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1 Department of Ecological Microbiology, University of Bayreuth, 95440 Bayreuth, Germany
2 Electron Microscopy Laboratory, University of Bayreuth, 95440 Bayreuth, Germany
3 Department of Biological Sciences, University of South Carolina, Columbus, SC 29208, USA
Correspondence
Harold L. Drake
HLD{at}Uni-Bayreuth.De
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of RC (1480 bp sequenced) and RST (1387 bp sequenced) are AM158323 and AM158322, respectively.
Present address: Limnology Research Group, Institute for Ecology, Friedrich Schiller University Jena, 07745 Jena, Germany.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Drake, 1994). These organisms have been classically thought of as obligate anaerobes, and most acetogens have been isolated from habitats having relatively stable anoxic conditions (Drake et al., 2004). However, acetogens also occur in (i) aerated litter and soils (Kuhner et al., 1997; Küsel et al., 1999a; Peters & Conrad, 1995; Wagner et al., 1996), (ii) termite guts that have steep O2 gradients (Brune et al., 1995; Tholen & Brune, 1999), and (iii) the rhizosphere of the sea grass Halodule wrightii (Küsel et al., 1999b, 2001), a habitat subject to transient periods of oxidation due in part to the transport of photosynthesis-derived O2 to the root from which the O2 can diffuse to the rhizoplane and adjacent sediments (Armstrong et al., 1991; Kraemer & Alberte, 1995). The colonization of such habitats by acetogens indicates that these so-called obligate anaerobes can tolerate periods of oxygenation. Indeed, several acetogens have been recently shown to be capable of survival and growth under mildly oxic conditions (Küsel et al., 2001; Karnholz et al., 2002; Drake et al., 2002; Boga & Brune, 2003).
The occurrence of acetogens in the rhizoplane and root cortex cells of H. wrightii (Küsel et al., 1999b) and the detection of formyltetrahydrofolate synthetase genes (indicative of acetogens: Lovell, 1994) from the roots of various salt marsh plants (Leaphart et al., 2003) suggests that acetogens have heretofore unknown trophic links in the rhizospheres of marine and estuarine plants. The main objectives of the present study were to (i) determine if acetogens were associated with the roots of black needlerush Juncus roemerianus, a common subdominant plant in marshes along the Altantic and northern Gulf of Mexico coasts of temperate north America, (ii) isolate and characterize an acetogen from J. roemerianus roots, and (iii) determine the physiological responses and protective mechanisms of this acetogen to oxidative stress.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Piceno et al., 1999). Low elevations of the North Inlet salt marsh estuary are dominated by Spartina alterniflora. Other wetlands plants occur in mixed and monotypic stands between the terrestrial biome and the short form S. alterniflora zone. These species include Spartina patens, Salicornia virginica, Borrichia frutescens and J. roemerianus (Juncus hereafter).
Medium composition and cultivation.
Anoxic, undefined medium (UM) contained yeast extract, vitamins, trace metals and reducing agents (Daniel et al., 1990). Media were dispensed under CO2 into 27 ml culture tubes (7 ml medium per tube) or 1 litre infusion bottles (500 ml medium per bottle, used for preparation of cell extracts), which were then sealed and autoclaved; the pH approximated 6·7. Anoxic, filter-sterilized stock solutions of substrates (prepared under argon) were added by syringe injection using O2-free techniques. Unless otherwise indicated, culture tubes and bottles were incubated in a horizontal, static position, cultivation was in UM, and the incubation temperature was 30 °C. Sterile O2 was added to the headspace of culture tubes to the concentrations (v/v) indicated. Shaken cultures were maintained on a rotary shaker at 200 r.p.m.
Root homogenates.
Juncus ramets with intact root zone sediments were collected from a monotypic stand at the Crab Haul Creek site of the North Inlet Microbial Observatory (http://www.nimo-sc.org). Roots were shaken and rinsed with sterile deionized water to remove sediment, then immediately frozen on dry ice. The frozen roots were shipped to the University of Bayreuth and stored at –80 °C. Frozen roots were transferred into a Mecaplex (Grenchen, Switzerland) O2-free chamber (100 % N2 gas phase; room temperature). Root homogenates and serial dilutions of root homogenates were prepared as previously described (Küsel et al., 2001). In brief, (i) 3 g (fresh weight) of roots were ground using a sterile glass grinder, (ii) the product was transferred to a bottle (containing 27 ml dilution buffer) that was sealed, (iii) the headspace of the bottle was replaced with sterile CO2, and (iv) the contents of the bottle were mixed using an end-over-end shaker for 1 h. Such root homogenates were used for the preparation of dilution series and subsequent inoculation of media in enumeration studies.
