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1.
mBio ; 12(4): e0226521, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465028

RESUMO

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 oxidizes the PCA at a rate that is environmentally relevant. We compared this terminal electron acceptor-dependent PCA-oxidizing activity of C. portucalensis MBL to that of several other gammaproteobacteria with various capacities to respire nitrate. We found that PCA oxidation by these strains in a nitrate-dependent manner is decoupled from growth and strain dependent. We infer that bacterial PCA oxidation is widespread and 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 MBL's 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. IMPORTANCE Phenazines 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.

2.
Nat Rev Microbiol ; 2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531577

RESUMO

Secondary metabolites profoundly affect microbial physiology, metabolism and stress responses. Increasing evidence suggests that these molecules can modulate microbial susceptibility to commonly used antibiotics; however, secondary metabolites are typically excluded from standard antimicrobial susceptibility assays. This may in part account for why infections by diverse opportunistic bacteria that produce secondary metabolites often exhibit discrepancies between clinical antimicrobial susceptibility testing results and clinical treatment outcomes. In this Review, we explore which types of secondary metabolite alter antimicrobial susceptibility, as well as how and why this phenomenon occurs. We discuss examples of molecules that opportunistic and enteric pathogens either generate themselves or are exposed to from their neighbours, and the nuanced impacts these molecules can have on tolerance and resistance to certain antibiotics.

3.
Science ; 373(6556)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385369

RESUMO

Capturing the heterogeneous phenotypes of microbial populations at relevant spatiotemporal scales is highly challenging. Here, we present par-seqFISH (parallel sequential fluorescence in situ hybridization), a transcriptome-imaging approach that records gene expression and spatial context within microscale assemblies at a single-cell and molecule resolution. We applied this approach to the opportunistic pathogen Pseudomonas aeruginosa, analyzing about 600,000 individuals across dozens of conditions in planktonic and biofilm cultures. We identified numerous metabolic- and virulence-related transcriptional states that emerged dynamically during planktonic growth, as well as highly spatially resolved metabolic heterogeneity in sessile populations. Our data reveal that distinct physiological states can coexist within the same biofilm just several micrometers away, underscoring the importance of the microenvironment. Our results illustrate the complex dynamics of microbial populations and present a new way of studying them at high resolution.


Assuntos
Pseudomonas aeruginosa/genética , Transcriptoma , Biofilmes/crescimento & desenvolvimento , Proteínas de Fímbrias/genética , Flagelina/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Hibridização in Situ Fluorescente , Fenótipo , Plâncton/genética , Plâncton/crescimento & desenvolvimento , Plâncton/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Piocinas/biossíntese , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula Única , Análise Espaço-Temporal , Virulência/genética
4.
Appl Environ Microbiol ; 87(17): e0070621, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34190605

RESUMO

A strain of Geobacter sulfurreducens, an organism capable of respiring solid extracellular substrates, lacking four of five outer membrane cytochrome complexes (extABCD+ strain) grows faster and produces greater current density than the wild type grown under identical conditions. To understand cellular and biofilm modifications in the extABCD+ strain responsible for this increased performance, biofilms grown using electrodes as terminal electron acceptors were sectioned and imaged using electron microscopy to determine changes in thickness and cell density, while parallel biofilms incubated in the presence of nitrogen and carbon isotopes were analyzed using NanoSIMS (nanoscale secondary ion mass spectrometry) to quantify and localize anabolic activity. Long-distance electron transfer parameters were measured for wild-type and extABCD+ biofilms spanning 5-µm gaps. Our results reveal that extABCD+ biofilms achieved higher current densities through the additive effects of denser cell packing close to the electrode (based on electron microscopy), combined with higher metabolic rates per cell compared to the wild type (based on increased rates of 15N incorporation). We also observed an increased rate of electron transfer through extABCD+ versus wild-type biofilms, suggesting that denser biofilms resulting from the deletion of unnecessary multiheme cytochromes streamline electron transfer to electrodes. The combination of imaging, physiological, and electrochemical data confirms that engineered electrogenic bacteria are capable of producing more current per cell and, in combination with higher biofilm density and electron diffusion rates, can produce a higher final current density than the wild type. IMPORTANCE Current-producing biofilms in microbial electrochemical systems could potentially sustain technologies ranging from wastewater treatment to bioproduction of electricity if the maximum current produced could be increased and current production start-up times after inoculation could be reduced. Enhancing the current output of microbial electrochemical systems has been mostly approached by engineering physical components of reactors and electrodes. Here, we show that biofilms formed by a Geobacter sulfurreducens strain producing ∼1.4× higher current than the wild type results from a combination of denser cell packing and higher anabolic activity, enabled by an increased rate of electron diffusion through the biofilms. Our results confirm that it is possible to engineer electrode-specific G. sulfurreducens strains with both faster growth on electrodes and streamlined electron transfer pathways for enhanced current production.


