Extrachromosomal Elements for in vivo genetic engineering

Abstract

This research was undertaken for the isolation of extra-chromosomal elements (plasmids) and transposons that can be used for in vivo genetic engineering of gram negative bacteria of plant origin. The process of in vivo genetic engineerings, in bacteria, involves the use of transposons. The transposons can only be introduced into a bacterial cell after their insertion into a plasmid, followed by conjugal transfer of the plasmid transposon complex into the bacterial cell. Thus, the process of in vivo genetic engineering depends on two major factors: (1) a suitable transposon that can express its function into a desired bacterium and (2) a suitable plasmid that can be used as a vehicle to carry the transposon into the bacterium. In this regard a wide-host-range plasmid (a plasmid that can be transferred to a large variety of bacteria) serves as a better vehicle. With a view to detect/isolate plasmids and transposons from bacteria associated with plants, we have screened 194 becteria for their resistance to the following eight antibiotics: ampicillin, agrimycin, chloramphenicol, gentamycin, kanamycin, neomycin, streptomycin and tetracycline. The screened bacteria, which included species of plant pathogenic bacteria (e.g. Xanthomonas, Erwinia, Pseudomonas etc.) as well2 as plant symbiotic bacteria (e.g. Rhizobium) exhibited antibiotic resistance in different patterns and combinations. These bacteria were tested for the presence of plasmids (R plasmids) by conjugating them with standard Escherichia coli recipients and observing whether the resistances borne by them were transferable. From the experiments conducted, it appears that the tested bacteria either lack plasmids or contain plasmids that are conjugally non-transferable. At times, an abortive transfer of resistance was observed indicating that the plasmid was although transmitted but could not be stably maintained in Escherichia coli. A great difficulty in detecting the conjugal transmission was ineffective donor elimination in most cases. Some of the plant pathogenic/associated bacteria exhibited the production of bacteriocins (known to be usually associated with the presence of bacteriocinogenic plasmids.) However, none of these bacteria transferred its bacteriocinogenic plasmid by conjugation. The difficulty was again ineffective donor elimination. As a parallel attempt, we have studied whether plasmids of other gram negative bacteria could be used for in vivo genetic engineering of plant pathogenic/associated bacteria. In this regards, twelve R plasmids were isolated from gram negative enteric bacteria, screened for resistance to seven different antibiotics. The R plasmids, that carriedJ different patterns of antibiotic resistance, were then studied for their conjugal transmission to plant pathogenic/associated bacteria including: Xanthomonas, Erwinia, Citrobacter, Rhizobium, Agarobacterium and Pseudomonas. Some of these R plasmids could be conjugally transmitted to as many as four different bacteria of plant origin. However, some could be transmitted to three, two or one plant pathogenic/associated bacteria. The R plasmids were not only transmitted to plant pathogenic/ associated bacteria but also expressed all their resistances in these bacteria. Studies were also made to see whether these R plasmids are stably maintained in their new hosts. Most of them (R plasmids) were found to be either completely or partly stable indicating that they could be used for in vivo genetic engineering of plant pathogenic/assocaited bacteria. Retransfer of the R plasmids from plant pathogenic/ associated bacteria to enteric bacteria could not be detected due to some technical difficulties. Similarly, the R plasmids were not found to carry any transposons during studies conducted so far. Conclusively, we can say that we have isolated some broad-host-range R plasmids that could be used as vehicles for in vivo genetic engineering (by using the available transposons, such as bacteriophage Mu) of plant pathogenic/associated bacteria.