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Nasopharyngeal carriage of staphylococci spreads potentially pathogenic strains into (peri)oral regions and increases the chance of cross-infections. Some laboratory strains can also move rapidly on hydrated agar surfaces, but the biological relevance of these observations is not clear. Using soft-agar [0.3% (wt/vol)] plate assays, we demonstrate the rapid surface dispersal of (peri)oral isolates of Staphylococcus aureus and Staphylococcus epidermidis and closely related laboratory strains in the presence of mucin glycoproteins. Mucin-induced dispersal was a stepwise process initiated by the passive spreading of the growing colonies followed by their rapid branching (dendrites) from the colony edge. Although most spreading strains used mucin as a growth substrate, dispersal was primarily dependent on the lubricating and hydrating properties of the mucins. Using S. aureus JE2 as a genetically tractable representative, we demonstrate that mucin-induced dendritic dispersal, but not colony spreading, is facilitated by the secretion of surfactant-active phenol-soluble modulins (PSMs) in a process regulated by the agr quorum-sensing system. Furthermore, the dendritic dispersal of S. aureus JE2 colonies was further stimulated in the presence of surfactant-active supernatants recovered from the most robust (peri)oral spreaders of S. aureus and S. epidermidis. These findings suggest complementary roles for lubricating mucins and staphylococcal PSMs in the active dispersal of potentially pathogenic strains from perioral to respiratory mucosae, where gel-forming, hydrating mucins abound. They also highlight the impact that interspecies interactions have on the co-dispersal of S. aureus with other perioral bacteria, heightening the risk of polymicrobial infections and the severity of the clinical outcomes. IMPORTANCE: Despite lacking classical motility machinery, nasopharyngeal staphylococci spread rapidly in (peri)oral and respiratory mucosa and cause cross-infections. We describe laboratory conditions for the reproducible study of staphylococcal dispersal on mucosa-like surfaces and the identification of two dispersal stages (colony spreading and dendritic expansion) stimulated by mucin glycoproteins. The mucin type mattered as dispersal required the surfactant activity and hydration provided by some mucin glycoproteins. While colony spreading was a passive mode of dispersal lubricated by the mucins, the more rapid and invasive form of dendritic expansion of Staphylococcus aureus and Staphylococcus epidermidis required additional lubrication by surfactant-active peptides (phenol-soluble modulins) secreted at high cell densities through quorum sensing. These results highlight a hitherto unknown role for gel-forming mucins in the dispersal of staphylococcal strains associated with cross-infections and point at perioral regions as overlooked sources of carriage and infection by staphylococci.
Assuntos
Mucinas , Percepção de Quorum , Staphylococcus aureus , Staphylococcus epidermidis , Staphylococcus epidermidis/fisiologia , Mucinas/metabolismo , Staphylococcus aureus/fisiologia , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Humanos , Infecções Estafilocócicas/microbiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Toxinas Bacterianas/metabolismoRESUMO
As part of society-wide efforts to promote open access in science, the American Society for Microbiology journals are piloting the publication of companion articles highlighting rigorous data resources. The simultaneous publication of original research and data resource articles will increase awareness of, and access to, verified data sets that are critical to scientific progress. Companion articles in Microbiology Resource Announcements and two research journals, mSystems and Applied and Environmental Microbiology, will serve as an initial experiment to promote open and reproducible science.
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As part of society-wide efforts to promote open access in science, the American Society for Microbiology journals are piloting the publication of companion articles highlighting rigorous data resources. The simultaneous publication of original research and data resource articles will increase awareness of, and access to, verified data sets that are critical to scientific progress. Companion articles in Microbiology Resource Announcements and two research journals, mSystems and Applied and Environmental Microbiology, will serve as an initial experiment to promote open and reproducible science.
RESUMO
As part of society-wide efforts to promote open access in science, the American Society for Microbiology journals are piloting the publication of companion articles highlighting rigorous data resources. The simultaneous publication of original research and data resource articles will increase awareness of, and access to, verified data sets that are critical to scientific progress. Companion articles in Microbiology Resource Announcements and two research journals, mSystems and Applied and Environmental Microbiology, will serve as an initial experiment to promote open and reproducible science.
