Naphthalene and Donor Cell Density Influence Field Conjugation of Naphthalene Catabolism Plasmids

We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur.     

Conjugation mapping of Pseudomonas mendocina bacteria

Donor strains of the Hfr type were isolated using plasmid pRK2013 with transposons Tn10 and Tn5 as a chromosome-mobilizing factor. The isolated strains were shown to promote transfer of donor chromosome from different origins in different directions during isogenic matings of Pseudomonas mendocina bacteria. The created collection of donors and polyauxotrophic recipient bacteria permitted mapping 26 genetic determinants on the bacterial chromosome and identifying the genome of these microorganisms as a circular DNA molecule.     

Conjugational Mapping of Bacteria Pseudomonas mendocina

Donor strains of the Hfr type were isolated using plasmid pRK2013 with transposons Tn10 and B21 as a chromosome-mobilizing factor. The isolated strains were shown to promote transfer of donor chromosome from different origins in different directions during isogenic matings of Pseudomonas mendocina bacteria. The created collection of donors and polyauxotrophic recipient bacteria permitted mapping 26 genetic determinants on the bacterial chromosome and identifying the genome of these microorganisms as a circular DNA molecule.     

Horizontal gene transfer in nematodes: a catalyst for plant parasitism?

The origin of plant parasitism within the phylum Nematoda is intriguing. The ability to parasitize plants has originated independently at least three times during nematode evolution and, as more molecular data has emerged, it has become clear that multiple instances of horizontal gene transfer (HGT) from bacteria and fungi have played a crucial role in the nematode's adaptation to this new lifestyle. The first reported HGT cases in plant-parasitic nematodes were genes encoding plant cell wall-degrading enzymes. Other putative examples of HGT were subsequently described, including genes that may be involved in the modulation of the plant's defense system, the establishment of a nematode feeding site, and the synthesis or processing of nutrients. Although, in many cases, it is difficult to pinpoint the donor organism, candidate donors are usually soil dwelling and are either plant-pathogenic or plant-associated microorganisms, hence occupying the same ecological niche as the nematodes. The exact mechanisms of transfer are unknown, although close contacts with donor microorganisms, such as symbiotic or trophic interactions, are a possibility. The widespread occurrence of horizontally transferred genes in evolutionarily independent plant-parasitic nematode lineages suggests that HGT may be a prerequisite for successful plant parasitism in nematodes.     

Transfer and incorporation of genes controlling beta-D-galactosidase synthesis from Hfr and F' donors of Escherichia coli

Ganesan, Ann K. (Syntex Institute of Molecular Biology, Palo Alto, Calif.), and Boris Rotman. Transfer and incorporation of genes controlling beta-d-galactosidase synthesis from Hfr and F' donors of Escherichia coli. J. Bacteriol. 92:1378-1382. 1966.-Comparisons were made between Hfr(1) and F(13) donors with respect to the frequency of transfer and incorporation of genes controlling beta-d-galactosidase synthesis. The Hfr(1) donor transfers these genes as part of the chromosome, and the F(13) donor transfers them by F-duction. The criterion used for gene transfer was the acquisition by recipient cells of the ability to synthesize the enzyme, beta-d-galactosidase, measured by fluorogenic assays at the single-cell level. The criterion for incorporation was the formation of lac(+) recombinant colonies. It was found that the two types of donor showed the same frequency of gene transfer, but the probability of incorporation was 10-fold higher in F(13) matings than in Hfr(1) matings. In the former, between 46 and 97% of the merozygotes produced recombinant colonies; in the latter, 2 to 6% did so.     

Retrotransfer in Escherichia coli conjugation: bidirectional exchange or de novo mating?

