Identification of a Serratia entomophila genetic locus encoding amber disease in New Zealand grass grub (Costelytra zealandica).
Serratia entomophila UC9 (A1MO2), which causes amber disease in the New Zealand grass grub Costelytra zealandica, was subjected to transposon (TnphoA)-induced mutagenesis. A mutant (UC21) was found to be nonpathogenic (Path-) to grass grub larvae in bioassays and was shown, by Southern hybridization, to contain a single TnphoA insertion. This mutant failed to adhere to the gut wall (Adn-) of the larvae and also failed to produce pili (Pil-). A comparative study of the total protein profiles of wild-type S. entomophila UC9 and mutant UC21 revealed that the mutant lacked an approximately 44-kDa protein and overexpressed an approximately 20-kDa protein. Transfer of cosmids containing homologous wild-type sequences into mutant strain UC21 restored wild-type phenotypes (Path+, Pil+, and Adn+). One of the complementing cosmids (pSER107) conferred piliation on Pil- Escherichia coli HB101. The TnphoA insertion in UC21 was mapped within an 8.6-kb BamHI fragment common to the complementing cosmids, and we designated this gene locus amb-1. Six gene products with molecular masses of 44, 36, 34, 33, 20, and 18 kDa were detected in E. coli minicells exclusive to the cloned 8.6-kb fragment (pSER201A). The 44-kDa gene product was not detected in E. coli minicells containing the cloned mutant fragment. Saturation mutagenesis of this fragment produced four unlinked insertional mutations with active fusions to TnphoA. These active fusions disrupted the expression of one or more gene products encoded by amb-1. The 8.6-kb fragment cloned in the opposite orientation (pSER201B) expressed only a 20-kDa protein. We propose that these are the products of structural and/or regulatory genes involved in adhesion and/or piliation which are prerequisites in the S. entomophila-grass grub interaction leading to amber disease.
Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand.
N M
NM
Upadhyaya
T R
TR
Glare
H K
HK
Mahanty
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002448:Cell Adhesion / Q000235:genetics*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics; D004252:DNA Mutational Analysis; D004064:Digestive System / Q000382:microbiology; D010861:Fimbriae, Bacterial / Q000502:physiology; D005798:Genes, Bacterial / Q000235:genetics*; D005816:Genetic Complementation Test; D007313:Insecta / Q000382:microbiology*; D007814:Larva / Q000382:microbiology; D016254:Mutagenesis, Insertional; D011993:Recombinant Fusion Proteins; D012705:Serratia / Q000235:genetics* / Q000472:pathogenicity / Q000648:ultrastructure; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins