Broth Mating Research Articles

Conjugal transfer of recombinant transposon Tn916 from Escherichia coli to Bacillus stearothermophilus

A gene for thermostable amylase has been inserted at the BstXI site of Tn916. Mating experiments demonstrated that unlike Tn916, the recombinant transposon, designated Tn916A, could transfer from Escherichia coli to Bacillus stearothermophilus BR219 in broth matings, resulting in chromosomal integration of the transposon and expression of the amylase at significant levels.

Transfer of plasmids and chromosomal genes amongst subspecies ofBacillus thuringiensis

The plasmids pBC16 and pC194 fromBacilus thuringiensis subsp.israelensis strains A084-16-194 were transferred to 25 subspecies ofB. thuringiensis by a conjugation-like process using broth mating technique. The frequencies of transfer varied considerably between different mating pairs, ranging from 1.1×10−9 to 9.8×10−5. Additionally, chromosomal transfer could also be demonstrated in tenB. thuringiensis subspecies with very low frequencies (4.3×10−9 to 3.7×10−7). The intersubspecies matings within a group of eight subspecies strains gave higher frequencies of transfer than the matings between the subspecies. Furthermore, the results indicated that the capability to transfer plasmids among these various subspecies did not depend on the presence of large plasmid.

Mutants of Enterococcus faecalis deficient as recipients in mating with donors carrying pheromone-inducible plasmids

From Enterococcus faecalis cells containing random chromosomal insertions of Tn 916, strains resistant to a lytic phage were selected and tested for conjugal mating ability. The phageresistant strains all showed decreased recipient ability (Con −) in broth matings with donors carrying pheromone-inducible plasmids. These strains were normal with respect to donor ability in broth matings and recipient ability in filter matings. The data suggest that the mutants are deficient in the binding substance receptor for the pheromone-induced donor aggregation substance. These mutants contained multiple insertions of Tn 916, and none of the individual insertions from the mutant strains were capable of generating the phenotype. Analysis of cell envelope lipoteichoic acids and protein revealed changes in both associated with the Con − phenotype.

Transfer of transposon Tn916 from Bacillus subtilis to Thermus aquaticus

Broad host range conjugating transposon Tn916 has been introduced into the extreme thermophile Thermus by transposon transformation and transposition into the Bacillus subtilis chromosome followed by broth mating with Thermus aquaticus ATC27634. Tetracycline resistant Thermus transconjugants were obtained at a frequency of 1.4×10−7 per donor and 1.2×10−7 per recipient. Transposon transfer from Thermus to Bacillus subtilis was also demonstrated in similar broth matings. Transfer characteristics were consistent with the conjugation mechanism described for Tn916 in mesophiles.

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

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.

Transferable or mobilisable antibiotic resistance in Shigella dysenteriae types 1, 2, 3, 4, 6 and 7 isolated in Ethiopia during 1974-85.

A total of 199 Shigella dysenteriae isolates resistant to one or more antibiotics and belonging to types 1, 2, 3, 4, 6 and 7 was examined by one-step broth mating with Escherichia coli K12 and, if non-conjugative, additionally by triparental crosses with the conjugative plasmids X and delta. Of the S. dysenteriae type 1 (Shiga's bacillus) strains, 96% harboured conjugative plasmids. During 1974-79, isolates of Shiga's bacillus carried conjugative plasmids coding for ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphonamide, tetracycline) resistance that transferred at low frequencies (less than 10(-4). After 1980, however, about 50% of isolates of Shiga's bacillus with this resistance (R)-type carried conjugative plasmids that transferred at high frequencies (10 degrees-10(-2)) and that expressed the ACT determinant only. The introduction of a new clone of Shiga's bacillus into Ethiopia in 1980 is suspected. Conjugative plasmids coding for SSuT resistance were detected in S. dysenteriae types 2, 3, and 4. Non-conjugative SSu determinants in S. dysenteriae type 3 were mobilised by conjugative plasmids X and delta. R-type CSSuT in strains of types 2 and 7, and R-type ACST in type-3 strains were neither transferable nor mobilisable and are probably determined chromosomally.

Introduction of the Streptococcus faecalis transposon Tn 916 into Bacillus thuringiensis subsp. israelensis

The conjugative Streptococcus faecalis transposon Tn 916 was introduced into Bacillus thuringiensis subsp. israelensis by filter matings with S. faecalis. B. thuringiensis transconjugants resistant to tetracycline (Tet r) were detected at a frequency of approximately 7.0 × 10 −7 per recipient cell during filter matings, whereas transfer of Tn 916 was not observed in broth matings. The Tet r phenotype in subsp. israelensis was stable in the absence of antibiotic selection. Southern hybridization analysis revealed that Tn 916 had inserted into several different sites on the B. thuringiensis subsp. israelensis chromosome but insertion into plasmid DNA was not observed. Movement of Tn 916 was demonstrated when Tet r B. thuringiensis transconjugants were mated with isogenic recipients. Southern hybridizations, however, showed that the resulting Tet r isolates contained Tn 916 junction fragments that were nearly identical to the donor, suggesting that this movement resulted from transfer of chromosomal DNA from donor to recipient or from a fusion of mating cells, rather than conjugative transposition of the Tn element.

