Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered the biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa.

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP's many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we quantify the motion of chromosomal loci and cytoplasmic tracer particles. In the absence of polyP and upon starvation, we observe a 2- to 10-fold increase in mean cytoplasmic diffusivity. Tracer particles reveal that polyP also modulates the partitioning between a "more mobile" and a "less mobile" population: Small particles in cells unable to make polyP are more likely to be "mobile" and explore more of the cytoplasm, particularly during starvation. Concomitant with this larger freedom of motion in polyP-deficient cells, we observe decompaction of the nucleoid and an increase in the steady-state concentration of ATP. The dramatic polyP-dependent effects we observe on cytoplasmic transport properties occur under nitrogen starvation, but not carbon starvation, suggesting that polyP may have distinct functions under different types of starvation.

Uncultivated DPANN archaea are ubiquitous inhabitants of global oxygen-deficient zones with diverse metabolic potential.

Archaea belonging to the DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota) superphylum have been found in an expanding number of environments and perform a variety of biogeochemical roles, including contributing to carbon, sulfur, and nitrogen cycling. Generally characterized by ultrasmall cell sizes and reduced genomes, DPANN archaea may form mutualistic, commensal, or parasitic interactions with various archaeal and bacterial hosts, influencing the ecology and functioning of microbial communities. While DPANN archaea reportedly comprise a sizeable fraction of the archaeal community within marine oxygen-deficient zone (ODZ) water columns, little is known about their metabolic capabilities in these ecosystems. We report 33 novel metagenome-assembled genomes (MAGs) belonging to the DPANN phyla Nanoarchaeota, Pacearchaeota, Woesearchaeota, Undinarchaeota, Iainarchaeota, and SpSt-1190 from pelagic ODZs in the Eastern Tropical North Pacific and the Arabian Sea. We find these archaea to be permanent, stable residents of all three major ODZs only within anoxic depths, comprising up to 1% of the total microbial community and up to 25%-50% of archaea as estimated from read mapping to MAGs. ODZ DPANN appear to be capable of diverse metabolic functions, including fermentation, organic carbon scavenging, and the cycling of sulfur, hydrogen, and methane. Within a majority of ODZ DPANN, we identify a gene homologous to nitrous oxide reductase. Modeling analyses indicate the feasibility of a nitrous oxide reduction metabolism for host-attached symbionts, and the small genome sizes and reduced metabolic capabilities of most DPANN MAGs suggest host-associated lifestyles within ODZs. IMPORTANCE: Archaea from the DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota) superphylum have diverse metabolic capabilities and participate in multiple biogeochemical cycles. While metagenomics and enrichments have revealed that many DPANN are characterized by ultrasmall genomes, few biosynthetic genes, and episymbiotic lifestyles, much remains unknown about their biology. We report 33 new DPANN metagenome-assembled genomes originating from the three global marine oxygen-deficient zones (ODZs), the first from these regions. We survey DPANN abundance and distribution within the ODZ water column, investigate their biosynthetic capabilities, and report potential roles in the cycling of organic carbon, methane, and nitrogen. We test the hypothesis that nitrous oxide reductases found within several ODZ DPANN genomes may enable ultrasmall episymbionts to serve as nitrous oxide consumers when attached to a host nitrous oxide producer. Our results indicate DPANN archaea as ubiquitous residents within the anoxic core of ODZs with the potential to produce or consume key compounds.

The proteome is a terminal electron acceptor.

Microbial metabolism is impressively flexible, enabling growth even when available nutrients differ greatly from biomass in redox state. E. coli, for example, rearranges its physiology to grow on reduced and oxidized carbon sources through several forms of fermentation and respiration. To understand the limits on and evolutionary consequences of metabolic flexibility, we developed a mathematical model coupling redox chemistry with principles of cellular resource allocation. Our integrated model clarifies key phenomena, including demonstrating that autotrophs grow slower than heterotrophs because of constraints imposed by intracellular production of reduced carbon. Our model further indicates that growth is improved by adapting the redox state of biomass to nutrients, revealing an unexpected mode of evolution where proteins accumulate mutations benefiting organismal redox balance.

Intracellular Pseudomonas aeruginosa within the Airway Epithelium of Cystic Fibrosis Lung Tissues.

