Donor Strains of the Soft-rot Bacterium Erwinia Chrysanthemi and Conjugational Transfer of the Pectolytic Capacity

Donor strains of Erwinia chrysanthemi ICPB EC16, a member of the soft-rot (pectolytic) section of the enterobacterial genus Erwinia, were obtained by chromosomal integration of an F'lac(+) plasmid originating from Escherichia coli. These stable donor strains, selected from an unstable F'lac(+) heterogenote by repeated platings of single Lac(+) colonies on lactose minimal agar, do not segregate (as does the parent F'lac(+) heterogenote) into Lac(-) or F(-) clones, in either the presence or absence of acridine orange. One representative donor strain (from the 12 that have been selected) has been examined in more detail; it can transfer ade(+), gal(+), gtu(+) (utilization of galacturonate), his(+), lac(+), leu(+), lys(+), mcu(+) (multiple carbohydrate utilization), pat(+) (production of polygalacturonic acid trans-eliminase), thr(+), and trp(+) in a polarized manner to appropriate recipient strains of E. chrysanthemi; the frequencies of ade(+), leu(+), and thr(+) transfer were higher than those of the other markers tested to date. This donor strain transfers lac(+) genes during a 6-h mating on membranes; most of the Lac(+) recombinants are donors of chromosomal markers. The kinetics of entry as well as the frequencies of transfer of chromosomal markers indicate that thr(+) and leu(+) enter the recipient as proximal markers and that lac(+) enters as a distal marker. Analysis of the recombinants demonstrates close linkage between thr and leu, ade and thr, his and pat, and his and trp loci. The results suggest that the integration of F'lac(+) into the chromosome of E. chrysanthemi has occurred at a region adjacent to the leu-thr loci, and that the chromosome is transferred in the following sequence: origin----leu--thr--ade--lys--mcu--pat--his--trp--gal--gtu--lac--F. Plant-tissue maceration occurs in Pat(+) recombinants and not in Pat(-) recombinants, even though both form another pectolytic enzyme, hydrolytic polygalacturonase. This genetic evidence supports the idea that the E. chrysanthemi polygalacturonic acid trans-eliminase plays an essential role in bringing about plant-tissue maceration.