Determination of cellulose amounts.

<p>Strains MG1655, MG1655<i>ΔcarB::cat</i>, and MG1655<i>ΔpyrC::tet</i> were grown 48 hours at 30°C on either LB1/4 agar (no added uracil, Cellulose extraction and determination was performed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031252#pone.0031252-Gualdi2" target="_blank">[35]</a>. Data shown are the average of two independent experiments giving very similar results. For strains MG1655<i>ΔcarB::cat</i> and MG1655<i>ΔpyrC::tet</i> grown on LB1/4 agar no glucose was detectable in the assays. A value of 0.5 nmol glucose, corresponding to the lowest detectable concentration in the assay, as determined by a glucose standard curve, was thus arbitrarily assigned to these strains.</p

Congo red binding by <i>E. coli</i> strains deficient in UMP biosynthesis.

<p>The MG1655 strain and isogenic mutants deficient in UMP biosynthetic genes were spotted on either CR medium or CR(ura) medium (CR medium supplemented with 0.25 mM uracil) and grown for 24 hours at 30°C. Plates were incubated for 48 hours at 4°C to enhance Congo red binding.</p

<i>Escherichia coli</i> strains and plasmids used in this work.

<p><i>Escherichia coli</i> strains and plasmids used in this work.</p

Congo red binding by <i>E. coli</i> strains deficient in pyrimidine sensing (<i>cytR</i> and <i>rutR</i> mutants) and purine biosynthesis (<i>purH</i> mutant).

<p><b>4A.</b> The MG1655 strain and its isogenic mutants in the <i>purH</i>, <i>cytR</i> and <i>rutR</i> genes were spotted either on CR medium (left panel) or on CR(ura) medium (right panel) and grown for 24 hours at 30°C. Plates were incubated for 48 hours at 4°C to enhance Congo red binding. Determination of transcript levels. <b>4B.</b> Relative expression of either the <i>csgD</i> gene (left panel) or the <i>udp</i> gene (right panel) was determined by Real-Time PCR on RNA extracted from overnight cultures of MG1655 and of its isogenic <i>purH</i> and <i>cytR</i> mutants. 16S RNA transcript was used as reference gene. <i>Δ</i>Ct values between the genes of interest and 16S RNA were set at 1 for MG1655 in LB1/4 medium, and transcript levels in other strains and/or growth conditions are expressed as relative values. Experiments were repeated at least three times, each time in duplicate; standard deviations were always lower than 5%.</p

UMP biosynthetic pathways in <i>Escherichia coli</i>.

<p>Adapted from Ecocyc (<a href="http://ecocyc.org/" target="_blank">http://ecocyc.org/</a>).</p

Determination of curli production by Congo red binding.

<p>Phenotypes on CR medium of MG1655 (wild type strain), AM70 (<i>csgA</i> deletion mutant, unable to produce curli), MG1655<i>carB::Tn5kan</i>, MG1655<i>carB::Tn5kan ΔcsgA::cat</i> and MG1655<i>ΔcarB::cat</i>. Strains were grown either at 30°C (for 24 hours) or at 37°C (for 18 hours). Plates were incubated for 48 hours at 4°C to enhance Congo red binding.</p

Determination of gene expression levels.

<p>Relative expression of the <i>csgD</i>, <i>csgB</i>, <i>adrA</i> and <i>bcsA</i> genes determined by Real-Time PCR on RNA extracted from overnight cultures. 16S RNA transcript was used as reference gene. <i>Δ</i>Ct values between the genes of interest and 16S RNA were set at 100 for MG1655 in LB1/4 medium, and transcript levels in other strains and/or growth conditions are expressed as relative values. Experiments were repeated at least three times, each time in duplicate; standard deviations were always lower than 5%.</p

Table_3.XLSX

<p>The small RNA ErsA of Pseudomonas aeruginosa was previously suggested to be involved in biofilm formation via negative post-transcriptional regulation of the algC gene that encodes the virulence-associated enzyme AlgC, which provides sugar precursors for the synthesis of several polysaccharides. In this study, we show that a knock-out ersA mutant strain forms a flat and uniform biofilm, not characterized by mushroom-multicellular structures typical of a mature biofilm. Conversely, the knock-out mutant strain showed enhanced swarming and twitching motilities. To assess the influence of ErsA on the P. aeruginosa transcriptome, we performed RNA-seq experiments comparing the knock-out mutant with the wild-type. More than 160 genes were found differentially expressed in the knock-out mutant. Parts of these genes, important for biofilm formation and motility regulation, are known to belong also to the AmrZ transcriptional regulator regulon. Here, we show that ErsA binds in vitro and positively regulates amrZ mRNA at post-transcriptional level in vivo suggesting an interesting contribution of the ErsA-amrZ mRNA interaction in biofilm development at several regulatory levels.</p

Image_3.PDF

<p>The small RNA ErsA of Pseudomonas aeruginosa was previously suggested to be involved in biofilm formation via negative post-transcriptional regulation of the algC gene that encodes the virulence-associated enzyme AlgC, which provides sugar precursors for the synthesis of several polysaccharides. In this study, we show that a knock-out ersA mutant strain forms a flat and uniform biofilm, not characterized by mushroom-multicellular structures typical of a mature biofilm. Conversely, the knock-out mutant strain showed enhanced swarming and twitching motilities. To assess the influence of ErsA on the P. aeruginosa transcriptome, we performed RNA-seq experiments comparing the knock-out mutant with the wild-type. More than 160 genes were found differentially expressed in the knock-out mutant. Parts of these genes, important for biofilm formation and motility regulation, are known to belong also to the AmrZ transcriptional regulator regulon. Here, we show that ErsA binds in vitro and positively regulates amrZ mRNA at post-transcriptional level in vivo suggesting an interesting contribution of the ErsA-amrZ mRNA interaction in biofilm development at several regulatory levels.</p

Table_2.PDF

<p>The small RNA ErsA of Pseudomonas aeruginosa was previously suggested to be involved in biofilm formation via negative post-transcriptional regulation of the algC gene that encodes the virulence-associated enzyme AlgC, which provides sugar precursors for the synthesis of several polysaccharides. In this study, we show that a knock-out ersA mutant strain forms a flat and uniform biofilm, not characterized by mushroom-multicellular structures typical of a mature biofilm. Conversely, the knock-out mutant strain showed enhanced swarming and twitching motilities. To assess the influence of ErsA on the P. aeruginosa transcriptome, we performed RNA-seq experiments comparing the knock-out mutant with the wild-type. More than 160 genes were found differentially expressed in the knock-out mutant. Parts of these genes, important for biofilm formation and motility regulation, are known to belong also to the AmrZ transcriptional regulator regulon. Here, we show that ErsA binds in vitro and positively regulates amrZ mRNA at post-transcriptional level in vivo suggesting an interesting contribution of the ErsA-amrZ mRNA interaction in biofilm development at several regulatory levels.</p