Title:

Identification of Virulence Factors of "Serratia sp. SCBI"

A Serratia species of bacteria, termed South African Caenorhabditis briggsae Isolate (SCBI), was isolated from the nematode C. briggsae KT0001. This bacterium forms an early symbiotic relationship with its nematode host, altering the nematode behavior to kill and feed upon insects. Serratia sp. strain SCBI is nonpathogenic to Caenorhabditis nematodes, but is lethal to lepidopteran insects including Galleria mellonella (Greater Wax Moth). Comparative genomic analysis shows that Serratia sp. SCBI is closely related to the broad-host-range pathogen Serratia marcescens Db11 which kills Caenorhabditis nematodes. The overall goal of this project is to identify the virulence factors of insect pathogen Serratia sp. strain SCBI to gain deeper understanding of the transition from a mutualistic state to a pathogenic lifestyle, and to provide insight into the design of precision antibiotics. A transposon mutant library of 2,100 mutants was screened for defective lipase activity or siderophore production. Nine mutants (2H-10, 10H-2, 11G-9, 12D-7, 12D-11, 12G-7, 14A-12, 18E-6, and 19E-4) exhibited reduced production of one or both exoenzymes. These defects were confirmed and their effects on pathogenesis was tested via a G. mellonella bioassay, and up to sixty biological replicates were used per mutant. Three mutants (10H-2, 12D-7, and 14A-12) lost or nearly lost insecticidal activity, while two mutants (2H-10 and 18E-6) were insecticidal to a lesser degree than the wildtype. When retested, 2H-10 was not insecticidal, producing conflicting results. One mutant (19E-4) retained wild type level insecticidal activity, and one mutant (11G-9) could not grow in selective medium, suggesting that it does not contain a region of virulence. Mutants 12D-11 and 12G-7 had conflicting results. To identify the site of the transposon insertion, rescue cloning was used on three mutants (10H-2, 12D-7, and 14A-12). These clones are being sequenced to identify insertion sites within the Serratia sp. SCBI genome.

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