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Biological nitrogen fixation is an essential reaction in a major pathway for supplying nitrogen to terrestrial environments. Previous culture-independent analyses based on soil DNA/RNA/protein sequencing could globally detect the nitrogenase genes/proteins of Anaeromyxobacter (in the class Deltaproteobacteria ), commonly distributed in soil environments and predominant in paddy soils; this suggests the importance of Anaeromyxobacter in nitrogen fixation in soil environments. However, direct experimental evidence is lacking; there has been no research on the genetic background and ability of Anaeromyxobacter to fix nitrogen. Therefore, we verified the diazotrophy of Anaeromyxobacter based on both genomic and culture-dependent analyses using Anaeromyxobacter sp. strains PSR-1 and Red267 isolated from soils. Based on the comparison of nif gene clusters, strains PSR-1 and Red267 as well as strains Fw109-5, K, and diazotrophic Geobacter and Pelobacter in the class Deltaproteobacteria contain the minimum set of genes for nitrogenase ( nifBHDKEN ). These results imply that Anaeromyxobacter species have the ability to fix nitrogen. In fact, Anaeromyxobacter PSR-1 and Red267 exhibited N 2 -dependent growth and acetylene reduction activity (ARA) in vitro Transcriptional activity of the nif gene was also detected when both strains were cultured with N 2 gas as a sole nitrogen source, indicating that Anaeromyxobacter can fix and assimilate N 2 gas by nitrogenase. In addition, PSR-1- or Red267-inoculated soil showed ARA activity and the growth of the inoculated strains on the basis of RNA-based analysis, demonstrating that Anaeromyxobacter can fix nitrogen in the paddy soil environment. Our study provides novel insights into the pivotal environmental function, i.e., nitrogen fixation, of Anaeromyxobacter , which is a common soil bacterium. IMPORTANCE Anaeromyxobacter is globally distributed in soil environments, especially predominant in paddy soils. Current studies based on environmental DNA/RNA analyses frequently detect gene fragments encoding nitrogenase of Anaeromyxobacter from various soil environments. Although the importance of Anaeromyxobacter as a diazotroph in nature has been suggested by culture-independent studies, there has been no solid evidence and validation from genomic and culture-based analyses that Anaeromyxobacter fixes nitrogen. This study demonstrates that Anaeromyxobacter harboring nitrogenase genes exhibits diazotrophic ability; moreover, N 2 -dependent growth was demonstrated in vitro and in the soil environment. Our findings indicate that nitrogen fixation is important for Anaeromyxobacter to survive under nitrogen-deficient environments and provide a novel insight into the environmental function of Anaeromyxobacter , which is a common bacterium in soils., (Copyright © 2020 American Society for Microbiology.)

Corallococcus spp. are common soil-dwelling organisms which kill and consume prey microbes through the secretion of antimicrobial substances. Two species of Corallococcus have been described previously ( Corallococcus coralloides and Corallococcus exiguus ). A polyphasic approach, including biochemical analysis of fatty acid methyl esters, substrate utilization, and sugar assimilation assays, was taken to characterize eight Corallococcus species strains and the two type strains. The genomes of all strains, including that of C. exiguus DSM 14696 T (newly reported here), shared an average nucleotide identity below 95% and digital DNA-DNA hybridization scores of less than 70%, indicating that they belong to distinct species. In addition, we characterized the prey range and antibiotic resistance profile of each strain, illustrating the diversity of antimicrobial activity and, thus, the potential for drug discovery within the Corallococcus genus. Each strain gave a distinct profile of properties, which together with their genomic differences supports the proposal of the eight candidate strains as novel species. The eight candidates are as follows: Corallococcus exercitus sp. nov. (AB043A T = DSM 108849 T = NBRC 113887 T ), Corallococcus interemptor sp. nov. (AB047A T = DSM 108843 T = NBRC 113888 T ), Corallococcus aberystwythensis sp. nov. (AB050A T = DSM 108846 T = NBRC 114019 T ), Corallococcus praedator sp. nov. (CA031B T = DSM 108841 T = NBRC 113889 T ), Corallococcus sicarius sp. nov. (CA040B T = DSM 108850 T = NBRC 113890 T ), Corallococcus carmarthensis sp. nov. (CA043D T = DSM 108842 T = NBRC 113891 T ), Corallococcus llansteffanensis sp. nov. (CA051B T = DSM 108844 T = NBRC 114100 T ), and Corallococcus terminator sp. nov. (CA054A T = DSM 108848 T = NBRC 113892 T ). IMPORTANCE Corallococcus is a genus of predators with broad prey ranges, whose genomes contain large numbers of gene clusters for secondary metabolite biosynthesis. The physiology and evolutionary heritage of eight Corallococcus species strains were characterized using a range of analyses and assays. Multiple metrics confirmed that each strain belonged to a novel species within the Corallococcus genus. The strains exhibited distinct patterns of drug resistance and predatory activity, which mirrored their possession of diverse sets of biosynthetic genes. The breadth of antimicrobial activities observed within the Corallococcus genus highlights their potential for drug discovery and suggests a previous underestimation of both their taxonomic diversity and biotechnological potential. Taxonomic assignment of environmental isolates to novel species allows us to begin to characterize the diversity and evolution of members of this bacterial genus with potential biotechnological importance, guiding future bioprospecting efforts for novel biologically active metabolites and antimicrobials., (Copyright © 2020 American Society for Microbiology.)

