High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria

Abstract

We report on the mobilization of shuttle plasmids from gram-negative Escherichia coli to gram-positive corynebacteria mediated by P-type transfer functions. Introduction of plasmids into corynebacteria was markedly enhanced after heat treatment of the recipient cells. High-frequency plasmid transfer was also observed when the restriction system of the recipient was mutated. On the basis of our data, we conclude that efficient DNA transfer from gram-negative to gram-positive bacteria, at least to coryneform bacteria, is conceivable in certain natural ecosystems.Conjugal transfer of broad-host-range IncP-type resistance plasmids within gram-negative bacterial species is well known (3, 9, 18). Non-self-transmissible plasmids carrying the appropriate origin of transfer (oriT) can be mobilized by IncP plasmids (28). Recent studies have shown that conjugation is a nonspecific process and accounts for most horizontal gene transfer between even phylogenetically remote organisms (7,13,25,26).To investigate the possibility of conjugal plasmid transfer between Escherichia coli and Corynebacterium glutamicum, we took advantage of a mobilization system previously developed for genetic engineering of a wide range of gramnegative bacteria (23). This strategy is based on the oriT and the transfer (Tra) functions of IncP-type broad-host-range plasmid RP4 (10) and consists of E. coli mobilizing strains and derivatives of conventional E. coli vectors (pSUP vectors [22,23]). A series of E. coli-C. glutamicum shuttle plasmids was constructed based on mobilizable E. coli vector pSUP102 (22). The 10.6-kilobase prototype shuttle vector pECM1 resulted from fusion of pSUP102 to C. glutamicum vector pCV35 (Fig. 1).Transfer of pECMl to C. glutamicum. For mating experiments, plasmid pECM1 was introduced by transformation into mobilizing strain E. coli S17-1 (23). E. coli S17-1 carries an RP4 derivative integrated into the chromosome which provides the transfer functions necessary for mobilization. By using this donor, plasmid pECM1 was transferred by conjugation as previously described (23) to a nalidixic acidresistant derivative of E. coli MM294 (14) at frequencies between 10-1 and 10-2 (Table 1) per donor cell. For conjugal transfer of pECM1 to coryneform recipient strains, donor strain S17-1(pECM1) was grown to the late-exponential phase in LB medium (15) containing 50 ,ug of kanamycin per ml. Recipient strains were grown in LB medium to an optical density at 580 nm of 3 to 4. About 7 x 108 donor and 3.5 x 109 recipient cells, corresponding to a ratio of 1:5, were mixed and pelleted by centrifugation at 20°C for a short time. The mating mixture was then carefully suspended in about 500 ,ll of LB medium and spread onto a 0.45-pum-pore-size cellulose acetate filter (Millipore Corp., Bedford, Mass.) placed on a prewarmed LB plate. After 20 h of incubation at 30°C, the cells were washed from the filter with 1 ml of LB medium and mechanical agitation. Transconjugants were * Corresponding author.

Expression of Vitreoscilla hemoglobin in Gordonia amarae enhances biosurfactant production

“…It is also possible that pSC160 can exist autonomously in G. amarae, although the likelihood for that event is unknown. Plasmid pSC160, however, is derived from plasmid RSF1010, which belongs to the Inc Q/P4 plasmid incompatibility group that can be propagated in a very extensive host range extending to Gram-positive bacteria and eukaryotes [26,34,37]. One of the recombinant strains (from electroporation using a recombinant plasmid with a 5.5 kb insert) was chosen for further studies.…”

Gene Technology and Gene Ecology of Infectious Diseases

“…It also depends on the DNA being resistant to the restriction enzymes in the host cell which break down foreign DNA. In one experiment, brief heat treatment (9 min at 48.5°C) dramatically increased the competence of cells for receiving foreign DNA by 4 orders of magnitude (73). This was attributed to the heat inactivation of restriction enzymes, thereby increasing the success of gene transfer.…”

“…The scope of horizontal gene transfer is essentially the entire biosphere (13,14,36,38,(71)(72)(73)(74)(75)(76)(77). Genes are found to have been transferred, not only among microorganisms and viruses, but among eukaryote species and across traditional Kingdoms of organisms.…”

Novel Channel Enzyme Fusion Proteins Confer Arsenate Resistance

“…Recombination was verified by PCR and sequencing of the junctions between the disruption cassettes and the fosmids. The fosmids were transferred by conjugation of the E. coli S17-1 mobilization strain (gift from Andreas Schlüter) (17) and S. tropica facilitated by oriT (RK2) sequences in the disruption cassettes (18). Resistant S. tropica colonies were restreaked on selective media four to five times before they were grown on nonselective medium.…”