Designing primers to fish auto-inducer synthase gene(s) of the quorum sensing system in Y -proteobacteria and their in-silico PCR validation

Quorum sensing is a well known phenomenon in bacteria that control diverse functions including colonization and formation of biofilm. The luxl gene, involved in quorum sensing of gram negative hacteria. codes tor auto-inducer synthase/ acylhomoserine lactone synthase. As loxI homologues are sequence-diversc it is dificult to identify its loci by hybridization technique in different bacteria whose whole genome sequence(s) are unknown. We have used diferent bioinformatics tools taking the existing genome database into cognizance to design manually suitable degenerate primers for amplifying huxl gene homologues from diverse representatives of gamma-proteobacteria. Two primer pairs, DeglF/Deg2R and Deg3F/Deg4R, were capable of in- silico PCR amplification from genome sequence() of Halothiobacillus neapolianus. Acinetobacter baunanii ATCC 17978, Acidiuhiobacillus ferrooxidans ATCC 53993 and Pseudomonas aeruginosa PAOI (with first primer pair); Edwardsiella ictaluri, Edwardsiella tarda, Enwinia tasmaneinsis, Serratia proteamaculans, Pectobacterium wasabiae, Pectobacterium carotovorum, Pectobacterium atrosepticum, Dickeya zeae and Yersinia pestis (with second primer pair). The phylogenetic trees derived from sequences of horl homologues and 16S rRNA gene sequences of the respective genomes were almost identical showing two distinct clusters. The degenerate primer pairs were also found to be cluster specific.