Mechanisms of chlorate toxicity and resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that often encounters hypoxic/anoxic environments within the host, which increases its tolerance to many conventional antibiotics. Toward identifying novel treatments, we explored the therapeutic potential of chlorate, a pro-drug that kills hypoxic/anoxic, antibiotic-tolerant P. aeruginosa populations. While chlorate itself is relatively nontoxic, it is enzymatically reduced to the toxic oxidizing agent, chlorite, by hypoxically induced nitrate reductase. To better assess chlorate's therapeutic potential, we investigated mechanisms of chlorate toxicity and resistance in P. aeruginosa. We used transposon mutagenesis to identify genes that alter P. aeruginosa fitness during chlorate treatment, finding that methionine sulfoxide reductases (Msr), which repair oxidized methionine residues, support survival during chlorate stress. Chlorate treatment leads to proteome-wide methionine oxidation, which is exacerbated in a ∆msrA∆msrB strain. In response to chlorate, P. aeruginosa upregulates proteins involved in a wide range of functions, including metabolism, DNA replication/repair, protein repair, transcription, and translation, and these newly synthesized proteins are particularly vulnerable to methionine oxidation. The addition of exogenous methionine partially rescues P. aeruginosa survival during chlorate treatment, suggesting that widespread methionine oxidation contributes to death. Finally, we found that mutations that decrease nitrate reductase activity are a common mechanism of chlorate resistance.

Visualization of mRNA Expression in Pseudomonas aeruginosa Aggregates Reveals Spatial Patterns of Fermentative and Denitrifying Metabolism.

Gaining insight into the behavior of bacteria at the single-cell level is important given that heterogeneous microenvironments strongly influence microbial physiology. The hybridization chain reaction (HCR) is a technique that provides in situ molecular signal amplification, enabling simultaneous mapping of multiple target RNAs at small spatial scales. To refine this method for biofilm applications, we designed and validated new probes to visualize the expression of key catabolic genes in Pseudomonas aeruginosa aggregates. In addition to using existing probes for the dissimilatory nitrate reductase (narG), we developed probes for a terminal oxidase (ccoN1), nitrite reductase (nirS), nitrous oxide reductase (nosZ), and acetate kinase (ackA). These probes can be used to determine gene expression levels across heterogeneous populations such as biofilms. Using these probes, we quantified gene expression across oxygen gradients in aggregate populations grown using the agar block biofilm assay (ABBA). We observed distinct patterns of catabolic gene expression, with upregulation occurring in particular ABBA regions both within individual aggregates and over the aggregate population. Aerobic respiration (ccoN1) showed peak expression under oxic conditions, whereas fermentation (ackA) showed peak expression in the anoxic cores of high metabolic activity aggregates near the air-agar interface. Denitrification genes narG, nirS, and nosZ showed peak expression in hypoxic and anoxic regions, although nirS expression remained at peak levels deeper into anoxic environments than other denitrification genes. These results reveal that the microenvironment correlates with catabolic gene expression in aggregates, and they demonstrate the utility of HCR in unveiling cellular activities at the microscale level in heterogeneous populations. IMPORTANCE To understand bacteria in diverse contexts, we must understand the variations in behaviors and metabolisms they express spatiotemporally. Populations of bacteria are known to be heterogeneous, but the ways this variation manifests can be challenging to characterize due to technical limitations. By focusing on energy conservation, we demonstrate that HCR v3.0 can visualize nuances in gene expression, allowing us to understand how metabolism in Pseudomonas aeruginosa biofilms responds to microenvironmental variation at high spatial resolution. We validated probes for four catabolic genes, including a constitutively expressed oxidase, acetate kinase, nitrite reductase, and nitrous oxide reductase. We showed that the genes for different modes of metabolism are expressed in overlapping but distinct subpopulations according to oxygen concentrations in a predictable fashion. The spatial transcriptomic technique described here has the potential to be used to map microbial activities across diverse environments.

