Photorhabdus asymbiotica kingscliff
New plasmids and putative virulence factors from the draft genome of an Australian clinical isolate of Photorhabdus asymbiotica
Genomic DNA (gDNA) was prepared from a single colony of P. asymbiotica Kingscliff by inoculating 500 ml of 2XYT culture media. The culture was incubated for 48 h at 37ºC with shaking (350 rpm). Genomic DNA was extracted using a Fermentas Life Sciences Genomic DNA purification kit, according to the manufacturer’s instructions.    The genomic DNA was used to create a paired end library and a fragment library using the standard Roche FLX 454 library Preparation kit. The fragment library was made by physical fragmentation, attachment of adapter sequences, refinement of the ends and selection of adapted molecules. The paired end library was generated by hydroshear shearing, circularisation, addition of adapters and selection of adapted molecules.    For the Illumina paired end sequencing, a library was prepared using 10 µg of gDNA in 200 µl TE for fragmentation by sonication using a Diogenode Biorupter.  End-repair and phosporylation, A-tailing, and adapter ligation were performed using a Paired-End DNA Sample Prep Kit; the resulting DNA fragments were concentrated and purified using QIAquick spin columns according to the manufacturers' instructions. Size fractionation and purification of ligation products was performed using a 5% polyacrylamide gel run in Tris borate EDTA at 180 V for 120 min. Gel slices containing DNA fragments in the 400-500 bp range were cut and DNA was extracted using 0.3M sodium acetate and 2mM EDTA (pH 8.0), followed by ethanol precipitation. Using 18 PCR cycles with primers PE1.1 and PE2.1 supplied by Illumina, Extension and enrichment of the adapter ligated fragments were performed. Library concentration and size range was assessed using 6% PAGE and Nanodrop ND-1000 Spectrophotometer. A 10 nM stock was prepared in 0.1% Tween and kept at -20C until used. The flow cell was prepared according to the manufacturer's instructions using Paired-End Cluster Generation Kit (PE-203-2001) on the Cluster Station.
Clinical isolates of Photorhabdus asymbiotica have been recovered from patients in both the United States of America and Australia, and the full sequence of P. asymbiotica ATCC43949 from the USA has recently been reported.  In contrast to other bacteria in the genus which only infect insects, P. asymbiotica strains are able to infect both insects and humans.  Using a combination of Solexa (Illumina) and 454 Life Sciences (Roche) sequence data in different assembly pipelines, we report on a draft genome sequence of a strain of P. asymbiotica recovered from a patient from Kingscliff, Australia.  The best assembly yielded an N50 scaffold size of 288,627 bp with >88.6% of the predicted genome covered by scaffolds over 100,000 bp.  One of the central differences found between this Australian isolate and the USA isolate is the presence of an additional plasmid, pPAA2.  This plasmid is similar to pCRY from Yersinia pestis, the causative agent of bubonic plague, and the presence of pPAA2 may account for the increased virulence of Australian isolates both against tissue culture cells and infected patients.  The genome of the Kingscliff strain also contains several genomic differences from the USA isolate whose potential significance in virulence against both man and insects is discussed.