Microbial Ecology of Flavobacteria in Soil
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Microbial ecology of flavobacteria in soil – potential implications for greenhouse gas production: Flavobacterium denitrificans as transient inhabitant of the earthworm gut Marcus A. Horn, Julian Ihssen, Carola Matthies, Pia K. Wüst, Peter Depkat-Jakob, Harold L. Drake Department of Ecological Microbiology, University of Bayreuth, Dr. Hans-Frisch-Str. 1-3, D- 95440 Bayreuth, Germany Correspondence: M.A. Horn ([email protected]) Species of the genus Flavobacterium include facultative aerobes and active members of the soil microbial community (1, 7). The soil microbial community transiently becomes the earthworm gut microbial biome upon ingestion of soil into the earthworm gut. Microorganisms capable of anaerobiosis are selectively activated in the anoxic and substrate-rich gut environment (3, 12). Earthworms supplement the ingested soil with mucopolysaccharides that are hypothesized to activate certain microorganisms ingested with the worm feed. The activated anaerobic microorganisms secrete exoenzymes that help the worm in digesting organic matter. The earthworm digestive system is thus referred to as ‘mutualistic digestive system’ (12). The earthworm gut is anoxic, moist, pH neutral, rich in proteins and carbohydrates derived from mucus and feed, which might serve as substrates for the ingested microorganisms and favor anaerobic microorganisms (5). Various mono- and disaccharides (like glucose and isomaltose) occur in the gut summing up to concentrations of up to 10 mM (13). More than 100 mM monosaccharides are detected after hydrolysis of polysaccharides, indicating that most saccharides in the earthworm gut are polymeric. Concentrations of monosaccharides decrease along the earthworm alimentary channel, indicating that monosaccharides are subjected to consumption by microorganisms or the earthworm (13). Up to 30 mM short chain fatty acids likewise occur in the alimentary canal. Earthworms emit molecular hydrogen and up to 10 µM H2 are detected in the gut, indicating ongoing fermentation. Indeed, microorganisms capable of fermentations are abundant anaerobes in the earthworm gut (6). The earthworm gut contains soil derived nitrate and nitrite, emit the greenhouse gas nitrous oxide due to activation of denitrification in their gut, and denitrifiers are 10 to 1000 x more abundant in the gut compared to soil (8, 10). Denitrification is the sequential reduction of nitrate to dinitrogen via nitrite and nitrous oxide, and is catalyzed by only a few Flavobacterium strains known so far (1, 7, 14). Concentrations of N2O in the earthworm alimentary channel are highest in the gut, indicating active dentrification. Consequently, soil denitrifiers might be activated upon ingestion in to the earthworm gut. Thus, it was hypothesized that soil denitrifiers ingested by the worm contribute to N2O-production and carbon cycling in the gut of earthworms. The objectives were to isolate N2O-producers from the earthworm gut, and to comparatively assess soil and gut microbiota by 16S rRNA and structural gene analyses. 136 N2O-producing isolates were obtained by serially dilution of gut content of the earthworm Aporrectodea caliginosa and repeated streak plating of isolated colonies on nitrate or nitrite supplemented, anoxic, diluted tryptic soy broth or nutrient broth (4, 6). Isolates were denitrifiers or dissimilatory nitrate reducers and were affiliated with the common soil genera Pseudomonas, Buttiauxella, Enterobacter, Aeromonas, Dechloromonas, Ralstonia, Sinorhizobium, Paenibacillus, Bacillus, Clostridium, and Flavobacterium on the basis of their 1 16S rRNA gene sequences, indicating a diverse, soil-derived N2O-producing community in the earthworm gut. The flavobacterial strain was capable of denitrification and named Flavobacterium denitrificans ED5 (DSM 15936T). F. denitrificans converted nitrate 7 stochiometrically to N2. 4.9 nmol N2O per hour and 10 cells were initially produced. Initial per cell nitrous oxide production rates of F. denitrificans under gut-like conditions approximated those obtained in culture media. F. denitrificans was isolated from the gut of the earthworm Aporrectodea caliginosa from a 10-4 dilution of gut content, indicating that this organism is an 6 abundant N2O producer in the earthworm gut. Given the most probable numbers of 10 per gram dry weight gut content obtained for denitrifiers, F. denitrificans might account for 1% of cultured denitrifiers from the earthworm gut. F. denitrificans was a facultative aerobe that produced yellow-pigmented colonies, and was flexirubin-reaction positive. The closest and validly named relative on the basis of comparative 16S rRNA gene analysis was F. johnsoniae ATCC 17061T. 16S rRNA gene similarities