The rubber oxygenase (RoxA) of Xanthomonas sp. strain 35Y (RoxA(Xsp)) is so far the only known extracellular c-type diheme cytochrome that is able to cleave poly(cis-1,4-isoprene). All other rubber-degrading bacteria described are Gram positive and employ a nonheme protein (latex-clearing protein [Lcp]) for the postulated primary attack of polyisoprene. Here, we identified RoxA orthologs in the genomes of Haliangium ochraceum, Myxococcus fulvus, Corallococcus coralloides, and Chondromyces apiculatus. The roxA orthologs of H. ochraceum (RoxA(Hoc)), C. coralloides BO35 (RoxA(Cco)), and M. fulvus (RoxA(Mfu)) were functionally expressed in a ΔroxA Xanthomonas sp. 35Y background. All RoxA orthologs oxidatively cleaved polyisoprene, as revealed by restoration of clearing-zone formation and detection of 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD) as a cleavage product. RoxA(Xsp), RoxA(Mfu), and RoxA(Cco) were purified and biochemically characterized. The optimal temperature of RoxA(Cco) and RoxA(Mfu) was between 22 and 30°C. All RoxA orthologs as isolated showed an oxidized UV-visible spectrum. Chemical reduction of RoxA(Cco) and RoxA(Mfu) indicated the presence of two slightly different heme centers with absorption maxima between 549 and 553 nm, similar to RoxA(Xsp). Sequence analysis and modeling of the three-dimensional structures of the RoxA orthologs revealed a high degree of similarity to the recently solved RoxA(Xsp) structure and included several conserved residues, notably, W302, F317, and a MauG motif at about H517. Lcp-like sequences were not detected in the genomes of the Xanthomonas sp. 35Y, H. ochraceum, M. fulvus, and C. coralloides. No RoxA orthologs were found in Gram-positive bacteria, and this first description of functional RoxA in Gram-negative bacteria other than Xanthomonas proves that RoxA is more common among rubber degraders than was previously assumed.