Model Systems to Study the Chronic, Polymicrobial Infections in Cystic Fibrosis: Current Approaches and Exploring Future Directions.

A recent workshop titled "Developing Models to Study Polymicrobial Infections," sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 35+ investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally.

Heat-shock proteases promote survival of Pseudomonas aeruginosa during growth arrest.

When nutrients in their environment are exhausted, bacterial cells become arrested for growth. During these periods, a primary challenge is maintaining cellular integrity with a reduced capacity for renewal or repair. Here, we show that the heat-shock protease FtsH is generally required for growth arrest survival of Pseudomonas aeruginosa, and that this requirement is independent of a role in regulating lipopolysaccharide synthesis, as has been suggested for Escherichia coli We find that ftsH interacts with diverse genes during growth and overlaps functionally with the other heat-shock protease-encoding genes hslVU, lon, and clpXP to promote survival during growth arrest. Systematic deletion of the heat-shock protease-encoding genes reveals that the proteases function hierarchically during growth arrest, with FtsH and ClpXP having primary, nonredundant roles, and HslVU and Lon deploying a secondary response to aging stress. This hierarchy is partially conserved during growth at high temperature and alkaline pH, suggesting that heat, pH, and growth arrest effectively impose a similar type of proteostatic stress at the cellular level. In support of this inference, heat and growth arrest act synergistically to kill cells, and protein aggregation appears to occur more rapidly in protease mutants during growth arrest and correlates with the onset of cell death. Our findings suggest that protein aggregation is a major driver of aging and cell death during growth arrest, and that coordinated activity of the heat-shock response is required to ensure ongoing protein quality control in the absence of growth.

Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid Pseudomonas aeruginosa Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone.

It is well appreciated that oxygen- and other nutrient-limiting gradients characterize microenvironments within chronic infections that foster bacterial tolerance to treatment and the immune response. However, determining how bacteria respond to these microenvironments has been limited by a lack of tools to study bacterial functions at the relevant spatial scales in situ Here, we report the application of the hybridization chain reaction (HCR) v3.0 to provide analog mRNA relative quantitation of Pseudomonas aeruginosa single cells as a step toward this end. To assess the potential for this method to be applied to bacterial populations, we visualized the expression of genes needed for the production of alginate (algD) and the dissimilatory nitrate reductase (narG) at single-cell resolution within laboratory-grown aggregates. After validating new HCR probes, we quantified algD and narG expression across microenvironmental gradients within both single aggregates and aggregate populations using the agar block biofilm assay (ABBA). For mucoid and nonmucoid ABBA populations, narG was expressed in hypoxic and anoxic regions, while alginate expression was restricted to the hypoxic zone (∼40 to 200 µM O2). Within individual aggregates, surface-adjacent cells expressed alginate genes at higher levels than interior cells, revealing that alginate expression is not constitutive in mucoid P. aeruginosa but instead varies with oxygen availability. These results establish HCR v3.0 as a versatile and robust tool to resolve subtle differences in gene expression at spatial scales relevant to microbial assemblages. This advance has the potential to enable quantitative studies of microbial gene expression in diverse contexts, including pathogen activities during infections.IMPORTANCE A goal for microbial ecophysiological research is to reveal microbial activities in natural environments, including sediments, soils, or infected human tissues. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to quantitatively measure microbial gene expression in situ at single-cell resolution in bacterial aggregates. Using quantitative image analysis of thousands of Pseudomonas aeruginosa cells, we validated new P. aeruginosa HCR probes. Within in vitroP. aeruginosa aggregates, we found that bacteria just below the aggregate surface are the primary cells expressing genes that protect the population against antibiotics and the immune system. This observation suggests that therapies targeting bacteria growing with small amounts of oxygen may be most effective against these hard-to-treat infections. More generally, this proof-of-concept study demonstrates that HCR v3.0 has the potential to identify microbial activities in situ at small spatial scales in diverse contexts.

Effects of childhood setting and interaction with nature on academic performance in introductory college-level courses in the environmental sciences.

