In vitro and in situ conjugal transfer of an R-plasmid among enterobacteriaceae species isolated from meat samples

Abstract An R-plasmid donor strain of Escherichia coli isolated from a meat sample was mated with potential bacterial recipients belonging to the family Enterobacteriaceae isolated from ground beef and chicken samples. Nine different strains having different plasmid profiles were used as recipients in broth conjugation experiments. The recipients were identified as Enterobacter cloacae, Hafnia alvei, E. coli, Klebsiella pneumoniae and K. oxytoca . Of 1250 ampicillin resistant, tetracycline sensitive colonies tested, the incidence of recipients was estimated to be 3% (in ground beef) and 11% (in chicken) of the bacteria population. Two of the recipients, E. coli and K. Oxytoca also behaved as donors and transferred their R-plasmids to a laboratory recipient strain of E. coli K12-711. In vitro R-plasmid transfer frequencies varied within a wide range, from 10⁻² to 10⁻⁷ among recipients. Generally, frequencies of plasmid transfer were highest at 30°C and declined with decreasing temperature. Three of the recipient isolates, E. cloacae, H. alvei and E. coli displayed transfer of R-plasmids at 10°C in broth matings. Similar trends in R-plasmid transfer frequencies also were observed under in situ mating conditions in raw ground beef and pasteurized milk samples.     

一步法生产1,5-戊二胺谷氨酸棒杆菌基因工程菌的构建

1,5-戊二胺是一种重要的化工原料,发酵法生产1,5-戊二胺是一条新颖且具有潜在竞争力的生产途径。以蜂房哈夫尼菌(Hafnia alvei)AS1.1009基因组为模板,通过PCR扩增,得到大小约为2.2kb的赖氨酸脱羧酶基因ldc。以大肠杆菌(Escherichia coli)/谷氨酸棒杆菌(Corynebacterium glutamicum)穿梭质粒pXMJl9为载体,将扩增得到的目的基因片段克隆至谷氨酸棒杆菌C.glutamicum TK260512,获得重组菌株C.glutamicum TK260512/pXMJl9-ldc.在摇瓶发酵水平上,通过IPTG诱导ldc基因的表达,并采用反相高效液相色谱方法测定了发酵液中1,5-戊二胺的含量,结果显示,经36h发酵,工程菌C.glutamicum TK260512/pXMJ19-ldc的1,5-戊二胺产量为0.96g/L。     

Virulence plasmid pJM1 prevents the conjugal entry of plasmid DNA into the marine fish pathogen Vibrio anguillarum 775.

Studies involving the introduction of cloned homologous genes into Vibrio anguillarum revealed that several plasmids could not be conjugally introduced into V. anguillarum 775(pJM1), but were transmissible to the pJM1-cured derivative H775-3. Recombinant pBR322 plasmids containing V. anguillarum genomic DNA inserts were mobilized from Escherichia coli donors, using pRK2013, into V. anguillarum H775-3 recipients at frequencies of 10(-6) to 10(-5) per recipient. When identical matings were performed with V. anguillarum 775(pJM1) recipients, the infrequent exconjugants recovered carried the pBR322-based plasmid but had lost the large virulence plasmid pJM1. Similar studies were carried out with plasmid RP4 and with recombinant derivatives of the closely related broad-host-range plasmid pRK290. While RP4 was transmissible from E. coli to V. anguillarum H775-3 at frequencies of 6.7 x 10(-2) per recipient, transmission to V. anguillarum 775(pJM1) recipients occurred at frequencies of only 2.5 x 10(-7). When pRK290 contained V. anguillarum DNA inserts, the only exconjugants recovered had lost pJM1, or contained pJM1 and a deletion derivative of the recombinant pRK290 plasmid where all of the DNA insert had been deleted. The use of Dam-, Dcm-, or EcoK- methylation-deficient E. coli donor strains failed to result in appreciable numbers of V. anguillarum 775(pJM1) exconjugants that contained the desired transferred plasmids. Following UV mutagenesis, a derivative of V. anguillarum 775(pJM1) was isolated that would accept conjugally transferred plasmid DNAs at frequencies similar to those observed when using V. anguillarum H775-3 recipients. These data suggest that virulence plasmid pJM1 mediates a restriction system that prevents conjugal transmission of plasmid DNA from E. coli donors into V. anguillarum 775(pJM1). This putative restriction system appears not to be directed towards Dam-, Dcm-, or EcoK-methylated DNA, and appears not to involve a Type II restriction endonuclease.     