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The identification of a diverse microbiome in otic secretions from healthy young adults challenged the entrenched dogma of middle ear sterility and underscored previously unknown roles for oral commensals in the seeding of otic biofilms. We gained insights into the selective forces that enrich for specific groups of oral migrants in the middle ear mucosa by investigating the phylogeny and physiology of 19 strains isolated previously from otic secretions and representing otic commensals (Streptococcus) or transient migrants (Staphylococcus, Neisseria and actinobacterial Micrococcus and Corynebacterium). Phylogenetic analyses of full length 16S rRNA sequences recovered from partially sequenced genomes resolved close relationships between the isolates and (peri)oral commensals. Physiological functions that facilitate mucosal colonization (swarming motility, surfactant production) and nutrition (mucin and protein degradation) were also widespread among the cultivars, as was their ability to grow in the presence or absence of oxygen. Yet, streptococci stood out for their enhanced biofilm-forming abilities under oxic and anoxic conditions and ability to ferment host-derived mucosal substrates into lactate, a key metabolic intermediate in the otic trophic webs. Additionally, the otic streptococci inhibited the growth of common otopathogens, an antagonistic interaction that could exclude competitors and protect the middle ear mucosa from infections. These adaptive traits allow streptococcal migrants to colonize the otic mucosa and grow microcolonies with syntrophic anaerobic partners, establishing trophic interactions with other commensals that mirror those formed by the oral ancestors in buccal biofilms.
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The ability of some metal-reducing bacteria to produce a rough (no O-antigen) lipopolysaccharide (LPS) could facilitate surface interactions with minerals and metal reduction. Consistent with this, the laboratory model metal reducer Geobacter sulfurreducens PCA produced two rough LPS isoforms (with or without a terminal methyl-quinovosamine sugar) when growing with the soluble electron acceptor fumarate but expressed only the shorter and more hydrophilic variant when reducing iron oxides. We reconstructed from genomic data conserved pathways for the synthesis of the rough LPS and generated heptosyltransferase mutants with partial (ΔrfaQ) or complete (ΔrfaC) truncations in the core oligosaccharide. The stepwise removal of the LPS core sugars reduced the hydrophilicity of the cell and increased outer membrane vesiculation. These changes in surface charge and remodeling did not substantially impact planktonic growth but disrupted the developmental stages and structure of electroactive biofilms. Furthermore, the mutants assembled conductive pili for extracellular mineralization of the toxic uranyl cation but were unable to prevent permeation and mineralization of the radionuclide in the cell envelope. Hence, not only does the rough LPS promote cell-cell and cell-mineral interactions critical to biofilm formation and metal respiration but it also functions as a permeability barrier to toxic metal cations. In doing so, the rough LPS maximizes the extracellular reduction of soluble and insoluble metals and preserves cell envelope functions critical to the environmental survival of Geobacter bacteria in metal-rich environments and their performance in bioremediation and bioenergy applications. IMPORTANCE Some metal-reducing bacteria produce an LPS without the repeating sugars (O-antigen) that decorate the surface of most Gram-negative bacteria, but the biological significance of this adaptive feature was not previously investigated. Using the model representative Geobacter sulfurreducens strain PCA and mutants carrying stepwise truncations in the LPS core sugars, we demonstrate the importance of the rough LPS in the control of cell surface chemistry during the respiration of iron minerals and the formation of electroactive biofilms. Importantly, we describe hitherto overlooked roles for the rough LPS in metal sequestration and outer membrane vesiculation that are critical for the extracellular reduction and detoxification of toxic metals and radionuclides. These results are of interest for the optimization of bioremediation schemes and electricity-harvesting platforms using these bacteria.
