Glycerol metabolism impacts biofilm phenotypes and virulence in Pseudomonas aeruginosa via the Entner-Doudoroff pathway.

Pseudomonas aeruginosa is a ubiquitous bacterium and a notorious opportunistic pathogen that forms biofilm structures in response to many environmental cues. Biofilm formation includes attachment to surfaces and the production of the exopolysaccharide Pel, which is present in both the PAO1 and PA14 laboratory strains of P. aeruginosa. Biofilms help protect bacterial cells from host defenses and antibiotics and abet infection. The carbon source used by the cells also influences biofilm, but these effects have not been deeply studied. We show here that glycerol, which can be liberated from host surfactants during infection, encourages surface attachment and magnifies colony morphology differences. We find that glycerol kinase is important but not essential for glycerol utilization and relatively unimportant for biofilm behaviors. Among downstream enzymes predicted to take part in glycerol utilization, Edd stood out as being important for glycerol utilization and for enhanced biofilm phenotypes in the presence of glycerol. Thus, gluconeogenesis and catabolism of anabolically produced glucose appear to impact not only the utilization of glycerol but also glycerol-stimulated biofilm phenotypes. Finally, waxworm moth larvae and nematode infection models reveal that interruption of the Entner-Doudoroff pathway, but not abrogation of glycerol phosphorylation, unexpectedly increases P. aeruginosa lethality in both acute and chronic infections, even while stimulating a stronger immune response by Caenorhabditis elegans.IMPORTANCEPseudomonas aeruginosa, the ubiquitous environmental bacterium and human pathogen, forms multicellular communities known as biofilms in response to various stimuli. We find that glycerol, a common carbon source that bacteria can use for energy and biosynthesis, encourages biofilm behaviors such as surface attachment and colony wrinkling by P. aeruginosa. Glycerol can be derived from surfactants that are present in the human lungs, a common infection site. Glycerol-stimulated biofilm phenotypes do not depend on phosphorylation of glycerol but are surprisingly impacted by a glucose breakdown pathway, suggesting that it is glycerol utilization, and not its mere presence or cellular import, that stimulates biofilm phenotypes. Moreover, the same mutations that block glycerol-stimulated biofilm phenotypes also impact P. aeruginosa virulence in both acute and chronic animal models. Notably, a glucose-breakdown mutant (Δedd) counteracts biofilm phenotypes but shows enhanced virulence and stimulates a stronger immune response in Caenorhabditis elegans.

Temporal σB stress-response profiles impact Bacillus subtilis fitness.

The Gram-positive model organism Bacillus subtilis responds to environmental stressors by activating the alternative sigma factor σB. The sensing apparatus upstream of σB activation is thought to consist of cytoplasmic stressosomes-megadalton-sized protein complexes that include five paralogous proteins known as RsbRs. The RsbRs are presumed to be involved in stress sensing and the subsequent response. Perturbations to the RsbR complement in stressosomes by engineering cells that produce only one of the RsbR paralogs ("single-RsbR strains") lead to altered σB response dynamics with respect to timing and magnitude. Here, we asked whether such changes to σB response dynamics impact the relative fitness of a strain. We competed strain pairs with different RsbR complements under ethanol and sodium chloride stress and found not only differences in relative fitness among wild-type and single-RsbR strains but also different relative fitness values in the two different stressors. We found that the presence of RsbRA, which dominates the wild-type σB response, enhances fitness in ethanol but is detrimental to fitness in NaCl. Meanwhile, RsbRD-only cells were among the most fit in NaCl. Strains producing hybrid RsbR fusion proteins displayed different fitness values that depended on the RsbR proteins from which they were derived. Our results here suggest that σB response dynamics can impact fitness, highlighting the physiological importance of the unusual stressosome-based general stress response system of B. subtilis. IMPORTANCE: The model bacterium Bacillus subtilis uses cytoplasmic multiprotein complexes, termed stressosomes, to activate the alternative sigma factor σB when facing environmental stresses. We have previously shown that genetically manipulating the complement of putative sensor proteins in stressosomes can alter the dynamics of the σB response in terms of its magnitude and timing. However, it is unknown whether these response dynamics impact the fitness of cells challenged by environmental stressors. Here, we examine the fitness of strains with different σB responses by competing strain pairs in exponential-phase co-cultures under environmental stress. We find that strains with different response dynamics show different competitive indices that differ by stressor. These results suggest that the dynamics of the σB response can affect the fitness of cells facing environmental stress, highlighting the relevance of different σB dynamics.

Bacillus subtilis Stressosome Sensor Protein Sequences Govern the Ability To Distinguish among Environmental Stressors and Elicit Different σB Response Profiles.

Bacteria use a variety of systems to sense stress and mount an appropriate response to ensure fitness and survival. Bacillus subtilis uses stressosomes-cytoplasmic multiprotein complexes-to sense environmental stressors and enact the general stress response by activating the alternative sigma factor σB. Each stressosome includes 40 RsbR proteins, representing four paralogous (RsbRA, RsbRB, RsbRC, and RsbRD) putative stress sensors. Population-level analyses suggested that the RsbR paralogs are largely redundant, while our prior work using microfluidics-coupled fluorescence microscopy uncovered differences among the RsbR paralogs' σB response profiles with respect to timing and intensity when facing an identical stressor. Here, we use a similar approach to address the question of whether the σB responses mediated by each paralog differ in the presence of different environmental stressors: can they distinguish among stressors? Wild-type cells (with all four paralogs) and RsbRA-only cells activate σB with characteristic transient response timing irrespective of stressor but show various response magnitudes. However, cells with other individual RsbR paralogs show distinct timing and magnitude in their responses to ethanol, salt, oxidative, and acid stress, implying that RsbR proteins can distinguish among stressors. Experiments with hybrid fusion proteins comprising the N-terminal half of one paralog and the C-terminal half of another argue that the N-terminal identity influences response magnitude and that determinants in both halves of RsbRA are important for its stereotypical transient σB response timing. IMPORTANCE Bacterial survival depends on appropriate responses to diverse stressors. The general stress-response system in the environmental model bacterium Bacillus subtilis is constantly poised for an immediate response and uses unusual stress-sensing protein complexes called stressosomes. Stressosomes typically contain four different types of putative sensing protein. We asked whether each type of sensor has a distinct role in mediating response dynamics to different environmental stressors. We find that one sensor type always mediates a transient response, while the others show distinct response magnitude and timing to different stressors. We also find that a transient response is exceptional, as several engineered hybrid proteins did not show strong transient responses. Our work reveals functional distinctions among subunits of the stressosome complex and represents a step toward understanding how the general stress response of B. subtilis ensures its survival in natural environmental settings.