Experimental design for competition experiments.

<p>Mixtures of <i>Vibrio fischeri</i> wild type (MJ1) and <i>luxI</i> mutant (MJ211) strains at optical density based starting ratios of 1∶1 or 1∶10 were inoculated (100 µl) into 5 ml of fresh media in triplicate. Each replicate culture was subsequently subcultured daily for ten days and wild type to mutant ratios determined every second day.</p

Impact of <i>luxR</i> complementation on bioluminescene during long term culturing of <i>Vibrio fischeri</i>.

<p>Relative bioluminescence output from the dark <i>V. fischeri</i> MJ1 Lineage 10 with a <i>luxR</i> mutation complimented with a functional <i>luxR</i> gene under control of its own promoter on pLS6luxR. Complementation with <i>luxR</i> initially restored the bioluminescence phenotype to wild type levels, however bioluminescence was observed to decrease during subsequent subculturing for 120 days.</p

Impact of <i>luxI</i> deletion on competition in <i>Vibrio fischeri</i>.

<p>Percentage of luminescence colonies plated from mixed <i>V. fischeri</i> MJ1 (wild type) and MJ211 (<i>luxI</i> mutant) cultures subcultured daily for ten days. The starting ratio of MJ1 to MJ211 was either 1∶1 (Panel A) or 1∶9 (Panel B). Regardless of the initial ratio of wild type (MJ1) to mutant (MJ211) cells, the wild type lineage dominated the cultures within days suggesting the <i>luxI</i> gene represents a selective advantage independent of the bioluminescence phenotype. Average values from triplicate cultures are presented. Error bars represent standard deviation.</p

Impact of <i>luxI</i> deletion on growth of <i>Vibrio fischeri</i> in isolation.

<p>Batch culture growth of <i>V. fischeri</i> wild type strain MJ1 (circles) and <i>luxI</i> deficient mutant strain MJ211 (squares) in the absence (A) and presence (B) of 5 µM <i>N</i>-3-oxohexanoyl-L-homoserine lactone. Values presented are averages of triplicate cultures. Error bars represent standard deviation. Small statistically significant differences were late in the logarithmic phase of growth.</p

Primers used for sequencing <i>V. fischeri</i> bioluminescence regulatory genes.

a<p>Primers and base numbering based on <i>luxR</i> and <i>luxI</i> (Genbank AF170104.1), ^bPrimers and numbering based on <i>ainR</i> and <i>ainS</i> (Genbank L37404), ^c<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067443#pone.0067443-Dunn1" target="_blank">[38]</a>.</p

Fate of the bioluminescence phenotype during long term culturing in <i>Vibrio fischeri</i>.

<p>Relative bioluminescence output from ten <i>V. fischeri</i> MJ1 cultures, subcultured daily over 325 days. Large fluctuations are observed in bioluminescence in all cultures. Half of the cultures (4, 5, 6, 8 and 10) irreversibly lost the bioluminescence phenotype during the course of the experiment. Quorum sensing regulatory genes (<i>ainS</i>, <i>ainR</i>, <i>luxI</i> and <i>luxR</i>) and the <i>luxI</i>-<i>luxR</i> intergenic region were sequenced to investigate the cause of the loss of phenotype.</p