RESUMEN
Bacteria possess various regulatory mechanisms to detect and coordinate a response to elemental nutrient limitation. In pseudomonads, the two-component system regulators CbrAB, NtrBC and PhoBR, are responsible for regulating cellular response to carbon (C), nitrogen (N) and phosphorus (P) respectively. Phosphonates are reduced organophosphorus compounds produced by a broad range of biota and typified by a direct C-P bond. Numerous pseudomonads can use the environmentally abundant phosphonate species 2-aminoethylphosphonate (2AEP) as a source of C, N, or P, but only PhoBR has been shown to play a role in 2AEP utilization. On the other hand, utilization of 2AEP as a C and N source is considered substrate inducible. Here, using the plant-growth-promoting rhizobacterium Pseudomonas putida BIRD-1 we present evidence that 2AEP utilization is under dual regulation and only occurs upon depletion of C, N, or P, controlled by CbrAB, NtrBC, or PhoBR respectively. However, the presence of 2AEP was necessary for full gene expression, i.e. expression was substrate inducible. Mutation of a LysR-type regulator, termed AepR, upstream of the 2AEP transaminase-phosphonatase system (PhnWX), confirmed this dual regulatory mechanism. To our knowledge, this is the first study identifying coordination between global stress response and substrate-specific regulators in phosphonate metabolism.
Asunto(s)
Organofosfonatos , Pseudomonas putida , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Regulación Bacteriana de la Expresión Génica , Nitrógeno/metabolismo , Organofosfonatos/metabolismo , Fósforo/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismoRESUMEN
Infections caused by antimicrobial-resistant bacterial pathogens are fast becoming an important global health issue. Strains of Escherichia coli are common causal agents of urinary tract infection and can carry multiple resistance genes. This includes the gene blaCTX-M-15, which encodes an extended-spectrum beta-lactamase (ESBL). While studying antimicrobial resistance (AMR) in the environment, we isolated several strains of E. coli ST131 downstream of a wastewater treatment plan (WWTP) in a local river. These isolates were surviving in the river sediment, and characterization proved that a multiresistant phenotype was evident. Here, we show that E. coli strain 48 (river isolate ST131) provided a protective effect against a third-generation cephalosporin (cefotaxime) for susceptible E. coli strain 33 (river isolate ST3576) through secretion of a functional ESBL into the growth medium. Furthermore, extracellular ESBL activity was stable for at least 24 h after secretion. Proteomic and molecular genetic analyses identified CTX-M-15 as the major secreted ESBL responsible for the observed protective effect. In contrast to previous studies, outer membrane vesicles (OMVs) were not the route for CTX-M-15 secretion. Indeed, mutation of the type I secretion system led to a significant reduction in the growth of the ESBL-producing strain as well as a significantly reduced ability to confer protective effect. We speculate that CTX-M-15 secretion, mediated through active secretion using molecular machinery, provides a public goods service by facilitating the survival of otherwise susceptible bacteria in the presence of cefotaxime.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli , Antibacterianos/farmacología , Escherichia coli/genética , Infecciones por Escherichia coli/tratamiento farmacológico , Genotipo , Humanos , Proteómica , beta-Lactamasas/genéticaRESUMEN
Bovine tuberculosis (bTB) is an economically important disease affecting the cattle industry in England and Wales. bTB, caused by Mycobacterium bovis, also causes disease in the Eurasian badger (Meles meles), a secondary maintenance host. Disease transmission between these two species is bidirectional. Infected badgers shed M. bovis in their feces. The Animal and Plant Health Agency (APHA) of the United Kingdom organized a comparative trial to determine the performance of tests in detecting M. bovis in badger feces for the Department for Environment, Food, and Rural Affairs (DEFRA). Here, we assessed the performance of the existing Warwick Fast24-qPCR test and its modified version based on a high-throughput DNA extraction method (Fast96-qPCR). We found Fast24-qPCR to have a sensitivity of 96.7% (95% confidence interval [CI], 94.5 to 99%; n = 244) and a specificity of 99% (95% CI, 97.8 to 100%; n = 292). Fast96-qPCR requires further optimization. Determining the disease status of badger social groups requires multiple tests per group. Therefore, to increase specificity further, we independently repeated the Fast24-qPCR test on positive samples, increasing stringency by requiring a second positive result. Fast24-qPCR with repeat testing had a sensitivity of 87.3% (95% CI, 83.1 to 91.5%; n = 244), and a specificity of 100% (95% CI, 100 to 100; n = 201) on an individual-sample level. At the social-group level, this repeat testing gives Fast24-qPCR high herd specificity, while testing multiple samples per group provides high herd sensitivity. With Fast24-qPCR, we provide a social-group-level test with sufficient specificity and sensitivity to monitor shedding in badgers via latrine sampling, delivering a potentially valuable tool to measure the impacts of bTB control measures.
