Isolation and Characterization of GFP-Tagged Transposon Mutants of Pectobacterium Carotovorum Subsp. Carotovorum KD100

Bacterial soft rot is a very common disease affecting 50% angiosperm plant orders. Every year substantial (15-35%) amount of yield is lost due to soft rot around the globe. Soft-rot diseases is caused by Pectobacterium, Erwinia and Dickeya genera of bacteria. Transposable elements are the sequence of DNA that move from one location of a genome to another location. This unique feature allows the generation of random mutations to for genome-wide studies. Transposon mutagenesis offers higher mutation frequency and a lower chance of killing the organism than other methods. The mini-transposon, pCKD100 is derived from wild type and has several unique features such as multiple cloning sites, reporter gene, marker genes. The gene for the enzyme that does the transposition (transposase) is located outside the transposon. Expression of GFP and antibiotic resistance genes by pCKD100-derived mutants indicates promoter activity of the truncated host gene. To identify virulence genes in genetically amenable nalidixic resistant P. carotovorum strain KD100, seventy transposon Tn5 mutants from a pool of 5272 were selected based on extracellular protease activity and GFP expression in celery and potato extract-supplemented media. The mutants were altered in their production of exoenzymes and pathogenicity compared to the parent. Five mutants (I2, I3, E6, E9 and E21) produced four times higher pectate lyases (Pel) than the parental strain. Mutant E21, with higher promoter activity, consistently produced more Pel and macerated more host tissues. Motility of mutants was not different from parent. However, motility of both mutants and parent were significantly induced in celery and potato extract media. Four mutants (E6, E9, E11 and E21) formed more biofilm than parental strain KD100