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Root-associated microorganisms play an important role in plant health, such as plant growth-promoting rhizobacteria (PGPR) from the Bacillus and Pseudomonas genera. Although bacterial consortia including these two genera would represent a promising avenue to efficient biofertilizer formulation, we observed that Bacillus subtilis root colonization is decreased by the presence of Pseudomonas fluorescens and Pseudomonas protegens. To determine if B. subtilis can adapt to the inhibitory effect of Pseudomonas on roots, we conducted adaptative laboratory evolution experiments with B. subtilis in mono-association or co-cultured with P. fluorescens on tomato plant roots. Evolved isolates with various colony morphology and stronger colonization capacity of both tomato plant and Arabidopsis thaliana roots emerged rapidly from the two evolution experiments. Certain evolved isolates also had better fitness on the root in the presence of other Pseudomonas species. In all independent lineages, whole-genome resequencing revealed non-synonymous mutations in genes ywcC or sinR encoding regulators involved in repressing biofilm development, suggesting their involvement in enhanced root colonization. These findings provide insights into the molecular mechanisms underlying B. subtilis adaptation to root colonization and highlight the potential of directed evolution to enhance the beneficial traits of PGPR.IMPORTANCEIn this study, we aimed to enhance the abilities of the plant-beneficial bacterium Bacillus subtilis to colonize plant roots in the presence of competing Pseudomonas bacteria. To achieve this, we conducted adaptive laboratory experiments, allowing Bacillus to evolve in a defined environment. We successfully obtained strains of Bacillus that were more effective at colonizing plant roots than the ancestor strain. To identify the genetic changes driving this improvement, we sequenced the genomes of these evolved strains. Interestingly, mutations that facilitated the formation of robust biofilms on roots were predominant. Many of these evolved Bacillus isolates also displayed the remarkable ability to outcompete Pseudomonas species. Our research sheds light on the mutational paths selected in Bacillus subtilis to thrive in root environments and offers exciting prospects for improving beneficial traits in plant growth-promoting microorganisms. Ultimately, this could pave the way for the development of more effective biofertilizers and sustainable agricultural practices.
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Arabidopsis , Bacillus , Pseudomonas fluorescens , Bacillus subtilis/genética , Biopelículas , Arabidopsis/genéticaRESUMEN
Siderophores are soluble or membrane-embedded molecules that bind the oxidized form of iron, Fe(III), and play roles in iron acquisition by microorganisms. Fe(III)-bound siderophores bind to specific receptors that allow microbes to acquire iron. However, certain soil microbes release a compound (pulcherriminic acid, PA) that, upon binding to Fe(III), forms a precipitate (pulcherrimin) that apparently functions by reducing iron availability rather than contributing to iron acquisition. Here, we use Bacillus subtilis (PA producer) and Pseudomonas protegens as a competition model to show that PA is involved in a peculiar iron-managing system. The presence of the competitor induces PA production, leading to precipitation of Fe(III) as pulcherrimin, which prevents oxidative stress in B. subtilis by restricting the Fenton reaction and deleterious ROS formation. In addition, B. subtilis uses its known siderophore bacillibactin to retrieve Fe(III) from pulcherrimin. Our findings indicate that PA plays multiple roles by modulating iron availability and conferring protection against oxidative stress during inter-species competition.
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Hierro , Sideróforos , Hierro/metabolismo , Sideróforos/metabolismo , Estrés Oxidativo , Interacciones MicrobianasRESUMEN
Plant growth-promoting rhizobacteria (PGPR) such as the root colonizers Bacillus spp. may be ideal alternatives to chemical crop treatments. This work sought to extend the application of the broadly active PGPR UD1022 to Medicago sativa (alfalfa). Alfalfa is susceptible to many phytopathogens resulting in losses of crop yield and nutrient value. UD1022 was cocultured with four alfalfa pathogen strains to test antagonism. We found UD1022 to be directly antagonistic toward Collectotrichum trifolii, Ascochyta medicaginicola (formerly Phoma medicaginis), and Phytophthora medicaginis, and not toward Fusarium oxysporum f. sp. medicaginis. Using mutant UD1022 strains lacking genes in the nonribosomal peptide (NRP) and biofilm pathways, we tested antagonism against A. medicaginicola StC 306-5 and P. medicaginis A2A1. The NRP surfactin may have a role in the antagonism toward the ascomycete StC 306-5. Antagonism toward A2A1 may be influenced by B. subtilis biofilm pathway components. The B. subtilis central regulator of both surfactin and biofilm pathways Spo0A was required for the antagonism of both phytopathogens. The results of this study indicate that the PGPR UD1022 would be a good candidate for further investigations into its antagonistic activities against C. trifolii, A. medicaginicola, and P. medicaginis in plant and field studies.