Assuntos
Geobacter/metabolismo , Lipopolissacarídeos/metabolismo , Urânio/metabolismo , Biofilmes/crescimento & desenvolvimento , Geobacter/genética , Geobacter/fisiologia , Lipopolissacarídeos/genética , Oxirredução , Urânio/toxicidadeRESUMO
Oxidative stress greatly limits current harvesting from anode biofilms in bioelectrochemical systems yet insufficient knowledge of the antioxidant responses of electricigens prevents optimization. Using Geobacter sulfurreducens PCA as a model electricigen, we demonstrated enhanced oxygen tolerance and reduced electron losses as the biofilms grew in height on the anode. To investigate the molecular basis of biofilm tolerance, we developed a genetic screening and isolated 11 oxygen-tolerant (oxt) strains from a library of transposon-insertion mutants. The aggregative properties of the oxt mutants promoted biofilm formation and oxygen tolerance. Yet, unlike the wild type, none of the mutants diverted respiratory electrons to oxygen. Most of the oxt mutations inactivated pathways for the detoxification of reactive oxygen species that could have triggered compensatory chronic responses to oxidative stress and inhibit aerobic respiration. One of the mutants (oxt10) also had a growth advantage with Fe(III) oxides and during the colonization of the anode electrode. The enhanced antioxidant response in this mutant reduced the system's start-up and promoted current harvesting from bioanodes even in the presence of oxygen. These results highlight a hitherto unknown role of oxidative stress responses in the stability and performance of current-harvesting biofilms of G. sulfurreducens and identify biological and engineering approaches to grow electroactive biofilms with the resilience needed for practical applications.
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Geobacter biofilms synthesize an electroactive exopolysaccharide matrix with conductive pili and c-cytochromes that spatially organizes cells optimally for growth and electron transport to iron oxide substrates, soluble metal contaminants, and current-harvesting electrodes. Despite its relevance to bioremediation and bioenergy applications, little is known about the developmental stages leading to the formation of mature (>20 µm thick) electroactive biofilms. Thus, we developed a transposon mutagenesis method and a high-throughput screening assay and identified mutants of Geobacter sulfurreducens PCA interrupted in the initial stages of surface colonization (attachment and monolayer formation) and the vertical growth and maturation of multilayered biofilms. The molecular dissection of biofilm formation demonstrated that cells undergo a regulated developmental program to first colonize the surface to saturation and then synthesize an electroactive matrix to support optimal cell growth within structured communities. Transitioning from a monolayer to a multilayered, mature biofilm required the expression of conductive pili, consistent with the essential role of these extracellular protein appendages as electronic conduits across all layers of the biofilms. The genetic screening also identified cell envelope processes, regulatory pathways, and electron transport components not previously linked to biofilm formation. These genes provide much-needed understanding of the cellular reprogramming needed to build electroactive biofilms. Importantly, they serve as predictive markers of the physiology and reductive capacity of Geobacter biofilms during the bioremediation of toxic metals and radionuclides and current harvesting in bioelectrochemical systems.
Assuntos
Geobacter , Biofilmes , Transporte de Elétrons , Fímbrias Bacterianas/genética , Testes Genéticos , Geobacter/genética , OxirreduçãoRESUMO
The entrenched dogma of a sterile middle ear mucosa in health is incongruent with its periodic aeration and seeding with saliva aerosols. To test this, we sequenced 16S rRNA-V4 amplicons from otic secretions collected at the nasopharyngeal orifice of the tympanic tube and, as controls, oropharyngeal and buccal samples. The otic samples harbored a rich diversity of oral keystone genera and similar functional traits but were enriched in anaerobic genera in the Bacteroidetes (Prevotella and Alloprevotella), Fusobacteria (Fusobacterium and Leptotrichia) and Firmicutes (Veillonella) phyla. Facultative anaerobes in the Streptococcus genus were also abundant in the otic and oral samples but corresponded to distinct, and sometimes novel, cultivars, consistent with the ecological diversification of the oral migrants once in the middle ear microenvironment. Neutral community models also predicted a large contribution of oral dispersal to the otic communities and the positive selection of taxa better adapted to growth and reproduction under limited aeration. These results challenge the traditional view of a sterile middle ear in health and highlight hitherto unknown roles for oral dispersal and episodic ventilation in seeding and diversifying otic biofilms.