RATIONALE: Pseudomonas aeruginosa (P.a.) is the major bacterial pathogen colonizing the airways of adult cystic fibrosis (CF) patients and causes chronic infections that persist despite antibiotic therapy. Intracellular bacteria may represent an unrecognized reservoir of bacteria that evades the immune system and antibiotic therapy. While the ability of P.a. to invade and survive within epithelial cells has been described in vitro in different epithelial cell models, evidence of this intracellular lifestyle in human lung tissues is currently lacking. OBJECTIVES: To detect and characterize intracellular P.a. in CF airway epithelium from human lung explant tissues. METHODS: We sampled the lung explant tissues from CF patients undergoing lung transplantation and non-CF lung donor control. We analyzed lung tissue sections for the presence of intracellular P.a. by quantitative culture and microscopy, in parallel to histopathology and airway morphometry. MEASUREMENTS AND MAIN RESULTS: P.a. was isolated from the lungs of 7 CF patients undergoing lung transplantation. Microscopic assessment revealed the presence of intracellular P.a. within airway epithelial cells in 3 out of the 7 patients analyzed, at a varying but low frequency. We observed those events occurring in lung regions with high bacterial burden. CONCLUSION: This is the first study describing the presence of intracellular P.a. in CF lung tissues. While intracellular P.a. in airway epithelial cells are likely relatively rare events, our findings highlight the plausible occurrence of this intracellular bacterial reservoir in chronic CF infections.

Engineering the Soil Bacterium Pseudomonas synxantha 2-79 into a Ratiometric Bioreporter for Phosphorus Limitation.

Microbial bioreporters hold promise for addressing challenges in medical and environmental applications. However, the difficulty in ensuring their stable persistence and function within the target environment remains a challenge. One strategy is to integrate information about the host strain and target environment into the design-build-test cycle of the bioreporter itself. Here, we present a case study for such an environmentally motivated design process by engineering the wheat commensal bacterium Pseudomonas synxantha 2-79 into a ratiometric bioreporter for phosphorus limitation. Comparative analysis showed that an exogenous P-responsive promoter outperformed its native counterparts. This reporter can selectively sense and report phosphorus limitation at plant-relevant concentrations of 25-100 µM without cross-activation from carbon or nitrogen limitation or high cell densities. Its performance is robust over a field-relevant pH range (5.8-8), and it responds only to inorganic phosphorus, even in the presence of common soil organic P. Finally, we used fluorescein-calibrated flow cytometry to assess whether the reporter's performance in shaken liquid culture predicts its performance in soil, finding that although the reporter is still functional at the bulk level, its variability in performance increases when grown in a soil slurry as compared to planktonic culture, with a fraction of the population not expressing the reporter proteins. Together, our environmentally aware design process provides an example of how laboratory bioengineering efforts can generate microbes with a greater promise to function reliably in their applied contexts.

Uncultivated DPANN archaea are ubiquitous inhabitants of global oxygen deficient zones with diverse metabolic potential.

Archaea belonging to the DPANN superphylum have been found within an expanding number of environments and perform a variety of biogeochemical roles, including contributing to carbon, sulfur, and nitrogen cycling. Generally characterized by ultrasmall cell sizes and reduced genomes, DPANN archaea may form mutualistic, commensal, or parasitic interactions with various archaeal and bacterial hosts, influencing the ecology and functioning of microbial communities. While DPANN archaea reportedly comprise 15-26% of the archaeal community within marine oxygen deficient zone (ODZ) water columns, little is known about their metabolic capabilities in these ecosystems. We report 33 novel metagenome-assembled genomes belonging to DPANN phyla Nanoarchaeota, Pacearchaeota, Woesarchaeota, Undinarchaeota, Iainarchaeota, and SpSt-1190 from pelagic ODZs in the Eastern Tropical North Pacific and Arabian Sea. We find these archaea to be permanent, stable residents of all 3 major ODZs only within anoxic depths, comprising up to 1% of the total microbial community and up to 25-50% of archaea. ODZ DPANN appear capable of diverse metabolic functions, including fermentation, organic carbon scavenging, and the cycling of sulfur, hydrogen, and methane. Within a majority of ODZ DPANN, we identify a gene homologous to nitrous oxide reductase. Modeling analyses indicate the feasibility of a nitrous oxide reduction metabolism for host-attached symbionts, and the small genome sizes and reduced metabolic capabilities of most DPANN MAGs suggest host-associated lifestyles within ODZs.

Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

Widespread detoxifying NO reductases impart a distinct isotopic fingerprint on N2O under anoxia.

