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  • Bacterial strains and media. All V. harveyi strains used in this study were derived from the wild-type strain BB120 [43] and grown aerobically at 30 °C in Autoinducer Bioassay (AB) broth. E. coli S17–1λpir was used for general DNA manipulation and grown with aeration at 37 °C in LB (Luria-Bertani) broth. The relevant strains and plasmids are listed in Table S1. DNA manipulations. DNA manipulation was performed using standard procedures [44]. Phusion DNA polymerase was used for PCR reactions. dNTPs, restriction enzymes, and T4 DNA ligase were obtained from New England Biolabs. DNA purification kits were provided by Qiagen. E. coli was transformed by electroporation using a Bio-Rad Micro Pulser. Plasmids were introduced into V. harveyi by conjugation [15] and exconjugants were selected using the antibiotic resistances carried on the plasmids together with polymyxin B. Fluorescent protein reporter construction. A cat-resistance cassette from pKD3 [45] was cloned into vector pCMW1 [7] downstream of gfp at the BamH1 site, making pTL3. The GFP-Cmr fragment from this construct was subsequently amplified by PCR and recombined using the λ red technique [45] into a cosmid to replace the wild-type qrr4 gene, producing pTL20. Lastly, PQrr4-GFP-Cmr was introduced onto the chromosome to replace qrr4 by allelic recombination. Ptac-mCherry was amplified from the vector pEVS143-mCherry containing an IPTG inducible mCherry gene and cloned into pKD13 [45] at the NheI site, resulting in pTL82. The cosmid, pTL83, was constructed using the λ red technique by recombining the Ptac-mCherry-Kanr fragment into the intergenic region downstream of the entire lux operon. Final insertion of Ptac-mCherry-Kanr onto the V. harveyi chromosome was accomplished by allelic recombination. V. harveyi strain construction. To construct the various V. harveyi sensor mutants, pKM780 carrying ΔluxS::Cmr, pJMH291 carrying ΔluxN::Cmr, pDLS100 carrying ΔluxPQ::Cmr, pJMH244 carrying ΔcqsS::Cmr, and pKM705 carrying ΔluxR::Kanr were used to sequentially delete the corresponding wild-type genes by allelic recombination. Following each gene deletion, the plasmid pTL18 containing an IPTG-inducible FLP recombinase, derived from pEVS143 and pCP20 [45], was introduced into the V. harveyi strain to eliminate the antibiotic resistance marker on the chromosome. Fluorescence assays. For dose–response experiments, V. harveyi strains LuxN+ (TL87), LuxPQ+ (TL88), and LuxN+ LuxPQ+ (TL89) were grown in AB medium for 8∼12 h. Growth was monitored by measuring optical density at 600 nm. Cultures were diluted to OD600 = 10−6 ∼ 10−7, and exogenous autoinducers were added at the specified concentrations. Following growth to steady state (13∼14 h; OD600 = 0.005 ∼ 0.05), cells were concentrated by centrifugation and maintained on ice until measurements were made. One μl of cell culture was spread on a glass slide and covered with a 1% AB agarose pad as well as a coverslip. Microscopy. Phase-contrast and fluorescent images were taken at room temperature using a Nikon TE-2000U inverted microscope. Custom Basic code was written to control the microscope. Images were acquired using a 100× oil objective and a cooled CCD camera (−65 °C, Andor iXon). Segmentation of individual cells was performed on phase-contrast images. Background and cellular auto-fluorescence values were subtracted from the green and red channels, respectively. Total fluorescence intensity of each cell was obtained by summing all pixels and fractions of pixels in the segmented cell region. Normalized GFP values for each cell were calculated by normalizing total green to total red fluorescence intensity.
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