Biology subjects

Newman, D. K.

Publications and source records attributed to Newman, D. K..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

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 Earths 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 biological 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. ImportanceWhen 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.

microbiology↗

bioRxiv2022

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 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 both in liquid culture and in 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 was much stronger in anoxic environments compared to other denitrification genes. These results reveal that the microenvironment correlates with catabolic gene expression in aggregates, and demonstrate the utility of HCR in unveiling cellular activities at the microscale in heterogeneous populations. ImportanceTo 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: 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.

microbiology↗

bioRxiv2021

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 polyPs 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 characterize the motion of chromosomal loci and free tracer particles in the cytoplasm. In the absence of polyP and upon starvation, we observe an increase in mobility both for chromosomal loci and for tracer particles. 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. We speculate that this larger freedom of motion may be a consequence of nucleoid decompaction, which we also observe in starved cells deficient in polyP. Our observations suggest that polyP limits cytoplasmic mobility and accessibility during nitrogen starvation, which may help to explain the pleiotropic phenotypes observed in the absence of polyP.

biophysics↗

bioRxiv2021

The double-edged role of nitric oxide drives predictable microbial community organization according to the microenvironment

Microbial assemblages are omnipresent in the biosphere, forming communities on the surfaces of roots, 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 principal maps to the microscale. More generally, this work underscores the importance of considering the contrasting and microenvironmentally tuned roles redox-active metabolites can play in shaping microbial communities.

microbiology↗

bioRxiv2021

Redox-active secondary metabolites act as interspecies modulators of antibiotic resilience

Bacterial opportunistic pathogens make a wide range of 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 two redox-active secondary metabolites, pyocyanin and toxoflavin, as interspecies modulators of antibiotic resilience. We find that these molecules dramatically change susceptibility levels of diverse bacteria to clinical antibiotics. Pyocyanin is made by Pseudomonas aeruginosa, while toxoflavin is made by Burkholderia gladioli, organisms that infect cystic fibrosis and other immunocompromised patients. Both 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 drugs 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.

microbiology↗

bioRxiv2021

Prevalence and correlates of phenazine resistance in culturable bacteria from a dryland wheat field

Phenazines are a class of bacterially-produced redox-active natural antibiotics that have demonstrated potential as a sustainable alternative to traditional pesticides for the biocontrol of fungal crop diseases. However, the prevalence of bacterial resistance to agriculturally-relevant phenazines is poorly understood, limiting both the understanding of how these molecules might shape rhizosphere bacterial communities and the ability to perform risk assessment for off-target effects. Here, we describe profiles of susceptibility to the antifungal agent phenazine-1-carboxylic acid (PCA) across more than 100 bacterial strains isolated from a wheat field where PCA producers are indigenous and abundant. We find that Gram-positive bacteria are typically more sensitive to PCA than Gram-negative bacteria, but that there is also significant variability in susceptibility both within and across phyla. Phenazine-resistant strains are more likely to be isolated from the wheat rhizosphere, where PCA producers are also more abundant, compared to bulk soil. Furthermore, PCA toxicity is pH-dependent for most susceptible strains and broadly correlates with PCA reduction rates, suggesting that uptake and redox-cycling are important determinants of phenazine toxicity. Our results shed light on which classes of bacteria are most likely to be susceptible to phenazine toxicity in acidic or neutral soils. In addition, the taxonomic and phenotypic diversity of our strain collection represents a valuable resource for future studies on the role of natural antibiotics in shaping wheat rhizosphere communities. ImportanceMicrobial communities contribute to crop health in important ways. For example, phenazine metabolites are a class of redox-active molecules made by diverse soil bacteria that underpin the biocontrol of wheat and other crops. Their physiological functions are nuanced: in some contexts they are toxic, in others, beneficial. While much is known about phenazine production and the effect of phenazines on producing strains, our ability to predict how phenazines might shape the composition of environmental microbial communities is poorly constrained; that phenazine prevalence in the rhizosphere is predicted to increase in arid soils as the climate changes provides an impetus for further study. As a step towards gaining a predictive understanding of phenazine-linked microbial ecology, we document the effects of phenazines on diverse bacteria that were co-isolated from a wheat rhizosphere and identify conditions and phenotypes that correlate with how a strain will respond to phenazines.