Nitrous oxide (N2O), a potent greenhouse gas, can be generated by compositionally complex microbial populations in diverse contexts. Accurately tracking the dominant biological sources of N2O has the potential to improve our understanding of N2O fluxes from soils as well as inform the diagnosis of human infections. Isotopic "Site Preference" (SP) values have been used towards this end, as bacterial and fungal nitric oxide reductases produce N2O with different isotopic fingerprints. Here we show that flavohemoglobin, a hitherto biogeochemically neglected yet widely distributed detoxifying bacterial NO reductase, imparts a distinct SP value onto N2O under anoxic conditions that correlates with typical environmental N2O SP measurements. We suggest a new framework to guide the attribution of N2O biological sources in nature and disease.

Pyocyanin-dependent electrochemical inhibition of Pseudomonas aeruginosa biofilms is synergistic with antibiotic treatment.

Pseudomonas aeruginosa biofilms are common in chronic wound infections and recalcitrant to treatment. Survival of cells within oxygen-limited regions in these biofilms is enabled by extracellular electron transfer (EET), whereby small redox active molecules act as electron shuttles to access distal oxidants. Here, we report that electrochemically controlling the redox state of these electron shuttles, specifically pyocyanin (PYO), can impact cell survival within anaerobic P. aeruginosa biofilms and can act synergistically with antibiotic treatment. Prior results demonstrated that under anoxic conditions, an electrode poised at sufficiently oxidizing potential (+100 mV vs Ag/AgCl) promotes EET within P. aeruginosa biofilms by re-oxidizing PYO for reuse by the cells. Here, when a reducing potential (-400 mV vs Ag/AgCl) was used to disrupt PYO redox cycling by maintaining PYO in the reduced state, we observed a 100-fold decrease in colony forming units within these biofilms compared with those exposed to electrodes poised at +100 mV vs Ag/AgCl. Phenazine-deficient Δphz* biofilms were unaffected by the potential applied to the electrode but were re-sensitized by adding PYO. The effect at -400 mV was exacerbated when biofilms were treated with sub-MICs of a range of antibiotics. Most notably, addition of the aminoglycoside gentamicin in a reductive environment almost completely eradicated wild-type biofilms but had no effect on the survival of Δphz* biofilms in the absence of phenazines. These data suggest that antibiotic treatment combined with the electrochemical disruption of PYO redox cycling, either through the toxicity of accumulated reduced PYO or the disruption of EET, or both, can lead to extensive killing. IMPORTANCE Biofilms provide a protective environment but also present challenges to the cells living within them, such as overcoming nutrient and oxygen diffusion limitations. Pseudomonas aeruginosa overcomes oxygen limitation by secreting soluble redox active phenazines, which act as electron shuttles to distal oxygen. Here, we show that electrochemically blocking the re-oxidation of one of these electron shuttles, pyocyanin, decreases cell survival within biofilms and acts synergistically with gentamicin to kill cells. Our results highlight the importance of the role that the redox cycling of electron shuttles fulfills within P. aeruginosa biofilms.

A phenazine-inspired framework for identifying biological functions of microbial redox-active metabolites.

While the list of small molecules known to be secreted by environmental microbes continues to grow, our understanding of their in situ biological functions remains minimal. The time has come to develop a framework to parse the meaning of these "secondary metabolites," which are ecologically ubiquitous and have direct applications in medicine and biotechnology. Here, we focus on a particular subset of molecules, redox active metabolites (RAMs), and review the well-studied phenazines as archetypes of this class. We argue that efforts to characterize the chemical, physical and biological makeup of the microenvironments, wherein these molecules are produced, coupled with measurements of the molecules' basic chemical properties, will enable significant progress in understanding the precise roles of novel RAMs.

The chemical ecology of coumarins and phenazines affects iron acquisition by pseudomonads.

Secondary metabolites are important facilitators of plant-microbe interactions in the rhizosphere, contributing to communication, competition, and nutrient acquisition. However, at first glance, the rhizosphere seems full of metabolites with overlapping functions, and we have a limited understanding of basic principles governing metabolite use. Increasing access to the essential nutrient iron is one important, but seemingly redundant role performed by both plant and microbial Redox-Active Metabolites (RAMs). We used coumarins, RAMs made by the model plant Arabidopsis thaliana, and phenazines, RAMs made by soil-dwelling pseudomonads, to ask whether plant and microbial RAMs might each have distinct functions under different environmental conditions. We show that variations in oxygen and pH lead to predictable differences in the capacity of coumarins vs phenazines to increase the growth of iron-limited pseudomonads and that these effects depend on whether pseudomonads are grown on glucose, succinate, or pyruvate: carbon sources commonly found in root exudates. Our results are explained by the chemical reactivities of these metabolites and the redox state of phenazines as altered by microbial metabolism. This work shows that variations in the chemical microenvironment can profoundly affect secondary metabolite function and suggests plants may tune the utility of microbial secondary metabolites by altering the carbon released in root exudates. Together, these findings suggest that RAM diversity may be less overwhelming when viewed through a chemical ecological lens: Distinct molecules can be expected to be more or less important to certain ecosystem functions, such as iron acquisition, depending on the local chemical microenvironments in which they reside.

