Construction and Verification of luxS Deletion Mutant and Complemented Strains

All plasmids and primers used for the construction of luxS deletion mutant and complemented strains were listed in Table 1. The upstream (566 bp) and downstream (553 bp) fragments of luxS gene from HPS2 genome, and kanamycin resistance cassette (909 bp) from pSHK3 plasmid were amplified using primer pairs HPS-LuxS-u F/R, H-LuxS-d F/R, and Kan-F/R, respectively. The overlap extension method was used in these three fragments to construct a new fragment UKD (luxS upstream sequence, kanamycin resistance cassette sequence and luxS downstream sequence), then, the obtained UKD fragment was inserted into pk18mobsacB plasmid with EcoRI and XbaI restriction enzymes to generate recombinant plasmid pk18-UKD. The recombinant plasmid was introduced into HPS2 by natural transformation method as described in previous studies with a simple modification (Zhang et al., 2012; Wang et al., 2013; Zou et al., 2013). Briefly, 20 μL of cAMP (8 mM) was added to 20 μL of recipient bacterial suspension in logarithmic phase (OD₆₀₀ = 0.9). Ten minutes later, 2 μg of donor DNA plasmid was added to the bacterial mixture. Afterwards, the cells were added to T/V/S plate and incubated at 37°C for 6 h. Subsequently, cells were transferred to a kanamycin selective plate. At last, the cells were incubated at 37°C for 48 h.

The complemented plasmid pSHK3-C-luxS was constructed by amplifying a 660 bp luxS open reading frame (ORF) and its promoter with primers C-LuxS-F/R. Subsequently, the amplicon was inserted into pSHK3-Gm plasmid derived from the framework of pSHK3-Kan with its kanamycin gene (909 bp) replaced by gentamicin gene (534 bp). The complemented plasmid was then introduced into the luxS deletion mutant by electroporation (2.5 kv, 5 ms) (Wang et al., 2013).

To verify the construction results of the deletion mutant and complemented strains, luxS, gentamicin gene, kanamycin gene, upstream (HAPS_0059) and downstream (HAPS_0064) genes of luxS were amplified and verified by sequencing.