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Endophytic bacterial populations are well-positioned to provide benefits to their host plants such as nutrient acquisition and plant hormone level manipulation. Actinorhizal plants such as alders are well known for their microbial symbioses that allow them to colonize harsh environments whether natural or anthropized. Although the nitrogen-fixing actinobacterium Frankia sp. is the main endophyte found in alder root nodules, other bacterial genera, whose roles remain poorly defined, inhabit this niche. In this study, we isolated a diverse panel of non-Frankia nodular endophytes (NFNE). Some NFNE were isolated from alders grown from surface-sterilized seeds and maintained in sterile conditions, suggesting these may have been seed-borne. In vitro testing of 24 NFNE revealed some possessed putative plant growth promotion traits. Their genomes were also sequenced to identify genes related to plant growth promotion traits. This study highlights the complexity of the alder nodular microbial community. It paves the way for further understanding of the biology of nodules and could help improve land reclamation practices that involve alders.
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Alnus , Endófitos , Endófitos/genética , Alnus/microbiología , Simbiosis , Plantas/microbiología , Bacterias , GenómicaRESUMEN
In the present study, we report the in vitro interactions between Frankia sp. ACN10a and non-Frankia nodular endophytes (NFNE) isolated from alder. The supernatant of NFNE grown in nitrogen-replete medium had neutral or negative effects on Frankia growth; none had a stimulatory effect. Inhibitory effects were observed for supernatants of some NFNE, notably Micromonospora, Pseudomonas, Serratia and Stenotrophomonas isolates. However, some NFNE-Frankia coculture supernatants could stimulate Frankia growth when used as a culture medium supplement. This was observed for supernatants of Frankia cocultured with Microvirga and Streptomyces isolates. In nitrogen-limited conditions, cocultures of Frankia with some NFNE, including some rhizobia and Cytobacillus, resulted in higher total biomass than Frankia-only cultures, suggesting cooperation, while other NFNE were strongly antagonistic. Microscopic observation of cocultures also revealed compromised Frankia membrane integrity, and some differentiation into stress resistance-associated morphotypes such as sporangia and reproductive torulose hyphae (RTH). Furthermore, the coculture of Frankia with Serratia sp. isolates resulted in higher concentrations of the auxinic plant hormone indole-3-acetic acid and related indolic compounds in the culture supernatant. This study sheds new light on the breadth of microbial interactions that occur amongst bacteria that inhabit the understudied ecological niche of the alder nodule.
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Alnus , Frankia , Endófitos , Alnus/microbiología , Simbiosis , Fijación del Nitrógeno , NitrógenoRESUMEN
Horizontal gene transfer (HGT) by integrative and conjugative elements (ICEs) is an important mechanism in the spread of antibiotic resistance genes. However, little is known about the spatiotemporal dynamic of ICE propagation in bacterial biofilms, which are multicellular structures ubiquitous in natural and clinical environments. We report here that a high level of biofilm matrix production favors ICEBs1 acquisition. Also, using a fluorescently marked ICEBs1, we observed that conjugation appears restricted to clusters of bacteria in a close neighborhood in which a high level of ICEBs1 transfer occurs. These conjugative clusters are heterogeneously distributed in the biofilm, forming close to the air-biofilm interface. Importantly, we established that transconjugant cells are the main contributors to ICEBs1 propagation in biofilms. Our findings provide a novel spatiotemporal understanding of ICEs propagation in biofilms, which should have an important role in our understanding of horizontal gene transfer in relevant settings. IMPORTANCE The transfer of mobile genetic elements between bacteria is the main cause of the spread of antibiotic resistance genes. While biofilms are the predominant bacterial lifestyle both in the environment and in clinical settings, their impact on the propagation of mobile genetic elements is still poorly understood. In this study, we examined the spatiotemporal propagation of the well-characterized ICEBs1. Using the Gram-positive Bacillus subtilis, we observed that the main actors of ICEBs1 propagation in biofilms are the newly formed transconjugants that allow rapid transfer of ICEBs1 to new recipients. Our study provides a better understanding of the spatiotemporal dynamic of conjugative transfer in biofilms.
