The NarX-NarL two-component system is a global regulator of biofilm formation, natural product biosynthesis, and host-associated survival in Burkholderia pseudomallei

Abstract In the environment, Burkholderia pseudomallei exists as a saprophyte inhabiting soils and surface waters where denitrification is important for anaerobic respiration. As an opportunistic pathogen, B. pseudomallei transitions from the environment to infect human and animal hosts where respiratory nitrate reduction enables replication in anoxic conditions. We have previously shown that B. pseudomallei responds to nitrate and nitrite in part by inhibiting biofilm formation and altering cyclic di-GMP signaling. Here, we describe the global transcriptomic response to nitrate and nitrite to characterize the nitrosative stress response relative to biofilm inhibition. To better understand the roles of nitrate-sensing in the biofilm inhibitory phenotype of B. pseudomallei, we created in-frame deletions of narX (Bp1026b_I1014) and narL (Bp1026b_I1013), which are adjacent components of the conserved nitrate-sensing two-component system. Through differential expression analysis of RNA-seq data, we observed that key components of the biofilm matrix are downregulated in response to nitrate and nitrite. In addition, several gene loci associated with the stringent response, central metabolism dysregulation, antibiotic tolerance, and pathogenicity determinants were significantly altered in their expression. Some of the most differentially expressed genes were nonribosomal peptide synthases (NRPS) and/or polyketide synthases (PKS) encoding the proteins for the biosynthesis of bactobolin, malleilactone, and syrbactin, in addition to an uncharacterized cryptic NRPS biosynthetic cluster. We also observed reduced expression of ribosomal structural and biogenesis loci, and gene clusters associated with translation and DNA replication, indicating modulation of growth rate and metabolism under nitrosative stress conditions. The differences in expression observed under nitrosative stress were reversed in narX and narL mutants, suggesting that nitrate sensing is an important checkpoint for regulating the diverse metabolic changes occurring in the biofilm inhibitory phenotype. Moreover, in a macrophage model of infection, narX and narL mutants were attenuated in intracellular replication, suggesting that nitrate sensing is important for host survival. Author Summary Burkholderia pseudomallei is a saprophytic bacterium inhabiting soils and surface waters throughout the tropics causing severe disease in humans and animals. Environmental signals such as the accumulation of inorganic ions mediates the biofilm forming capabilities and survival of B. pseudomallei. In particular, nitrate metabolism inhibits B. pseudomallei biofilm formation through complex regulatory cascades that relay environmental cues to intracellular second messengers that modulate bacterial physiology. Nitrates are common environmental contaminants derived from artificial fertilizers and byproducts of animal wastes that can be readily reduced by bacteria capable of denitrification. In B. pseudomallei 1026b, biofilm dynamics are in part regulated by a gene pathway involved in nitrate sensing, metabolism, and transport. This study investigated the role of a two-component nitrate sensing system, NarX-NarL, in regulating gene expression, biofilm formation, and cellular invasion. Global gene expression analyses in the wild type, as compared to Δ narX and Δ narL mutant strains with nitrate or nitrite implicate the NarX-NarL system in the regulation of biofilm components as well as B. pseudomallei host-associated survival. This study characterizes a conserved nitrate sensing system that is important in environmental and host-associated contexts and aims to bridge a gap between these two important B. pseudomallei lifestyles.