DNA can be transferred among eubacteria and to plants and fungi by related, plasmid-mediated processes collectively referred to as bacterial conjugation. Conjugation occurs between cells in contact with one another and results in the unidirectional delivery of DNA from a bacterial donor to a recipient. Recent experiments that have reexamined the directionality of DNA flow during conjugation have come to different conclusions, some suggesting that genetic material also flows from recipient cells into the donor and that this process, termed retrotransfer, is likewise directed by donor-encoded functions. Given that bacteria are perhaps united with all living creatures by conjugation, the possibility of gene flow into donor bacteria during conjugation raises interesting evolutionary and biocontainment issues. Here we report that plasmid transmission from bacterial recipients to donors is not a donor-mediated event. Movement of genetic material from recipients to donors was inhibited by streptomycin, which does not inhibit the conjugative donor, indicating that retrotransfer requires gene expression in recipients. Furthermore, retrotransfer was reduced in matings mediated by plasmids that encode strong entry exclusion, to a similar degree as matings between two donors. Therefore we suggest that retrotransfer is in fact newly initiated conjugation between transconjugants and donors.     

Effects of different nitrogen sources on lignocellulose degradation and APPL production

Naphthalene utilizing bacteria were isolated from several sites above and below the discharge from a coking plant. The distribution of the bacteria was influenced by the effluent. Of these isolates, 11·4% obtained from the effluent discharge site, contained plasmids. No conjugal transfer of naphthalene utilizing ability was observed in over 1000 matings. Curing and transformation experiments demonstrated that one plasmid pNB33 (101 kb) was concerned with naphthalene catabolism.     

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.     

Studying plasmid horizontal transfer in situ: a critical review

This review deals with the prospective, experimental documentation of horizontal gene transfer (HGT) and its role in real-time, local adaptation. We have focused on plasmids and their function as an accessory and/or adaptive gene pool. Studies of the extent of HGT in natural environments have identified certain hot spots, and many of these involve biofilms. Biofilms are uniquely suited for HGT, as they sustain high bacterial density and metabolic activity, even in the harshest environments. Single-cell detection of donor, recipient and transconjugant bacteria in various natural environments, combined with individual-based mathematical models, has provided a new platform for HGT studies.     

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

From Enterococcus faecalis cells containing random chromosomal insertions of Tn916, strains resistant to a lytic phage were selected and tested for conjugal mating ability. The phage-resistant 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 Tn916, 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.     

Conjugal retrotransfer of chromosomal markers inAzotobacter vinelandii

The IncP plasmids R68.45 and pJB3JI mediate retrotransfer (i.e., transfer of chromosomal markers from the recipient bacterium to the original donor) in homologous matings withAzotobacter vinelandii. Retrotransfer is not an early event inAzotobacter. On the contrary, it begins after genetic transfer in the usual direction, i.e., from the donor bacterium to the recipient, has been taking place for some time. Transfer of chromosomal markers mediated by the RP4/Tn5-Mob system does not undergo retrotransfer. The simplest hypothesis compatible with our results is that IncP plasmids that confer a high chromosome-mobilizing ability promote retrotransfer when transferred to the recipient bacteria.     

Survival of Pseudomonas putida UWC1 containing cloned catabolic genes in a model activated-sludge unit

The possibility of the accidental or deliberate release of genetically engineered microorganisms into the environment has accentuated the need to study their survival in, and effect on, natural habitats. In this study, Pseudomonas putida UWC1 harboring a non-self-transmissible plasmid, pD10, encoding the breakdown of 3-chlorobenzoate was shown to survive in a fully functioning laboratory-scale activated-sludge unit (ASU) for more than 8 weeks. The ASU maintained a healthy, diverse protozoal population throughout the experiment, and the introduced strain did not adversely affect the functioning of the unit. Although plasmid pD10 was stably maintained in the host bacterium, the introduced strain did not enhance the degradation of 3-chlorobenzoate in the ASU. When reisolated from the ASU, derivatives of strain UWC1 (pD10) were identified which were able to transfer plasmid pD10 to a recipient strain, P. putida PaW340, indicating the in situ transfer of mobilizing plasmids from the indigenous population to the introduced strain. Results from plate filter matings showed that bacteria present in the activated-sludge population could act as recipients for plasmid pD10 and actively expressed genes carried on the plasmid. Some of these activated-sludge transconjugants gave higher rates of 3-chlorobenzoate breakdown than did strain UWC1(pD10) in batch culture.     

Survival of Pseudomonas putida UWC1 containing cloned catabolic genes in a model activated-sludge unit.