Enumeration and acetogenic enrichment protocols.
Most probable number (MPN) tubes were incubated in the dark and scored positive based on consumption of substrate or formation of products (Küsel et al., 1999b). MPN values are the means of three or four replicates and were calculated from standard tables (Alef, 1991). The protocol for the acetogenic enrichment from which strains RC and RST were obtained was as previously described (Küsel et al., 1999b, 2001). Inoculum for the acetogenic enrichment was obtained from a highest growth-positive H2/CO2 MPN tube; the initial H2/CO2 enrichment contained 15 mM bromoethanesulfonate to inhibit the growth of methanogens (Sparling & Daniels, 1987). The H2/CO2 enrichment was streaked onto solidified medium, and colonies were picked and transferred to liquid medium for subsequent cultivation on the substrates indicated. Analysis of acetate confirmed the acetogenic conversion of substrates.
Preparation of cell-free extracts, enzyme assays and redox difference spectra.
Cell-free extracts were prepared under anoxic conditions (Kuhner et al., 2000). Membranous and cytoplasmic fractions were prepared under oxic conditions (Fröstl et al., 1996). Redox-difference spectra were obtained with a Uvikon 930 (Kontron Instruments) double-beam recording spectrophotometer (Kuhner et al., 1997). Hydrogenase and formate dehydrogenase activities were assayed spectrophotometrically (Daniel et al., 1990); units for these enzyme activities are µmol substrate oxidized min–1. Catalase, peroxidase, NADH oxidase and superoxide dismutase activities were assayed according to standard protocols (Beauchamp & Fridovich, 1971; Beers & Sizers, 1952; Stanton & Jensen, 1993; Stellmach et al., 1988), and units for these activities are, respectively: 1 µmol H2O2 consumed min–1, 1 mg pyrogallol oxidized min–1, 1 µmol NADH oxidized min–1, and 1 µmol nitrotetrazolium blue chloride not reduced min–1. RC was grown in UM that lacked reducing agents and contained 10 mM glucose and 3 % (v/v) O2 in the headspace when these enzymes were evaluated. RST was grown in UM that lacked reducing agents and contained 10 mM N-acetylglucosamine and 0·3 % (v/v) O2 in the headspace when these enzymes were evaluated. Cell extracts of Escherichia coli K-12 (DSM 423) [cultivated in peptone-beef extract medium (5 g l–1; Difco) at pH 7·0] were used as positive controls.
Phylogenetic analysis.
Genomic DNA was purified from RC and RST using the Promega Wizard Genomic DNA Purification Kit. Near full-length 16S rRNA gene sequences were then PCR amplified using the Bacteria domain-specific oligonucleotide primers 27F (forward primer) and 1492R (reverse primer) (Lane, 1991). The PCR reaction system consisted of the following: 1 ng template DNA µl–1, 0·025 U Expand High Fidelity DNA polymerase (Boehringer Mannheim) µl–1, 1x Expand HF buffer, 1·5 mM MgCl2, 0·2 mM each deoxynucleoside triphosphate, and 0·25 pmol each primer µl–1, in a final volume of 25 µl. The thermal cycling programme was described previously (Dang & Lovell, 2000). Cloning and amplified rRNA gene restriction analysis of cloned amplicons were as previously described (Dang & Lovell, 2000) with the following exceptions: (i) Taq DNA polymerase (Qiagen) was used instead of AmpliTaq Gold DNA polymerase (Perkin-Elmer) and (ii) amplicons were digested with HaeIII and MspI.
16S rRNA gene sequencing.