This study explored the relationships between student background and academic performance in college introductory environmental science (ES) courses at a large U.S. research university with the premise that this analysis may inform teaching practices, curricula, and efforts to increase retention. We surveyed over 700 students across eleven introductory ES courses and used multiple linear mixed-effects regressions to model the data. We found that students who grew up in rural settings or who had frequent childhood interactions with natural environments earned higher grades, on average, than students from urban settings or with fewer childhood interactions with natural environments. Our results indicate that students reporting frequent childhood interactions with forests, for example, were projected to earn grades up to 1.5 letter grades higher in these courses than students with no such interactions. In addition, students with frequent childhood interactions with nature were likelier to report that such interactions helped them in their ES course, suggesting that these students may recognize the value of these experiences. Greater interest in the subject matter also correlated with higher ES course grades, whereas amount of prior ES coursework did not. We discuss the possible implications of these correlations for ES academic performance and educational practice.

Chlorate Specifically Targets Oxidant-Starved, Antibiotic-Tolerant Populations of Pseudomonas aeruginosa Biofilms.

Nitrate respiration is a widespread mode of anaerobic energy generation used by many bacterial pathogens, and the respiratory nitrate reductase, Nar, has long been known to reduce chlorate to the toxic oxidizing agent chlorite. Here, we demonstrate the antibacterial activity of chlorate against Pseudomonas aeruginosa, a representative pathogen that can inhabit hypoxic or anoxic host microenvironments during infection. Aerobically grown P. aeruginosa cells are tobramycin sensitive but chlorate tolerant. In the absence of oxygen or an alternative electron acceptor, cells are tobramycin tolerant but chlorate sensitive via Nar-dependent reduction. The fact that chlorite, the product of chlorate reduction, is not detected in culture supernatants suggests that it may react rapidly and be retained intracellularly. Tobramycin and chlorate target distinct populations within metabolically stratified aggregate biofilms; tobramycin kills cells on the oxic periphery, whereas chlorate kills hypoxic and anoxic cells in the interior. In a matrix populated by multiple aggregates, tobramycin-mediated death of surface aggregates enables deeper oxygen penetration into the matrix, benefiting select aggregate populations by increasing survival and removing chlorate sensitivity. Finally, lasR mutants, which commonly arise in P. aeruginosa infections and are known to withstand conventional antibiotic treatment, are hypersensitive to chlorate. A lasR mutant shows a propensity to respire nitrate and reduce chlorate more rapidly than the wild type does, consistent with its heightened chlorate sensitivity. These findings illustrate chlorate's potential to selectively target oxidant-starved pathogens, including physiological states and genotypes of P. aeruginosa that represent antibiotic-tolerant populations during infections.IMPORTANCE The anaerobic growth and survival of bacteria are often correlated with physiological tolerance to conventional antibiotics, motivating the development of novel strategies targeting pathogens in anoxic environments. A key challenge is to identify drug targets that are specific to this metabolic state. Chlorate is a nontoxic compound that can be reduced to toxic chlorite by a widespread enzyme of anaerobic metabolism. We tested the antibacterial properties of chlorate against Pseudomonas aeruginosa, a pathogen that can inhabit hypoxic or anoxic microenvironments, including those that arise in human infection. Chlorate and the antibiotic tobramycin kill distinct metabolic populations in P. aeruginosa biofilms, where chlorate targets anaerobic cells that tolerate tobramycin. Chlorate is particularly effective against P. aeruginosalasR mutants, which are frequently isolated from human infections and more resistant to some antibiotics. This work suggests that chlorate may hold potential as an anaerobic prodrug.

Different Functions of Phylogenetically Distinct Bacterial Complex I Isozymes.