The possibility of the accidental or deliberate release of genetically engineered microorganisms into the environment has accentuated the need to study their survival in, and effect on, natural habitats. In this study, Pseudomonas putida UWC1 harboring a non-self-transmissible plasmid, pD10, encoding the breakdown of 3-chlorobenzoate was shown to survive in a fully functioning laboratory-scale activated-sludge unit (ASU) for more than 8 weeks. The ASU maintained a healthy, diverse protozoal population throughout the experiment, and the introduced strain did not adversely affect the functioning of the unit. Although plasmid pD10 was stably maintained in the host bacterium, the introduced strain did not enhance the degradation of 3-chlorobenzoate in the ASU. When reisolated from the ASU, derivatives of strain UWC1 (pD10) were identified which were able to transfer plasmid pD10 to a recipient strain, P. putida PaW340, indicating the in situ transfer of mobilizing plasmids from the indigenous population to the introduced strain. Results from plate filter matings showed that bacteria present in the activated-sludge population could act as recipients for plasmid pD10 and actively expressed genes carried on the plasmid. Some of these activated-sludge transconjugants gave higher rates of 3-chlorobenzoate breakdown than did strain UWC1(pD10) in batch culture.     

Factors affecting the kinetics of progeny formation with F′lac in Escherichia coli K12

In matings of F′lac donors with an excess of recipient cells, different donor cells mated at different times. The concentration dependence of mating is incompatible with bimolecular reaction kinetics. In exponentially growing cultures, F′lac transfer from each donor cell continues to occur about once per generation. The establishment of F′lac in some recipient cells may take more than five generations.     

Natural transformation in river epilithon.

Natural transformation was demonstrated in unenclosed experiments incubated in river epilithon. Strains of Acinetobacter calcoaceticus were transformed to prototrophy by either free DNA (lysates) or live donor cells. The sources of transforming DNA and recipient culture were immobilized on filters, secured to stones, and incubated midstream in the river. The transfer frequency generally increased with temperature. No transfer was detected in the river Taff below 10 degrees C. The age of the recipient culture affected the transformation frequencies in situ but did not significantly affect the transfer frequency on laboratory media. Transformation of recipient cultures which had been incorporated into the natural epilithic biofilm and transformation of the plasmid pQM17 in situ were also demonstrated. This study provides the first direct evidence of natural transformation in situ of bacteria incorporated into an indigenous community.     

Transfer of resistance plasmids from Staphylococcus epidermidis to Staphylococcus aureus: evidence for conjugative exchange of resistance.

The ability of Staphylococcus epidermidis to transfer antimicrobial resistance to Staphylococcus aureus was tested by mixed culture on filter membranes. Two of six clinical isolates examined were able to transfer resistance to S. aureus strains 879R4RF, RN450RF, and UM1385RF. Subsequent S.aureus transconjugants resulting from matings with S. epidermidis donors were able to serve as donors to other S. aureus strains at similar frequencies. Cell-free and mitomycin C-induced filtrates of donors and transconjugants showed no plaque-forming ability. Addition of DNase I, citrate, EDTA, calcium chloride, and human sera to mating mixes and agar showed no effect on transfer. Nonviable donor cells were unable to transfer resistance and transfer did not occur at 4 degrees C. Cell-to-cell contact was required since transfer did not occur in broth or when filters of donor and recipient, respectively, were placed back-to-back so cells were not in direct contact. Analysis of DNA from S. epidermidis isolate UM899, its subsequent S. aureus transconjugants, and cured derivatives demonstrated that all resistance markers which transferred resided on plasmids. Mating experiments suggested a central role for the gentamicin plasmid pAM899-1 in the transfer process. It is concluded that our results are consistent with a conjugative transfer of resistance from S. epidermidis to S. aureus analogous to plasmid transfer demonstrated in streptococcal species for plasmids such as pAM beta 1. This represents a novel mechanism for gene exchange among staphylococci.     

Transferable lincosamide-macrolide resistance in Bacteroides.