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Bacillus subtilis , Conjugación Genética , Bacillus subtilis/genética , Transferencia de Gen Horizontal , Biopelículas , Farmacorresistencia MicrobianaRESUMEN
Bacterial biofilms are involved in chronic infections and confer 10 to 1,000 times more resistance to antibiotics compared with planktonic growth, leading to complications and treatment failure. When transitioning from a planktonic lifestyle to biofilms, some Gram-positive bacteria are likely to modulate several cellular pathways, including central carbon metabolism, biosynthesis pathways, and production of secondary metabolites. These metabolic adaptations might play a crucial role in biofilm formation by Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecalis. Here, we performed a transcriptomic approach to identify cellular pathways that might be similarly regulated during biofilm formation in these bacteria. Different strains and biofilm-inducing media were used to identify a set of regulated genes that are common and independent of the environment or accessory genomes analyzed. Our approach highlighted that the de novo purine biosynthesis pathway was upregulated in biofilms of both species when using a tryptone soy broth-based medium but not so when a brain heart infusion-based medium was used. We did not identify other pathways commonly regulated between both pathogens. Gene deletions and usage of a drug targeting a key enzyme showed the importance of this pathway in biofilm formation of S. aureus. The importance of the de novo purine biosynthesis pathway might reflect an important need for purine during biofilm establishment, and thus could constitute a promising drug target. IMPORTANCE Biofilms are often involved in nosocomial infections and can cause serious chronic infections if not treated properly. Current anti-biofilm strategies rely on antibiotic usage, but they have a limited impact because of the biofilm intrinsic tolerance to drugs. Metabolism remodeling likely plays a central role during biofilm formation. Using comparative transcriptomics of different strains of Staphylococcus aureus and Enterococcus faecalis, we determined that almost all cellular adaptations are not shared between strains and species. Interestingly, we observed that the de novo purine biosynthesis pathway was upregulated during biofilm formation by both species in a specific medium. The requirement for purine could constitute an interesting new anti-biofilm target with a wide spectrum that could also prevent resistance evolution. These results are also relevant to a better understanding of the physiology of biofilm formation.
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Biopelículas , Medios de Cultivo/metabolismo , Enterococcus faecalis/fisiología , Purinas/biosíntesis , Staphylococcus aureus/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Medios de Cultivo/química , Enterococcus faecalis/genética , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genéticaRESUMEN
BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease ultimately manifesting as clinical dementia. Despite considerable effort and ample experimental data, the role of neuroinflammation related to systemic inflammation is still unsettled. While the implication of microglia is well recognized, the exact contribution of peripheral monocytes/macrophages is still largely unknown, especially concerning their role in the various stages of AD. OBJECTIVES: AD develops over decades and its clinical manifestation is preceded by subjective memory complaints (SMC) and mild cognitive impairment (MCI); thus, the question arises how the peripheral innate immune response changes with the progression of the disease. Therefore, to further investigate the roles of monocytes/macrophages in the progression of AD we assessed their phenotypes and functions in patients at SMC, MCI and AD stages and compared them with cognitively healthy controls. We also conceptualised an idealised mathematical model to explain the functionality of monocytes/macrophages along the progression of the disease. RESULTS: We show that there are distinct phenotypic and functional changes in monocyte and macrophage populations as the disease progresses. Higher free radical production upon stimulation could already be observed for the monocytes of SMC patients. The most striking results show that activation of peripheral monocytes (hyperactivation) is the strongest in the MCI group, at the prodromal stage of the disease. Monocytes exhibit significantly increased chemotaxis, free radical production, and cytokine production in response to TLR2 and TLR4 stimulation. CONCLUSION: Our data suggest that the peripheral innate immune system is activated during the progression from SMC through MCI to AD, with the highest levels of activation being in MCI subjects and the lowest in AD patients. Some of these parameters may be used as biomarkers, but more holistic immune studies are needed to find the best period of the disease for clinical intervention.