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Mustelidae , Mycobacterium bovis , Tuberculosis Bovina , Animales , Bovinos , Reservorios de Enfermedades , Inglaterra , Mycobacterium bovis/genética , Tuberculosis Bovina/diagnóstico , Reino Unido , GalesRESUMEN
The origin of carbapenem-hydrolyzing metallo-ß-lactamases (MBLs) acquired by clinical bacteria is largely unknown. We investigated the frequency, host range, diversity, and functionality of MBLs in the soil microbiota. Twenty-five soil samples of different types and geographical origins were analyzed by antimicrobial selective culture, followed by phenotypic testing and expression of MBL-encoding genes in Escherichia coli, and whole-genome sequencing of MBL-producing strains was performed. Carbapenemase activity was detected in 29 bacterial isolates from 13 soil samples, leading to identification of seven new MBLs in presumptive Pedobacter roseus (PEDO-1), Pedobacter borealis (PEDO-2), Pedobacter kyungheensis (PEDO-3), Chryseobacterium piscium (CPS-1), Epilithonimonas tenax (ESP-1), Massilia oculi (MSI-1), and Sphingomonas sp. (SPG-1). Carbapenemase production was likely an intrinsic feature in Chryseobacterium and Epilithonimonas, as it occurred in reference strains of different species within these genera. The amino acid identity to MBLs described in clinical bacteria ranged between 40 and 69%. Remarkable features of the new MBLs included prophage integration of the encoding gene (PEDO-1), an unusual amino acid residue at a key position for MBL structure and catalysis (CPS-1), and overlap with a putative OXA ß-lactamase (MSI-1). Heterologous expression of PEDO-1, CPS-1, and ESP-1in E. coli significantly increased the MICs of ampicillin, ceftazidime, cefpodoxime, cefoxitin, and meropenem. Our study shows that MBL producers are widespread in soil and include four genera that were previously not known to produce MBLs. The MBLs produced by these bacteria are distantly related to MBLs identified in clinical samples but constitute resistance determinants of clinical relevance if acquired by pathogenic bacteria.
