A c-di-GMP-Modulating Protein Regulates Swimming Motility of Burkholderia cenocepacia in Response to Arginine and Glutamate

Abstract

Burkholderia cenocepacia is an opportunistic bacterium that can thrive in different environments, including the amino acid-rich mucus of the cystic fibrosis (CF) lung. B. cenocepacia responds to the nutritional conditions that mimic the CF sputum by increasing flagellin expression and swimming motility. Individual amino acids also induce swimming but not flagellin expression. Here, we show that modulation of the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) levels by the PAS-containing c-di-GMP phosphodiesterase, BCAL1069 (CdpA), regulates the swimming motility of B. cenocepacia K56-2 in response to CF sputum nutritional conditions. Heterologous expression of WspR, a diguanylate cyclase, in B. cenocepacia K56-2 caused an increase in c-di-GMP levels and reduced swimming motility but did not affect flagellin expression or flagellar biosynthesis. After insertional mutagenesis of 12 putative genes encoding c-di-GMP metabolizing enzymes, one mutant of the locus BCAL1069 (cdpA), exhibited decreased swimming motility independent of flagellin expression in CF sputum nutritional conditions and an increase in intracellular c-di-GMP levels. The reduced swimming motility phenotype of the BCAL1069 mutant was observed in the presence of arginine and glutamate, but not of histidine, phenylalanine, or proline. The B. cenocepacia CdpA was also found to be involved in regulation of protease activity but not in biofilm formation. Altogether, these results highlight a role of B. cenocepacia BCAL1069 (CdpA) in sensing the nutritional conditions of the CF sputum and eliciting a pathogenic response that includes swimming motility toward amino acids and an increase in protease activity.

Keywords: BCAL1069; Burkholderia cenocepacia; Burkholderia cepacia complex; SCFM; c-di-GMP; cdpA; cystic fibrosis; motility.

Figure 1

The effect of c-di-GMP modulating conditions on swimming motility, flagellin expression, and flagella biosynthesis of B. cenocepacia K56-2 WT. (A) Motility of the strains was examined in semi-solid SCFM 0.3% agar plates after 24 h at 37°C. Experiments were performed three-times independently in duplicate. ^*Denotes significant p-values (p < 0.01). (B) Whole cell lysates were used to separate proteins by 12% SDS-PAGE followed by detection of flagellin protein using anti-flagellin primary antibody and alkaline phosphatase crosslinked to secondary antibody. From two Western blots, one representative experiment is shown. (C) Electron micrographs of uranyl acetate stained WT strains displaying flagella under c-di-GMP varying conditions.

Figure 2

Predicted domains coded by c-di-GMP metabolic proteins and their role in swimming motility of B. cenocepacia K56-2 WT. (A) Putative proteins with c-di-GMP modulating and signal transduction domains as predicted by SMART. Amino acid sequences were obtained from the genome of B. cenocepacia J2315. EAL, putative diguanylate phosphodiesterase; GG(D/E)EF, putative diguanylate cyclase; PAS, Per/ARNT/Sim; GAF, cGMP-specific phosphodiesterase/Adenyly cyclase/FhlA; REC, cheY-homologous receiver domain; Cache, CAlcium channels and CHEmotaxis receptors; HAMP, Histidine kinases/Adenylate cyclases/Methyl accepting proteins/Phosphatases; Vertical bars, transmembrane domain. Domains are not drawn to scale. (B) The swimming motility of the mutants was examined in semi-solid SCFM 0.3% agar plates after 24 h at 37°C. Three independent experiments were performed in duplicates (^*p < 0.01; ^**p < 0.001).

Figure 3

Role of BCAL1069 (cdpA) motility in swimming motility. (A) The swimming motility of the strains was examined in semi-solid SCFM 0.3% agar plates at 37°C after 24 h. The motility assay was performed three-times independently in duplicates (^*p < 0.01). (B) To detect flagellin protein, proteins from whole cell lysates of strains were separated on 12% SDS-PAGE followed by flagellin detection using anti-flagellin primary antibody and alkaline phosphatase cross linked to secondary antibody. Western blots were performed twice. One representative experiment is shown. (C) The presence of flagella in the WT and mutant strains were observed using transmission electron microscope.

Figure 4

Multiple sequence alignment of residues of signature motifs of BCAL1069 (CdpA) and determination of intracellular levels of c-di-GMP in the BCAL1069 mutant. (A) Partial alignment of BCAL1069 amino acid sequence with GGDEF and EAL domain-containing proteins, retrieved from the UniProt database. The amino acids in the GGDEF and EAL motifs are highlighted in bold. Other conserved amino acids are shaded in gray. The numbers show the position of the amino acid in the protein. BCAL1069, UniProt Id B4ED05; WspR, UniProt Id Q9HXT9; RpfR, UniProt Id B4EKM4; PleD, UniProt Id Q9A515; AdrA, UniProt Id Q9L401; RocR, UniProt Id Q9HX69; YhjH, UniProt Id P37646; VieA, UniProt Id O68318. (B) Relative intracellular c-di-GMP levels in the WT:BCAL1069 mutant. Percentage represents area under the c-di-GMP curve of the strain relative to the WT strain. Three independent experiments were performed. ^*Denotes significant p-values (p < 0.01).

Figure 5

The effect of individual amino acids on the swimming motility of WT and WT::BCAL1069 mutant. (A–G) The motility halos of the indicated strains were examined in MOPS-glucose 5 mM with individual amino acids at the same concentration as present in SCFM, after incubation for 24 h at 37°C. Each experiment was performed three times independently in duplicate (^*p < 0.01). (H) Growth curves of WT, WT::BCAL1069 and WT::BCAL1069/pBCAL1069 in MOPS-glucose 5 mM with individual amino acid at the same concentration as present in SCFM. The figure is representative of one from two independent experiments.

Figure 6

Protease activity of B. cenocepacia K56-2 WT, WT::BCAL1069 c-di-GMP mutant and complemented mutant. (A) Protease activity of strains on 2% skim milk agar plates after 24 and 48 h at 37°C. One representative of four independent experiments is shown. (B) Quantitative analysis of proteolytic activity after 48 h on 2% skim milk agar plates. Protease activity is represented in percentage relative to the WT strain. The protease assay was performed four times independently with three technical replicates per experiment (^*p < 0.01).