microbiology↗

bioRxiv2021

Hopanoids confer robustness to physicochemical variability in the niche of the plant symbiont Bradyrhizobium diazoefficiens

Climate change poses a threat to soil health and agriculture, but the potential effects of climate change on soil bacteria that can help maintain soil health are understudied. Rhizobia are a group of bacteria that increase soil nitrogen content through a symbiosis with legume plants. The soil and symbiosis are potentially stressful environments, and the soil will likely become even more stressful as the climate changes. Many rhizobia within the bradyrhizobia 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 similarly successful symbioses compared to the wild type, despite being delayed in root nodule initiation. Here, we show that in B. diazoefficiens, the in vitro 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, we show that extended hopanoids confer robustness to environmental variability. These results help explain the discrepancy between previous in vitro 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 environment within root nodules. To improve the legume-rhizobia symbiosis through either bioengineering or strain selection, it will be important to consider the full lifecycle of rhizobia, from the soil to the symbiosis. ImportanceRhizobia, 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 reentered the spotlight 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 towards improving the legume-rhizobia 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.

microbiology↗

bioRxiv2021

Nitrate reduction stimulates and is stimulated by phenazine-1-carboxylic acid oxidation by Citrobacter portucalensis MBL

Phenazines are secreted metabolites that microbes use in diverse ways, from quorum sensing to antimicrobial warfare to energy conservation. Phenazines are able to contribute to these activities due to their redox activity. The physiological consequences of cellular phenazine reduction have been extensively studied, but the counterpart phenazine oxidation has been largely overlooked. Phenazine-1-carboxylic acid (PCA) is common in the environment and readily reduced by its producers. Here, we describe its anaerobic oxidation by Citrobacter portucalensis strain MBL, which was isolated from topsoil in Falmouth, MA, and which does not produce phenazines itself. This activity depends on the availability of a suitable terminal electron acceptor, specifically nitrate. When C. portucalensis MBL is provided reduced PCA and nitrate, it rapidly oxidizes the PCA. We compared this terminal electron acceptor-dependent PCA-oxidizing activity of C. portucalensis MBL to that of several other {gamma}-proteobacteria with varying capacities to respire nitrate. We found that PCA oxidation by these strains in a nitrate-dependent manner is decoupled from growth and correlated with their possession of the periplasmic nitrate reductase Nap. We infer that bacterial PCA oxidation is widespread and propose that it may be genetically determined. Notably, oxidizing PCA enhances the rate of nitrate reduction to nitrite by C. portucalensis MBL beyond the stoichiometric exchange of electrons from PCA to nitrate, which we attribute to C. portucalensis MBLs ability to also reduce oxidized PCA, thereby catalyzing a complete PCA redox cycle. This bidirectionality highlights the versatility of PCA as a biological redox agent. IMPORTANCEPhenazines are increasingly appreciated for their roles in structuring microbial communities. These tricyclic aromatic molecules have been found to regulate gene expression, be toxic, promote antibiotic tolerance, and promote survival under oxygen starvation. In all of these contexts, however, phenazines are studied as electron acceptors. Even if their utility arises primarily from being readily reduced, they need to be oxidized in order to be recycled. While oxygen and ferric iron can oxidize phenazines abiotically, biotic oxidation of phenazines has not been studied previously. We observed bacteria that readily oxidize phenazine-1-carboxylic acid (PCA) in a nitrate-dependent fashion, concomitantly increasing the rate of nitrate reduction to nitrite. Because nitrate is a prevalent terminal electron acceptor in diverse anoxic environments, including soils, and phenazine-producers are widespread, this observation of linked phenazine and nitrogen redox cycling suggests an underappreciated role for redox-active secreted metabolites in the environment.

microbiology↗

bioRxiv2021

Paraburkholderia edwinii protects Aspergillus sp. from phenazines by acting as a toxin sponge