Asunto(s)
Chryseobacterium/enzimología , Pedobacter/enzimología , Microbiología del Suelo , Sphingomonas/enzimología , Resistencia betalactámica/genética , beta-Lactamasas/genética , Secuencia de Aminoácidos , Antibacterianos/farmacología , Carbapenémicos/farmacología , Chryseobacterium/efectos de los fármacos , Chryseobacterium/genética , Chryseobacterium/aislamiento & purificación , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Europa (Continente) , Expresión Génica , Hidrólisis , Datos de Secuencia Molecular , Pedobacter/efectos de los fármacos , Pedobacter/genética , Pedobacter/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sphingomonas/efectos de los fármacos , Sphingomonas/genética , Sphingomonas/aislamiento & purificación , beta-Lactamasas/metabolismoRESUMEN
ß-Lactamase production increasingly threatens the effectiveness of ß-lactams, which remain a mainstay of antimicrobial chemotherapy. New activities emerge through both mutation of previously known ß-lactamases and mobilization from environmental reservoirs. The spread of metallo-ß-lactamases (MBLs) represents a particular challenge because of their typically broad-spectrum activities encompassing carbapenems, in addition to other ß-lactam classes. Increasingly, genomic and metagenomic studies have revealed the distribution of putative MBLs in the environment, but in most cases their activity against clinically relevant ß-lactams and, hence, the extent to which they can be considered a resistance reservoir remain uncharacterized. Here we characterize the product of one such gene, blaRm3, identified through functional metagenomic sampling of an environment with high levels of biocide exposure. blaRm3 encodes a subclass B3 MBL that, when expressed in a recombinant Escherichia coli strain, is exported to the bacterial periplasm and hydrolyzes clinically used penicillins, cephalosporins, and carbapenems with an efficiency limited by high Km values. An Rm3 crystal structure reveals the MBL superfamily αß/ßα fold, which more closely resembles that in mobilized B3 MBLs (AIM-1 and SMB-1) than other chromosomal enzymes (L1 or FEZ-1). A binuclear zinc site sits in a deep channel that is in part defined by a relatively extended N terminus. Structural comparisons suggest that the steric constraints imposed by the N terminus may limit its affinity for ß-lactams. Sequence comparisons identify Rm3-like MBLs in numerous other environmental samples and species. Our data suggest that Rm3-like enzymes represent a distinct group of B3 MBLs with a wide distribution and can be considered an environmental reservoir of determinants of ß-lactam resistance.
Asunto(s)
Metagenómica/métodos , beta-Lactamasas/química , beta-Lactamasas/genética , Cristalografía por Rayos X , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Filogenia , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Lactamasas/metabolismoRESUMEN
Bacteria that inhabit the rhizosphere of agricultural crops can have a beneficial effect on crop growth. One such mechanism is the microbial-driven solubilization and remineralization of complex forms of phosphorus (P). It is known that bacteria secrete various phosphatases in response to low P conditions. However, our understanding of their global proteomic response to P stress is limited. Here, exoproteomic analysis of Pseudomonas putida BIRD-1 (BIRD-1), Pseudomonas fluorescens SBW25 and Pseudomonas stutzeri DSM4166 was performed in unison with whole-cell proteomic analysis of BIRD-1 grown under phosphate (Pi) replete and Pi deplete conditions. Comparative exoproteomics revealed marked heterogeneity in the exoproteomes of each Pseudomonas strain in response to Pi depletion. In addition to well-characterized members of the PHO regulon such as alkaline phosphatases, several proteins, previously not associated with the response to Pi depletion, were also identified. These included putative nucleases, phosphotriesterases, putative phosphonate transporters and outer membrane proteins. Moreover, in BIRD-1, mutagenesis of the master regulator, phoBR, led us to confirm the addition of several novel PHO-dependent proteins. Our data expands knowledge of the Pseudomonas PHO regulon, including species that are frequently used as bioinoculants, opening up the potential for more efficient and complete use of soil complexed P.
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Fósforo/metabolismo , Pseudomonas fluorescens/genética , Pseudomonas putida/genética , Pseudomonas stutzeri/genética , Microbiología del Suelo , Productos Agrícolas/crecimiento & desarrollo , Productos Agrícolas/microbiología , Genómica , Fosfatos/metabolismo , Proteómica , Pseudomonas fluorescens/metabolismo , Pseudomonas putida/metabolismo , Pseudomonas stutzeri/metabolismo , Regulón , RizosferaRESUMEN
Dichelobacter nodosus (D. nodosus) is the causative agent of footrot in sheep; one of the most important health and welfare issues of sheep worldwide. For control programmes to be effective, it is essential that the transmission cycle of D. nodosus is understood and bacterial reservoirs in the environment are better defined. This study evaluated the survival of D. nodosus in different soils using soil microcosms. Cultivation independent and dependent methods were used to detect D. nodosus over 40 days from seeding in soil. A D. nodosus specific probe was used for quantification by qPCR and viability was assessed by cell permeability to an intercalating dye, PMA, and by culture. Survival varied dramatically depending on soil type, matric potential (MP) and temperature. Our findings indicate that D. nodosus survival was higher at 5 °C compared with 25 °C in all soils and significantly longer at both temperatures in clay soil (>44% clay) compared with other soil types. Survival under all conditions was longer than 30 days for both culture independent and dependent methods, this is substantially longer than previous studies and, if this is an infectious dose, longer than the current recommendation of resting a field for 14 days to prevent onward infection.
