Disruption of c-di-GMP Signaling Networks Unlocks Cryptic Expression of Secondary Metabolites during Biofilm Growth in Burkholderia pseudomallei

Abstract

The regulation and production of secondary metabolites during biofilm growth of Burkholderia spp. is not well understood. To learn more about the crucial role and regulatory control of cryptic molecules produced during biofilm growth, we disrupted c-di-GMP signaling in Burkholderia pseudomallei, a soilborne bacterial saprophyte and the etiologic agent of melioidosis. Our approach to these studies combined transcriptional profiling with genetic deletions that targeted key c-di-GMP regulatory components to characterize responses to changes in temperature. Mutational analyses and conditional expression studies of c-di-GMP genes demonstrates their contribution to phenotypes such as biofilm formation, colony morphology, motility, and expression of secondary metabolite biosynthesis when grown as a biofilm at different temperatures. RNA-seq analysis was performed at various temperatures in a ΔII2523 mutant background that is responsive to temperature alterations resulting in hypobiofilm- and hyperbiofilm-forming phenotypes. Differential regulation of genes was observed for polysaccharide biosynthesis, secretion systems, and nonribosomal peptide and polyketide synthase (NRPS/PKS) clusters in response to temperature changes. Deletion mutations of biosynthetic gene clusters (BGCs) 2, 11, 14 (syrbactin), and 15 (malleipeptin) in parental and ΔII2523 backgrounds also reveal the contribution of these BGCs to biofilm formation and colony morphology in addition to inhibition of Bacillus subtilis and Rhizoctonia solani. Our findings suggest that II2523 impacts the regulation of genes that contribute to biofilm formation and competition. Characterization of cryptic BGCs under different environmental conditions will allow for a better understanding of the role of secondary metabolites in the context of biofilm formation and microbe-microbe interactions. IMPORTANCE Burkholderia pseudomallei is a saprophytic bacterium residing in the environment that switches to a pathogenic lifestyle during infection of a wide range of hosts. The environmental cues that serve as the stimulus to trigger this change are largely unknown. However, it is well established that the cellular level of c-di-GMP, a secondary signal messenger, controls the switch from growth as planktonic cells to growth as a biofilm. Disrupting the signaling mediated by c-di-GMP allows for a better understanding of the regulation and the contribution of the surface associated and secreted molecules that contribute to the various lifestyles of this organism. The genome of B. pseudomallei also encodes cryptic biosynthetic gene clusters predicted to encode small molecules that potentially contribute to growth as a biofilm, adaptation, and interactions with other organisms. A better understanding of the regulation of these molecules is crucial to understanding how this versatile pathogen alters its lifestyle.

Keywords: Burkholderia pseudomallei; biofilm; c-di-GMP; diguanylate cyclase; malleipeptin; syrbactin.

FIG 1

Biofilm formation of B. pseudomallei 1026b c-di-GMP deletion mutants. Wild-type and single, double, triple, and quadruple mutant strains were grown statically at 30°C (A) and 37°C (B) for 24 h. The data are representative of three independent experiments. Asterisks indicate a significance difference as determined with a Student t test utilizing the Bonferroni correction (P < 0.002) to account for multiple comparisons (n = 13).

FIG 2

Quantification of c-di-GMP levels of B. pseudomallei 1026b and ΔII2523 grown statically at 30 and 37°C. P < 0.01. The statistical significance was determined using the Sidak-Bonferroni method across multiple Student t tests (**, P < 0.01). Error bars indicate the standard errors for three technical replicates. c-di-GMP extractions were repeated on separate days using two biological replicate cultures for each strain and temperature condition, with three technical replicates each.

FIG 3

Swimming motility of B. pseudomallei c-di-GMP deletion mutants. Swimming motility of the wild type and single, double, triple, and quadruple mutant strains in 0.3% agar plates. The plates were incubated at 30°C (A) and 37°C (B) for 24 h. Asterisks indicate a significance difference determined using a Student t test with the Bonferroni correction (P < 0.002) to account for multiple comparisons (n = 13).

FIG 4

Biofilm formation, swimming motility, and colony morphology of B. pseudomallei Bp82 strains conditionally expressing cdpA, I2285, cdpA I2285, II0885, and II2523. Biofilm assays were incubated at 28°C (A) or 37°C (B) for 24 h. Swim assays were incubated at either 28°C (C) or 37°C (D) for 24 h. (E) Colony morphology on NAP-A plates incubated at 37°C. Images were taken after 3 days. Conditional expression of c-di-GMP genes was achieved by the addition of 1 mM IPTG.

FIG 5

Site-directed mutations in cdpA, I2285, and II2523 identify amino acids that are important for swim and biofilm phenotypes in Bp82. (A and B) Swim (A) and biofilm (B) phenotypes of CdpA PAS4 (S72A) and EAL mutants. (C and D) Swim (C) and biofilm (D) phenotypes of I2285 mutants. (D and E) Swim (E) and biofilm (F) phenotypes of II2523 PAS4 (N32A) mutant. We used 1 mM IPTG to induce conditional expression. All assays were done at 37°C.

FIG 6

(A and B) Volcano plots of genes differentially regulated in the Bp82 parental strain (A) and Bp82 ΔII2523 (B) cells statically grown at 37°C versus 28°C. (C and D) Volcano plots of ΔII2523 at 37°C versus the parental strain at 37°C (C) and ΔII2523 at 28°C versus the parental strain at 28°C (D). Dashed lines represent cutoffs for a log₂-fold change of <−1 or >1 (vertical) and an adjusted P value significance of <0.01 (horizontal).

FIG 7

WoPPER analysis of gene clusters differentially regulated in the Bp82 parental strain and Bp82 ΔII2523 by chromosome. (A to D) Comparisons of the parental cells at 37°C versus 28°C (A), ΔII2523 at 37°C versus 28°C (B), ΔII2523 at 37°C versus the parental strain at 37°C (C), and ΔII2523 at 28°C versus the parental strain at 28°C (D). Yellow indicates gene clusters that were upregulated, and blue indicates gene clusters that were downregulated.

FIG 8

Gene expression (qRT-PCR) of fliC (I3555), cdpA (I2284), I2285, bce-II, (II1799), I2928 (hybrid), I2235 (DGC), I1579 (PDE), capsule II (II0478), capsule III (II1965), and capsule IV (I0530) of biofilm cells from ΔII2523 versus the parental strain grown at either 28 or 37°C. RNA samples for qRT-PCR assays were isolated after 24 h under conditions identical to those used for the RNA-seq data sets.

FIG 9

Colony morphology of NRPS/PKS mutants in the Bp82 parental strain or Bp82 ΔII2523 backgrounds on different media. Spots were grown on YEM (A) or NAP-A (B) at 28 or 37°C. Images were taken after 4 days of growth. Scale bar, 2 mm.

FIG 10

Biofilm formation of BGC Bp82 mutants either 28°C (A) or 37°C (B) for 24 h. Significance: a, statistical difference from the parental strain; b, statistical difference from ΔII2523.

FIG 11

(A and B) Growth inhibition of B. subtilis (A) and R. solani (B). The zones of inhibition of B. subtilis and R. solani by the Bp82 parental strain or the Bp82 ΔII2523 NRPS deletion mutants are depicted. The B. subtilis images were taken after 24 h, whereas the R. solani images were taken after 5 days. All experiments were performed at 28°C.