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ABSTRACT Azospirillum brasilenseis plant-growth promoting rhizobacteria that produces the phytohormone indole-3-acetic acid (IAA) to induce changes in plant root architecture. The major pathway for IAA biosynthesis inA. brasilenseconverts tryptophan into indole-3-pyruvic acid (I3P) and then, through the rate-limiting enzyme, indole-3-pyruvate decarboxylase (IpdC), into IAA. Here, we characterize the potential role for IAA biosynthesis in the physiology of these bacteria by characterizing the expression pattern of theipdCpromoter, analyzing anA. brasilense ipdCmutant using multiple physiological assays and characterizing the effect of I3P, which likely accumulates in the absence ofipdCand affects bacterial physiology. We found that theipdCmutant derivative has a reduced growth rate and an altered physiology, including reduced translation activity as well as a more depolarized membrane potential compared to the parent strain. Similar effects could be recapitulated in the parent strain by exposing these cells to increasing concentrations of I3P, as well as other indole intermediates of IAA biosynthesis. Our results also indicate a protective role for IAA against the harmful effects of indole derivatives, with exogenous IAA restoring the membrane potential of cells exposed to indole derivatives for prolonged periods. These protective effects appeared to restore cell physiology, including in the wheat rhizosphere. Together, our data suggest that the IAA biosynthesis pathway plays a major role inA. brasilensephysiology by maintaining membrane potential homeostasis and regulating translation, likely to mitigate the potential membrane-damaging effects of indoles that accumulate during growth under stressful conditions.IMPORTANCEIAA is widely synthesized in bacteria, particularly in soil and rhizosphere bacteria, where it functions as a phytohormone to modulate plant root architecture. IAA as a secondary metabolite has been shown to serve as a signaling molecule in several bacterial species, but the role of IAA biosynthesis in the physiology of the producing bacterium remains seldom explored. Results obtained here suggest that IAA serves to protectA. brasilensefrom the toxic effect of indoles, including metabolite biosynthetic precursors of IAA, on membrane potential homeostasis. Given the widespread production of IAA in soil bacteria, this protective effect of IAA may be conserved in diverse soil bacteria. 

ABSTRACT Environmental microorganisms have evolved a variety of strategies to survive fluctuations in environmental conditions, including the production of biofilms and differentiation into spores.Myxococcus xanthusare ubiquitous soil bacteria that produce starvation-induced multicellular fruiting bodies filled with environmentally resistant spores (a specialized biofilm). Isolated spores have been shown to be more resistant than vegetative cells to heat, ultraviolet radiation, and desiccation. The evolutionary advantage of producing spores inside fruiting bodies is not clear. Here, we examine a hypothesis that the fruiting body provides additional protection from environmental insults. We developed a high-throughput method to compare the recovery (outgrowth) of distinct cell types (vegetative cells, free spores, and spores within intact fruiting bodies) after exposure to ultraviolet radiation or desiccation. Our data indicate that haystack-shaped fruiting bodies protect spores from extended UV radiation but do not provide additional protection from desiccation. Perturbation of fruiting body morphology strongly impedes recovery from both UV exposure and desiccation. These results hint that the distinctive fruiting bodies produced by different myxobacterial species may have evolved to optimize their persistence in distinct ecological niches.IMPORTANCEEnvironmental microorganisms play an important role in the production of greenhouse gases that contribute to changing climate conditions. It is imperative to understand how changing climate conditions feedback to influence environmental microbial communities. The myxobacteria are environmentally ubiquitous social bacteria that influence the local microbial community composition. Defining how these bacteria are affected by environmental insults is a necessary component of predicting climatic feedback effects. When starved, myxobacteria produce multicellular fruiting bodies filled with spores. As spores are resistant to a variety of environmental insults, the evolutionary advantage of building a fruiting body is not clear. Using the model myxobacterium,Myxococcus xanthus, we demonstrate that the tall, haystack-shaped fruiting body morphology enables significantly more resistance to UV exposure than the free spores. In contrast, fruiting bodies are slightly detrimental to recovery from extended desiccation, an effect that is strongly exaggerated if fruiting body morphology is perturbed. These results suggest that the variety of fruiting body morphologies observed in the myxobacteria may dictate their relative resistance to changing climate conditions. 