Asunto(s)
Dichelobacter nodosus/fisiología , Panadizo Interdigital/microbiología , Viabilidad Microbiana , Enfermedades de las Ovejas/microbiología , Microbiología del Suelo , Animales , Antiinfecciosos/farmacología , Azidas/farmacología , ADN Bacteriano , Dichelobacter nodosus/clasificación , Dichelobacter nodosus/aislamiento & purificación , Propidio/análogos & derivados , Propidio/farmacología , OvinosRESUMEN
Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications.
Asunto(s)
Variación Genética , Metagenoma , Péptido Sintasas/genética , Sintasas Poliquetidas/genética , Sphagnopsida/microbiología , Biología Computacional , Pruebas Genéticas , Sphagnopsida/crecimiento & desarrolloRESUMEN
Bacteria in the genus Streptomyces are soil-dwelling oligotrophs and important producers of secondary metabolites. Previously, we showed that global messenger RNA expression was subject to a series of metabolic and regulatory switches during the lifetime of a fermentor batch culture of Streptomyces coelicolor M145. Here we analyze the proteome from eight time points from the same fermentor culture and, because phosphate availability is an important regulator of secondary metabolite production, compare this to the proteome of a similar time course from an S. coelicolor mutant, INB201 (ΔphoP), defective in the control of phosphate utilization. The proteomes provide a detailed view of enzymes involved in central carbon and nitrogen metabolism. Trends in protein expression over the time courses were deduced from a protein abundance index, which also revealed the importance of stress pathway proteins in both cultures. As expected, the ΔphoP mutant was deficient in expression of PhoP-dependent genes, and several putatively compensatory metabolic and regulatory pathways for phosphate scavenging were detected. Notably there is a succession of switches that coordinately induce the production of enzymes for five different secondary metabolite biosynthesis pathways over the course of the batch cultures.
Asunto(s)
Aclimatación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Mutación/genética , Fosfatos/metabolismo , Streptomyces coelicolor/metabolismo , Técnicas de Cultivo Celular por Lotes , Biomarcadores/metabolismo , Células Cultivadas , Cromatografía Liquida , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteoma/análisis , Proteómica , ARN Bacteriano/genética , ARN Mensajero/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Streptomyces coelicolor/crecimiento & desarrolloRESUMEN
BACKGROUND: Plastics pollution and antimicrobial resistance (AMR) are two major environmental threats, but potential connections between plastic associated biofilms, the 'plastisphere', and dissemination of AMR genes are not well explored. RESULTS: We conducted mesocosm experiments tracking microbial community changes on plastic surfaces transitioning from wastewater effluent to marine environments over 16 weeks. Commonly used plastics, polypropylene (PP), high density polyethylene (HDPE), low density polyethylene (LDPE) and polyethylene terephthalate (PET) incubated in wastewater effluent, river water, estuarine water, and in the seawater for 16 weeks, were analysed via 16S rRNA gene amplicon and shotgun metagenome sequencing. Within one week, plastic-colonizing communities shifted from wastewater effluent-associated microorganisms to marine taxa, some members of which (e.g. Oleibacter-Thalassolituus and Sphingomonas spp., on PET, Alcanivoracaceae on PET and PP, or Oleiphilaceae, on all polymers), were selectively enriched from levels undetectable in the starting communities. Remarkably, microbial biofilms were also susceptible to parasitism, with Saprospiraceae feeding on biofilms at late colonisation stages (from week 6 onwards), while Bdellovibrionaceae were prominently present on HDPE from week 2 and LDPE from day 1. Relative AMR gene abundance declined over time, and plastics did not become enriched for key AMR genes after wastewater exposure. CONCLUSION: Although some resistance genes occurred during the mesocosm transition on plastic substrata, those originated from the seawater organisms. Overall, plastic surfaces incubated in wastewater did not act as hotspots for AMR proliferation in simulated marine environments.