UNLABELLED: NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethyl sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroidescomplex I enzymes (complex IA and complex IE) in maintaining a cellular redox state during photoheterotrophic growth. We propose that the function of each isozyme during photoheterotrophic growth is either NADH synthesis (complex IA) or NADH oxidation (complex IE). The canonical alphaproteobacterial complex I isozyme (complex IA) was also shown to be important for routing electrons to nitrogenase-mediated H2 production, while the horizontally acquired enzyme (complex IE) was dispensable in this process. Unlike the singular role of complex I in mitochondria, we predict that the phylogenetically distinct complex I enzymes found across bacterial species have evolved to enhance the functions of their respective electron transport chains. IMPORTANCE: Cells use a proton motive force (PMF), NADH, and ATP to support numerous processes. In mitochondria, complex I uses NADH oxidation to generate a PMF, which can drive ATP synthesis. This study analyzed the function of complex I in bacteria, which contain more-diverse and more-flexible electron transport chains than mitochondria. We tested complex I function in Rhodobacter sphaeroides, a bacterium predicted to encode two phylogenetically distinct complex I isozymes. R. sphaeroides cells lacking both isozymes had growth defects during all tested modes of growth, illustrating the important function of this enzyme under diverse conditions. We conclude that the two isozymes are not functionally redundant and predict that phylogenetically distinct complex I enzymes have evolved to support the diverse lifestyles of bacteria.

Phylogenomic analysis and predicted physiological role of the proton-translocating NADH:quinone oxidoreductase (complex I) across bacteria.

UNLABELLED: The proton-translocating NADH:quinone oxidoreductase (complex I) is a multisubunit integral membrane enzyme found in the respiratory chains of both bacteria and eukaryotic organelles. Although much research has focused on the enzyme's central role in the mitochondrial respiratory chain, comparatively little is known about its role in the diverse energetic lifestyles of different bacteria. Here, we used a phylogenomic approach to better understand the distribution of complex I across bacteria, the evolution of this enzyme, and its potential roles in shaping the physiology of different bacterial groups. By surveying 970 representative bacterial genomes, we predict complex I to be present in ~50% of bacteria. While this includes bacteria with a wide range of energetic schemes, the presence of complex I is associated with specific lifestyles, including aerobic respiration and specific types of phototrophy (bacteria with only a type II reaction center). A phylogeny of bacterial complex I revealed five main clades of enzymes whose evolution is largely congruent with the evolution of the bacterial groups that encode complex I. A notable exception includes the gammaproteobacteria, whose members encode one of two distantly related complex I enzymes predicted to participate in different types of respiratory chains (aerobic versus anaerobic). Comparative genomic analyses suggest a broad role for complex I in reoxidizing NADH produced from various catabolic reactions, including the tricarboxylic acid (TCA) cycle and fatty acid beta-oxidation. Together, these findings suggest diverse roles for complex I across bacteria and highlight the importance of this enzyme in shaping diverse physiologies across the bacterial domain. IMPORTANCE: Living systems use conserved energy currencies, including a proton motive force (PMF), NADH, and ATP. The respiratory chain enzyme, complex I, connects these energy currencies by using NADH produced during nutrient breakdown to generate a PMF, which is subsequently used for ATP synthesis. Our goal is to better understand the role of complex I in bacteria, whose energetic diversity allows us to view its function in a range of biological contexts. We analyzed sequenced bacterial genomes to predict the presence, evolution, and function of complex I in bacteria. We identified five main classes of bacterial complex I and predict that different classes participate in different types of respiratory chains (aerobic and anaerobic). We also predict that complex I helps maintain a cellular redox state by reoxidizing NADH produced from central metabolism. Our findings suggest diverse roles for complex I in bacterial physiology, highlighting the need for future laboratory-based studies.

Pathways involved in reductant distribution during photobiological H(2) production by Rhodobacter sphaeroides.

We used global transcript analyses and mutant studies to investigate the pathways that impact H(2) production in the photosynthetic bacterium Rhodobacter sphaeroides. We found that H(2) production capacity is related to the levels of expression of the nitrogenase and hydrogenase enzymes and the enzymes of the Calvin-Benson-Bassham pathway.