pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora

Abstract

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+, ilv+, rbs+, ser+, thr+; E. chrysanthemi: arg+, his+, ilv+, leu+; E. carotovora subsp. atroseptica: arg+, gua+, leu+, lys+, pur+, trp+; E. carotovora subsp. carotovora: arg+, gua+, leu+, lys+, out+ [export of enzymes], pur+, trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the Pstl and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.

A cloned cellulase gene from Erwinia carotovora subsp. carotovora is expressed in Rhizobium fredii but does not influence nodulation of cowpea

“…Although rhizobia containing the vector alone produced no detectable activity, pHKCl-containing cells of R. fredii, R. meliloti and R. tropici were surrounded by broad zones of clearing (Fig. 1A), an indication of cellulase degradation by extracellular enzymes [10,19]. The observation with R.…”

“…An agar plate assay for cellulase activity was modified after that of Chatterjee et al [19]. Rhizobia were cultured for 2 days in liquid YEM medium at 30°C.…”

Releasing genetically modified organisms: will any harm outweigh any advantage?*

“…Plasmid-mediated conjugation may also be an important way in which chromosomal genes are transported among dierent genera. This is because some plasmids can interact with chromosomes and pick-up' chromosomal genes that can then be moved among bacteria in matings mediated by the plasmids concerned (Chatterjee et al 1985;Holloway 1986). This phenomenon allows chromosomal genes to be inherited without the need for homology and recombination, but it does not obviate the need for the genes concerned to confer a selective advantage to the host if they are to be maintained in the host organisms.…”

The extreme C‐terminus is required for secretion of both the native polygalacturonase (PehA) and PehA‐Bla hybrid proteins in Erwinia carotovora subsp. carotovora

“…The ability to secrete enzymes is essential for virulence, as mutations affecting either their production or secretion cause reduced virulent or avirulent phenotypes (Pirhonen et aL, 1991;Saarilahti et aL, 1992). The pectinases and the cellulases appear to employ a common secretion pathway because in secretion-deficient (Out-) mutants these proteins accumulate in the periplasmic space (Chatterjee et aL, 1985;Murata et aL, 1990;Pirhonen et aL, 1991). Secretion through the Out pathway requires the participation of at least 13 accessory secretion proteins which share a high degree of similarity with the corresponding components of secretion mechanisms recently identified both in the related species E~winia chrysanthemi (Echr) and in many other bacterial genera, such as Aeromonas, Klebsiella, Pseudomonas and Xanthomonas .…”