Nitrate Sensing and Metabolism Inhibit Biofilm Formation in the Opportunistic Pathogen Burkholderia pseudomallei by Reducing the Intracellular Concentration of c-di-GMP

Abstract

The opportunistic pathogen Burkholderia pseudomallei is a saprophytic bacterium and the causative agent of melioidosis, an emerging infectious disease associated with high morbidity and mortality. Although melioidosis is most prevalent during the rainy season in endemic areas, domestic gardens and farms can also serve as a reservoir for B. pseudomallei during the dry season, in part due to irrigation and fertilizer use. In the environment, B. pseudomallei forms biofilms and persists in soil near plant root zones. Biofilms are dynamic bacterial communities whose formation is regulated by extracellular cues and corresponding changes in the nearly universal secondary messenger cyclic dimeric GMP. Recent studies suggest B. pseudomallei loads are increased by irrigation and the addition of nitrate-rich fertilizers, whereby such nutrient imbalances may be linked to the transmission epidemiology of this important pathogen. We hypothesized that exogenous nitrate inhibits B. pseudomallei biofilms by reducing the intracellular concentration of c-di-GMP. Bioinformatics analyses revealed B. pseudomallei 1026b has the coding capacity for nitrate sensing, metabolism, and transport distributed on both chromosomes. Using a sequence-defined library of B. pseudomallei 1026b transposon insertion mutants, we characterized the role of denitrification genes in biofilm formation in response to nitrate. Our results indicate that the denitrification pathway is implicated in B. pseudomallei biofilm growth dynamics and biofilm formation is inhibited by exogenous addition of sodium nitrate. Genomics analysis identified transposon insertional mutants in a predicted two-component system (narX/narL), a nitrate reductase (narGH), and a nitrate transporter (narK-1) required to sense nitrate and alter biofilm formation. Additionally, the results presented here show that exogenous nitrate reduces intracellular levels of the bacterial second messenger c-di-GMP. These results implicate the role of nitrate sensing in the regulation of a c-di-GMP phosphodiesterase and the corresponding effects on c-di-GMP levels and biofilm formation in B. pseudomallei 1026b.

The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of Escherichia coli

“…Several terminal dehydrogenase components ( sdhA–D ) produced altered CR binding phenotypes ( Table S1 ), where loss of the catalytic domains, sdhAB, lead to lower CsgA levels and the loss of stabilizing domains, sdhCD , lead to higher CsgA levels. Additionally, narQ , the primary sensor of the presence of nitrate, has been shown to alter biofilm formation and motility [ 101 , 102 ], and in our study yielded decreased CsgA and CsgD levels ( Table S1 ). Taken together, these respiration mutants point to the involvement of the electron transport chain in curli production.…”

Occurrence and diversity of the oxidative hydroxyhydroquinone pathway for the anaerobic degradation of aromatic compounds in nitrate‐reducing bacteria

“…Only the genes for RehL and RehS were conserved in the genome of seven Burkholderia species; the remaining proteins returned hits with less than 30% identity with the corresponding homologue and located far away from the rehLS genes. Although Burkholderia have been described as obligate aerobes, some pathogenic strains are capable of nitrate respiration (Mangalea et al, ) and the presence of the gene complement for dissimilatory nitrate reduction was suggested for plant‐beneficial‐environmental Burkholderia (Suárez‐Moreno et al, ). In five of the seven Burkholderia species bearing RehLS homologues, these two genes formed a cluster with four additional genes that probably constituted an operon, with an identical organization in all of them.…”

Inactivation of bpsl1039-1040 ATP-binding cassette transporter reduces intracellular survival in macrophages, biofilm formation and virulence in the murine model of Burkholderia pseudomallei infection

“…Anaerobic growth by bacteria, including B . pseudomallei , is accomplished through a denitrification enzymes pathway that catalyse the sequential reduction of nitrate to nitrogen gas [ 24 , 25 ]. Measurement of the nitrate reduction to nitrite in unpermeabilised and permeabilised cell suspensions of the B .…”