Genetic transfer of pAP38, pAP39, and pAP41 plasmids into E. coli, Erwinia, and Hafnia strains

Genetic transfer factors pAP38, pAP39 and pAP41 can be transferred to E. coli, both typed and untypecd, as well as to Erwinia and Hafnia strains, with different frequency (10(-2) to 10(-8)). The transconjugates thus obtained possess donor activity and can transfer the factors they have received to recipient E. coli strains. After transfer these factors are stably maintained in a new host for at least 10 days. Under the action of ethidium bromide or elevated temperature the elimination of the transfer factors from host bacteria is observed. The studied transfer factors pAP38, pAP39 and pAP41 (F-like factors) are plasmids fi+.     

Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria

The mobilizable shuttle cloning vectors, pAT18 and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAM beta 1; (ii) an erythromycin-resistance-encoding gene expressed in Gram- and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the multiple cloning site and the lacZ alpha reporter gene of pUC18 (pAT18) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique cloning sites that allow screening of derivatives containing DNA inserts by alpha-complementation in Escherichia coli carrying the lacZ delta M15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E. coli donors. Plasmids pAT18, pAT19 and recombinant derivatives have been successfully transferred by conjugation from E. coli to Bacillus subtilis, Bacillus thuringiensis, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus at frequencies ranging from 10(-6) to 10(-9). The presence of a restriction system in the recipient dramatically affects (by three orders of magnitude) the efficiency of conjugal transfer of these vectors from E. coli to Gram+ bacteria.     

Physical characterization of the plasmid pON5300

Summary The antibiotic resistance plasmid Rldrd19Km-which has spontaneously lost its kanamycin resistance marker, and its derivative pON5300, were analysed using the restriction endonucleases SalI, BamHI, HindIII and EcoRI. The fragment patterns were compared with that of the Rldrd19 and the fragments responsible for kanamycin resistance were found to be missing in Rldrd19Km ^– and pON5300. In these plasmids a 7 Mg/mol EcoRI fragment was observed instead of the D (6.3 Mg/mol) fragment of Rldrd19. Further a new 6 Mg/mol EcoRI restriction fragment was observed in pON5300. Using double digestions it was shown that the new fragment does not carry restriction sites for HindIII, BamHI and SalI endonucleases. The non-homology of the analysed plasmid was proved electron microscopically by heteroduplex techniques. The possibility of amplification in the regulatory region for the expression of R-determinants in pON5300 is discussed.     

Transfer and maintenance of IncP and IncW group plasmids into and between extra-slow-growing Bradyrhizobium japonicum strains

Abstract IncP group plasmid pRL180 was conjugally transferred from Agrobacterium tumefaciens LBA928 into extra-slow-growing (ESG) Bradyrhizobium japonicum strains and between ESG strains, RJ17W and RJ12S. pRL180 was integrated into the chromosome of RJ12S, RJ17W and RJ19FY. ESG strains efficiently transferred pRL180 into Escherichia coli at about a 3 × 10⁻⁵ frequency. IncW group plasmid pTY97 was transferred in intergeneric matings from E. coli into ESG strains at a high frequency of 2.5 × 10⁻³; between RJ17W and RJ12S transfer was about 5.6 × 10⁻⁴. pTY97 was maintained as an R' plasmid in RJ12S. The R' plasmid was resolved upon transfer into E. coli C where only pTY97 was autonomously replicated.     

Shuttle plasmids constructed by the transformation of an Escherichia coli cloning vector into two Deinococcus radiodurans plasmids

An Escherichia coli plasmid that confers kanamycin resistance (Kmr) was inserted into the large Deinococcus radiodurans cryptic plasmids pUE10 and pUE11, yielding pS28 and pS19. The method of insertion involved both in vitro splicing and the natural transformation of D. radiodurans and yielded full-length clones in E. coli of pUE10 and pUE11. Both pS28 and pS19 replicated and expressed Kmr in E. coli and D. radiodurans. In both pS28 and pS19, D. radiodurans plasmid sequences were immediately upstream from the Kmr determinant. Transformation experiments suggested that Kmr expression in D. radiodurans was initiated in upstream D. radiodurans sequences. Restriction maps of pS28 and pS19 showed that each plasmid contained three MraI sites. Both pS28 and pS19 transformed the MraI-producing D. radiodurans strain R1 at low frequencies. D. radiodurans strain Sark, which naturally contains pUE10 and pUE11, was transformed by pS28 and pS19 at much higher frequencies. A Sark derivative that was cured for pUE10 was isolated by screening Sark/pS28 subisolates for loss of kanamycin resistance.     