Many environmentally and clinically important fungi are sensitive to toxic, bacterially-produced, redox-active molecules called phenazines. Despite being vulnerable to phenazine-assault, fungi inhabit microbial communities that contain phenazine producers. Because many fungi cannot withstand phenazine challenge, but some bacterial species can, we hypothesized that bacterial partners may protect fungi in phenazine-replete environments. In the first soil sample we collected, we co-isolated several such physically associated pairings. We discovered the novel species Paraburkholderia edwinii and demonstrated it can protect a co-isolated Aspergillus species from phenazine-1-carboxylic acid (PCA) by sequestering it, acting as a toxin sponge; in turn, it also gains protection. When challenged with PCA, P. edwinii changes its morphology, forming aggregates within the growing fungal colony. Further, the fungal partner triggers P. edwinii to sequester PCA and maintains conditions that limit PCA toxicity by promoting an anoxic and highly reducing environment. A mutagenic screen revealed this program depends on the stress-inducible transcriptional repressor HrcA. We show that one relevant stressor in response to PCA challenge is fungal acidification and that acid stress causes P. edwinii to behave as though the fungus were present. Finally, we reveal this phenomenon as widespread among Paraburkholderia with moderate specificity among bacterial and fungal partners, including plant and human pathogens. Our discovery suggests a common mechanism by which fungi can gain access to phenazine-replete environments, and provides a tractable model system for its study. These results have implications for how rhizosphere microbial communities as well as plant and human infection sites are policed for fungal membership.

microbiology↗

bioRxiv2021

In situ single-cell activities of microbial populations revealed by spatial transcriptomics

Microbial populations and communities are heterogeneous, yet capturing their diverse activities has proven challenging at the relevant spatiotemporal scales. Here we present par-seqFISH, a targeted transcriptome-imaging approach that records both gene-expression and spatial context within microscale assemblies at a single-cell and molecule resolution. We apply this approach to the opportunistic bacterial pathogen, Pseudomonas aeruginosa, analyzing [~]600,000 individuals across dozens of physiological conditions in planktonic and biofilm cultures. We explore the phenotypic landscape of this bacterium and identify metabolic and virulence related cell-states that emerge dynamically during growth. We chart the spatial context of biofilm-related processes including motility and kin-exclusion mechanisms and identify extensive and highly spatially-resolved metabolic heterogeneity. We find that distinct physiological states can co-exist within the same biofilm, just a few microns away, underscoring the importance of the microenvironment. Together, our results illustrate the complexity of microbial populations and present a new way of studying them at high-resolution.

microbiology↗

bioRxiv2020

Bidirectional redox cycling of phenazine-1-carboxylic acid by Citrobacter portucalensis MBL drives increased nitrate reduction

Phenazines are secreted metabolites that microbes use in diverse ways, from quorum sensing to antimicrobial warfare to energy conservation. Phenazines are able to contribute to these activities due to their redox activity. The physiological consequences of cellular phenazine reduction have been extensively studied, but the counterpart phenazine oxidation has been largely overlooked. Phenazine-1-carboxylic acid (PCA) is common in the environment and readily reduced by its producers. Here, we describe its anaerobic oxidation by Citrobacter portucalensis strain MBL, which was isolated from topsoil in Falmouth, MA, and which does not produce phenazines itself. This activity depends on the availability of a suitable terminal electron acceptor, specifically nitrate or fumarate. When C. portucalensis MBL is provided reduced PCA and either nitrate or fumarate, it continuously oxidizes the PCA. We compared this terminal electron acceptor-dependent PCA-oxidizing activity of C. portucalensis MBL to that of several other {gamma}-proteobacteria with varying capacities to respire nitrate and/or fumarate. We found that PCA oxidation by these strains in a fumarate-or nitrate-dependent manner is decoupled from growth and correlated with their possession of the fumarate or periplasmic nitrate reductases, respectively. We infer that bacterial PCA oxidation is widespread and genetically determined. Notably, reduced PCA enhances the rate of nitrate reduction to nitrite by C. portucalensis MBL beyond the stoichiometric prediction, which we attribute to C. portucalensis MBLs ability to also reduce oxidized PCA, thereby catalyzing a complete PCA redox cycle. This bidirectionality highlights the versatility of PCA as a biological redox agent. IMPORTANCEPhenazines are increasingly appreciated for their roles in structuring microbial communities. These tricyclic aromatic molecules have been found to regulate gene expression, be toxic, promote antibiotic tolerance, and promote survival under oxygen starvation. In all of these contexts, however, phenazines are studied as electron acceptors. Even if their utility arises primarily from being readily reduced, they would need to be oxidized in order to be recycled. While oxygen and ferric iron can oxidize phenazines abiotically, biotic oxidation of phenazines has not been studied previously. We observed bacteria that readily oxidize phenazine-1-carboxylic acid (PCA) in a nitrate-dependent fashion, concomitantly increasing the rate of nitrate reduction to nitrite. Because nitrate is a prevalent terminal electron acceptor in diverse anoxic environments, including soils, and phenazine-producers are widespread, this observation of linked phenazine and nitrogen redox cycling suggests an underappreciated role for redox-active secreted metabolites in the environment.