Assuntos
Biofilmes , Espaço Extracelular/metabolismo , Geobacter/química , Geobacter/fisiologia , Eletricidade , Eletrodos , Transporte de Elétrons , Espaço Extracelular/química , Geobacter/crescimento & desenvolvimento
5.
Environ Microbiol ; 23(6): 2906-2918, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33989442

RESUMO

Bacteria are a globally sustainable source of fixed nitrogen, which is essential for life and crucial for modern agriculture. Many nitrogen-fixing bacteria are agriculturally important, including bacteria known as rhizobia that participate in growth-promoting symbioses with legume plants throughout the world. To be effective symbionts, rhizobia must overcome multiple environmental challenges: from surviving in the soil, to transitioning to the plant environment, to maintaining high metabolic activity within root nodules. Climate change threatens to exacerbate these challenges, especially through fluctuations in soil water potential. Understanding how rhizobia cope with environmental stress is crucial for maintaining agricultural yields in the coming century. The bacterial outer membrane is the first line of defence against physical and chemical environmental stresses, and lipids play a crucial role in determining the robustness of the outer membrane. In particular, structural remodelling of lipid A and sterol-analogues known as hopanoids are instrumental in stress acclimation. Here, we discuss how the unique outer membrane lipid composition of rhizobia may underpin their resilience in the face of increasing osmotic stress expected due to climate change, illustrating the importance of studying microbial membranes and highlighting potential avenues towards more sustainable soil additives.


Assuntos
Fabaceae , Rhizobium , Mudança Climática , Fixação de Nitrogênio , Simbiose
6.
Science ; 371(6533): 1033-1037, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674490

RESUMO

Microbial production of antibiotics is common, but our understanding of their roles in the environment is limited. In this study, we explore long-standing observations that microbes increase the production of redox-active antibiotics under phosphorus limitation. The availability of phosphorus, a nutrient required by all life on Earth and essential for agriculture, can be controlled by adsorption to and release from iron minerals by means of redox cycling. Using phenazine antibiotic production by pseudomonads as a case study, we show that phenazines are regulated by phosphorus, solubilize phosphorus through reductive dissolution of iron oxides in the lab and field, and increase phosphorus-limited microbial growth. Phenazines are just one of many examples of phosphorus-regulated antibiotics. Our work suggests a widespread but previously unappreciated role for redox-active antibiotics in phosphorus acquisition and cycling.


Assuntos
Antibacterianos/biossíntese , Fenazinas/metabolismo , Fósforo/metabolismo , Pseudomonas/metabolismo , Técnicas de Cultura Celular por Lotes , Disponibilidade Biológica , Oxirredução , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento
7.
PLoS Biol ; 19(3): e3001093, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33690640

RESUMO

Bacterial opportunistic human pathogens frequently exhibit intrinsic antibiotic tolerance and resistance, resulting in infections that can be nearly impossible to eradicate. We asked whether this recalcitrance could be driven by these organisms' evolutionary history as environmental microbes that engage in chemical warfare. Using Pseudomonas aeruginosa as a model, we demonstrate that the self-produced antibiotic pyocyanin (PYO) activates defenses that confer collateral tolerance specifically to structurally similar synthetic clinical antibiotics. Non-PYO-producing opportunistic pathogens, such as members of the Burkholderia cepacia complex, likewise display elevated antibiotic tolerance when cocultured with PYO-producing strains. Furthermore, by widening the population bottleneck that occurs during antibiotic selection and promoting the establishment of a more diverse range of mutant lineages, PYO increases apparent rates of mutation to antibiotic resistance to a degree that can rival clinically relevant hypermutator strains. Together, these results reveal an overlooked mechanism by which opportunistic pathogens that produce natural toxins can dramatically modulate the efficacy of clinical antibiotics and the evolution of antibiotic resistance, both for themselves and other members of clinically relevant polymicrobial communities.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Bactérias/genética , Burkholderia cepacia/efeitos dos fármacos , Burkholderia cepacia/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Tolerância a Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/metabolismo , Piocianina/farmacologia
8.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33723058

