Fig 1.
B. pseudomallei T24 transposon mutants impaired in biofilm formation.
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.
Fig 2.
Biofilm-associated exopolysaccharide gene cluster from B. pseudomallei and comparative analysis with B. cenocepacia.
The putative exopolysaccharide gene clusters from the sequenced genomes of B. pseudomallei 1026b (top) and B. cenocepacia J2315 (bottom). A total of 18 loci spanning Bp1026b_I2907-Bp1026b_I2927 (becA-R) on chromosome I of B. pseudomallei 1026b, not including a cluster of three pseudogenes, are aligned with 21 loci spanning BCAM1330-BCAM1350 on chromosome II of B. cenocepacia 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.
Fig 3.
Swimming motility of B. pseudomallei T24 transposon mutants.
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.
Fig 4.
Growth of B. pseudomallei T24 transposon mutants on NAP-A plates.
The wild type and transposon mutants were replica plated onto NAP-A agar plates using a pin replicator. Plates were incubated at 37°C for two days.
Table 1.
Summary of phenotypes (biofilm, motility, growth, and colony morphology) along with genetic annotations for selected biofilm-deficient transposon insertion mutants.
Fig 5.
Complementation of I1954, I2907 (becA), and II2527 FRT mutants in the static biofilm assay and colony morphology on NAP-A agar plates.
(A) Biofilm formation and (B) colony morphology of complemented I1954, I2907 (becA), 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.
Fig 6.
Deletion of biofilm exopolysaccharide gene cluster alters biofilm formation and growth on NAP-A plates, but not motility.
(A) Biofilm formation of wild type, ΔbecA-R (biofilm EPS deficient), ΔwcbR-A (CPSI deficient), and ΔwcbR-A ΔbecA-R (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.
Fig 7.
Western blot and GC-MS of the exopolysaccharide deletion mutant.
Detection of polysaccharides with mAb 3015 (A) or the CPSI capsular polysaccharide-specific mAb 4C4 (B) from wild type (Bp82), ΔbecA-R (biofilm EPS deficient), ΔwcbR-A (CPSI deficient), ΔwcbR-A ΔbecA-R (CPSI and biofilm EPS deficient) preparations, and purified CPSI (0.5, 1.0, and 2.0 μg) from Bp82. (C) Typical GC-MS total ion chromatograms of monosaccharides (standards peaks: 1-rhamnose, 2-ribose, 3-fucose, 4-arabinose, 5-xylose, 6-inositol, 7-mannose, 8-glucose, and 9-galactose. IS = internal standard (3-O-methyl-glucose)). (D) Relative abundance of sugars from Bp82 exopolysaccharide extracts. Each monosaccharide was normalized with an internal standard (PAR = peak area ratio) and normalized against sample protein content.
Fig 8.
Biofilm formation, swimming, and growth of published biofilm B. pseudomallei mutants.
(A) Biofilm formation of the wild type and T24 transposon mutants in genes previously reported to be involved in biofilm production and four efflux pump deletion mutants grown at 37°C for 24 h. (B) The wild type and transposon/deletion mutants were inoculated into 0.3% agar plates, incubated at 37°C for 24 h, and diameters measured. (C) Growth curves of transposon/deletion mutants over 48 h at 37°C. Asterisks indicate a significant difference as obtained with a paired Student’s t-test 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.
Table 2.
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.