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Bacteria in the genus Geobacter thrive in iron- and manganese-rich environments where the divalent cobalt cation (CoII) accumulates to potentially toxic concentrations. Consistent with selective pressure from environmental exposure, the model laboratory representative Geobacter sulfurreducens grew with CoCl2 concentrations (1 mM) typically used to enrich for metal-resistant bacteria from contaminated sites. We reconstructed from genomic data canonical pathways for CoII import and assimilation into cofactors (cobamides) that support the growth of numerous syntrophic partners. We also identified several metal efflux pumps, including one that was specifically upregulated by CoII. Cells acclimated to metal stress by downregulating non-essential proteins with metals and thiol groups that CoII preferentially targets. They also activated sensory and regulatory proteins involved in detoxification as well as pathways for protein and DNA repair. In addition, G. sulfurreducens upregulated respiratory chains that could have contributed to the reductive mineralization of the metal on the cell surface. Transcriptomic evidence also revealed pathways for cell envelope modification that increased metal resistance and promoted cell-cell aggregation and biofilm formation in stationary phase. These complex adaptive responses confer on Geobacter a competitive advantage for growth in metal-rich environments that are essential to the sustainability of cobamide-dependent microbiomes and the sequestration of the metal in hitherto unknown biomineralization reactions.
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Type IV pili (T4P) are bacterial appendages used for cell adhesion and surface motility. In metal-reducing bacteria in the genus Geobacter, they have the unique property of being conductive and essential to wire cells to extracellular electron acceptors and other cells within biofilms. These electroactive bacteria use a conserved pathway for biological assembly and disassembly of a short and aromatic dense peptide subunit (pilin). The polymerization of the pilins clusters aromatic residues optimally for charge transport and exposes ligands for metal immobilization and reduction. The simple design yet unique functionalities of conductive T4P afford opportunities for the scaled-up production of recombinant pilins and their in vitro assembly into electronic biomaterials of biotechnological interest. This review summarizes current knowledge of conductive T4P biogenesis and functions critical to actualize applications in bioelectronics, bioremediation, and nanotechnology.
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Biotecnologia , Fímbrias Bacterianas , Nanofios , Biofilmes , Biologia , Condutividade Elétrica , Proteínas de Fímbrias/análise , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/química , Fímbrias Bacterianas/metabolismo , Geobacter/metabolismo , Metais/metabolismo , Nanofios/química , Peptídeos/metabolismoRESUMO
Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca2+-independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca2+-dependent enzymes, suggesting convergent evolution.
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Proteínas de Bactérias/metabolismo , Citocromos a1/metabolismo , Citocromos c1/metabolismo , Geobacter/metabolismo , Nitrato Redutases/metabolismo , Compostos de Amônio/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Citocromos a1/química , Citocromos a1/genética , Citocromos c1/química , Citocromos c1/genética , Geobacter/química , Geobacter/genética , Cinética , Modelos Moleculares , Nitrato Redutases/química , Nitrato Redutases/genética , Nitratos/metabolismo , Filogenia , Conformação ProteicaRESUMO
Nutrients and pesticides in agricultural runoff contribute to the degradation of water resources. Nitrates and phosphates can be remediated through the use of treatment systems such as woodchip bioreactors and adsorbent aggregate filters; however, concerns remain over potential effects of pesticides on nutrient removal efficiency in these systems. To test this, we designed laboratory-scale woodchip bioreactors equipped with secondary adsorbent aggregate filters and investigated the capacity of these systems to remediate nutrients when operated under two hydraulic retention times (HRT) and in the presence of commonly used pesticides. The woodchip bioreactors effectively removed over 99% of nitrate per day when operated under a 72 h hydraulic retention time, with the secondary expanded shale aggregate filters consistently reducing phosphate concentrations by 80-87%. Treatment efficacy of both systems was maintained in the presence of the insecticide chlorpyrifos. Reducing HRT in the bioreactors to 21 min decreased nitrate removal efficiency; however, the insecticides bifenthrin, chlorpyrifos, and the herbicide oxyfluorfen were reduced by 76%, 63%, and 31%, respectively. Cultivation approaches led to the isolation of 45 different species from the woodchip bioreactors operated under a 21 min HRT, with Bacillus species being the most prevalent throughout the treatment. By contrast, pesticide application decreased the number and diversity of Bacillus isolates and enriched for Pseudomonas and Exiguobacterium species. Woodchip bioreactors and adsorbent aggregate filters provide effective treatment platforms to remediate agrochemicals, where they maintain treatment efficacy in the presence of pesticides and can be modulated through HRT management to achieve environmental and operational water quality goals.