Capacity of aquatic bacteria to act as recipients of plasmid DNA.

A total of 68 gram-negative freshwater bacterial isolates were screened for their ability to receive and express plasmids from Pseudomonas aeruginosa donors. The plate mating technique identified 26 of the isolates as recipient active for the self-transmissible wide-host-range plasmid R68; 10 were recipient active by R68 mobilization for the wide-host-range plasmid cloning vector R1162. Frequencies of transfer were compared by using three conjugal transfer procedures: broth, plate, and filter mating. For every recipient tested, a solid environment was superior to a liquid environment for transfer. The broth mating technique failed to demonstrate R68 transfer in 63% of the recipient-active isolates. Filter mating, in general, yielded the highest transfer frequencies. The more-rapid plate mating procedure, however, was just as sensitive for testing the capacity of natural isolates to participate in conjugal plasmid transfer.

In situ conjugal transfer of plasmid-encoded multiple antibiotic resistance in Escherichia coli isolated from heat

Three different Escherichia coli strains harboring transferable R-plasmids were isolated from raw meats. All three isolated were capable of R-plasmid transfer in ground meat, summer sausage, and milk. Transfer frequencies in ground beef and milk were significantly higher than in summer sausages and that observed under standard broth matings at both 25°C and 30°C. In situ plasmid transfer decreased with decreasing temperatures. All isolates were able to transfer multiple antibiotic resistance at 10°C but not at 5°C.

Transfer functions of the Streptococcus faecalis plasmid pAD1: organization of plasmid DNA encoding response to sex pheromone.

The conjugative plasmid pAD1 (59.6 kilobases) of Streptococcus faecalis shows a 10,000-fold increase in transfer frequency following induction by the sex pheromone cAD1. Mutagenesis of the plasmid with transposon Tn917 was undertaken to determine the region(s) of pAD1 required for the mating response. The relevant genetic material was found to be distributed over a 31.2-kilobase contiguous region of the plasmid. Although insertions in two previously identified regions (traA and traB) exhibited increased transfer frequencies, insertions in five new regions (D, E, F, G, and H) decreased the ability of pAD1 to transfer. Insertions in region H allowed the cells to form visible mating aggregates, but the plasmid transfer frequency was decreased to levels below detection during a 1-h broth mating. Mutants with mutations in region G were able to form aggregates; however, insertions in regions D, E, and F prevented aggregate formation. Insertions in region C decreased the sensitivity of the cell to exogenous cAD1 and exhibited increased activity of the pheromone inhibitor iAD1. Surface protein profiles produced by a number of these mutants were examined, and in some cases were found to be different from those of the wild type. A map showing the various regions is presented, and related aspects of the regulation of the pAD1 mating response are discussed.

R-Plasmid transfer in non-sterile agricultural soil

Summary R-plasmid transfer via conjugation in Escherichia coli was investigated in non-sterile agricultural soil to better understand conjugation in the soil environment. The number of transconjugants recovered from the soil was usually 10 to 57-fold lower than the number of transconjugants detected in broth matings. Also, R-plasmid transfer was higher in aerobically incubated soil at 22 and 30°C, than anaerobically incubated soil. R-plasmid transfer was influenced by soil moisture, but not by soil pH in the range from 6.15 to 7.45.

A procedure to increase R-plasmid transfer during conjugation in Escherichia coli

An increased frequency (5.4-fold at 22°C after 5 h using donor to recipient ratio of 1:1) of R-plasmid transfer in Escherichia coli was observed in nutrient broth supplemented with 0.02% (w/v) agarose, as compared to standard broth matings. Similar trends were also observed with donor to recipient cell ratios of 1:10 and 1:100 under identical incubation conditions. The use of this technique to increase transconjugant numbers in conjugation experiments may be beneficial in situations where the frequency of plasmid transfer is normally low (10 −8).

A possible role of R plasmids in bacterial permeability for beta-lactam antibiotics.