NADH dehydrogenases are the predominant phenazine reductases in the electron transport chain of Pseudomonas aeruginosa.

Phenazines are redox-active secondary metabolites produced by diverse bacteria including the opportunistic pathogen Pseudomonas aeruginosa. Extracellular electron transfer via phenazines enhances anaerobic survival by serving as an electron sink for glucose catabolism. However, the specific phenazine reductase(s) used to support this catabolism are unknown. Because electron transport chain components have been previously implicated in phenazine reduction, we sought to determine which of them possess phenazine reductase activity. We show that phenazine-1-carboxamide (PCN) and pyocyanin (PYO) are reduced at the highest rate by cells and are localized to the cell envelope while reduced. Using a coupled genetic and biochemical approach, we show that phenazine reductase activity in membrane fractions is attributable to the three NADH dehydrogenases present in P. aeruginosa and that their order of phenazine reductase activity is Nqr > Nuo > Ndh. In mutants possessing only one functional NADH dehydrogenase, whole cell reduction rates of PCN, but not PYO, recapitulate the pattern of biochemical results, implying that PYO reduction is predominantly occurring in the cytosol. Lastly, we show that ubiquinone rapidly and non-enzymatically oxidizes reduced phenazines, demonstrating that phenazines have the capability to serve in a redox loop between the NADH and ubiquinone pools, a finding that carries bioenergetic implications.

The contrasting roles of nitric oxide drive microbial community organization as a function of oxygen presence.

Microbial assemblages are omnipresent in the biosphere, forming communities on the surfaces of roots and rocks and within living tissues. These communities can exhibit strikingly beautiful compositional structures, with certain members reproducibly occupying particular spatiotemporal microniches. Despite this reproducibility, we lack the ability to explain these spatial patterns. We hypothesize that certain spatial patterns in microbial communities may be explained by the exchange of redox-active metabolites whose biological function is sensitive to microenvironmental gradients. To test this, we developed a simple community consisting of synthetic Pseudomonas aeruginosa strains with a partitioned denitrification pathway: a strict consumer and strict producer of nitric oxide (NO), a key pathway intermediate. Because NO can be both toxic or beneficial depending on the amount of oxygen present, this system provided an opportunity to investigate whether dynamic oxygen gradients can tune metabolic cross-feeding and fitness outcomes in a predictable fashion. Using a combination of genetic analysis, controlled growth environments, and imaging, we show that oxygen availability dictates whether NO cross-feeding is deleterious or mutually beneficial and that this organizing principle maps to the microscale. More generally, this work underscores the importance of considering the double-edged and microenvironmentally tuned roles redox-active metabolites can play in shaping microbial communities.

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.

Optical O2 Sensors Also Respond to Redox Active Molecules Commonly Secreted by Bacteria.

From a metabolic perspective, molecular oxygen (O2) is arguably the most significant constituent of Earth's atmosphere. Nearly every facet of microbial physiology is sensitive to the presence and concentration of O2, which is the most favorable terminal electron acceptor used by organisms and also a dangerously reactive oxidant. As O2 has such sweeping implications for physiology, researchers have developed diverse approaches to measure O2 concentrations in natural and laboratory settings. Recent improvements to phosphorescent O2 sensors piqued our interest due to the promise of optical measurement of spatiotemporal O2 dynamics. However, we found that our preferred bacterial model, Pseudomonas aeruginosa PA14, secretes more than one molecule that quenches such sensors, complicating O2 measurements in PA14 cultures and biofilms. Assaying supernatants from cultures of 9 bacterial species demonstrated that this phenotype is common: all supernatants quenched a soluble O2 probe substantially. Phosphorescent O2 probes are often embedded in solid support for protection, but an embedded probe called O2NS was quenched by most supernatants as well. Measurements using pure compounds indicated that quenching is due to interactions with redox-active small molecules, including phenazines and flavins. Uncharged and weakly polar molecules like pyocyanin were especially potent quenchers of O2NS. These findings underscore that optical O2 measurements made in the presence of bacteria should be carefully controlled to ensure that O2, and not bacterial secretions, is measured, and motivate the design of custom O2 probes for specific organisms to circumvent sensitivity to redox-active metabolites. IMPORTANCE When they are closely packed, as in biofilms, colonies, and soils, microbes can consume O2 faster than it diffuses. As such, O2 concentrations in natural environments can vary greatly over time and space, even on the micrometer scale. Wetting soil, for example, slows O2 diffusion higher in the soil column, which, in concert with microbial respiration, greatly diminishes [O2] at depth. Given that variation in [O2] has outsized implications for microbial physiology, there is great interest in measuring the dynamics of [O2] in microbial cultures and biofilms. We demonstrate that certain classes of bacterial metabolites frustrate optical measurement of [O2] with phosphorescent sensors, but also that some species (e.g., E. coli) do not produce problematic secretions under the conditions tested. Our work therefore offers a strategy for identifying organisms and culture conditions in which optical quantification of spatiotemporal [O2] dynamics with current sensors is feasible.