Structural and functional stability of IncP plasmids during stepwise transmission by trans-kingdom mating: promiscuous conjugation of Escherichia coli and Saccharomyces cerevisiae

In order to establish a gene transfer system for yeast by promiscuous conjugation, we constructed plasmid pAY101 which contained an oriT sequence derived from RK2 (IncP) and the yeast TRP1 and ARS1 genes. A conjugation mixture consisted of yeast Saccharomyces cerevisiae, E. coli harboring pAY101, and E. coli carrying a helper plasmid with mob and tra. In the conjugation mixture a tryptophan-requiring yeast mutant (trp1) was converted to be prototrophic for tryptophan at a frequency of about 10(-5) to 10(-3) per recipient cell. This E. coli-yeast conjugation system required the mob, tra, oriT, TRP1 and ARS1 genes. The mob and tra genes were trans-acting elements as in an E. coli conjugation system. The mobilization was inhibited by nalidixic acid as in a typical bacterial conjugation. DNA analysis indicated that the plasmid pAY101 was transferred from E. coli to S. cerevisiae, and retained its original structure and function in yeast host cells.     

Gene transfer in enteric bacteria through the formation of R-prime plasmids by an RP4: :mini-Mu element.

Gene transfer in seven pathogenic enteric bacteria was studied using an RP4: :mini-Mu element, the plasmid pULB113. From the E. coli K-12 host strain the plasmid could be efficiently transferred to these enteric bacteria, but its transfer back to E. coli K-12 was not as efficient, being detected only in Shigella dysenteriae 1, S. flexneri and the 'smooth' variant of S. sonnei. In these three species, transposition of chromosomal fragments into the plasmid to produce R-prime plasmid was also detected at a frequency of approximately 10(-5). Transposition was random as suggested by the recovery at approximately the same frequency (10(-5) to 10(-6)) of R-primes involving 20 different auxotrophic markers from widely separated chromosomal locations. Formation of R-prime plasmids expressing toxicity in the E. coli K-12 recipient strain was also efficient in S. dysenteriae 1 but the toxin-activity was rapidly lost from these R-primes. In our experiments, the plasmid pULB113 incorporated relatively small amounts of chromosomal DNA as determined by restriction endonuclease digestion. For a Thy+ R-prime that we analyzed, the amount of cloned DNA was approximately 15 kb.     

Transposition of the phage D3112 genome in Escherichia coli cells

It has been demonstrated that the genome of phage D3112 of Preudomonas aeruginosa can be transposed into Escherichia coli chromosome as a component of the hybrid plasmid RP4 TcrKms::D3112. Also, transposition of D3112 from E. coli (D3112) chromosome into RP4 plasmid occurs. The phage stimulates the chromosome mobilizing activity of RP4 plasmid, similar to other transposons. E. coli (RP4::D3112) cells were previously shown to form no colonies at 30 degrees C. Auxotrophic mutants and mutants incapable of utilizing different carbohydrates were found among E. coli clones survived after a long incubation at 30 degrees C (at frequencies approximately 10(-3) - 10(-4). These mutants inherited stably the capability to produce D3112 phage. E. coli auxotrophic mutants have arisen indeed as a consequence of phage integration into the E. coli chromosome, since prototrophic transductants derived from these mutants after their treatment with generalized transducing P1 phage have lost the ability to produce D3112 phage. Clones with mutations in Km or Tc genes of RP4 plasmid, occurring at high frequencies (about 3%) were found after introduction of RP4 into E. coli (D3112). These mutant RP4 plasmids carry insertions of D3112 genomes. Clones of E. coli which lost mutant plasmids still produce D3112 and retain their initial auxotrophic mutations.     

Resistance plasmids in Pseudomonas cepacia 4G9.