microbiology↗

bioRxiv2020

Global landscape of phenazine biosynthesis and biodegradation reveals species-specific colonization patterns in agricultural soils and crop microbiomes

Phenazines are natural bacterial antibiotics that can protect crops from disease. However, for most crops it is unknown which producers and specific phenazines are ecologically relevant, and whether phenazine biodegradation can counter their effects. To better understand their ecology, we developed and environmentally-validated a quantitative metagenomic approach to mine for phenazine biosynthesis and biodegradation genes, applying it to >800 soil and plant-associated shotgun-metagenomes. We discover novel producer-crop associations and demonstrate that phenazine biosynthesis is prevalent across habitats and preferentially enriched in rhizospheres, whereas biodegrading bacteria are rare. We validate an association between maize and Dyella japonica, a putative producer abundant in crop microbiomes. D. japonica upregulates phenazine biosynthesis during phosphate limitation and robustly colonizes maize seedling roots. This work provides a global picture of phenazines in natural environments and highlights plant-microbe associations of agricultural potential. Our metagenomic approach may be extended to other metabolites and functional traits in diverse ecosystems.

microbiology↗

bioRxiv2020

Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics

As antibiotic-resistant infections become increasingly prevalent worldwide, understanding the factors that lead to antimicrobial treatment failure is essential to optimizing the use of existing drugs. Opportunistic human pathogens in particular typically exhibit high levels of intrinsic antibiotic resistance and tolerance1, leading to chronic infections that can be nearly impossible to eradicate2. We asked whether the recalcitrance of these organisms to antibiotic treatment could be driven in part by their evolutionary history as environmental microbes, which frequently produce or encounter natural antibiotics3,4. Using the opportunistic pathogen Pseudomonas aeruginosa as a model, we demonstrate that the self-produced natural antibiotic pyocyanin (PYO) activates bacterial defenses that confer collateral tolerance to certain synthetic antibiotics, including in a clinically-relevant growth medium. Non-PYO-producing opportunistic pathogens isolated from lung infections similarly display increased antibiotic tolerance when they are co-cultured with PYO-producing P. aeruginosa. Furthermore, we show that beyond promoting bacterial survival in the presence of antibiotics, PYO can increase the apparent rate of mutation to antibiotic resistance by up to two orders of magnitude. Our work thus suggests that bacterial production of natural antibiotics in infections could play an important role in modulating not only the immediate efficacy of clinical antibiotics, but also the rate at which antibiotic resistance arises in multispecies bacterial communities.

microbiology↗

bioRxiv2019

Extracellular DNA promotes efficient extracellular electron transfer by pyocyanin in Pseudomonas aeruginosa biofilms

Extracellular electron transfer (EET), the process whereby cells access electron acceptors or donors that reside many cell lengths away, enables metabolic activity by microorganisms, particularly under oxidant-limited conditions that occur in multicellular bacterial biofilms. Although different mechanisms underpin this process in select organisms, a widespread strategy involves extracellular electron shuttles, redox-active metabolites that are secreted and recycled by diverse bacteria. How these shuttles catalyze electron transfer within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazine electron shuttles mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms, which are important in nature and disease. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by binding to eDNA. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and phenazines can participate directly in redox reactions through DNA; the biofilm eDNA can also support rapid electron transfer between redox active intercalators. Electrochemical measurements of biofilms indicate that retained PYO supports an efficient redox cycle with rapid EET and slow loss from the biofilm. Together, these results establish that eDNA facilitates phenazine metabolic processes in P. aeruginosa biofilms, suggesting a model for how extracellular electron shuttles achieve retention and efficient EET in biofilms.