Versaphilic Anaeromyxobacter dehalogenans strains implicated in hexavalent uranium reduction and immobilization are present in the fractured saprolite subsurface environment at the U.S. Department of Energy Integrated Field-Scale Subsurface Research Challenge (IFC) site near Oak Ridge, TN. To provide insight into the in situ distribution of Anaeromyxobacter strains in this system with a nonuniform groundwater flow, 16S rRNA gene-targeted primers and linear hybridization (TaqMan) probes were designed for Oak Ridge IFC Anaeromyxobacter isolates FRC-D1 and FRC-W, along with an Anaeromyxobacter genus-targeted probe and primer set. Multiplex quantitative real-time PCR (mqPCR) was applied to samples collected from Oak Ridge IFC site areas 1 and 3, which are not connected by the primary groundwater flow paths; however, transport between them through cross-plane fractures is hypothesized. Strain FRC-W accounted for more than 10% of the total quantifiable Anaeromyxobacter community in area 1 soils, while strain FRC-D1 was not detected. In FeOOH-amended enrichment cultures derived from area 1 site materials, strain FRC-D1 accounted for 30 to 90% of the total Anaeromyxobacter community, demonstrating that this strain was present in situ in area 1. The area 3 total Anaeromyxobacter abundance exceeded that of area 1 by 3 to 5 orders of magnitude, but neither strain FRC-W- nor FRC-D1-like sequences were quantifiable in any of the 33 area 3 groundwater or sediment samples tested. The Anaeromyxobacter community in area 3 increased from <10(5) cells/g sediment outside the ethanol biostimulation treatment zone to 10(8) cells/g sediment near the injection well, and 16S rRNA gene clone library analysis revealed that representatives of a novel phylogenetic cluster dominated the area 3 Anaeromyxobacter community inside the treatment loop. The combined applications of genus- and strain-level mqPCR approaches along with clone libraries provided novel information on patterns of microbial variability within a bacterial group relevant to uranium bioremediation.

Acetate is the most abundant intermediate of organic matter degradation in anoxic rice field soil and is converted to CH(4) and/or CO(2). Aceticlastic methanogens are the primary microorganisms dissimilating acetate in the absence of sulfate and reducible ferric iron. In contrast, very little is known about bacteria capable of assimilating acetate under methanogenic conditions. Here, we identified active acetate-assimilating microorganisms by using a combined approach of frequent label application at a low concentration and comparative RNA-stable isotope probing with (13)C-labeled and unlabeled acetate. Rice field soil was incubated anaerobically at 25 degrees C for 12 days, during which (13)C-labeled acetate was added at a concentration of 500 muM every 3 days. (13)C-labeled CH(4) and CO(2) were produced from the beginning of the incubation and accounted for about 60% of the supplied acetate (13)C. RNA was extracted from the cells in each sample taken and separated by isopycnic centrifugation according to molecular weight. Bacterial and archaeal populations in each density fraction were screened by reverse transcription-PCR-mediated terminal restriction fragment polymorphism analysis. No differences in the bacterial populations were observed throughout the density fractions of the unlabeled treatment. However, in the heavy fractions of the (13)C treatment, terminal restriction fragments (T-RFs) of 161 bp and 129 bp in length predominated. These T-RFs were identified by cloning and sequencing of 16S rRNA as from a Geobacter sp. and an Anaeromyxobacter sp., respectively. Apparently these bacteria, which are known as dissimilatory iron reducers, were able to assimilate acetate under methanogenic conditions, i.e., when CO(2) was the predominant electron acceptor. We hypothesize that ferric iron minerals with low bioavailability might have served as electron acceptors for Geobacter spp. and Anaeromyxobacter spp. under these conditions.

Seven strains of the myxobacterium Chondromyces crocatus, isolated from widely separated geographic regions, were investigated for the presence of an associate gram-negative, rod-shaped companion bacterium that is phylogenetically related to the genus Sphingobacterium and has been named "Candidatus comitans" (C. A. Jacobi, E. Stackebrandt, H. Reichenbach, and B. J. Tindall, Int. J. Syst. Bacteriol. 46:119-122, 1996). Five of the Chondromyces strains were found to be associated with a companion bacterium, and one strain lost its companion during the study. A 16S ribosomal DNA (16S rDNA) clone library was generated for each Chondromyces culture. Sequence similarity was > 99.1% for all but one strain of C. crocatus and all but one strain of "Candidatus comitans". The three analyzed 16S rDNA clone sequences of the companion of Cm c7 indicated that this companion strain is slightly less related to the other companion strains. The association between the companion and the myxobacterium including the sporangioles was determined by in situ hybridization with fluorescently labeled rRNA probes and scanning confocal laser microscopy. Based on these results, there are indications that the companion strains may survive environmental stress by inclusion in the aggregates and in the sporangioles of the myxobacterium.