RESUMEN
BACKGROUND: The widespread nature of plastic pollution has given rise to wide scientific and social concern regarding the capacity of these materials to serve as vectors for pathogenic bacteria and reservoirs for Antimicrobial Resistance Genes (ARG). In- and ex-situ incubations were used to characterise the riverine plastisphere taxonomically and functionally in order to determine whether antibiotics within the water influenced the ARG profiles in these microbiomes and how these compared to those on natural surfaces such as wood and their planktonic counterparts. RESULTS: We show that plastics support a taxonomically distinct microbiome containing potential pathogens and ARGs. While the plastisphere was similar to those biofilms that grew on wood, they were distinct from the surrounding water microbiome. Hence, whilst potential opportunistic pathogens (i.e. Pseudomonas aeruginosa, Acinetobacter and Aeromonas) and ARG subtypes (i.e. those that confer resistance to macrolides/lincosamides, rifamycin, sulfonamides, disinfecting agents and glycopeptides) were predominant in all surface-related microbiomes, especially on weathered plastics, a completely different set of potential pathogens (i.e. Escherichia, Salmonella, Klebsiella and Streptococcus) and ARGs (i.e. aminoglycosides, tetracycline, aminocoumarin, fluoroquinolones, nitroimidazole, oxazolidinone and fosfomycin) dominated in the planktonic compartment. Our genome-centric analysis allowed the assembly of 215 Metagenome Assembled Genomes (MAGs), linking ARGs and other virulence-related genes to their host. Interestingly, a MAG belonging to Escherichia -that clearly predominated in water- harboured more ARGs and virulence factors than any other MAG, emphasising the potential virulent nature of these pathogenic-related groups. Finally, ex-situ incubations using environmentally-relevant concentrations of antibiotics increased the prevalence of their corresponding ARGs, but different riverine compartments -including plastispheres- were affected differently by each antibiotic. CONCLUSIONS: Our results provide insights into the capacity of the riverine plastisphere to harbour a distinct set of potentially pathogenic bacteria and function as a reservoir of ARGs. The environmental impact that plastics pose if they act as a reservoir for either pathogenic bacteria or ARGs is aggravated by the persistence of plastics in the environment due to their recalcitrance and buoyancy. Nevertheless, the high similarities with microbiomes growing on natural co-occurring materials and even more worrisome microbiome observed in the surrounding water highlights the urgent need to integrate the analysis of all environmental compartments when assessing risks and exposure to pathogens and ARGs in anthropogenically-impacted ecosystems. Video Abstract.
Asunto(s)
Antibacterianos , Microbiota , Antibacterianos/farmacología , Bacterias/genética , Lincosamidas , Genes Bacterianos/genética , Microbiota/genética , AguaRESUMEN
BACKGROUND: The rhizosphere is a hotspot for microbial activity and contributes to ecosystem services including plant health and biogeochemical cycling. The activity of microbial viruses, and their influence on plant-microbe interactions in the rhizosphere, remains undetermined. Given the impact of viruses on the ecology and evolution of their host communities, determining how soil viruses influence microbiome dynamics is crucial to build a holistic understanding of rhizosphere functions. RESULTS: Here, we aimed to investigate the influence of crop management on the composition and activity of bulk soil, rhizosphere soil, and root viral communities. We combined viromics, metagenomics, and metatranscriptomics on soil samples collected from a 3-year crop rotation field trial of oilseed rape (Brassica napus L.). By recovering 1059 dsDNA viral populations and 16,541 ssRNA bacteriophage populations, we expanded the number of underexplored Leviviricetes genomes by > 5 times. Through detection of viral activity in metatranscriptomes, we uncovered evidence of "Kill-the-Winner" dynamics, implicating soil bacteriophages in driving bacterial community succession. Moreover, we found the activity of viruses increased with proximity to crop roots, and identified that soil viruses may influence plant-microbe interactions through the reprogramming of bacterial host metabolism. We have provided the first evidence of crop rotation-driven impacts on soil microbial communities extending to viruses. To this aim, we present the novel principal of "viral priming," which describes how the consecutive growth of the same crop species primes viral activity in the rhizosphere through local adaptation. CONCLUSIONS: Overall, we reveal unprecedented spatial and temporal diversity in viral community composition and activity across root, rhizosphere soil, and bulk soil compartments. Our work demonstrates that the roles of soil viruses need greater consideration to exploit the rhizosphere microbiome for food security, food safety, and environmental sustainability. Video Abstract.