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Alzheimer's disease (AD) is the most common form of dementia and aging is the most common risk factor for developing the disease. The etiology of AD is not known but AD may be considered as a clinical syndrome with multiple causal pathways contributing to it. The amyloid cascade hypothesis, claiming that excess production or reduced clearance of amyloid-beta (Aß) and its aggregation into amyloid plaques, was accepted for a long time as the main cause of AD. However, many studies showed that Aß is a frequent consequence of many challenges/pathologic processes occurring in the brain for decades. A key factor, sustained by experimental data, is that low-grade infection leading to production and deposition of Aß, which has antimicrobial activity, precedes the development of clinically apparent AD. This infection is chronic, low grade, largely clinically silent for decades because of a nearly efficient antimicrobial immune response in the brain. A chronic inflammatory state is induced that results in neurodegeneration. Interventions that appear to prevent, retard or mitigate the development of AD also appear to modify the disease. In this review, we conceptualize further that the changes in the brain antimicrobial immune response during aging and especially in AD sufferers serve as a foundation that could lead to improved treatment strategies for preventing or decreasing the progression of AD in a disease-modifying treatment.
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Iron (Fe) is one of the most important micronutrients for most life forms on earth. While abundant in soil, Fe bioavailability in oxic soil is very low. Under environmental conditions, bacteria need to acquire sufficient Fe to sustain growth while limiting the energy cost of siderophore synthesis. Biofilm formation might mitigate this Fe stress, since it was shown to accumulate Fe in certain Gram-negative bacteria and that this Fe could be mobilized for uptake. However, it is still unclear if, and to what extent, the amount of Fe accumulated in the biofilm can sustain growth and if the mobilization of this local Fe pool is modulated by the availability of environmental Fe (i.e., Fe outside the biofilm matrix). Here, we use a nondomesticated strain of the ubiquitous biofilm-forming soil bacterium Bacillus subtilis and stable Fe isotopes to precisely evaluate the origin of Fe during growth in the presence of tannic acid and hydroxides, used as proxies for different environmental conditions. We report that this B. subtilis strain can accumulate a large quantity of Fe in the biofilm, largely exceeding Fe associated with cells. We also report that only a fraction of biofilm-bound Fe is available for uptake in the absence of other sources of Fe in the vicinity of the biofilm. We observed that the availability of environmental Fe modulates the usage of this pool of biofilm-bound Fe. Finally, our data suggest that consumption of biofilm-bound Fe relates to the efficacy of B. subtilis to transport Fe from the environment to the biofilm, possibly through siderophores.IMPORTANCE Recent pieces of evidence suggest that Fe bound to the biofilm could assume at least two important functions, a local source of Fe for uptake and a support to extracellular metabolism, such as extracellular electron transfer. Our results show that B. subtilis can use biofilm-bound Fe for uptake only if it does not compromise Fe homeostasis of the biofilm, i.e., maintains a minimum Fe concentration in the biofilm for extracellular purposes. We propose a theoretical framework based on our results and recent literature to explain how B. subtilis manages biofilm-bound Fe and Fe uptake in response to environmental Fe availability. These results provide important insights into the management of biofilm-bound and environmental Fe by B. subtilis in response to Fe stress.
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Bacillus subtilis/fisiología , Biopelículas , Hierro/metabolismo , Bacillus subtilis/crecimiento & desarrollo , Transporte BiológicoRESUMEN
Bacillus subtilis and Bacillus velezensis are used in organic agriculture as an alternative to chemical pesticides to fight against phytopathogen organisms. These Gram-positive soil-dwelling bacteria are able to resist harsh conditions and survive by differentiating into endospores. Few studies have examined how bacterial populations change on plants over time, and whether they remain active or enter a dormant state. Nonetheless, these characteristics are strikingly important to determine the usage of B. subtilis and B. velezensis and their efficacy in environmental conditions. Here, we investigated the population dynamics of B. subtilis NCIB3610 and B. velezensis QST713 when applied as spores on different ornamental plants. We report that on all the plants studied (Echinacea purpurea 'Salsa red', Echinacea purpurea 'Fatal attraction', and Lavandula angustifolia 'Hidecote blue'), spores rapidly germinated and colonized the rhizoplane, maintaining a relatively low proportion of spores in the population over time, whereas the bacterial population on the leaves rapidly declined. Bacteria in the surrounding soil did not germinate and persisted as spores. Taken together, these results suggest that only cells found at the rhizosphere remain metabolically active to allow the formation of a lasting relationship with the plant, making possible beneficial effects from the inoculated bacteria.