Pseudomonas cepacia 4G9 utilizes 2-tridecanone as its sole carbon source and has been shown to be resistant to a variety of antibiotics. To ascertain whether any of these characteristics were plasmid mediated, Escherichia coli HB101 was transformed with plasmid DNA isolated from Pseudomonas cepacia 4G9. No 2-tridecanone-utilizing transformants were obtained. Tetracycline (Tc)- and ampicillin (Ap)- resistant transformants were obtained at a low frequency. Plasmid deoxyribonucleic acid from antibiotic-resistant E. coli HB101 transformants had molecular weights of 2.9 x 10(6) for pJW2 Tcr and 5.4 x 10(6) for pJW3 Apr as determined by electron microscopy. Electron microscopy of plasmid deoxyribonucleic acid from P. cepacia 4G9 revealed a single plasmid species, pJW1 of 1.78 x 10(6). Tetracycline resistance in both P. cepacia 4G9 and E. coli HB101(pJW2) was inducible, whereas ampicillin resistance in P. cepacia 4G9 was constitutive. The level of ampicillin resistance coded by pJW3 was lower in P. cepacia 4G9 than in the transformant E. coli HB101(pJW3).     

Transformation of Escherichia coli in foodstuffs.

The plasmid transfer by transformation of Escherichia coli in 12 foods was investigated under conditions commonly found in processing and storage of food. Transformation occurred in all foods with frequencies of at least 10(-8) when a simplified standard transformation protocol with non-growing cells was applied. Higher rates (ca. 10(-7)) were found in milk, soy drink, tomato and orange juice. Furthermore, E. coli became transformed at temperatures below 5 degrees C, i.e. under conditions highly relevant in storage of perishable foods. In soy drink this condition resulted in frequencies which were even higher than those determined after application of a temperature shift to 37 degrees C. The transformation of cells growing in milk and carrot juice at a constantly kept temperature of 37 degrees C provides evidence for the potential of E. coli to become transformed naturally. With purified DNA frequencies were determined in these substrates of ca. 2.5 x 10(-7) and 2.5 x 10(-8), respectively. Similar frequencies were also obtained in milk containing the crude nucleic acids of homogenised cell suspensions of E. coli (pUC18). Moreover, the release of plasmid DNA from E. coli during food processing and the subsequent uptake of this DNA by growing E. coli cells was shown to take place after homogenisation in milk indicating a horizontal plasmid transfer by transformation of E. coli.     

Development and use of cloning systems for Bacteroides fragilis: cloning of a plasmid-encoded clindamycin resistance determinant.

Chimeric plasmids able to replicate in Bacteroides fragilis or in B. fragilis and Escherichia coli were constructed and used as molecular cloning vectors. The 2.7-kilobase pair (kb) cryptic Bacteroides plasmid pBI143 and the E. coli cloning vector pUC19 were the two replicons used for these constructions. Selection of the plasmid vectors in B. fragilis was made possible by ligation to a restriction fragment bearing the clindamycin resistance (Ccr) determinant from a Bacteroides R plasmid, pBF4;Ccr was not expressed in E. coli. The chimeric plasmids ranged from 5.3 to 7.3 kb in size and contained at least 10 unique restriction enzyme recognition sites suitable for cloning. Transformation of B. fragilis with the chimeric plasmids was dependent upon the source of the DNA; generally 10(5) transformants micrograms-1 of DNA were recovered when plasmid purified from B. fragilis was used. When the source of DNA was E. coli, there was a 1,000-fold decrease in the number of transformants obtained. Two of the shuttle plasmids not containing the pBF4 Ccr determinant were used in an analysis of the transposon-like structure encoding Ccr in the R plasmid pBI136. This gene encoding Ccr was located on a 0.85-kb EcoRI-HaeII fragment and cloned nonselectively in E. coli. Recombinants containing the gene inserted in both orientations at the unique ClaI site within the pBI143 portion of the shuttle plasmids could transform B. fragilis to clindamycin resistance. These results together with previous structural data show that the gene encoding Ccr lies directly adjacent to one of the repeated sequences of the pBI136 transposon-like structure.     

