Using mini-Tn5Cm:: , a transposon that allows transcriptional fusions to a promoterless β-glucuronidase gene, a mutant of subsp. SCC3193 deficient in extracellular protease production and soft-rot pathogenicity in plants was isolated. The mutant, designated SCC6004, produced normal levels of pectate lyase, polygalacturonase and cellulase. The region of the transposon insertion was partially sequenced to permit the design of specific oligonucleotide primers to amplify a 2·7 kb fragment from subsp. SCC3193. The DNA sequence of the cloned fragment contained two complete and one partial ORFs. One of the complete ORFs (ORF1) was designated and encodes a secreted protease. The deduced amino acid sequence of PrtW showed a high overall identity of 60-66% to the previously described proteases, but no homology to other proteases isolated from different strains. Downstream from ORF1, a further complete ORF (ORF2) and a partial ORF (ORF3) were found, with deduced peptide sequences that have significant similarity to the Inh and PrtD proteins, respectively, from , which are involved in protease secretion. Gene fusion to the reporter was employed to characterize the regulation of The gene was found to be strongly induced in the presence of plant extracts. The mutant exhibited reduced virulence, suggesting that PrtW enhances the ability of strain SCC3193 to macerate plant tissue.


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