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Bacillus subtilis/crecimiento & desarrollo , Bacillus/crecimiento & desarrollo , Plantas/microbiología , Recuento de Colonia Microbiana , Hojas de la Planta/microbiología , Rizosfera , Esporas Bacterianas/crecimiento & desarrolloRESUMEN
Alzheimer's disease (AD) is the most prevalent dementia in the world. Its cause(s) are presently largely unknown. The most common explanation for AD, now, is the amyloid cascade hypothesis, which states that the cause of AD is senile plaque formation by the amyloid ß peptide, and the formation of neurofibrillary tangles by hyperphosphorylated tau. A second, burgeoning theory by which to explain AD is based on the infection hypothesis. Much experimental and epidemiological data support the involvement of infections in the development of dementia. According to this mechanism, the infection either directly or via microbial virulence factors precedes the formation of amyloid ß plaques. The amyloid ß peptide, possessing antimicrobial properties, may be beneficial at an early stage of AD, but becomes detrimental with the progression of the disease, concomitantly with alterations to the innate immune system at both the peripheral and central levels. Infection results in neuroinflammation, leading to, and sustained by, systemic inflammation, causing eventual neurodegeneration, and the senescence of the immune cells. The sources of AD-involved microbes are various body microbiome communities from the gut, mouth, nose, and skin. The infection hypothesis of AD opens a vista to new therapeutic approaches, either by treating the infection itself or modulating the immune system, its senescence, or the body's metabolism, either separately, in parallel, or in a multi-step way.
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Enfermedad de Alzheimer/tratamiento farmacológico , Antiinfecciosos/uso terapéutico , Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Humanos , Inmunidad Innata/efectos de los fármacos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Placa Amiloide/tratamiento farmacológico , Placa Amiloide/metabolismoRESUMEN
Plant growth-promoting rhizobacteria (PGPR) have enormous potential for solving some of the myriad challenges facing our global agricultural system. Intense research efforts are rapidly moving the field forward and illuminating the wide diversity of bacteria and their plant beneficial activities. In the development of better crop solutions using these PGPR, producers are including multiple different species of PGPR in their formulations in a "consortia" approach. While the intention is to emulate more natural rhizomicrobiome systems, the aspect of bacterial interactions has not been properly regarded. By using a tri-trophic model of Medicago truncatula A17 Jemalong, its nitrogen (N)-fixing symbiont Sinorhizobium meliloti Rm8530, and the PGPR Bacillus subtilis UD1022, we demonstrate indirect influences between the bacteria affecting their plant growth-promoting activities. Co-cultures of UD1022 with Rm8530 significantly reduced Rm8530 biofilm formation and downregulated quorum sensing (QS) genes responsible for symbiotically active biofilm production. This work also identifies the presence and activity of a quorum quenching lactonase in UD1022 and proposes this as the mechanism for non-synergistic activity of this model "consortium." These interspecies interactions may be common in the rhizosphere and are critical to understand as we seek to develop new sustainable solutions in agriculture.
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Secondary metabolites have an important impact on the biocontrol potential of soil-derived microbes. In addition, various microbe-produced chemicals have been suggested to impact the development and phenotypic differentiation of bacteria, including biofilms. The non-ribosomal synthesized lipopeptide of Bacillus subtilis, surfactin, has been described to impact the plant promoting capacity of the bacterium. Here, we investigated the impact of surfactin production on biofilm formation of B. subtilis using the laboratory model systems; pellicle formation at the air-medium interface and architecturally complex colony development, in addition to plant root-associated biofilms. We found that the production of surfactin by B. subtilis is not essential for pellicle biofilm formation neither in the well-studied strain, NCIB 3610, nor in the newly isolated environmental strains, but lack of surfactin reduces colony expansion. Further, plant root colonization was comparable both in the presence or absence of surfactin synthesis. Our results suggest that surfactin-related biocontrol and plant promotion in B. subtilis strains are independent of biofilm formation.
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Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer-ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.