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Desnitrificação , Praguicidas , Reatores Biológicos , Nitratos , NutrientesRESUMO
The family Geobacteraceae, with its only valid genus Geobacter, comprises deltaproteobacteria ubiquitous in soil, sediments, and subsurface environments where metal reduction is an active process. Research for almost three decades has provided novel insights into environmental processes and biogeochemical reactions not previously known to be carried out by microorganisms. At the heart of the environmental roles played by Geobacter bacteria is their ability to integrate redox pathways and regulatory checkpoints that maximize growth efficiency with electron donors derived from the decomposition of organic matter while respiring metal oxides, particularly the often abundant oxides of ferric iron. This metabolic specialization is complemented by versatile metabolic reactions, respiratory chains, and sensory networks that allow specific members to adaptively respond to environmental cues to integrate organic and inorganic contaminants in their oxidative and reductive metabolism, respectively. Thus, Geobacteraceae are important members of the microbial communities that degrade hydrocarbon contaminants under iron-reducing conditions and that contribute, directly or indirectly, to the reduction of radionuclides, toxic metals, and oxidized species of nitrogen. Their ability to produce conductive pili as nanowires for discharging respiratory electrons to solid-phase electron acceptors and radionuclides, or for wiring cells in current-harvesting biofilms highlights the unique physiological traits that make these organisms attractive biological platforms for bioremediation, bioenergy, and bioelectronics application. Here we review some of the most notable physiological features described in Geobacter species since the first model representatives were recovered in pure culture. We provide a historical account of the environmental research that has set the foundation for numerous physiological studies and the laboratory tools that had provided novel insights into the role of Geobacter in the functioning of microbial communities from pristine and contaminated environments. We pay particular attention to latest research, both basic and applied, that has served to expand the field into new directions and to advance interdisciplinary knowledge. The electrifying physiology of Geobacter, it seems, is alive and well 30 years on.
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Condutividade Elétrica , Geobacter/fisiologia , Biodegradação Ambiental , Biofilmes/crescimento & desenvolvimento , Biotecnologia , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Transporte de Elétrons/fisiologia , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Geobacter/classificação , Metais/metabolismo , OxirreduçãoRESUMO
Geobacter bacteria assemble a helical peptide of the Type IVa pilin subclass as conductive pili decorated with metal binding and reduction sites. We used recombinant techniques to synthesize thiolated pilin derivatives and self-assembled them on gold electrodes as a monolayer that concentrated the metal traps at the liquid interface. Cyclic and step potential voltammetry demonstrated the conductivity of the pilin films and their ability to bind and reductively precipitate divalent cobalt (Co2+) in a diffusion-controlled reaction characterized by fast binding kinetics, efficient charge transfer, and three-dimensional nanoparticle growth at discreet sites. Furthermore, cobalt oxidation at the pilin film was slower than on bare gold, consistent with a peptide optimized for metal immobilization. These properties make recombinant pilins attractive building blocks for the synthesis of novel biomaterials for the immobilization of toxic cationic metals that, like Co2+, are sparingly soluble and, thus, less mobile and bioavailable as reduced species.
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Cobalto/química , Geobacter/química , Nanofios , Condutividade Elétrica , Eletrodos , Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Óxidos/químicaRESUMO
Geobacter bacteria are the only microorganisms known to produce conductive appendages or pili to electronically connect cells to extracellular electron acceptors such as iron oxide minerals and uranium. The conductive pili also promote cell-cell aggregation and the formation of electroactive biofilms. The hallmark of these electroactive biofilms is electronic heterogeneity, mediated by coordinated interactions between the conductive pili and matrix-associated cytochromes. Collectively, the matrix-associated electron carriers discharge respiratory electrons from cells in multilayered biofilms to electron-accepting surfaces such as iron oxide coatings and electrodes poised at a metabolically oxidizable potential. The presence of pilus nanowires in the electroactive biofilms also promotes the immobilization and reduction of soluble metals, even when present at toxic concentrations. This review summarizes current knowledge about the composition of the electroactive biofilm matrix and the mechanisms that allow the wired Geobacter biofilms to generate electrical currents and participate in metal redox transformations.