Chromosomal Location And Deleted Strain Construction Of The Novel Gene R049in Uropathogenic Escherichia Coli

ObjectiveTo investigate the chromosomal location of the novel gene R049from uropathogenic Escherichia coli(UPEC)132,and construct a R049gene deleted mutant strain and lay a foundation of further study on the function of R049gene.Methods1. Identificated UPEC132, Escherichia coli K-12MG1655by Gram stain,test of biochemical reaction,papC and R049PCR.Harvested the cells after an overnight culture at37°C,and isolated the bacterial chromosome.2. UPEC132chromosome was fragmented by a high-pressure nitrogen nebulization.Obtained the single-stranded DNA library,after size selection and alkaline denaturation.3. Used magnetic beads to capture the single-stranded DNA library,and one fragment fixed to one bead. Distributed the beads into single emulsion droplet for PCR,which called emPCR.Finally one bead had tens of thousands of identical copies of fragment fixed to it.4. Added the amplified DNA library with beads into PicoTiterPlate,then was pyrosequenced using a454Life Sciences GS-FLX sequencer(Roche).The454reads were assembled with Newbler program (Roche).5. Analyzed the sequencing data using the bioinformatics methods to find the R049-associated specific contig and its characteristics.Preliminary location the contig and R049-ORF in UPEC132chromosome.6. Inserted a Zeocin resistance gene into pKD46and pCP20using PCR cloning techniques,and modificated their antibiotic marker. Deleted R049gene using Red system,and constructed a lacking R049mutant strain of multiple antibiotic-resistant UPEC132.Results1. The biochemical reaction of UPEC132conformed to the biochemical pattern of Escherichia coli. The results of papC and R049PCR showed the prospectively strip.2. A shotgun single-stranded DNA library of UPEC132was constructed with the300bp to1000bp size fraction of DNA fragments and an average size of598bp,which meeted the requirements.3. The sequencing data were assembled using the454Inc Newbler program produced133contigs and coveraged approximately48times of UPEC132chromosome.The R049-contig was169022bp.4. Compared with the chromosome sequence of UPEC536strain, the R049-contig sequence was very similar to the233074base to451502base of the former.A20773bp region including gene R049replaced the pathogenicity island PAIⅢ536of UPEC536,which had a lower GC content (46.97%) and16bp direct repeats in two ends. Significantly it also was adjacented to thrW tRNA, insertion element and genes coding integrase. Thus the20773bp fragment was named R049genome island(R049-GI).There were25ORFs in R049-GI, and gene R049was located in the thirteenth ORF.5. Used the plasmid pPICZaA as a template,and PCR amplificated the Zeocin resistance gene with its promoter(459bp). Introduced this resistance gene within the bla resistance gene(Ampr) of pKD46and pCP20.And named these antibiotic resistance markers modificated plasmids:pKD46-zeo and pCP20-zeo.6. Used the Red system with pKD46-zeo, pKD3and pCP20-zeo to delete the R049gene from UPEC132chromosome.PCR products sequencing result identificated that the R049gene has been deleted and only one FRT site was saved in the chromosome of UPEC132.The mutant strain was named UPEC132ΔR049.Conclusion1. Gene R049was a component of the genome island in UPEC132chromosome acquired by horizontal gene transfer.2. The Red homologous recombination system with pKD46-zeo and pCP20-zeo was suitable for gene deletions in clinical multidrug-resistant strains.3. Constructed a R049gene deleted mutant strain and provided a foundation of further experiments about the function of R049gene.