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Regulación Bacteriana de la Expresión Génica , Magnesio/fisiología , Riboswitch , Bacillus subtilis/química , Bacillus subtilis/genética , Transferencia Resonante de Energía de Fluorescencia , Ligandos , Magnesio/análisis , Pliegue del ARN , Transcripción GenéticaRESUMEN
Bacillus subtilis is a Gram-positive plant-growth-promoting rhizobacterium exerting many beneficial effects on plant health. Because they secrete antimicrobial compounds and elicit induced systemic resistance, B. subtilis and phylogenetically related species are of particular interest as antifungals in organic agriculture. These bacteria are also known for their capacity to differentiate phenotypically into endospores able to withstand many environmental stresses. However, although B. subtilis is often inoculated on plants as spores, dynamics of germination and sporulation on roots remain unexplored. Using a hydroponic culture system and a soil system for Arabidopsis thaliana, we observed that B. subtilis spores germinate rapidly on contact with plants. However, the vegetative cells are abundant on roots for only a few days before reversing back to spores. We observed that the germinant receptor GerK and sporulation kinases KinA and KinB identified in vitro control sporulation dynamics on plants. Surprisingly, when plants are inoculated with B. subtilis, free-living cells sporulate more rapidly than plant-associated cells. However, direct contact between plant and bacteria is required for the induction of sporulation in the surrounding B. subtilis. This study has fundamental implications for our understanding of interactions between Bacillus spp. and plants, and particularly for a more efficient usage of B. subtilis as a biofertilizer or biofungicide.
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Arabidopsis , Bacillus subtilis , Interacciones Huésped-Patógeno , Plantones , Esporas Bacterianas , Arabidopsis/microbiología , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas , Plantones/microbiología , Esporas Bacterianas/crecimiento & desarrolloRESUMEN
Iron (Fe) is the most important metal in biology. Despite its abundance, Fe is mostly present as a ferric form in soils, strongly limiting its bioavailability. To overcome the challenge of Fe acquisition, many microorganisms produce siderophores to retrieve Fe from natural sources. Another ubiquitous feature of bacteria in natural environments is biofilm formation. Previous studies showed that external Fe strongly influenced biofilm formation in several bacteria, suggesting that this microenvironment plays a mechanistic role in micronutrient acquisition for bacteria. Here, we applied a complementary set of analytical methods and deletion mutants to evaluate the role of biofilm formation, siderophore production, and their interaction in Fe homeostasis in Bacillus subtilis We observed that Fe homeostasis, i.e., active growth at a constant intracellular Fe concentration, requires both siderophore production and biofilm formation. Also, we report that in B. subtilis, both biofilm formation and siderophore production are required to achieve active Fe acquisition from the medium and to sustain normal growth. Furthermore, we provide evidence that the formation of biofilm slightly enhances the kinetics of Fe complexation by catechol siderophores and markedly improves siderophore use efficiency. These results provide new perspectives on the mechanism underlying siderophore-based acquisition of Fe in biofilm-forming bacteria.IMPORTANCE Iron acquisition is of fundamental importance for microorganisms, since this metal is generally poorly bioavailable under natural conditions. In the environment, most bacteria are found tightly packed within multicellular communities named biofilms. Here, using the soil Gram-positive bacterium Bacillus subtilis, we show that biofilm formation and the production of siderophores, i.e., small molecules specifically binding metals, are both essential to ensure Fe uptake from the medium and maintain cellular Fe homeostasis. The biofilm matrix appears to play an important role favoring the efficient usage of siderophores. Taken together, our results demonstrate a close link between biofilm formation and iron acquisition in B. subtilis, allowing a better comprehension of how bacteria can cope with metal limitation under environmental conditions.
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Bacillus subtilis/fisiología , Biopelículas , Hierro/metabolismo , Sideróforos/biosíntesis , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , HomeostasisRESUMEN
Horizontal gene transfer by integrative and conjugative elements (ICEs) is a very important mechanism for spreading antibiotic resistance in various bacterial species. In environmental and clinical settings, most bacteria form biofilms as a way to protect themselves against extracellular stress. However, much remains to be known about ICE transfer in biofilms. Using ICEBs1 from Bacillus subtilis, we show that the natural conjugation efficiency of this ICE is greatly affected by the ability of the donor and recipient to form a biofilm. ICEBs1 transfer considerably increases in biofilm, even at low donor/recipient ratios. Also, while there is a clear temporal correlation between biofilm formation and ICEBs1 transfer, biofilms do not alter the level of ICEBs1 excision in donor cells. Conjugative transfer appears to be favored by the biophysical context of biofilms. Indeed, extracellular matrix production, particularly from the recipient cells, is essential for biofilms to promote ICEBs1 transfer. Our study provides basic new knowledge on the high rate of conjugative transfer of ICEs in biofilms, a widely preponderant bacterial lifestyle in the environment, which could have a major impact on our understanding of horizontal gene transfer in natural and clinical environments.IMPORTANCE Transfer of mobile genetic elements from one bacterium to another is the principal cause of the spread of antibiotic resistance. However, the dissemination of these elements in environmental contexts is poorly understood. In clinical and environmental settings, bacteria are often found living in multicellular communities encased in a matrix, a structure known as a biofilm. In this study, we examined how forming a biofilm influences the transmission of an integrative and conjugative element (ICE). Using the model Gram-positive bacterium B. subtilis, we observed that biofilm formation highly favors ICE transfer. This increase in conjugative transfer is due to the production of extracellular matrix, which creates an ideal biophysical context. Our study provides important insights into the role of the biofilm structure in driving conjugative transfer, which is of major importance since biofilm is a widely preponderant bacterial lifestyle for clinically relevant bacterial strains.