RESUMO

Pseudomonas aeruginosa is an opportunistic human pathogen that develops difficult-to-treat biofilms in immunocompromised individuals, cystic fibrosis patients, and in chronic wounds. P. aeruginosa has an arsenal of physiological attributes that enable it to evade standard antibiotic treatments, particularly in the context of biofilms where it grows slowly and becomes tolerant to many drugs. One of its survival strategies involves the production of the redox-active phenazine, pyocyanin, which promotes biofilm development. We previously identified an enzyme, PodA, that demethylated pyocyanin and disrupted P. aeruginosa biofilm development in vitro. Here, we asked if this protein could be used as a potential therapeutic for P. aeruginosa infections together with tobramycin, an antibiotic typically used in the clinic. A major roadblock to answering this question was the poor yield and stability of wild-type PodA purified from standard Escherichia coli overexpression systems. We hypothesized that the insufficient yields were due to poor packing within PodA's obligatory homotrimeric interfaces. We therefore applied the protein design algorithm, AffiLib, to optimize the symmetric core of this interface, resulting in a design that incorporated five mutations leading to a 20-fold increase in protein yield from heterologous expression and purification and a substantial increase in stability to environmental conditions. The addition of the designed PodA with tobramycin led to increased killing of P. aeruginosa cultures under oxic and hypoxic conditions in both the planktonic and biofilm states. This study highlights the potential for targeting extracellular metabolites to assist the control of P. aeruginosa biofilms that tolerate conventional antibiotic treatment.


Assuntos
Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Oxirredutases N-Desmetilantes/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/metabolismo , Tobramicina/farmacologia , Desenho de Fármacos , Sinergismo Farmacológico , Humanos , Oxirredutases N-Desmetilantes/farmacologia , Engenharia de Proteínas , Pseudomonas aeruginosa/fisiologia
9.
Microbiol Resour Announc ; 10(1)2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33414314

RESUMO

Here, we report the genome sequence of Tenacibaculum mesophilum strain ECR, which was isolated from the river/ocean interface at Trunk River in Falmouth, Massachusetts. The isolation and sequencing were performed as part of the 2016 and 2018 Microbial Diversity courses at the Marine Biological Laboratory in Woods Hole, Massachusetts.

10.
Curr Biol ; 30(19): R1131-R1137, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33022255

RESUMO

The role of microbes in sustaining agricultural plant growth has great potential consequences for human prosperity. Yet we have an incomplete understanding of the basic function of rhizosphere microbial communities and how they may change under future stresses, let alone how these processes might be harnessed to sustain or improve crop yields. A reductionist approach may aid the generation and testing of hypotheses that can ultimately be translated to agricultural practices. With this in mind, we ask whether some rhizosphere microbial communities might be governed by 'keystone metabolites', envisioned here as microbially produced molecules that, through antibiotic and/or growth-promoting properties, may play an outsized role in shaping the development of the community spatiotemporally. To illustrate this point, we use the example of redox-active metabolites, and in particular phenazines, which are produced by many bacteria found in agricultural soils and have well-understood catalytic properties. Phenazines can act as potent antibiotics against a variety of cell types, yet they also can promote the acquisition of essential inorganic nutrients. In this essay, we suggest the ways these metabolites might affect microbial communities and ultimately agricultural productivity in two specific scenarios: firstly, in the biocontrol of beneficial and pathogenic fungi in increasingly arid crop soils and, secondly, through promotion of phosphorus bioavailability and sustainable fertilizer use. We conclude with specific proposals for future research.

11.
Elife ; 92020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32930660

RESUMO

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.


Assuntos
Produtos Agrícolas/microbiologia , Metagenômica , Microbiota , Fenazinas/metabolismo , Microbiologia do Solo , Poluentes do Solo/metabolismo , Solo/química , Agricultura , Biodegradação Ambiental , Gammaproteobacteria/fisiologia , Especificidade da Espécie , Regulação para Cima , Zea mays/microbiologia
12.
Cell ; 182(4): 919-932.e19, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32763156

RESUMO

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm.


