title: Penicillin-binding proteins from Erwinia amylovora: mutants lacking PBP2 are avirulent.
abstract: Radiolabelled penicillin G was used to examine penicillin-binding proteins (PBPs) from Erwinia amylovora (OT1). This procedure identified seven PBPs with molecular masses ranging from 22 to 83 kDa. E. amylovora PBPs were compared with those from Escherichia coli (JM101) and from two spherical, avirulent TnphoA mutants derived from OT1. Radiolabelled penicillin G bound to only six proteins from the spherical mutants which lacked a 69-kDa PBP. The spherical mutants could be complemented by the cloned E. coli pbpA-rodA operon, which restored both cell shape and virulence to apple seedlings. This suggested that the E. amylovora 69-kDa PBP is probably the functional equivalent of the E. coli PBP2 protein. Southern blot analysis using the E. coli rodA and pbpA genes as radiolabelled probes showed that TnphoA had inserted into the E. amylovora equivalent of the E. coli rodA-pbpA operon. Southern blots to chromosomal DNAs of the two spherical mutants, using the cloned hrp and dsp genes from E. amylovora as radiolabelled probes, confirmed that the TnphoA insertions were not located in the region of the E. amylovora chromosome postulated to encode known virulence factors. Both of the spherical TnphoA mutants synthesized amounts of extracellular polysaccharide equivalent to those synthesized by the wild-type strain (OT1), were resistant to lysis in distilled water and to lysozyme, and elicited the hypersensitive response on nonhost plants. These results indicate a possible role for cell shape in the virulence of this plant pathogen.
authors.affiliation: Department of Microbiology, University of Leicester, United Kingdom.
authors.forename: J S
authors.initials: JS
authors.lastname: Milner
authors.forename: D
authors.initials: D
authors.lastname: Dymock
authors.forename: R M
authors.initials: RM
authors.lastname: Cooper
authors.forename: I S
authors.initials: IS
authors.lastname: Roberts
mesh_terms: D001426:Bacterial Proteins; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004885:Erwinia / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005838:Genotype; D006602:Hexosyltransferases / Q000235:genetics*; D009097:Multienzyme Complexes / Q000235:genetics*; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010400:Penicillin G / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases / Q000235:genetics*; D010944:Plants / Q000382:microbiology; D010957:Plasmids; D015183:Restriction Mapping; D014169:Transformation, Bacterial; D014774:Virulence / Q000235:genetics / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D010400:Penicillin G