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Bacillus subtilis/fisiología , Biopelículas/crecimiento & desarrollo , Conjugación Genética , Transferencia de Gen Horizontal , Bacillus subtilis/genética , ADN Bacteriano/genética , Farmacorresistencia Bacteriana , Matriz Extracelular/genéticaRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of deadly hospital-acquired infections. The discovery of anti-Staphylococcus antibiotics and new classes of drugs not susceptible to the mechanisms of resistance shared among bacteria is imperative. We recently showed that tomatidine (TO), a steroidal alkaloid from solanaceous plants, possesses potent antibacterial activity against S. aureus small-colony variants (SCVs), the notoriously persistent form of this bacterium that has been associated with recurrence of infections. Here, using genomic analysis of in vitro-generated TO-resistant S. aureus strains to identify mutations in genes involved in resistance, we identified the bacterial ATP synthase as the cellular target. Sequence alignments were performed to highlight the modified sequences, and the structural consequences of the mutations were evaluated in structural models. Overexpression of the atpE gene in S. aureus SCVs or introducing the mutation found in the atpE gene of one of the high-level TO-resistant S. aureus mutants into the Bacillus subtilis atpE gene provided resistance to TO and further validated the identity of the cellular target. FC04-100, a TO derivative which also possesses activity against non-SCV strains, prevents high-level resistance development in prototypic strains and limits the level of resistance observed in SCVs. An ATP synthesis assay allowed the observation of a correlation between antibiotic potency and ATP synthase inhibition. The selectivity index (inhibition of ATP production by mitochondria versus that of bacterial ATP synthase) is estimated to be >105-fold for FC04-100.
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Antibacterianos/farmacología , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Tomatina/análogos & derivados , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/metabolismo , Pruebas de Sensibilidad Microbiana , ATPasas de Translocación de Protón Mitocondriales/genética , Mutación , Tomatina/farmacologíaRESUMEN
Colonization of plant roots by Bacillus subtilis is mutually beneficial to plants and bacteria. Plants can secrete up to 30% of their fixed carbon via root exudates, thereby feeding the bacteria, and in return the associated B. subtilis bacteria provide the plant with many growth-promoting traits. Formation of a biofilm on the root by matrix-producing B. subtilis is a well-established requirement for long-term colonization. However, we observed that cells start forming a biofilm only several hours after motile cells first settle on the plant. We also found that intact chemotaxis machinery is required for early root colonization by B. subtilis and for plant protection. Arabidopsis thaliana root exudates attract B. subtilis in vitro, an activity mediated by the two characterized chemoreceptors, McpB and McpC, as well as by the orphan receptor TlpC. Nonetheless, bacteria lacking these chemoreceptors are still able to colonize the root, suggesting that other chemoreceptors might also play a role in this process. These observations suggest that A. thaliana actively recruits B. subtilis through root-secreted molecules, and our results stress the important roles of B. subtilis chemoreceptors for efficient colonization of plants in natural environments. These results demonstrate a remarkable strategy adapted by beneficial rhizobacteria to utilize carbon-rich root exudates, which may facilitate rhizobacterial colonization and a mutualistic association with the host. IMPORTANCE: Bacillus subtilis is a plant growth-promoting rhizobacterium that establishes robust interactions with roots. Many studies have now demonstrated that biofilm formation is required for long-term colonization. However, we observed that motile B. subtilis mediates the first contact with the roots. These cells differentiate into biofilm-producing cells only several hours after the bacteria first contact the root. Our study reveals that intact chemotaxis machinery is required for the bacteria to reach the root. Many, if not all, of the B. subtilis 10 chemoreceptors are involved in the interaction with the plant. These observations stress the importance of root-bacterium interactions in the B. subtilis lifestyle.