Assuntos
Biofilmes/crescimento & desenvolvimento , DNA/química , Pseudomonas aeruginosa/fisiologia , Piocianina/química , DNA/metabolismo , Técnicas Eletroquímicas , Eletrodos , Transporte de Elétrons/efeitos dos fármacos , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Oxirredução , Fenazinas/química , Fenazinas/metabolismo , Fenazinas/farmacologia , Piocianina/metabolismo
13.
Microbiol Resour Announc ; 9(32)2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32763937

RESUMO

We grew a soil enrichment culture to identify organisms that anaerobically oxidize phenazine-1-carboxylic acid. A strain of Citrobacter portucalensis was isolated from this enrichment and sequenced by both Illumina and PacBio technologies. It has a genome with a length of 5.3 Mb, a G+C content of 51.8%, and at least one plasmid.

14.
J Bacteriol ; 202(11)2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32071098

RESUMO

Classifying microorganisms as "obligate" aerobes has colloquially implied death without air, leading to the erroneous assumption that, without oxygen, they are unable to survive. However, over the past few decades, more than a few obligate aerobes have been found to possess anaerobic energy conservation strategies that sustain metabolic activity in the absence of growth or at very low growth rates. Similarly, studies emphasizing the aerobic prowess of certain facultative aerobes have sometimes led to underrecognition of their anaerobic capabilities. Yet an inescapable consequence of the affinity both obligate and facultative aerobes have for oxygen is that the metabolism of these organisms may drive this substrate to scarcity, making anoxic survival an essential skill. To illustrate this, we highlight the importance of anaerobic survival strategies for Pseudomonas aeruginosa and Streptomyces coelicolor, representative facultative and obligate aerobes, respectively. Included among these strategies, we describe a role for redox-active secondary metabolites (RAMs), such as phenazines made by P. aeruginosa, in enhancing substrate-level phosphorylation. Importantly, RAMs are made by diverse bacteria, often during stationary phase in the absence of oxygen, and can sustain anoxic survival. We present a hypothesis for how RAMs may enhance or even unlock energy conservation pathways that facilitate the anaerobic survival of both RAM producers and nonproducers.


Assuntos
Pseudomonas aeruginosa/metabolismo , Streptomyces coelicolor/metabolismo , Aerobiose , Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Oxirredução , Oxigênio/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Streptomyces coelicolor/genética , Streptomyces coelicolor/crescimento & desenvolvimento
15.
Proc Natl Acad Sci U S A ; 117(8): 4358-4367, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32029587

RESUMO

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.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , Viabilidade Microbiana , Peptídeo Hidrolases/genética , Pseudomonas aeruginosa/genética
16.
mBio ; 10(6)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848278

RESUMO

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 vitro P. 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.


Assuntos
Alginatos/metabolismo , Expressão Gênica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Biofilmes , Biomarcadores , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Mucosa Intestinal/fisiologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/patologia
17.
PLoS One ; 14(12): e0226578, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31851705

RESUMO

The bacterial growth environment within cystic fibrosis (CF) sputum is complex, dynamic, and shaped by both host and microbial processes. Characterization of the chemical parameters within sputum that stimulate the in vivo growth of airway pathogens (e.g. Pseudomonas aeruginosa) and their associated virulence factors may lead to improved CF treatment strategies. Motivated by conflicting reports of the prevalence and abundance of P. aeruginosa-derived metabolites known as phenazines within CF airway secretions, we sought to quantify these metabolites in sputum using quadrupole time-of-flight mass spectrometry. In contrast to our previous work, all phenazines tested (pyocyanin (PYO), phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide, and 1-hydroxyphenazine) were below detection limits of the instrument (0.1 µM). Instead, we identified a late-eluting compound that shared retention time and absorbance characteristics with PCA, yet generated mass spectra and a fragmentation pattern consistent with ferriprotoporphyrin IX, otherwise known as heme B. These data suggested that UV-vis chromatographic peaks previously attributed to PCA and PYO in sputum were mis-assigned. Indeed, retrospective analysis of raw data from our prior study found that the heme B peak closely matched the peaks assigned to PCA, indicating that the previous study likely uncovered a positive correlation between pulmonary function (percent predicted forced expiratory volume in 1 second, or ppFEV1) and heme B, not PCA or any other phenazine. To independently test this observation, we performed a new tandem mass-spectrometry analysis of 71 additional samples provided by the Mountain West CF Consortium Sputum Biomarker study and revealed a positive correlation (ρ = -0.47, p<0.001) between sputum heme concentrations and ppFEV1. Given that hemoptysis is strongly associated with airway inflammation, pulmonary exacerbations and impaired lung function, these new data suggest that heme B may be a useful biomarker of CF pathophysiology.


