Phosphorylated LuxO activates transcription of five regulatory sRNAs (Qrr1-5), four of which, together with the chaperone Hfq, destabilize the mRNA for the master regulator LuxR. (C) In the presence of AIs, LuxO is dephosphorylated, and LuxR is produced. LuxR activates genes responsible for bioluminescence, biofilm formation and exoproteolytic activity,

and represses genes involved in type III secretion and siderophore production V. harveyi is an opportunistic pathogen mainly for shrimps, but also for fish, squids and lobsters [25–27] and causes #Emricasan ic50 randurls[1|1|,|CHEM1|]# major losses in shrimp aquaculture [28]. The response to QS signals is of interest in this context, because genes regulated by QS encode proteins required for biofilm formation [3]

and virulence factors, such as siderophores [29], type III secretion (e.g. vscP) [30] and exoproteolytic activity (e.g. vhp) [17, 31], in addition to bioluminescence (using the lux system) [32]. Here we focused on the single cell analysis of fluorescent reporter strains bearing plasmids containing promoter::gfp fusions, which allowed us to simultaneously monitor the expression of two AI-regulated genes in single cells. Results AI-regulated bioluminescence correlates well with the activity of the corresponding promoter::gfp fusion To expand our previous findings on heterogeneous behavior of a V. harveyi population found for bioluminescence [3] to other AI-regulated genes, we decided to construct promoter::gfp fusions. It was important to use a wild type DNA-PK inhibitor genetic background to monitor bioluminescence as a marker for an intact QS cascade in each strain. Therefore, all promoter::gfp fusions are plasmid based. To set up the reporter system we tested first a plasmid containing a promoter::gfp fusion of the constitutively expressed housekeeping gene recA to estimate the degree of heterogeneity

in the expression of this gene [33]. Wild type cells conjugated with this plasmid were grown to the exponential growth phase, stained with propidium iodide to identify dead cells (about 5%), and single cells in the same field of view were analyzed in phase contrast and fluorescence Glycogen branching enzyme modes. Images were analyzed using ImageJ. Luminescence and fluorescence intensities of each living cell are expressed as intensity values per cell after normalization to the same cell size. All living cells were fluorescent, indicating expression of recA in all cells. Fluorescence intensities were determined in about 1,400 cells. The average fluorescence intensity was calculated to be 1,017 a.u./cell [(a.u.) arbitrary units] with a standard deviation of 9.9% (data not shown). For comparison all living cells of strain BB120gfp containing a chromosomal encoded gfp were fluorescent and showed an average fluorescence intensity of 1,085 a.u./cell with a standard deviation of 10.5% (data not shown).