of F. denitrificans and F. johnsoniae were 96%. Close relatives were F. tegetincola, F. flevense, and F. hibernum, indicating that F. denitrificans is related to soil and fresh water Flavobacteria. F. denitrificans was a motile rod that grew in chains at 10-30°C and at pH 5.5-8.2, with optimal growth at 25°C and pH 7. c-type cytochromes occurred in its membranes. Cells were up to 0.8 x 3.0 µm The DNA G+C-content was 34.6 mol%. F. denitrificans utilized mono- and polymeric carbohydrates as well as proteins as electron donors, indicating hydrolytic enzyme activities (Table 1). Starch, inulin, pectin and gelatin were growth supportive. Closely related species likewise have the capability to hydrolyze biopolymers (Table 1), indicating that Flavobacteria might contribute to biopolymer hydrolysis and thus digestion in the worm gut. Carbohydrates and proteins are abundant in the gut of earthworms (5, 13). Oxygen, nitrate and nitrite were used as electron acceptors but not iron(III) and sulphate. Denitrifiers occur in diverse genera (14) and thus require a structural rather than a 16S rRNA gene based molecular analysis. The nosZ genes encode a nitrous oxide reductase that catalyzes the conversion of nitrous oxide to dinitrogen during denitrification. NosZ was amplified and sequenced from soil Table 1. Potential substrates of F. denitrificans and two closely related species. Symbols: +, growth positive; -, negative; (+), and guts of the earthworms A. weakly positive; ND, not determined. Data were compiled from (2). caliginosa, Lumbricus terrestris, and Lumbricus rubellus to assess Substrates F. denitrificans F. johnsoniae F. flevense whether gut denitrifers are indeed Glucose + + + soil derived. A total of 182 and 180 Lactose + (+) (+) nosZ were retrieved from gut and Cellobiose + ND ND soil, respectively. Most of the nosZ Chitin - + - Starch + + + sequences were hitherto unknown, Inulin + ND ND clustered with known soil derived Pectin + + + sequences, or were related to nosZ Gelatin + + - of Bradyrhizobium, Brucella, Dechloromonas, Pseudomonas, Ralstonia, Sinorhizobium and Flavobacterium. Phylogenies of gut and soil derived nosZ were highly similar, indicating that gut denitrifiers were indeed soil-derived. Total rRNA was extracted from the gut of the earthworm L. terrestris. Reverse transcription, PCR amplification, cloning and sequencing were performed. Acidobacteria, Planctomycetacia, Proteobacteria, Actinobacteria, and Flavobacteria were detected. 5% of sequences obtained from the earthworm gut were 94-97% similar to those of the genus Flavobacterium, indicating that a significant portion of the earthworm gut microbial biome are 2 Flavobacteria and that the earthworm gut constitutes a reservoir of hitherto uncultured Flavobacteria. The isolation of F. denitrificans and other Flavobacteria from A. caliginosa and L. terrestris as well as the detection of 16S rRNA genes indicative of such organisms in gut content (2, 9, 11) and cast are in accordance with the high abundance and thus potential relevance of flavobacterial 16S rRNA in the earthworm gut. Indeed, the detectability of Flavobacteria by fluorescence in situ hybridization in the alimentary canal of L. terrestris was much higher than in soil and increased along the alimentary canal, supporting that Flavobacteria are activated in the earthworm gut. Diverse Flavobacteria occur in soil, which are capable of biopolymer hydrolysis and denitrification. Denitrifiers in the earthworm gut are soil-derived. Thus, the potential relevance of Flavobacteria including F. denitrificans in the earthworm gut is to (i) contribute to the emission of nitrous oxide by earthworms, and (ii) help the worm in digestion of soil organic matter by providing hydrolytic enzyme activities in the gut. Whether there is a true mutualistic relationship rather than a opportunistic association remains to be resolved. References 1. Bernardet J-F, Bowman J. 2006. The genus Flavobacterium. In The prokaryotes., ed. M Dworkin, S Falkow, E Rosenberg, K-H Schleifer, E Stackebrandt, pp. 455-80. New York: Springer 2. Byzov BA, Nechitaylo TY, Bumazhkin BK, Kurakov AV, Golyshin PN, Zvyagintsev DG. 2009. Culturable microorgansims from the earthworm digestive tract. Microbiology 78: 360-8 3. Drake HL, Horn MA. 2007. As the worm turns: the earthworm gut as a transient habitat for soil microbial biomes. Annual Reviews in Microbiology 61: 169–89 4. Horn MA, Ihssen J, Matthies C, Schramm A, Acker G, Drake HL. 2005. Dechloromonas denitrificans sp nov., Flavobacterium denitrificans sp nov., Paenibacillus anaericanus sp. nov and Paenibacillus terrae strain MH72, N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa. International Journal of Systematic and Evolutionary Microbiology 55: 1255-65 5. Horn MA, Schramm A, Drake HL. 2003. The earthworm