microbiology↗

bioRxiv2019

Quantitative visualization of gene expression in Pseudomonas aeruginosa aggregates reveals peak expression of alginate in the hypoxic zone

It is well appreciated that oxygen- and 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 Pseudomonas aeruginosa aggregates as a step towards this end. As proof-of-principle, we visualize the expression of genes needed for the production of alginate (algD) and the dissimilatory nitrate reductase (narG). Using an inducible bacterial gene expression construct to calibrate the HCR signal, we were able to quantify algD and narG gene expression across microenvironmental gradients both within single aggregates and within aggregate populations using the Agar Block Biofilm Assay (ABBA). For the ABBA population, alginate gene expression was restricted to hypoxic regions within the environment (~40-200 M O2), as measured by an oxygen microelectrode. Within individual biofilm aggregates, cells proximal to the surface expressed alginate genes to a greater extent than interior cells. Lastly, mucoid biofilms consumed more oxygen than nonmucoid biofilms. These results establish that HCR has a sensitive dynamic range and can be used to resolve subtle differences in gene expression at spatial scales relevant to microbial assemblages. Because HCR v3.0 can be performed on diverse cell types, this methodological advance has the potential to enable quantitative studies of microbial gene expression in diverse contexts, including pathogen behavior in human chronic infections.\n\nImportanceThe visualization of microbial activities in natural environments is an important goal for numerous studies in microbial ecology, be the environment a sediment, soil, or infected human tissue. Here we report the application of the hybridization chain reaction (HCR) v3.0 to measure microbial gene expression in situ at single-cell resolution in aggregate biofilms. Using Pseudomonas aeruginosa with a tunable gene expression system, we show that this methodology is quantitative. Leveraging HCR v3.0 to measure gene expression within a P. aeruginosa aggregate, we find 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, HCR v3.0 has potential for broad application into microbial activities in situ at small spatial scales.

microbiology↗

bioRxiv2019

Aggregation of nontuberculous mycobacteria is regulated by carbon:nitrogen balance

Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens that form biofilms in environmental reservoirs such as household water systems and aggregate into phagocytosis-resistant clusters during infection. NTM constitutively aggregate in vitro, a phenotype typically considered to be a by-product of the mycolic-acid-rich cell wall. While culturing a model NTM, Mycobacterium smegmatis, in rich medium, we fortuitously discovered that planktonic cells accumulated in the culture after [~]3 days. By providing selective pressure for bacteria that disperse earlier, we isolated a strain with two mutations in the oligopeptide permease operon (opp). A mutant lacking the opp operon ({Delta}opp) dispersed earlier and more completely than wildtype (WT). We show that {Delta}opps aggregation defect was nutrient related; aggregation was restored by non-peptide carbon sources. Experiments with WT M. smegmatis revealed that growth as aggregates is favored when carbon is replete, while dispersal can be induced by carbon starvation. In addition, under conditions of low available carbon relative to available nitrogen, M. smegmatis grows as planktonic cells. By adjusting carbon and nitrogen sources in defined medium, we tuned the cellular C:N ratio such that M. smegmatis grows either as aggregates or planktonic cells. Lastly, we tested the effect of C:N balance on aggregation in clinically relevant NTM. Altogether, we show that NTM aggregation is a controlled process that is regulated by the relative availability of carbon and nitrogen for metabolism. Because NTM aggregation is correlated with increased virulence, these results may contribute to targeted anti-biofilm therapeutics.\n\nImportanceFree-living bacteria can assemble into multicellular aggregates called biofilms. Biofilms help bacteria tolerate multiple stresses, including antibiotics and the host immune system. Differing environmental pressures have resulted in biofilm architecture and regulation varying among bacterial species and strains. Nontuberculous mycobacteria are a group of emerging opportunistic pathogens that utilize biofilms to adhere to household plumbing and showerheads and to avoid phagocytosis by host immune cells. Mycobacteria harbor a unique cell wall built chiefly of long chain mycolic acids that confers hydrophobicity and has been thought to cause constitutive aggregation in liquid media. Here we show that aggregation is instead a regulated process dictated by the balance of available carbon and nitrogen. Understanding that mycobacteria utilize metabolic cues to regulate the transition between planktonic and aggregated cells reveals an inroad to controlling aggregation through targeted therapeutics.