Antimicrobial properties of the Escherichia coli R1 plasmid host killing peptide

The 52 amino acid host killing peptide (Hok) from the hok/sok post-segregational killer system of the Escherichia coli plasmid R1 was synthesized using Fmoc (9-fluorenylmethoxycarbonyl) chemistry, and its molecular weight was confirmed by mass spectroscopy. Hok kills cells by depolarizing the cytoplasmic membrane when it is made in the cytosol. Six microorganisms, E. coli, Bacillus subtilis, Pseudomonas aeruginosa, P. putida, Salmonella typhimurium, and Staphylococcus aureus were exposed to the purified peptide but showed no significant killing. However, electroporation of Hok (200 microgml(-1)) into E. coli cells showed a dramatic reduction (100000-fold) in the number of cells transformed with plasmid DNA which indicates that the synthetic Hok peptide killed cells. Electroporation of Hok into P. putida was also very effective with a 500-fold reduction in electrocompetent cells (100 microgml(-1)). Heat shock in the presence of Hok (380 microgml(-1)) resulted in a 5-fold reduction in E. coli cells but had no effect on B. subtilis. In addition, three Hok fragments (Hok(1-28), Hok(31-52) and Hok(16-52)) killed cells when electroporated into E. coli at 200 microgml(-1) (over 1000-fold killing for Hok(1-28), 50-fold killing for Hok(16-52) and over 1000-fold killing for Hok(31-52)). E. coli cells electroporated with Hok and visualized using transmission electron microscopy showed the same morphological changes as control cells to which Hok was induced using a plasmid inside the cell.     

Stability in Escherichia coli of an antibiotic resistance plasmid from Bacteroides fragilis.

A Bacteroides fragilis strain resistant to penicillin G, tetracycline, and clindamycin was screened for the presence of plasmid deoxyribonucleic acid (DNA). Agarose gel electrophoresis of ethanol-precipitated DNA from cleared lysates of this strain revealed two plasmid DNA bands. The molecular weights of the plasmids were estimated by their relative mobility in agarose gel and compared with standard plasmids with known molecular weights. The molecular weights were 3.40 +/- 0.20 x 10(6) and 1.95 +/- 0.05 x 10(6) for plasmids pBY1 and pBY2, respectively. Plasmid DNA purified by cesium chloride-ethidium bromide gradient centrifugation was used to transform a restriction- and modification-negative strain of Escherichia coli. Penicillin G- and tetracycline-resistant transformants were screened for the presence of plasmid DNA. A plasmid band corresponding to a molecular weight of 1.95 x 10(6) was present in all transformants tested. Curing experiments demonstrated that the plasmid, referred to as pBY22 when present in transformants, was responsible for penicillin G and tetracycline resistance. Plasmid pBY22 was mobilized and transferred to other E. coli strains by plasmid R1drd-19. Stability of pBY22 was examined in different E. coli strains and was shown to be stably maintained in both restriction-negative and restriction-positive strains. Unexpectedly, pBY2 and pBY22 were resistant to digestion by 12 different restriction endonucleases.     

Evidence that the clindamycin-erythromycin resistance gene of Bacteroides plasmid pBF4 is on a transposable element.

We constructed a shuttle vector, pE5-2, which can replicate in both Bacteroides spp. and Escherichia coli. pE5-2 contains a cryptic Bacteroides plasmid (pB8-51), a 3.8-kilobase (kb) EcoRI-D fragment from the 41-kb Bacteroides fragilis plasmid pBF4, and RSF1010, an IncQ E. coli plasmid. pE5-2 was mobilized by R751, an IncP E. coli plasmid, between E. coli strains with a frequency of 5 X 10(-2) to 3.8 X 10(-1) transconjugants per recipient. R751 also mobilized pE5-2 from E. coli donors to Bacteroides uniformis 0061RT and Bacteroides thetaiotaomicron 5482 with a frequency of 0.9 X 10(-6) to 2.5 X 10(-6). The Bacteroides transconjugants contained only pE5-2 and were resistant to clindamycin and erythromycin. Thus, the gene for clindamycin and erythromycin resistance must be located within the Eco RI-D fragment of BF4. A second recombinant plasmid, pSS-2, which contained 33 kb of pBF4 (including the EcoRI-D fragment and contiguous regions) could also be mobilized by R751 between E. coli strains. In some transconjugants, a 5.5-kb (+/- 0.3 kb) segment of the pBF4 portion of pSS2 was inserted into one of several sites on R751. In some other transconjugants this same 5.5-kb segment was integrated into the E. coli chromosome. This segment could transfer a second time onto R751. Transfer was RecA independent. The transferred segment included the entire EcoRI-D fragment, and thus the clindamycin-erythromycin resistance determinant, from pBF4.     

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.