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Cloning of a large gene cluster involved in Erwinia amylovora CFBP1430 virulence
Author(s) -
Barny M. A.,
Guinebretière M. H.,
Marçais B.,
Coissac E.,
Paulin J. P.,
Laurent J.
Publication year - 1990
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1990.tb00648.x
Subject(s) - biology , erwinia , cosmid , virulence , mutant , genetics , pseudomonas syringae , population , mutagenesis , gene , insertional mutagenesis , microbiology and biotechnology , demography , sociology
Summary Phage Mu d IIPR13 insertional mutagenesis of Erwinia amylovora CFBP1430 allowed us to isolate 6900 independent Cm R mutants. The frequencies of different auxotrophs in this population indicated that Mu d IIPR13 had inserted randomly in E. amylovora. Screening of 3500 Cm R mutants on (i) apple calli and (ii) pear and apple seedlings led to the isolation of 19 non‐pathogenic prototrophic single mutants, four of which expressed a LacZ + hybrid protein. Expression of the fusion proteins was temperature sensitive. The 19 mutants could be separated into two classes according to their behaviour on tobacco: 13 were unable to elicit the hypersensitive response on tobacco (Hrp − ) while six still could (Dsp − ). The 19 Mu d IIPR13 insertions all mapped in the same virulence region. The Mu d IIPR13 insertions of Hrp − mutants were all clustered on the left part of this region, white the Mu d IIPR13 insertions of Dsp − mutants were located on the right part. All of the mutants except one, which proved to have a large deletion of the entire virulence region, could be complemented functionally by cosmids from an E amylovora CFBP1430 genomic library. No hybridization was observed between the cosmid pPV130, which complemented 12 hrp ::Mu d IIPR13 mutations, and the hrp genes from Pseudomonas syringae pv. phaseolicola (Lindgren et al. , 1986), P. syringae pv. tomato (N. J. Panopoulos, unpublished data) or P. solanacearum (Boucher et al. , 1987). Further analysis of the large virulence region will allow mapping of the border of the virulence region and facilitate the study of the function and regulation of the hrp and dsp genes.