Biofilm formation by <i>luxS</i> mutant in conditioned media.

<p><i>luxS⁻</i> cells growing in MT medium during 24 h were reinoculated in wild type MT CM or MT+P CM and in <i>ppk⁻ppx⁻</i> MT CM from different growth times, as indicated. Biofilm formation was determined 24 h after the shift to CM. 48 h MT cultures were used as control (–). Different letters indicate significant differences according to Tukey's test with a <i>p</i>-value of 0.05.</p

Biofilm formation after reinoculation in CM.

*<p>Biofilm formation was determined 24 h after the cells transference to CM. 48 h MT cultures were used as control. Different letters indicate significant differences according to Tukey's test with a <i>p</i>-value of 0.05.</p

Biofilm formation after changes of phosphate concentration in stationary phase.

<p>Biofilm formation by MC4100 was measured at indicated times of growth in MT and in MT with the addition of 40 mM phosphate buffer pH 7 at 24 h (MT+24P) or at 48 h (MT+48P) (<b>left panel</b>), or in MT+P and in MT+P culture switched to fresh MT at 24 h (MT+P→24MT) (<b>right panel</b>). Result represents the mean ± SD of at least three independent experiments performed in triplicate. Different letters indicate significant differences according to Tukey's test with a <i>p</i>-value of 0.05.</p

Biofilm formation in MT and MT+P media.

<p><i>E. coli</i> wild type strains were grown in static conditions during 48 h at 30°C in the indicated media. The biofilm amount was determined by the crystal violet assay. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050368#s3" target="_blank">Results</a> represent the mean ± SD of four independent experiments. Different letters indicate significant differences according to Tukey's test with a <i>p</i>-value of 0.05.</p

Polyphosphate Degradation in Stationary Phase Triggers Biofilm Formation via LuxS Quorum Sensing System in <em>Escherichia coli</em>

<div><p>In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that <em>Escherichia coli</em> cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in <em>E. coli</em>. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in <em>ppk⁻ppx⁻</em> or <em>ppk⁻ppx⁻</em>/<em>ppk⁺</em> strains, deficient in polyP synthesis and hydrolysis, respectively. <em>luxS</em> and <em>lsrK</em> mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.</p> </div

PolyP levels and biofilm formation in different Pi concentrations media.

<p>The biofilm amount (white bars) and DAPI-polyP fluorescence (black dots) were determined at 48 h in the indicated <i>E. coli</i> strains grown in static conditions in MT medium modified with the indicated Pi concentrations. Data represent the mean ± SD of at least three independent experiments.</p

Biofilm formation by quorum sensing mutants.

<p>The biofilm amount was determined at 48 h in the indicated strains grown in static conditions in MT or MT+P media. Different letters indicate significant differences according to Tukey's test with a <i>p</i>-value of 0.05.</p

<i>E. coli</i> strains and plasmid used in this work.

<p><i>E. coli</i> strains and plasmid used in this work.</p