ABSTRACT Microbial communities perform various functions, many of which contribute to ecosystem-level nutrient cycling via decomposition. Factors influencing leaf detrital decomposition are well understood in terrestrial and aquatic ecosystems, but much less is known about arthropod detrital inputs. Here, we sought to infer how differences in arthropod detritus affect microbial-driven decomposition and community function in a carnivorous pitcher plant,Sarracenia purpurea. Using sterile mesh bags filled with different types of sterile arthropod prey, we assessed if prey type influenced the rate of decomposition in pitcher plants over 7 weeks. Additionally, we measured microbial community composition and function, including hydrolytic enzyme activity and carbon substrate use. When comparing decomposition rates, we found that ant and beetle prey with higher exoskeleton content lost less mass compared with fly prey. We observed the highest protease activity in the fly treatment, which had the lowest exoskeleton content. Additionally, we saw differences in the pH of the pitcher fluid, driven by the ant treatment which had the lowest pH. According to our results from 16S rRNA gene metabarcoding, prey treatments with the highest bacterial amplicon sequence variant (ASV) richness (ant and beetle) were associated with prey that lost a lower proportion of mass over the 7 weeks. Overall, arthropod detritus provides unique nutrient sources to decomposer communities, with different prey influencing microbial hydrolytic enzyme activity and composition. IMPORTANCEMicrobial communities play pivotal roles in nutrient cycling via decomposition and nutrient transformation; however, it is often unclear how different substrates influence microbial activity and community composition. Our study highlights how different types of insects influence decomposition and, in turn, microbial composition and function. We use the aquatic pools found in a carnivorous pitcher plant as small, discrete ecosystems that we can manipulate and study independently. We find that some insect prey (flies) breaks down faster than others (beetles or ants) likely because flies contain more things that are easy for microbes to eat and derive essential nutrients from. This is also reflected in higher enzyme activity in the microbes decomposing the flies. Our work bridges a knowledge gap about how different substrates affect microbial decomposition, contributing to the broader understanding of ecosystem function in a nutrient cycling context. 

ABSTRACT Marine macroalgae produce abundant and diverse polysaccharides, which contribute substantially to the organic matter exported to the deep ocean. Microbial degradation of these polysaccharides plays an important role in the turnover of macroalgal biomass. Various members of thePlanctomycetes-Verrucomicrobia-Chlamydia(PVC) superphylum are degraders of polysaccharides in widespread anoxic environments. In this study, we isolated a novel anaerobic bacterial strain NLcol2^Tfrom microbial mats on the surface of marine sediments offshore Santa Barbara, CA, USA. Based on 16S ribosomal RNA (rRNA) gene and phylogenomic analyses, strain NLcol2^Trepresents a novel species within thePontiellagenus in theKiritimatiellotaphylum (within the PVC superphylum). Strain NLcol2^Tis able to utilize various monosaccharides, disaccharides, and macroalgal polysaccharides such as agar and ɩ-carrageenan. A near-complete genome also revealed an extensive metabolic capacity for anaerobic degradation of sulfated polysaccharides, as evidenced by 202 carbohydrate-active enzymes (CAZymes) and 165 sulfatases. Additionally, its ability of nitrogen fixation was confirmed by nitrogenase activity detected during growth on nitrogen-free medium, and the presence of nitrogenases (nifDKH) encoded in the genome. Based on the physiological and genomic analyses, this strain represents a new species of bacteria that may play an important role in the degradation of macroalgal polysaccharides and with relevance to the biogeochemical cycling of carbon, sulfur, and nitrogen in marine environments. Strain NLcol2^T(= DSM 113125^T= MCCC 1K08672^T) is proposed to be the type strain of a novel species in thePontiellagenus, and the namePontiella agarivoranssp. nov. is proposed.IMPORTANCEGrowth and intentional burial of marine macroalgae is being considered as a carbon dioxide reduction strategy but elicits concerns as to the fate and impacts of this macroalgal carbon in the ocean. Diverse heterotrophic microbial communities in the ocean specialize in these complex polymers such as carrageenan and fucoidan, for example, members of theKiritimatiellotaphylum. However, only four type strains within the phylum have been cultivated and characterized to date, and there is limited knowledge about the metabolic capabilities and functional roles of related organisms in the environment. The new isolate strain NLcol2^Texpands the known substrate range of this phylum and further reveals the ability to fix nitrogen during anaerobic growth on macroalgal polysaccharides, thereby informing the issue of macroalgal carbon disposal. 

