpha by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into Acinetobacter sp., Ralstonia eutropha, Agrobacterium tumefaciens, and P. putida, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.     

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.     

Pervasive sign epistasis between conjugative plasmids and drug-resistance chromosomal mutations

Multidrug-resistant bacteria arise mostly by the accumulation of plasmids and chromosomal mutations. Typically, these resistant determinants are costly to the bacterial cell. Yet, recently, it has been found that, in Escherichia coli bacterial cells, a mutation conferring resistance to an antibiotic can be advantageous to the bacterial cell if another antibiotic-resistance mutation is already present, a phenomenon called sign epistasis. Here we study the interaction between antibiotic-resistance chromosomal mutations and conjugative (i.e., self-transmissible) plasmids and find many cases of sign epistasis (40%)--including one of reciprocal sign epistasis where the strain carrying both resistance determinants is fitter than the two strains carrying only one of the determinants. This implies that the acquisition of an additional resistance plasmid or of a resistance mutation often increases the fitness of a bacterial strain already resistant to antibiotics. We further show that there is an overall antagonistic interaction between mutations and plasmids (52%). These results further complicate expectations of resistance reversal by interdiction of antibiotic use.     

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.     

Vesicle-mediated transfer of virulence genes from Escherichia coli O157:H7 to other enteric bacteria

Membrane vesicles are released from the surfaces of many gram-negative bacteria during growth. Vesicles consist of proteins, lipopolysaccharide, phospholipids, RNA, and DNA. Results of the present study demonstrate that membrane vesicles isolated from the food-borne pathogen Escherichia coli O157:H7 facilitate the transfer of genes, which are then expressed by recipient Salmonella enterica serovar Enteritidis or E. coli JM109. Electron micrographs of purified DNA from E. coli O157:H7 vesicles showed large rosette-like structures, linear DNA fragments, and small open-circle plasmids. PCR analysis of vesicle DNA demonstrated the presence of specific genes from host and recombinant plasmids (hly, L7095, mobA, and gfp), chromosomal DNA (uidA and eaeA), and phage DNA (stx1 and stx2). The results of PCR and the Vero cell assay demonstrate that genetic material, including virulence genes, is transferred to recipient bacteria and subsequently expressed. The cytotoxicity of the transformed enteric bacteria was sixfold higher than that of the parent isolate (E. coli JM109). Utilization of the nonhost plasmid (pGFP) permitted the evaluation of transformation efficiency (ca. 10(3) transformants microg of DNA(-1)) and demonstrated that vesicles can deliver antibiotic resistance. Transformed E. coli JM109 cells were resistant to ampicillin and fluoresced a brilliant green. The role vesicles play in genetic exchange between different species in the environment or host has yet to be defined.     

Chromate-resistance genes in plasmids from antibiotic-resistant nosocomial enterobacterial isolates

The presence of chromate-resistance genes in enterobacteria was evaluated in a collection of 109 antibiotic-resistant nosocomial isolates from nine major cities in México. Results were compared with the presence of mercury-resistance genes. Susceptibility tests showed that 21% of the isolates were resistant to chromate (Cr(R)), whereas 36% were resistant to mercury (Hg(R)). Cr(R) levels were high in Klebsiella pneumoniae (61%), low in Enterobacter cloacae (12%) and Escherichia coli (4%), and null in Salmonella sp. isolates. Colony hybridization demonstrated that the majority of metal-resistant isolates hybridized with chrA gene (87% of Cr(R) isolates), encoding a CHR transporter homologue, and merA gene (74% of Hg(R) isolates), encoding MerA mercuric reductase, suggesting that most isolates expressed these widespread metal-resistance systems. Southern blot hybridization of Cr(R) isolates showed that plasmids of 80, 85, and 95 kb from K. pneumoniae isolates, and of 100 kb from an E. cloacae isolate, contained chrA-related sequences. These plasmids belonged to IncN or IncP incompatibility groups, and conferred Cr(R), as well as multiple antibiotic resistance, when transferred by conjugation to an E. coli standard strain. These data indicated that Cr(R) genes may be distributed among clinical enterobacteria via conjugative plasmids, probably by coselection with antibiotic-resistant genes.     

