Genome-scale analysis of the genes that contribute to <i>Burkholderia pseudomallei</i> biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster

<div><p><i>Burkholderia pseudomallei</i>, the causative agent of melioidosis, is an important public health threat due to limited therapeutic options for treatment. Efforts to improve therapeutics for <i>B</i>. <i>pseudomallei</i> infections are dependent on the need to understand the role of <i>B</i>. <i>pseudomallei</i> biofilm formation and its contribution to antibiotic tolerance and persistence as these are bacterial traits that prevent effective therapy. In order to reveal the genes that regulate and/or contribute to <i>B</i>. <i>pseudomallei</i> 1026b biofilm formation, we screened a sequence defined two-allele transposon library and identified 118 transposon insertion mutants that were deficient in biofilm formation. These mutants include transposon insertions in genes predicted to encode flagella, fimbriae, transcriptional regulators, polysaccharides, and hypothetical proteins. Polysaccharides are key constituents of biofilms and <i>B</i>. <i>pseudomallei</i> has the capacity to produce a diversity of polysaccharides, thus there is a critical need to link these biosynthetic genes with the polysaccharides they produce to better understand their biological role during infection. An allelic exchange deletion mutant of the entire <i>B</i>. <i>pseudomallei</i> biofilm-associated exopolysaccharide biosynthetic cluster was decreased in biofilm formation and produced a smooth colony morphology suggestive of the loss of exopolysaccharide production. Conversely, deletion of the previously defined capsule I polysaccharide biosynthesis gene cluster increased biofilm formation. Bioinformatics analyses combined with immunoblot analysis and glycosyl composition studies of the partially purified exopolysaccharide indicate that the biofilm-associated exopolysaccharide is neither cepacian nor the previously described acidic exopolysaccharide. The biofilm-associated exopolysaccharide described here is also specific to the <i>B</i>. <i>pseudomallei</i> complex of bacteria. Since this novel exopolysaccharide biosynthesis cluster is retained in <i>B</i>. <i>mallei</i>, it is predicted to have a role in colonization and infection of the host. These findings will facilitate further advances in understanding the pathogenesis of <i>B</i>. <i>pseudomallei</i> and improve diagnostics and therapeutic treatment strategies.</p></div

Summary of phenotypes (biofilm, motility, growth, and colony morphology) along with genetic annotations for selected biofilm-deficient transposon insertion mutants.

<p>Summary of phenotypes (biofilm, motility, growth, and colony morphology) along with genetic annotations for selected biofilm-deficient transposon insertion mutants.</p

Deletion of biofilm exopolysaccharide gene cluster alters biofilm formation and growth on NAP-A plates, but not motility.

<p>(A) Biofilm formation of wild type, Δ<i>becA-R</i> (biofilm EPS deficient), Δ<i>wcbR-A</i> (CPSI deficient), and Δ<i>wcbR-A</i> Δ<i>becA-R</i> (CPSI and biofilm EPS deficient) strains after 24 h at 37°C. (B) Pellicle formation of wild type and deletion mutants after six days at 37°C. (C) Swim zone diameters (cm) of the wild type and deletion mutants after 24 h at 37°C. (D) Overnight cultures were spotted onto NAP-A plates and grown for two day at 37°C. Asterisks indicate a significant difference as obtained with a paired Student’s t-test utilizing for the biofilm data and the Mann-Whitney test for the swim motility data utilizing a p-value of 0.001. Error bars indicate standard error of the mean.</p

<i>B</i>. <i>pseudomallei</i> T24 transposon mutants impaired in biofilm formation.

<p>The wild type and 37 transposon mutants were grown statically for 24 h at 37°C in polystyrene plates. Biofilm formation was quantified using crystal violet. All transposon mutants exhibited at least a 40% decrease and were tested at least twice in replicates of six. Error bars indicate standard error of the mean.</p

Swimming motility of <i>B</i>. <i>pseudomallei</i> T24 transposon mutants.

<p>Overnight cultures of the wild type and transposon mutants were used to inoculate 0.3% agar plates, incubated at 37°C, and swim zone diameter was measured at 24 h. Asterisks indicate a significant difference as obtained with the Mann-Whitney test utilizing the Bonferroni correction (p = 0.001) to account for multiple comparisons (n = 37). All mutants were tested at least twice in triplicate. Error bars indicate standard error of the mean.</p

Biofilm-associated exopolysaccharide gene cluster from <i>B</i>. <i>pseudomallei</i> and comparative analysis with <i>B</i>. <i>cenocepacia</i>.

<p>The putative exopolysaccharide gene clusters from the sequenced genomes of <i>B</i>. <i>pseudomallei</i> 1026b (top) and <i>B</i>. <i>cenocepacia</i> J2315 (bottom). A total of 18 loci spanning Bp1026b_I2907-Bp1026b_I2927 (<i>becA</i>-<i>R</i>) on chromosome I of <i>B</i>. <i>pseudomallei</i> 1026b, not including a cluster of three pseudogenes, are aligned with 21 loci spanning BCAM1330-BCAM1350 on chromosome II of <i>B</i>. <i>cenocepacia</i> J2315. Coding sequences are depicted by arrows per positive or negative strand orientation. Sizes of genes and intergenic regions are to scale. The results of BLASTN annotations with minimum identity of 60% and threshold E-value of 1E-3 are aligned to regions of similarity. Red bars depict sequence inversions and blue bars depict direct homology in a color density gradient.</p

Complementation of I1954, I2907 (<i>becA</i>), and II2527 <i>FRT</i> mutants in the static biofilm assay and colony morphology on NAP-A agar plates.

<p>(A) Biofilm formation and (B) colony morphology of complemented I1954, I2907 (<i>becA</i>), and II2527 FRT mutants. EV indicates empty vector. Complementation was induced with 1mM IPTG. Asterisks indicate a significant difference as obtained with a pairwise Student’s t-test utilizing a p-value of 0.001. Error bars indicate standard error of the mean.</p

Summary of phenotypes (biofilm, motility, growth, and colony morphology) for selected transposon insertion and deletion mutants in previously published genes that contribute to biofilm formation.

<p>Summary of phenotypes (biofilm, motility, growth, and colony morphology) for selected transposon insertion and deletion mutants in previously published genes that contribute to biofilm formation.</p