Asunto(s)
Bacteriófagos , Brassica napus , Microbiota , Virus ARN , Rizosfera , Microbiología del Suelo , Raíces de Plantas/microbiología , Microbiota/genética , Suelo/química , Bacterias/genética , Virus ARN/genética , Bacteriófagos/genética , ADNRESUMEN
Polyethylene (PE) is one of the most recalcitrant carbon-based synthetic materials produced and, currently, the most ubiquitous plastic pollutant found in nature. Over time, combined abiotic and biotic processes are thought to eventually breakdown PE. Despite limited evidence of biological PE degradation and speculation that hydrocarbon-degrading bacteria found within the plastisphere is an indication of biodegradation, there is no clear mechanistic understanding of the process. Here, using high-throughput proteomics, we investigated the molecular processes that take place in the hydrocarbon-degrading marine bacterium Alcanivorax sp. 24 when grown in the presence of low density PE (LDPE). As well as efficiently utilising and assimilating the leachate of weathered LDPE, the bacterium was able to reduce the molecular weight distribution (Mw from 122 to 83 kg/mol) and overall mass of pristine LDPE films (0.9 % after 34 days of incubation). Most interestingly, Alcanivorax acquired the isotopic signature of the pristine plastic and induced an extensive array of metabolic pathways for aliphatic compound degradation. Presumably, the primary biodegradation of LDPE by Alcanivorax sp. 24 is possible via the production of extracellular reactive oxygen species as observed both by the material's surface oxidation and the measurement of superoxide in the culture with LDPE. Our findings confirm that hydrocarbon-biodegrading bacteria within the plastisphere may in fact have a role in degrading PE.
Asunto(s)
Alcanivoraceae , Alcanivoraceae/metabolismo , Bacterias/metabolismo , Biodegradación Ambiental , Hidrocarburos/metabolismo , Plásticos/metabolismo , Polietileno/metabolismoRESUMEN
Advances in DNA sequencing technologies have drastically changed our perception of the structure and complexity of the plant microbiome. By comparison, our ability to accurately identify the metabolically active fraction of soil microbiota and its specific functional role in augmenting plant health is relatively limited. Important ecological interactions being performed by microbes can be investigated by analyzing the extracellular protein fraction. Here, we combined a unique protein extraction method and an iterative bioinformatics pipeline to capture and identify extracellular proteins (metaexoproteomics) synthesized in the rhizosphere of Brassica spp. We first validated our method in the laboratory by successfully identifying proteins related to a host plant (Brassica rapa) and its bacterial inoculant, Pseudomonas putida BIRD-1. This identified numerous rhizosphere specific proteins linked to the acquisition of plant-derived nutrients in P. putida. Next, we analyzed natural field-soil microbial communities associated with Brassica napus L. (oilseed rape). By combining metagenomics with metaexoproteomics, 1,885 plant, insect, and microbial proteins were identified across bulk and rhizosphere samples. Metaexoproteomics identified a significant shift in the metabolically active fraction of the soil microbiota responding to the presence of B. napus roots that was not apparent in the composition of the total microbial community (metagenome). This included stimulation of rhizosphere-specialized bacteria, such as Gammaproteobacteria, Betaproteobacteria, and Flavobacteriia, and the upregulation of plant beneficial functions related to phosphorus and nitrogen mineralization. Our metaproteomic assessment of the "active" plant microbiome at the field-scale demonstrates the importance of moving beyond metagenomics to determine ecologically important plant-microbe interactions underpinning plant health. IMPORTANCE Plant-microbe interactions are critical to ecosystem function and crop production. While significant advances have been made toward understanding the structure of the plant microbiome, learning about its full functional role is still in its infancy. This is primarily due to an incomplete ability to determine in situ plant-microbe interactions actively operating under field conditions. Proteins are the functional entities of the cell. Therefore, their identification and relative quantification within a microbial community provide the best proxy for which microbes are the most metabolically active and which are driving important plant-microbe interactions. Here, we provide the first metaexoproteomics assessment of the plant microbiome using field-grown oilseed rape as the model crop species, identifying key taxa responsible for specific ecological interactions. Gaining a mechanistic understanding of the plant microbiome is central to developing engineered plant microbiomes to improve sustainable agricultural approaches and reduce our reliance on nonrenewable resources.