Plasmid DNA was purified from each unique clone (Qiagen) and plasmid quality and quantity were determined by agarose gel electrophoresis and fluorometry, respectively. The DNA insert was sequenced in both directions using a SequiTherm EXCEL II Long-Read LC DNA sequencing kit (Epicentre Technologies) and a Li-Cor DNA4000LS sequencer. Reported 16S rRNA gene sequences most similar to those determined in this study were identified using the advanced BLAST search program (Altschul et al., 1990) of the US National Center for Biotechnology Information GenBank database (Bilofsky & Burks, 1988). Sequences were aligned using CLUSTAL W (Thompson et al., 1994). Distance matrices were constructed using Jukes–Cantor or Kimura models, with complete deletion of gaps and missing data, giving 1137 positions scored (MEGA version 2.1) (Kumar et al., 2001). Neighbour-joining and maximum-parsimony (utilizing the mini-mini heuristic search method) phylogenetic trees were built from the distance matrices. Reliability of tree topology was estimated using bootstrapping (1000 bootstrap replicates).
Additional methods.
Electron micrographs were obtained with a Zeiss CEM 902A (Oberkocken, Germany) (Gößner et al., 1999, Kuhner et al., 2000); cells were negatively stained with uranyl acetate (Valentine et al., 1968). Growth was measured as OD660; the optical path width (inner diameter of culture tubes) was 1·6 cm. Uninoculated medium was used as reference. Protein was determined colorimetrically (Bradford, 1976). Substrates and products were determined by high-performance liquid chromatography and gas chromatography (Daniel et al., 1990; Küsel & Drake, 1995; Matthies et al., 1993). Unless otherwise indicated, data are the means of duplicate experiments.
Accession numbers.
RC has been deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany, and the American Type Culture Collection (ATCC), Manassas, VA, USA, under accession numbers DSM 16614 and ATCC BAA-1027, respectively. RST has been deposited at the DSMZ and ATCC under accession numbers DSM 16652 and ATCC BAA-1028, respectively. The 16S rRNA gene sequences of RC (1480 bp sequenced) and RST (1387 bp sequenced) have been deposited at the EMBL Nucleotide Sequence Database (Cambridge, UK) under accession numbers AM158323 and AM158322, respectively.
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RESULTS |
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ST1, a mixed culture of two organisms, RC and RST
A colony that had been obtained from an acetogenic enrichment (see Methods) yielded an isolate (designated ST1) that displayed acetogenic activity on both glucose and H2/CO2. ST1 was thought to be a pure culture, as it was obtained after restreaking four times on solidified medium and was composed of weakly Gram-positive, rod-shaped cells that appeared to be uniform under a light microscope. Anaerobic growth of ST1 yielded acetate on H2, and butyrate and acetate on glucose (both butyrate and acetate are expected products of certain acetogens: Drake et al., 2004). However, two observations suggested that ST1 was composed of two organisms. (i) ST1 tolerated 21 % O2 in the gas phase of shaken broth cultures and produced lactate from glucose under highly oxic conditions. Although certain acetogens can tolerate and reduce low concentrations of O2 (Küsel et al., 2001; Karnholz et al., 2002; Drake et al., 2002; Boga & Brune, 2003), the level of tolerance for O2 by ST1 and production of lactate under oxic conditions suggested that it might contain an aerotolerant lactate-forming anaerobe, the lactate being subject to consumption by an acetogen under anoxic conditions, as has been documented for the trophic interaction of the fermentative aerophile Thermicanus aegyptius and the acetogen Moorella thermoacetica (Gößner et al., 1999). (ii) Two dissimilar partial 16S rRNA gene sequences were obtained from ST1 and related to species of Clostridium and Sporomusa (data not shown).
The two organisms were separated by culturing ST1 repeatedly on either 5 mM glucose with 12 % O2 in the headspace (four sequential transfers, each during the stationary phase of growth) or 5 mM lactate under anoxic conditions (four sequential transfers, each during the exponential phase of growth), after which each culture was serially diluted in the same medium. The highest growth-positive dilution of each dilution series was then examined for purity and growth properties. The oxic glucose dilution series yielded an isolate (hereafter referred to as RC) that grew anaerobically on glucose but not H2, and the anoxic lactate dilution series yielded an isolate [hereafter referred to as RST (T indicates type strain)] that grew anaerobically on H2 but not glucose.
