ABSTRACT
Spontaneous bacteriophage-resistant mutants of the phytopathogen Erwinia carotovora subsp. atroseptica (Eca) SCRI1043 were isolated and, out of 40, two were found to exhibit reduced virulence in planta. One of these mutants, A5/22, showed multiple cell surface defects including alterations in synthesis of outer membrane proteins, lipopolysaccharide (LPS), enterobacterial common antigen (ECA), and flagella. Mutant A5/22 also showed reduced synthesis of the exoenzymes pectate lyase (Pel) and cellulase (Cel), major virulence factors for this pathogen. Genetic analysis revealed the pronounced pleiotropic mutant phenotype to be due to a defect in a single gene (rffG) that, in Escherichia coli, is involved in the production of ECA. We also show that while other enteric bacteria possess duplicate homologues of this gene dedicated separately to synthesis of LPS and ECA, Eca has a single gene.
Subject(s)
Antigens, Bacterial/genetics , Erwinia/genetics , Erwinia/pathogenicity , Lipopolysaccharides/biosynthesis , Plants/microbiology , Antigens, Bacterial/biosynthesis , Bacterial Outer Membrane Proteins/biosynthesis , Bacterial Outer Membrane Proteins/genetics , Bacteriophages/physiology , Erwinia/immunology , Escherichia coli/genetics , Flagella/genetics , Genes, Plant , Molecular Sequence Data , Mutagenesis , Restriction Mapping , VirulenceABSTRACT
Using enrichment methods, a new bacteriophage (M1) was isolated, which is capable of generalized transduction in Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043. M1 is probably a virulent phage and contains double-stranded DNA of approximately 43 kb. Transduction frequencies for a number of chromosomal markers and plasmid pHCP2 were established, and conditions for transduction optimized. UV irradiation of the lysates prior to transduction enhanced the transduction frequency. M1 infected over 25% of Eca strains tested and so may be useful both for the genetic analysis of a number of Eca isolates and for the transductional transfer of selectable markers between strains.
ABSTRACT
Erwinia carotovora subsp. atroseptica was mutagenized and assayed for virulence in planta. Those mutants which exhibited reduced virulence (Rvi-) were assayed for growth rate, auxotrophy and extracellular enzyme secretion and seven mutants were found to be wild type for all of these phenotypes. When screened for other phenotypes, two were found to be non-motile. One mutant was complemented for motility by a heterologous gene library. A 2.7kb XmaIII-ClaI complementing fragment was sequenced and the gene products were found to have similarity to flagella biosynthesis gene products from several bacteria. Further similarity was found to a pathogenicity protein from the plant pathogen Xanthomonas campestris pv. glycines and to the Spa pathogenicity proteins of the human pathogen Shigella flexneri, which are involved in the surface presentation of antigens. These studies highlight the emergence of common themes in the molecular strategies employed by both plant and animal bacterial pathogens for the targeting of proteins involved in the elaboration of disease.
Subject(s)
Erwinia/genetics , Flagella/metabolism , Genes, Bacterial , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacteriophages/physiology , Base Sequence , Cell Movement/genetics , Erwinia/pathogenicity , Erwinia/physiology , Erwinia/ultrastructure , Gene Library , Genetic Complementation Test , Gram-Negative Bacteria/genetics , Molecular Sequence Data , Mutagenesis, Insertional , Phenotype , Plant Diseases/microbiology , Plants/microbiology , Sequence Alignment , Sequence Homology, Amino Acid , Solanum tuberosum/microbiology , Virulence/geneticsABSTRACT
The chemical mutagen ethylmethanesulphonate (EMS) has been used to generate mutants of Erwinia carotovora subspecies carotovora which are defective in the secretion of pectinases (Pel) and cellulases (Cel) but unaltered for protease (Prt) secretion. Such mutants, called Out-, still synthesize Pel and Cel but these enzymes accumulate within the periplasm. Cosmid clones carrying wild-type E. carotovora ssp. carotovora DNA, identified by their ability to restore the Out+ phenotype when transferred to some Out- mutants, were classified into six complementation groups using cosmids and cosmid derivatives. Analysis of the nucleotide sequence of a 12.7 kb DNA fragment, encompassing complementing cosmid inserts, revealed a coding capacity for 13 potential open reading frames (ORFs), and these were designated outC-outO. Some of the out gene products were visualized using a T7 gene 10 expression system. The predicted Out proteins are highly similar to components of extracellular enzyme secretion systems from a diverse range of eubacteria including Erwinia chrysanthemi, Klebsiella oxytoca, Aeromonas hydrophila, Pseudomonas aeruginosa and Xanthomonas campestris. Lower levels of similarity exist between Ecc Out proteins and components of macromolecular trafficking systems from Bacillus subtilis, Haemophilus influenzae, Agrobacterium tumefaciens, Yersinia pestis and a protein involved in the morphogenesis of filamentous bacteriophages such as M13.