Four strains of clinical isolates of Serratia marcescens (13039, 13090, 13093, 14093) harboring R plasmids were highly resistant to ampicillin (ABPC) and cephaloridine (CER). With elimination of R plasmids from these strains by acriflavine treatment, ABPC-resistance levels of these strains were markedly reduced. Reduction of CER-resistance levels was also demonstrated in strains 13039 and 13093, but not in strains 13090 and 14093. The permeability of the former strains for CER was also decreased, but not in the latter strains. At the same time, beta-lactamase activity of these strains also almost completely disappeared when the R plasmids were eliminated. By broth matings with these strains. The recipient strains of S. marcescens 13031 (rif), Escherichia coli K-12 (rif), and E coli 15046 (rif) all acquired a high permeability barrier against CER with inheritance of the R plasmids from strains 13039 and 13093, but not from strains 13090 and 14093. The transconjugant of strain 13031 that inherited R plasmid 13093 was resistant not only to CER but also to cefazolin, cephalothin, and cephalexin. Its permeability to these antibiotics was significantly lower than that of the original strain. This fact suggest the possibility that the R plasmid from strain 13093 may be involved not only in production of beta-lactamases, but also in regulation of bacterial permeability for cephalosporins.

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.

Acceptance by Erwinia spp. of R plasmid R68.45 and its ability to mobilize the chromosome of Erwinia chrysanthemi.

R plasmid R68.45 was transferred in broth matings from Escherichia coli to strains of Erwinia amylovora, E. carotovora subsp. atroseptica, E. chrysanthemi, and E. herbicola (Enterobacter agglomerans); the frequency of transfer ranged from 2 x 10(-8) to 5 x 10(-4) per input donor cell depending on the bacterial species. The drug resistance markers tet(+), amp(+), and kan(+) were stable in these Erwinia species. Transconjugants of Erwinia spp., but not of the wild-type parent Erwinia strains, acquired levels of antibiotic resistance (tetracycline, 50 mug/ml; ampicillin, 200 mug/ml; kanamycin 200 mug/ml) similar to those of the donor R68.45-bearing strain of Escherichia coli. Erwinia transconjugants (with one exception of E. carotovora subsp. atroseptica) were donors of the antibiotic resistance markers; the frequency of transfer was consistently higher with an E. coli strain than with Erwinia spp. as recipients, and when matings were done on a solid surface (membranes) rather than in liquid. Transfer of chromosomal markers ade(+), gal(+), gtu(+) (utilization of galacturonate), his(+), leu(+), lys(+), thr(+), and trp(+) occurred in crosses between E. chrysanthemi strains harboring R68.45 and appropriate recipient strains; the frequency of transfer ranged from 9.0 x 10(-8) to 2.0 x 10(-6) depending on the selective marker. Analysis of the coinheritance of unselected markers among various classes of recombinants revealed linkage between thr-leu-lys-ade and between trp and his, thus confirming earlier findings with the Hfr-type donor cells. Since R68.45 mobilized an array of chromosomal markers in the wild-type as well as genetically marked strains of E. chrysanthemi, the system, used in conjunction with the existing Hfr strains, should provide a useful tool to study the genetics of plant pathogenicity of this bacterial species. In contrast to E. chrysanthemi, R68.45 did not mobilize chromosomal markers ilv(+), his(+), rbs(+), ser(+), and thr(+) in E. amylovora EA178.

Self-transfer and genetic recombination mediated by P, the sex factor of Vibrio cholerae.

Vibrio cholerae cells, infected with the sex factor P, produce discrete, plaque-like clearings when plated on lawns of P(-) cells. We investigated the nature of these clearings and conclude that they are probably sites of active mating. We developed a quantitative assay for P(+) cells and used it to study the kinetics of sex factor spread in broth cultures. Both established and newly infected donor populations were efficient sex factor donors, indicating that P is not self-repressed. We also investigated the kinetics of recombinant formation in broth matings. In 1-hr matings, we routinely found recombination frequencies of 10(-6) per donor cell. Kinetic studies of recombinant formation showed that the markers tested all appeared at early times. Thus P, the V. cholerae sex factor, seems to resemble F in its transfer properties.

Transferable drug resistance in Pseudomonas aeruginosa.

Three strains of Pseudomonas aeruginosa were demonstrated to transfer double-drug resistance by conjugation to a P. aeruginosa recipient at frequencies of 10(-4) to 10(-2) per recipient cell. Two of the three strains also transferred to Escherichia coli at frequencies which were 10(3)- to 10(5)-fold lower, but the third strain could not be demonstrated to do so. The latter strain, however, conferred maleness on the Pseudomonas recipient. The transfer of streptomycin resistance was associated with the acquisition of streptomycin phosphorylase by both P. aeruginosa and E. coli recipients. Maximal broth mating frequencies were obtained with nonagitated cultures less than 1 mm in depth. A pyocine selection system based on donor sensitivity and recipient resistance is described and appears to have future value as a generalized selective device for use after matings.