Morphologies of RC and RST
Both RC and RST stained weakly Gram-positive and formed endospores; free refractile spores were occasionally seen in both cultures under a light microscope. Cells of RC were approximately 2·8 µm long and 0·6 µm wide (Fig. 1a). Cells of RST were slightly curved, flagellate, sometimes paired, and approximately 3·5 µm long and 0·8 µm wide (Fig. 1b, c).
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), and it was concluded that RC was a new isolate of C. intestinale. The two organisms yielding sequences most similar to that of RST were Sporomusa aerivorans and Sporomusa malonica (96 % and 95 % 16S rRNA gene sequence similarity to that of RST, respectively) (Fig. 2). It was concluded that RST constitutes a new species of Sporomusa; the name Sporomusa rhizae is proposed.
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Cellobiose, glucose, fructose and N-acetylglucosamine supported the growth of RC; acetate, butyrate, lactate, formate and H2 were fermentation products (Table 1, and data not shown). Ethanol was not detected in RC cultures. The formation of butyrate and H2 as the main reduced end products from saccharides under anoxic conditions was indicative of butyrate fermentation (Buckel, 2005; Dürre, 2005). However, the relatively low recovery of reductant in RC cultures suggested that RC might have produced an undetected end product or a storage compound. RC did not grow on stachyose, sucrose, raffinose, vanillate, syringate, citrate, xylose, butyrate, propionate, lactate, pyruvate, betaine, ethanol, acetate or H2.
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). Although high amounts of O2 curtailed the growth of RC, cells remained metabolically active under highly oxic conditions, as shown by the production of lactate as nearly the sole reduced end product detected in both static and shaken cultures containing approximately 20 % O2 in the headspace (Table 2).
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Vanillate, syringate, citrate, N-acetylglucosamine, betaine, lactate, formate and H2 supported the growth of RST (Table 3, and data not shown). Acetate was the main reduced end product formed by RST, a trait indicative of acetogens. Traces of butyrate, isobutyrate and propionate were also detected in cultures of RST. Although lactate was utilized, growth and acetate production with this substrate was minimal. Certain acetogens (e.g. M. thermoacetica) can use CO, but some (e.g. Thermoanaerobacter kivui) cannot (Daniel et al., 1990). CO was not utilized by RST, and growth in unsupplemented and vanillate-supplemented UM was inhibited by CO (Table 3). RST did not grow on raffinose, stachyose, sucrose, glucose, fructose, xylose, butyrate, propionate, ethanol, acetate, oxalate or glyoxylate.
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). A metabolic shift did not occur in response to O2. Under static conditions, N-acetylglucosamine-dependent growth of RST was less sensitive to O2 than was H2-dependent growth. Growth at the expense of N-acetylglucosamine or H2 was equally sensitive to O2 under shaken conditions. Acetate production ceased when the growth of RST was inhibited by elevated amounts of O2. The spores of RST appeared to be highly resistant to O2, as evidenced by the ability of cultures that had been incubated with 6 % O2 in the headspace for 12 months (during which time no growth occurred and no O2 was consumed) to be revived when transferred to anoxic conditions (data not shown).
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; Breznak et al., 1988; Kuhner et al., 1997; Boga et al., 2003). Cytochromes were not detected in the cytoplasmic fraction of RST. Cell-free extracts of RC and RST contained peroxidase activity (0·9 and 1·1 units per mg protein, respectively) and NADH oxidase activity (5·6 and 32·2 units per mg protein, respectively). Catalase and superoxide dismutase were not detected in extracts of RC or RST. Hydrogenase activity (measured in the direction of uptake) was present in cell-free extracts of RC and RST (87 and 2730 units per mg protein, respectively). Formate dehydrogenase activity was only detected in extracts of RST (7340 units per mg protein).