Hopanoids Confer Robustness to Physicochemical Variability in the Niche of the Plant Symbiont Bradyrhizobium diazoefficiens.

Rhizobia are a group of bacteria that increase soil nitrogen content through symbiosis with legume plants. The soil and symbiotic host are potentially stressful environments, and the soil will likely become even more stressful as the climate changes. Many rhizobia within the Bradyrhizobium clade, like Bradyrhizobium diazoefficiens, possess the genetic capacity to synthesize hopanoids, steroid-like lipids similar in structure and function to cholesterol. Hopanoids are known to protect against stresses relevant to the niche of B. diazoefficiens. Paradoxically, mutants unable to synthesize the extended class of hopanoids participate in symbioses with success similar to that of the wild type, despite being delayed in root nodule initiation. Here, we show that in B. diazoefficiens, the growth defects of extended-hopanoid-deficient mutants can be at least partially compensated for by the physicochemical environment, specifically, by optimal osmotic and divalent cation concentrations. Through biophysical measurements of lipid packing and membrane permeability, we show that extended hopanoids confer robustness to environmental variability. These results help explain the discrepancy between previous in-culture and in planta results and indicate that hopanoids may provide a greater fitness advantage to rhizobia in the variable soil environment than the more controlled environments within root nodules. To improve the legume-rhizobium symbiosis through either bioengineering or strain selection, it will be important to consider the full life cycle of rhizobia, from soil to symbiosis. IMPORTANCE Rhizobia, such as B. diazoefficiens, play an important role in the nitrogen cycle by making nitrogen gas bioavailable through symbiosis with legume plants. As climate change threatens soil health, this symbiosis has received increased attention as a more sustainable source of soil nitrogen than the energy-intensive Haber-Bosch process. Efforts to use rhizobia as biofertilizers have been effective; however, long-term integration of rhizobia into the soil community has been less successful. This work represents a small step toward improving the legume-rhizobium symbiosis by identifying a cellular component-hopanoid lipids-that confers robustness to environmental stresses rhizobia are likely to encounter in soil microenvironments as sporadic desiccation and flooding events become more common.

Phenazines and toxoflavin act as interspecies modulators of resilience to diverse antibiotics.

Bacterial opportunistic pathogens make diverse secondary metabolites both in the natural environment and when causing infections, yet how these molecules mediate microbial interactions and their consequences for antibiotic treatment are still poorly understood. Here, we explore the role of three redox-active secondary metabolites, pyocyanin, phenazine-1-carboxylic acid, and toxoflavin, as interspecies modulators of antibiotic resilience. We find that these molecules dramatically change susceptibility levels of diverse bacteria to clinical antibiotics. Pyocyanin and phenazine-1-carboxylic acid are made by Pseudomonas aeruginosa, while toxoflavin is made by Burkholderia gladioli, organisms that infect cystic fibrosis and other immunocompromised patients. All molecules alter the susceptibility profile of pathogenic species within the "Burkholderia cepacia complex" to different antibiotics, either antagonizing or potentiating their effects, depending on the drug's class. Defense responses regulated by the redox-sensitive transcription factor SoxR potentiate the antagonistic effects these metabolites have against fluoroquinolones, and the presence of genes encoding SoxR and the efflux systems it regulates can be used to predict how these metabolites will affect antibiotic susceptibility of different bacteria. Finally, we demonstrate that inclusion of secondary metabolites in standard protocols used to assess antibiotic resistance can dramatically alter the results, motivating the development of new tests for more accurate clinical assessment.

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.

Microbial communities: The metabolic rate is the trait.

Making sense of the metabolism of microbial communities is a daunting task. Using denitrification as a model metabolism, a new paper shows that the rate of denitrification can often be predicted from genome contents, and dynamical models can be composed to predict denitrification rates of communities of two to five species.