Inter- and intraspecies transfer of resistance to clindamycin, lincomycin, and erythromycin in the strict anaerobe, Bacteroides, is described. This lincosamide-macrolide resistance was found to be specified by a 27 × 10⁶-dalton plasmid, designated pBF4, originally identified in a clinical Bacteroides fragilis isolate. Transfer of this plasmid to a strain of Bacteroides uniformis was demonstrated to occur by a deoxyribonuclease insensitive process which required cell-to-cell contact. Chloroform sterilized donor cell supernatants or filtrates of donor cells did not mediate resistance transfer. Transfer of the antibiotic resistance and pBF4 plasmid deoxyribonucleic acid (DNA) were always coincident. Drug resistant progeny recovered from such matings were able to transfer the pBF4 plasmid and its associated resistance markers to a suitable B.fragilis recipient strain. Compared to interspecies matings, resistance transfer was 100- to 1000-fold greater between isogenic donor and recipient strains, suggesting the possibility of a host controlled restriction-modification system.     

Tetracycline enhances Tn916-mediated conjugal transfer.

Pregrowth of the donor on medium containing tetracycline increased conjugative transposition of Tn916 and the transposon-dependent mobilization of pC194 19- to 119-fold in matings between Bacillus subtilis and Bacillus thuringiensis subsp. israelensis. Tn916 and pC194 transferred independently under these conditions. When Enterococcus faecalis was the donor and B. thuringiensis subsp. israelensis the recipient, pregrowth in tetracycline increased the conjugative transposition frequency by approximately 15-fold. Tetracycline-enhanced conjugation appeared during matings as short as 3 h in length. Pregrowth in tetracycline did not enhance conjugation in Bacillus sphaericus x B. thuringiensis subsp. israelensis or B. thuringiensis subsp. israelensis x B. subtilis matings. Incorporation of tetracycline into the mating medium, at concentrations that did not inhibit growth of the B. thuringiensis subsp. israelensis recipient, resulted in conjugation frequencies similar to those obtained by pregrowth of the B. subtilis donors in antibiotic-containing medium. The data suggest stimulation of donor function by tetracycline.     

Role of surface exclusion genes in lethal zygosis in Escherichia coli K12 mating

Summary We find that diaminopimelic acid in the recipient membrane is released into the medium during bacterial matings, indicating that membrane damage was inflicted on the recipient by the donor, probably for forming a channel for DNA transfer. When the damage is extensive, as in matings with an excess of Hfr bacteria, the F^- bacteria are killed (lethal zygosis). The transfer of a large amount of DNA in Hfr matings appears to enhance the killing. In analogous F⁺xF^- (Nal^r) matings, on the other hand, killing of F^- bacteria does not occur unless F plasmid transfer is inhibited by a substance like nalidixic acid. The F^- bacteria are killed, suggesting that F plasmids contain genes that express immunity to lethal zygosis in the recipient. For example, bacteria containing surface exclusion-deficient mutants of F plasmids, such as traS ^- and traT ^-, induce lethal zygosis in F^- bacteria and are susceptible to it. Various tra ^- polar mutants that abolish surface exclusion are also susceptible to lethal zygosis when mated with Hfr bacteria. Kinetic experiments indicate that in F⁺ (wild type) x F^- matings, immunity to lethal zygosis is expressed in the F^- recipient within 1/4 division time, whereas a complete expression of surface exclusion requires more than 1 division time. Thus, a complete change in all receptor sites seems to be required for the expression of surface exclusion.     

Factors influencing success of embryo transfer in sheep and goats

The results of embryo transfers from 130 donor Angora goats and 60 sheep of 3 breeds are presented, and the data analyzed to determine some of the sources of variation in success rate. Of all adult donor goats programmed, 94.9% yielded embryos suitable for transfer and 93.4% yielded offspring from the transfers. Donor ewes yielded percentages of 76.8 and 46.7, respectively. Fertilization failure and/or degeneration of embryos in donors prior to flushing accounted for the lower recoveries of viable embryos from sheep, the incidence of both being greater in donors with higher ovulation rates. High ovulation rate of donors also decreased percentage survival of sheep but not goat embryos after transfer. Stage of embryo development, site of transfer (oviduct vs. uterus) or number of embryos transferred (1 vs. 2) per recipient did not affect survival of sheep embryos following transfer to appropriately synchronized recipients. In goats, survival was significantly better with two than with one embryo transferred per recipient. Super-ovulation failure and poor fertilization limited the yield of embryos obtained from donor goats and sheep less than 1 year of age. These could be overcome to some extent by use of progestagen sponge rather than prostaglandin in the superovulation treatment regimen.