Trophic interaction of RC and RST
RC utilized saccharides that RST did not, and RST grew at the expense of products formed by RC, i.e. H2, formate and lactate. These properties suggested that of RC and RST would be metabolically linked and yield acetate and butyrate under co-culture conditions. Indeed, consumption of approximately 5 mM glucose by glucose-maintained co-cultures was concomitant with the production of approximately 6 mM acetate and 3 mM butyrate [these values are corrected for the amounts of acetate (4·5 mM) and butyrate (0·1 mM) produced by co-cultures in unsupplemented UM] under anoxic conditions. H2 was a transient product in anoxic co-cultures, and lactate and formate were essentially not detected (Fig. 3a and data not shown). The maximum concentration of H2 in anoxic co-cultures approximated 30 % that of anoxic RC cultures (Fig. 3a, Table 1
). The high levels of hydrogenase and formate dehydrogenase in RST were consistent with this strain's apparent use of RC-produced H2 and formate in co-cultures. Glucose-maintained co-cultures consistently yielded approximately 50 % more butyrate than glucose-maintained RC cultures, and the recovery of glucose-derived reductant in co-cultures approximated 90 %, a value higher than that obtained in RC cultures (see above). These results suggested that the presence of H2-consuming RST enhanced the production of butyrate by RC.
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). H2 and butyrate were not apparent until the concentration of O2 had decreased, and lactate and H2 were subject to utilization when O2 reached negligible levels. Acetate and butyrate were the two main reduced end products of O2-enriched shaken co-cultures. These findings suggested that RC might aid vegetative cells of RST in survival under highly oxic conditions, because pure cultures of RST did not grow under such conditions (Table 4). |
DISCUSSION |
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). Glucose-dependent homoacetogenesis (i.e. the conversion of glucose to three molecules of acetate) is a hallmark of acetogens and is the basis by which the model acetogen M. thermoacetica was cultivated during the decades of research with it that resolved the acetyl-CoA pathway (Drake & Daniel, 2004). The inability of RST to utilize simple sugars demonstrates that it must be integrated to a food web via other trophic links under in situ conditions. The trophic interaction of RC and RST theoretically provides such a link (Fig. 4). H2 appeared to be the primary trophic link between RC and RST under anoxic conditions. In contrast, formate appears to be the primary trophic link in co-cultures of the thermophiles T. aegyptius and M. thermoacetica (Gößner et al., 1999).
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; Madigan & Martinko, 2005; Jones & Keis, 2005). RC's tolerance to highly oxic conditions illustrates that clostridial species in the rhizosphere of estuarine plants can have extraordinary tolerance to O2. Under highly oxic conditions, the catabolism of RC shifted to a nearly homolactate fermentation, a form of anaerobic catabolism utilized by a variety of aerotolerant fermenters (Jensen et al., 2001; van Niel et al., 2002; Matthies et al., 2004; Madigan & Martinko, 2005). Butyrate fermentation necessitates the use of O2-sensitive hydrogenase (Buckel, 2005), and RC's capacity to engage homolactate fermentation is likely the primary mechanism for its ability to cope with high amounts of O2. The type strain of C. intestinale (ATCC 49213) was isolated from cattle faeces and, like RC, is aerotolerant (Lee et al., 1989). However, the production of H2, metabolic shifts in response to O2, and quantitative product profiles have not been reported for C. intestinale (Lee et al., 1989; Hippe et al., 1992).
RST was identified as a new species of Sporomusa, and the name Sporomusa rhizae is proposed. All species of Sporomusa are acetogens. In contrast, clostridial acetogens do not form a monophyletic functional group (i.e. the closest phylogenetic neighbour of a clostridial acetogen might be a clostridial species that lacks the acetyl-CoA pathway) (Drake et al., 2004; Drake & Küsel, 2005). Species of Sporomusa have been obtained from a variety of habitats, including silage (Möller et al., 1984), the termite gut (Breznak et al., 1988; Boga et al., 2003) and soil (Kuhner et al., 1997). The isolation of a species of Sporomusa from the roots of an estuarine plant extends the habitat range and illustrates the versatility of this spore-forming acetogenic genus.