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Brassica napus , Microbiota , Rizosfera , Bacterias/genética , Microbiota/genética , Plantas , SueloRESUMEN
The continued emergence of bacterial pathogens presenting antimicrobial resistance is widely recognised as a global health threat and recent attention focused on potential environmental reservoirs of antibiotic resistance genes (ARGs). Freshwater environments such as rivers represent a potential hotspot for ARGs and antibiotic resistant bacteria as they are receiving systems for effluent discharges from wastewater treatment plants (WWTPs). Effluent also contains low levels of different antimicrobials including antibiotics and biocides. Sulfonamides are antibacterial chemicals widely used in clinical, veterinary and agricultural settings and are frequently detected in sewage sludge and manure in addition to riverine ecosystems. The impact of such exposure on ARG prevalence and diversity is unknown, so the aim of this study was to investigate the release of a sub-lethal concentration of the sulfonamide compound sulfamethoxazole (SMX) on the river bacterial microbiome using a flume system. This system was a semi-natural in vitro flume using river water (30 L) and sediment (6 kg) with circulation to mimic river flow. A combination of 'omics' approaches were conducted to study the impact of SMX exposure on the microbiomes within the flumes. Metagenomic analysis showed that the addition of low concentrations of SMX (<4 µg L-1) had a limited effect on the bacterial resistome in the water fraction only, with no impact observed in the sediment. Metaproteomics did not show differences in ARGs expression with SMX exposure in water. Overall, the river bacterial community was resilient to short term exposure to sub-lethal concentrations of SMX which mimics the exposure such communities experience downstream of WWTPs throughout the year.
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Microbiota , Sulfametoxazol , Antibacterianos/farmacología , Farmacorresistencia Microbiana , Genes Bacterianos , Ríos , Aguas ResidualesRESUMEN
Understanding the variables that influence microbiome studies is critical for successful translational research. Inflammatory bowel disease (IBD) is a complex group of diseases that can present at multiple locations within the Gastrointestinal tract. Here, using the FAMISHED study cohort, we aimed to investigate the relationship between IBD condition, IBD disease location, and the microbiome. Signatures of the microbiome, including measures of diversity, taxonomy, and functionality, all significantly differed across the three different IBD conditions, Crohn's disease (CD), ulcerative colitis (UC), and microscopic colitis (MC). Notably, when stratifying by disease location, patients with CD in the terminal ileum were more similar to healthy controls than patients with CD in the small bowel or colon, however no differences were observed at different disease locations across patients with UC. Change in taxonomic composition resulted in changes in function, with CD at each disease location, UC and MC all having unique functional dysbioses. CD patients in particular had deficiencies in Short-Chain Fatty Acid (SCFA) pathways. Our results demonstrate the complex relationship between IBD and the microbiome and highlight the need for consistent strategies for the stratification of clinical cohorts and downstream analysis to ensure results across microbiome studies and clinical trials are comparable.