Assuntos
Fibrose Cística/metabolismo , Heme/metabolismo , Pulmão/fisiopatologia , Escarro/metabolismo , Adulto , Feminino , Humanos , Masculino
18.
Elife ; 82019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31526475

RESUMO

The swimming larvae of many marine animals identify a location on the sea floor to undergo metamorphosis based on the presence of specific bacteria. Although this microbe-animal interaction is critical for the life cycles of diverse marine animals, what types of biochemical cues from bacteria that induce metamorphosis has been a mystery. Metamorphosis of larvae of the tubeworm Hydroides elegans is induced by arrays of phage tail-like contractile injection systems, which are released by the bacterium Pseudoalteromonas luteoviolacea. Here we identify the novel effector protein Mif1. By cryo-electron tomography imaging and functional assays, we observe Mif1 as cargo inside the tube lumen of the contractile injection system and show that the mif1 gene is required for inducing metamorphosis. Purified Mif1 is sufficient for triggering metamorphosis when electroporated into tubeworm larvae. Our results indicate that the delivery of protein effectors by contractile injection systems may orchestrate microbe-animal interactions in diverse contexts.


Assuntos
Proteínas de Bactérias/metabolismo , Interações entre Hospedeiro e Microrganismos , Metamorfose Biológica , Poliquetos/crescimento & desenvolvimento , Poliquetos/microbiologia , Pseudoalteromonas/metabolismo , Animais , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Poliquetos/efeitos dos fármacos , Transporte Proteico
19.
mBio ; 10(4)2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409683

RESUMO

Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens that colonize household water systems and cause chronic lung infections in susceptible patients. The ability of NTM to form surface-attached biofilms in the nonhost environment and corded aggregates in vivo is important to their ability to persist in both contexts. Underlying the development of these multicellular structures is the capacity of mycobacterial cells to adhere to one another. Unlike most other bacteria, NTM spontaneously and constitutively aggregate in vitro, hindering our ability to understand the transition between planktonic and aggregated cells. While culturing a model NTM, Mycobacterium smegmatis, in rich medium, we fortuitously discovered that planktonic cells accumulate after ∼3 days of growth. 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 (Δopp) disperses earlier than wild type (WT) due to a defect in nutrient uptake. Experiments with WT M. smegmatis revealed that growth as aggregates is favored when carbon is replete, but 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 as planktonic cells. C/N-mediated aggregation regulation is widespread among NTM with the possible exception of rough-colony Mycobacterium abscessus isolates. Altogether, we show that NTM aggregation is a controlled process that is governed by the relative availability of carbon and nitrogen for metabolism.IMPORTANCE Free-living bacteria can assemble into multicellular structures called biofilms. Biofilms help bacteria tolerate multiple stresses, including antibiotics and the host immune system. 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. Typically, bacteria regulate biofilm formation by controlling expression of adhesive structures to attach to surfaces and other bacterial 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 biofilm formation through targeted therapeutics.


Assuntos
Aderência Bacteriana , Carbono/metabolismo , Nitrogênio/metabolismo , Micobactérias não Tuberculosas/fisiologia , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Meios de Cultura , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/fisiologia , Micobactérias não Tuberculosas/crescimento & desenvolvimento , Micobactérias não Tuberculosas/metabolismo , Óperon
20.
Mol Plant Microbe Interact ; 32(10): 1415-1428, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31170026

RESUMO

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 hopanoid loss can be fully explained by a reduction in root nodule sizes rather than per-bacteroid nitrogen-fixation levels. Using a single-nodule tracking approach to quantify 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 the 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.


Assuntos
Bradyrhizobium , Fabaceae , Nódulos Radiculares de Plantas , Simbiose , Bradyrhizobium/fisiologia , Fabaceae/microbiologia , Cinética , Fixação de Nitrogênio/fisiologia , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/microbiologia
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