ABSTRACT On the roots of wetland plants such as rice, Fe(II) oxidation forms Fe(III) oxyhydroxide-rich plaques that modulate plant nutrient and metal uptake. The microbial roles in catalyzing this oxidation have been debated and it is unclear if these iron-oxidizers mediate other important biogeochemical and plant interactions. To investigate this, we studied the microbial communities, metagenomes, and geochemistry of iron plaque on field-grown rice, plus the surrounding rhizosphere and bulk soil. Plaque iron content (per mass root) increased over the growing season, showing continuous deposition. Analysis of 16S rRNA genes showed abundant Fe(II)-oxidizing and Fe(III)-reducing bacteria (FeOB and FeRB) in plaque, rhizosphere, and bulk soil. FeOB were enriched in relative abundance in plaque, suggesting FeOB affinity for the root surface. Gallionellaceae FeOBSideroxydanswere enriched during vegetative and early reproductive rice growth stages, while aGallionellawas enriched during reproduction through grain maturity, suggesting distinct FeOB niches over the rice life cycle. FeRBAnaeromyxobacterandGeobacterincreased in plaque later, during reproduction and grain ripening, corresponding to increased plaque iron. Metagenome-assembled genomes revealed that Gallionellaceae may grow mixotrophically using both Fe(II) and organics. TheSideroxydansare facultative, able to use non-Fe substrates, which may allow colonization of rice roots early in the season. FeOB genomes suggest adaptations for interacting with plants, including colonization, plant immunity defense, utilization of plant organics, and nitrogen fixation. Taken together, our results strongly suggest that rhizoplane and rhizosphere FeOB can specifically associate with rice roots, catalyzing iron plaque formation, with the potential to contribute to plant growth. IMPORTANCEIn waterlogged soils, iron plaque forms a reactive barrier between the root and soil, collecting phosphate and metals such as arsenic and cadmium. It is well established that iron-reducing bacteria solubilize iron, releasing these associated elements. In contrast, microbial roles in plaque formation have not been clear. Here, we show that there is a substantial population of iron oxidizers in plaque, and furthermore, that these organisms (SideroxydansandGallionella) are distinguished by genes for plant colonization and nutrient fixation. Our results suggest that iron-oxidizing and iron-reducing bacteria form and remodel iron plaque, making it a dynamic system that represents both a temporary sink for elements (P, As, Cd, C, etc.) as well as a source. In contrast to abiotic iron oxidation, microbial iron oxidation results in coupled Fe-C-N cycling, as well as microbe-microbe and microbe-plant ecological interactions that need to be considered in soil biogeochemistry, ecosystem dynamics, and crop management. 