A specific DNA probe for the identification of Campylobacter jejuni

A 6.1 kb DNA probe for the human enteric pathogen Campylobacter jejuni has been isolated from a genomic library constructed in the plasmid vector pBR322 in Escherichia coli. The DNA sequence used as a probe was identified from recombinant plasmids following immunological screening of transformants using polyclonal antisera to whole cells and to membrane antigens of C. jejuni. Restriction endonuclease fragment mapping of C. jejuni DNA inserts from three of the recombinant plasmids showed an overlapping DNA fragment. One of these recombinant plasmids, when used as a DNA probe in Southern hybridization, specifically hybridized with chromosomal DNA from all of the C. jejuni strains tested. Hybridization was not detected at high stringency between the DNA probe and chromosomal DNA from any other Campylobacter species tested except weakly with the chromosomal DNA of strains of Campylobacter coli. Hybridization was also not detected with chromosomal DNA from a range of other enteric bacteria likely to be encountered in faecal material. The intensity of hybridization with C. coli could be increased by reducing the stringency of hybridization.     

Infectious plasmid resistance and efflux pump mediated resistance

Various bacterial plasmids can be eliminated from bacterial species cultured as pure or mixed bacterial cultures by non-mutagenic heterocyclic compounds at subinhibitory concentrations. For plasmid curing, the replication should be inhibited at three different levels simultaneously: the intracellular replication of plasmid DNA, partition and intercellular transconjugal transfer. The antiplasmid action of the compounds depends on the chemical structure. The targets for antiplasmid compounds were analysed in detail. It was found that amplified extrachromosomal DNA in the superhelical state binds more drug molecules than does the linear or open-circular form of the plasmid or the chromosome, without stereospecificity which leads to functional inactivation of the extrachromosomal genetic code. Plasmid elimination also occurs in ecosystems containing numerous bacterial species simultaneously, but the elimination of antibiotic resistance-encoding plasmids from all individual cells of the population is never complete. The medical significance of plasmid elimination in vitro is, it provides a method to isolate plasmid-free bacteria for biotechnology without any risk of mutations, and it opens up a new perspective in rational drug design against bacterial plasmids. Hypothetically, the combination of antiplasmid drugs and antibiotics may improve the effectivity of antibiotics against resistant bacteria; therefore, the results cannot be exploited until the curing efficiency reaches 100%. Inhibition of the conjugational transfer of antibiotic resistance plasmids can be exploited to reduce the spreading of these plasmids in ecosystems.     

Self-transmissible plasmid in Zymomonas mobilis carrying antibiotic resistance.

The cryptic plasmid pRUT41 from Zymomonas mobilis was examined for its biological properties. This plasmid was found to be conjugally transferred from Z. mobilis CP4 to Escherichia coli BM21 and to carry genes for antibiotic resistance (gentamicin, kanamycin, and streptomycin). Covalently closed circular plasmid DNA was isolated from eight transconjugants of E. coli BM21. These plasmids were identical in mobility on agarose gels and exhibited the same restriction patterns as the native pRUT41 plasmid isolated from Z. mobilis. The plasmid location of the antibiotic resistance genes was further confirmed by transforming E. coli BM21 with isolated pRUT41 plasmid from strain CP4 and with plasmids from the transconjugants of BM21. Resistance to streptomycin, kanamycin, and gentamicin was tightly linked and transferred together in all cases.     

Self-transmissible plasmid in Zymomonas mobilis carrying antibiotic resistance

The cryptic plasmid pRUT41 from Zymomonas mobilis was examined for its biological properties. This plasmid was found to be conjugally transferred from Z. mobilis CP4 to Escherichia coli BM21 and to carry genes for antibiotic resistance (gentamicin, kanamycin, and streptomycin). Covalently closed circular plasmid DNA was isolated from eight transconjugants of E. coli BM21. These plasmids were identical in mobility on agarose gels and exhibited the same restriction patterns as the native pRUT41 plasmid isolated from Z. mobilis. The plasmid location of the antibiotic resistance genes was further confirmed by transforming E. coli BM21 with isolated pRUT41 plasmid from strain CP4 and with plasmids from the transconjugants of BM21. Resistance to streptomycin, kanamycin, and gentamicin was tightly linked and transferred together in all cases.