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Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino/microbiología , Estudios de Casos y Controles , Biología Computacional , Humanos , Ciencia Traslacional BiomédicaRESUMEN
The planktonic synthesis of reduced organophosphorus molecules, such as alkylphosphonates and aminophosphonates, represents one half of a vast global oceanic phosphorus redox cycle. Whilst alkylphosphonates tend to accumulate in recalcitrant dissolved organic matter, aminophosphonates do not. Here, we identify three bacterial 2-aminoethylphosphonate (2AEP) transporters, named AepXVW, AepP and AepSTU, whose synthesis is independent of phosphate concentrations (phosphate-insensitive). AepXVW is found in diverse marine heterotrophs and is ubiquitously distributed in mesopelagic and epipelagic waters. Unlike the archetypal phosphonate binding protein, PhnD, AepX has high affinity and high specificity for 2AEP (Stappia stellulata AepX Kd 23 ± 4 nM; methylphosphonate Kd 3.4 ± 0.3 mM). In the global ocean, aepX is heavily transcribed (~100-fold>phnD) independently of phosphate and nitrogen concentrations. Collectively, our data identifies a mechanism responsible for a major oxidation process in the marine phosphorus redox cycle and suggests 2AEP may be an important source of regenerated phosphate and ammonium, which are required for oceanic primary production.
Asunto(s)
Ácido Aminoetilfosfónico/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Minerales/metabolismo , Fósforo/metabolismo , Rhodobacteraceae/metabolismo , Agua de Mar/microbiología , Proteínas Bacterianas/metabolismo , Transporte Biológico , Regulación Bacteriana de la Expresión Génica , Cinética , Océanos y Mares , Oxidación-Reducción , Filogenia , Proteómica , Pseudomonas putida/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rhodobacteraceae/genéticaRESUMEN
Bacteroidetes are abundant pathogen-suppressing members of the plant microbiome that contribute prominently to rhizosphere phosphorus mobilisation, a frequent growth-limiting nutrient in this niche. However, the genetic traits underpinning their success in this niche remain largely unknown, particularly regarding their phosphorus acquisition strategies. By combining cultivation, multi-layered omics and biochemical analyses we first discovered that all plant-associated Bacteroidetes express constitutive phosphatase activity, linked to the ubiquitous possession of a unique phosphatase, PafA. For the first time, we also reveal a subset of Bacteroidetes outer membrane SusCD-like complexes, typically associated with carbon acquisition, and several TonB-dependent transporters, are induced during Pi-depletion. Furthermore, in response to phosphate depletion, the plant-associated Flavobacterium used in this study expressed many previously characterised and novel proteins targeting organic phosphorus. Collectively, these enzymes exhibited superior phosphatase activity compared to plant-associated Pseudomonas spp. Importantly, several of the novel low-Pi-inducible phosphatases and transporters, belong to the Bacteroidetes auxiliary genome and are an adaptive genomic signature of plant-associated strains. In conclusion, niche adaptation to the plant microbiome thus appears to have resulted in the acquisition of unique phosphorus scavenging loci in Bacteroidetes, enhancing their phosphorus acquisition capabilities. These traits may enable their success in the rhizosphere and also present exciting avenues to develop sustainable agriculture.
Asunto(s)
Microbiota , Fósforo , Bacteroidetes/genética , Raíces de Plantas , Plantas , RizosferaRESUMEN
BACKGROUND: The transition from exponential to stationary phase in Streptomyces coelicolor is accompanied by a major metabolic switch and results in a strong activation of secondary metabolism. Here we have explored the underlying reorganization of the metabolome by combining computational predictions based on constraint-based modeling and detailed transcriptomics time course observations. RESULTS: We reconstructed the stoichiometric matrix of S. coelicolor, including the major antibiotic biosynthesis pathways, and performed flux balance analysis to predict flux changes that occur when the cell switches from biomass to antibiotic production. We defined the model input based on observed fermenter culture data and used a dynamically varying objective function to represent the metabolic switch. The predicted fluxes of many genes show highly significant correlation to the time series of the corresponding gene expression data. Individual mispredictions identify novel links between antibiotic production and primary metabolism. CONCLUSION: Our results show the usefulness of constraint-based modeling for providing a detailed interpretation of time course gene expression data.