ABSTRACT While methane is typically produced under anoxic conditions, methane supersaturation in the presence of oxygen has been observed in both marine and fresh waters. The biological cleavage of methylphosphonate (MPn), which releases both phosphate and methane, is one pathway that may contribute to this paradox. Here, we explore the genomic and functional potential for oxic methane production (OMP) via MPn in Flathead Lake, a large oligotrophic freshwater lake in northwest Montana. Time series and depth profile measurements show that epilimnetic methane was persistently supersaturated despite high oxygen levels, suggesting a possiblein situoxic source. Metagenomic sequencing indicated that 10% of microorganisms in the lake, many of which are related to the Burkholderiales (Betaproteobacteria) and Actinomycetota, have the genomic capacity to cleave MPn. We experimentally demonstrated that these organisms produce methane stoichiometrically with MPn consumption across multiple years. However, methane was only produced at appreciable rates in the presence of MPn when a labile organic carbon source was added, suggesting that this process may be limited by both MPn and labile carbon supply. Members of the generaAcidovorax,Rhodoferax, andAllorhizobium, organisms which make up less than 1% of Flathead Lake communities, consistently responded to MPn addition. We demonstrate that the genomic and physiological potential for MPn use exists among diverse, resident members of Flathead Lake and could contribute to OMP in freshwater lakes when substrates are available. IMPORTANCEMethane is an important greenhouse gas that is typically produced under anoxic conditions. We show that methane is supersaturated in a large oligotrophic lake despite the presence of oxygen. Metagenomic sequencing indicates that diverse, widespread microorganisms may contribute to the oxic production of methane through the cleavage of methylphosphonate. We experimentally demonstrate that these organisms, especially members of the genusAcidovorax, can produce methane through this process. However, appreciable rates of methane production only occurred when both methylphosphonate and labile sources of carbon were added, indicating that this process may be limited to specific niches and may not be completely responsible for methane concentrations in Flathead Lake. This work adds to our understanding of methane dynamics by describing the organisms and the rates at which they can produce methane through an oxic pathway in a representative oligotrophic lake. 

ABSTRACT Sulfur-cycling microbial communities in salt marsh rhizosphere sediments mediate a recycling and detoxification system central to plant productivity. Despite the importance of sulfur-cycling microbes, their biogeographic, phylogenetic, and functional diversity remain poorly understood. Here, we use metagenomic data sets from Massachusetts (MA) and Alabama (AL) salt marshes to examine the distribution and genomic diversity of sulfur-cycling plant-associated microbes. Samples were collected from sediments underSporobolus alterniflorusandSporobolus pumilusin separate MA vegetation zones, and underS. alterniflorusandJuncus roemerianusco-occuring in AL. We grouped metagenomic data by plant species and site and identified 38 MAGs that included pathways for sulfate reduction or sulfur oxidation. Phylogenetic analyses indicated that 29 of the 38 were affiliated with uncultivated lineages. We showed differentiation in the distribution of MAGs between AL and MA, betweenS. alterniflorusandS. pumilusvegetation zones in MA, but no differentiation betweenS. alterniflorusandJ. roemerianusin AL. Pangenomic analyses of eight ubiquitous MAGs also detected site- and vegetation-specific genomic features, including varied sulfur-cycling operons, carbon fixation pathways, fixed single-nucleotide variants, and active diversity-generating retroelements. This genetic diversity, detected at multiple scales, suggests evolutionary relationships affected by distance and local environment, and demonstrates differential microbial capacities for sulfur and carbon cycling in salt marsh sediments. IMPORTANCESalt marshes are known for their significant carbon storage capacity, and sulfur cycling is closely linked with the ecosystem-scale carbon cycling in these ecosystems. Sulfate reducers are key for the decomposition of organic matter, and sulfur oxidizers remove toxic sulfide, supporting the productivity of marsh plants. To date, the complexity of coastal environments, heterogeneity of the rhizosphere, high microbial diversity, and uncultured majority hindered our understanding of the genomic diversity of sulfur-cycling microbes in salt marshes. Here, we use comparative genomics to overcome these challenges and provide an in-depth characterization of sulfur-cycling microbial diversity in salt marshes. We characterize communities across distinct sites and plant species and uncover extensive genomic diversity at the taxon level and specific genomic features present in MAGs affiliated with uncultivated sulfur-cycling lineages. Our work provides insights into the partnerships in salt marshes and a roadmap for multiscale analyses of diversity in complex biological systems. 