microbiology↗

bioRxiv2018

Extended hopanoid lipids promote bacterial motility, surface attachment, and root nodule development in the Bradyrhizobium diazoefficiens-Aeschynomene afraspera symbiosis

Hopanoids are steroid-like bacterial lipids that enhance membrane rigidity and promote bacterial growth under diverse stresses. Hopanoid biosynthesis genes are conserved in nitrogen-fixing plant symbionts, and we previously found that the extended (C35) class of hopanoids in Bradyrhizobium diazoefficiens are required for efficient symbiotic nitrogen fixation in the tropical legume host Aeschynomene afraspera. Here we demonstrate that the nitrogen fixation defect conferred by extended loss can fully be explained by a reduction in root nodule sizes rather than per-bacteroid nitrogen fixation levels. Using a single-nodule tracking approach to track A. afraspera nodule development, we provide a quantitative model of root nodule development in this host, uncovering both the baseline growth parameters for wild-type nodules and a surprising heterogeneity of extended hopanoid mutant developmental phenotypes. These phenotypes include a delay in root nodule initiation and presence of a subpopulation of nodules with slow growth rates and low final volumes, which are correlated with reduced motility and surface attachment in vitro and lower bacteroid densities in planta, respectively. This work provides a quantitative reference point for understanding the phenotypic diversity of ineffective symbionts in A. afraspera and identifies specific developmental stages affected by extended hopanoid loss for future mechanistic work.

microbiology↗

bioRxiv2018

The dormancy specific regulator, SutA, is an intrinsically-disordered protein that modulates transcription initiation in Pseudomonas aeruginosa

Though bacteria in nature are often nutritionally limited and growing slowly, most of our understanding of core cellular processes such as transcription comes from studies in a handful of model organisms doubling rapidly under nutrient-replete conditions. We previously identified a small protein of unknown function, called SutA, in a global screen of proteins synthesized in Pseudomonas aeruginosa under growth arrest (Babin BM, et al. (2016) SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa. PNAS 113(5):E597-605). SutA binds RNA polymerase (RNAP), causing widespread changes in gene expression, including upregulation of the ribosomal RNA (rRNA) genes. Here, using biochemical and structural methods, we examine how SutA interacts with RNAP and the functional consequences of these interactions. We show that SutA consists of a central -helix with unstructured N- and C-terminal tails, and binds to the {beta}1 domain of RNAP. It activates transcription from the P. aeruginosa rrn promoter by both the housekeeping sigma factor holoenzyme (E{sigma}70) and the general stress response sigma factor holoenzyme (E{sigma}S) in vitro, and its N-terminal tail is required for activation in both holoenzyme contexts. However, we find that the interaction between SutA and each holoenzyme is distinct, with the SutA C-terminal tail and an acidic loop unique to {sigma}70 playing the determining roles in these differences. Our results add SutA to a growing list of transcription regulators that use their intrinsically disordered regions to remodel transcription complexes.\n\nSIGNIFICANCELittle is known about how bacteria regulate their activities during periods of dormancy, yet growth arrest dominates bacterial existence in most environments and is directly relevant to the problem of physiological antibiotic tolerance. Though much is known about transcription in the model organism, Escherichia coli, even there, our understanding of gene expression during dormancy is incomplete. Here we explore how transcription under growth arrest is modulated in Pseudomonas aeruginosa by the small acidic protein, SutA. We show that SutA binds to RNA polymerase and controls transcription by a mechanism that is distinct from other known regulators. Our work underscores the potential for fundamental, mechanistic discovery in this important and understudied realm of bacterial physiology.

molecular biology↗