Clostridium glycolicum RD1, a clostridial acetogen isolated from root homogenates of the seagrass H. wrightii, can tolerate up to 6 % O2 in the gas phase and consume small amounts of O2 (Küsel et al., 2001; Drake & Küsel, 2005). RST likewise tolerated and reduced low concentrations of O2. Other sporomusal species are also capable of reductively consuming O2 (Drake et al., 2002; Karnholz et al., 2002; Boga et al., 2003). Acetogenesis is dependent upon the acetyl-CoA pathway, and many of the enyzmes involved in this reductive pathway are highly sensitive to oxidation (Wood & Ljungdahl, 1991; Ragsdale, 1991). Acetogens have several mechanisms for coping with oxidative stress. For example, C. glycolicum RD1 engages ethanol and lactate fermentations when challenged with O2 (Küsel et al., 2001). RST did not undergo such a metabolic shift and likewise had a higher sensitivity to O2 than C. glycolicum RD1. Acetogens possess a variety of oxidoreducatases that can reduce O2 and its toxic by-products peroxide and superoxide. For example, the model acetogen M. thermoacetica contains peroxidase, NADH oxidase, rubredoxin oxidoreductase (a superoxide reductase) and rubrerythrin (a peroxidase), and also has the ability to dissimilate nitrate to ammonium (a more redox-positive half-cell reaction than that of CO2/acetate) as an alternative terminal electron-accepting process (Das et al., 2001, 2005; Karnholz et al., 2002; Silaghi-Dumitrescu et al., 2003; Drake & Daniel, 2004).
Marine and estuarine plants grow in sediments that are constantly or frequently water saturated, and anoxia can occur at depths of only a few millimetres in flooded sediments. Plants growing in anoxic sediments ventilate their root systems to provide O2 for root respiration (Blom, 1999; Jackson & Armstrong, 1999). Ventilation occurs via the aerenchyma, a tissue containing interconnected intercellular gas-filled spaces that can occupy as much as 70 % of the plant tissue volume. Diffusive and convective airflows through the aerenchyma of stems, rhizomes and roots have been demonstrated for several species (Christensen et al., 1994; Hwang & Morris, 1991). In the case of plants having stomata, such as Juncus and other emergent grasses, ventilation is not dependent upon photosynthesis and thus occurs throughout the day–night cycle. Many of these ventilating plants appear to leak O2 from root surfaces (Lovell, 2005 and references therein). Thus, it is likely that anaerobic bacteria growing on and in the roots of ventilating plants (i) must be innately aerotolerant (as is RC), (ii) grow in association with O2-consuming organisms (as exemplified by RST's trophic growth with RC), or (iii) have other adaptations protecting them from O2 toxicity (e.g. through development of protective microenvironments, a mechanism exploited by anaerobes in a number of O2-rich environments: Jorgensen, 1977; Paerl & Pinckney, 1996).
In conclusion, this study provides an example of a symbiotic interaction between an aerotolerant fermentative Clostridium and an acetogenic Sporomusa that not only permits commensal utilization of saccharides with production of acetate, the key substrate for terminal carbon oxidation processes in marine and estuarine sediments (Gibson, 1990; Laanbroek & Pfennig, 1981; Sorensen et al., 1981), but also confers protection to the more O2-sensitive partner by the O2-tolerant partner (Fig. 4). Such symbiotic, protective interactions are relatively unexplored, but may be both commonplace and essential to the activities of acetogens and other anaerobes in environments subject to transient fluxes of O2.
Description of Sporomusa rhizae sp. nov.
Sporomusa rhizae [rhi'zae. N.L. gen. fem. n. rhizae (from Gr. fem. n. rhiza, root), of the root; to indicate a spore-forming banana (per epithet of Sporomusa) from roots]. An acetogenic bacterium. Cells are slightly curved flagellate rods (3·5x0·8 µm), form endospores, and contain a membranous b-type cytochrome. Grows at 15–40 °C (optimum at 35 °C) and at pH 5·5–9·0 (optimum at pH 7·5). Grows anaerobically on vanillate, syringate, citrate, N-acetylglucosamine, betaine, lactate, formate and H2. Acetate is primary reduced end product. Tolerates and consumes (i.e. reduces) small amounts of O2. Peroxidase, NADH oxidase, hydrogenase and formate dehydrogenase positive. The type strain RST (=DSM 16652T=ATCC BAA-1028T) (i) was isolated from a root homogenate prepared from the black needlerush Juncus roemerianus, and (ii) grows by trophic interaction with the aerotolerant fermentative Clostridium intestinale RC (fortuitously co-isolated with RST).
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ACKNOWLEDGEMENTS |
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Received 28 November 2005;
revised 29 December 2005;
accepted 14 January 2006.
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, OD660; 
, O2; 
, glucose; 
, lactate; 
, butyrate; 
, acetate; 
, H2.