Nitrogen gas (N2) fixation in the anode-respiring bacterium Geobacter sulfurreducens occurs through complex, multistep processes. Optimizing ammonium (NH4+) production from this bacterium in microbial electrochemical technologies (METs) requires an understanding of how those processes are regulated in response to electrical driving forces. In this study, we quantified gene expression levels (via RNA sequencing) of G. sulfurreducens growing on anodes fixed at two different potentials (−0.15 V and +0.15 V versus standard hydrogen electrode). The anode potential had a significant impact on the expression levels of N2 fixation genes. At −0.15 V, the expression of nitrogenase genes, such as nifH, nifD, and nifK, significantly increased relative to that at +0.15 V, as well as genes associated with NH4+ uptake and transformation, such as glutamine and glutamate synthetases. Metabolite analysis confirmed that both of these organic compounds were present in significantly higher intracellular concentrations at −0.15 V. N2 fixation rates (estimated using the acetylene reduction assay and normalized to total protein) were significantly larger at −0.15 V. Genes expressing flavin-based electron bifurcation complexes, such as electron-transferring flavoproteins (EtfAB) and the NADH-dependent ferredoxin:NADP reductase (NfnAB), were also significantly upregulated at −0.15 V, suggesting that these mechanisms may be involved in N2 fixation at that potential. Our results show that in energy-constrained situations (i.e., low anode potential), the cells increase per-cell respiration and N2 fixation rates. We hypothesize that at −0.15 V, they increase N2 fixation activity to help maintain redox homeostasis, and they leverage electron bifurcation as a strategy to optimize energy generation and use. 

Using a high-resolution sampling time series, this study is the first to demonstrate a persistent microbial community shift with quiescence (dormancy) in a marine organism, the temperate coralAstrangia poculata. Furthermore, during this period of community turnover, there is a shedding of putative pathogens and copiotrophs and an enhancement of the ammonia-oxidizing bacteria (Nitrosococcales) and archaea (“CandidatusNitrosopumilus”). 

ABSTRACT Microbe-mediated transformations of arsenic (As) often require As to be taken up into cells prior to enzymatic reaction. Despite the importance of these microbial reactions for As speciation and toxicity, understanding of how As bioavailability and uptake are regulated by aspects of extracellular water chemistry, notably dissolved organic matter (DOM), remains limited. Whole-cell biosensors utilizing fluorescent proteins are increasingly used for high-throughput quantification of the bioavailable fraction of As in water. Here, we present a mathematical framework for interpreting the time series of biosensor fluorescence as a measure of As uptake kinetics, which we used to evaluate the effects of different forms of DOM on uptake of trivalent arsenite. We found that thiol-containing organic compounds significantly inhibited uptake of arsenite into cells, possibly through the formation of aqueous complexes between arsenite and thiol ligands. While there was no evidence for competitive interactions between arsenite and low-molecular-weight neutral molecules (urea, glycine, and glyceraldehyde) for uptake through the aquaglyceroporin channel GlpF, which mediates transport of arsenite across cell membranes, there was evidence that labile DOM fractions may inhibit arsenite uptake through a catabolite repression-like mechanism. The observation of significant inhibition of arsenite uptake at DOM/As ratios commonly encountered in wetland pore waters suggests that DOM may be an important control on the microbial uptake of arsenite in the environment, with aspects of DOM quality playing an important role in the extent of inhibition. IMPORTANCE The speciation and toxicity of arsenic in environments like rice paddy soils and groundwater aquifers are controlled by microbe-mediated reactions. These reactions often require As to be taken up into cells prior to enzymatic reaction, but there is limited understanding of how microbial arsenic uptake is affected by variations in water chemistry. In this study, we explored the effect of dissolved organic matter (DOM) quantity and quality on microbial As uptake, with a focus on the role of thiol functional groups that are well known to form aqueous complexes with arsenic. We developed a quantitative framework for interpreting fluorescence time series from whole-cell biosensors and used this technique to evaluate effects of DOM on the rates of microbial arsenic uptake. We show that thiol-containing compounds significantly decrease rates of As uptake into microbial cells at environmentally relevant DOM/As ratios, revealing the importance of DOM quality in regulating arsenic uptake, and subsequent biotransformation, in the environment.