Source: MICHIGAN STATE UNIV submitted to
PHYSIOLOGY AND PHYLOGENETIC DIVERSITY OF TERMITE GUT SYMBIONTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0176656
Grant No.
(N/A)
Project No.
MICL01857
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Aug 1, 2002
Project End Date
Jul 31, 2007
Grant Year
(N/A)
Project Director
Breznak, J.
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
MICROBIOLOGY AND MOLECULAR GENETICS
Non Technical Summary
Termites are one of earth's most abundant and important terrestrial insects. In some locations they are also one of the our most destructive pests. The purpose of this project is to obtain a clearer understanding of their symbiotic gut microbiota (on which they depend for nutrition and vitality). Such understanding will enhance our appreciation of their positive roles in our biosphere; may suggest ways to manage termites for increasing N2 fixation and, ultimately, soil fertility; and could suggest rational, environmentally-safe methods for controlling them and their destructive activities.
Animal Health Component
(N/A)
Research Effort Categories
Basic
95%
Applied
(N/A)
Developmental
5%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053110101020%
3053110104020%
3053110107020%
3053110110020%
3053110110320%
Goals / Objectives
The objectives of this project are to test the following hypotheses: 1. Spirochetes are primarily responsible for H2/CO2 acetogenesis in termite hindguts; 2. H2/CO2-acetogenic spirochetes (Treponema strain ZAS-2) interact synergistically with H2-producing spirochetes (Treponema strain ZAS-9) in acetate production, via interspecies H2- and N-compound transfer; 3. Spirochetes exhibit cooperative interactions with folate-secreting microbes; 4. Compounds inhibitory to methanogenesis are present in termite hindguts and are lumazine-like pterins, which may be produced by spirochetes; 5. Termite gut spirochetes fix N2 and excrete fixed N-compounds under in situ conditions; 6. The nitrogenase of termite gut Treponema ZAS-9 has a high intrinsic resistance to inhibition by H2; 7. Spirochetes are major N2-fixing organisms in termite guts; and 8. The microoxic periphery of termite hindguts is home to novel microaerophiles important to termite nutrition and vitality.
Project Methods
A combination physiological, biochemical, and molecular biological methods will now be used to test each (correspondingly numbered) hypothesis, as follows: (1) Combining microradiography with 16SrRNA-targeted, fluorescent probes to identify cells in gut homogenates that carry out H2-dependent fixation of 14CO2 to an acid-stable, soluble form, which we know from previous studies is primarily 14C-acetate; (2) Perform fermentation balances with termite gut Treponema strain ZAS-9 (which ferments sugars to acetate, ethanol, H2 and CO2) growing in monoculture, and growing in co-culture with strain ZAS-2 (which is an H2-consuming homoacetogen); (3) Screen for folate-secretors by using Enterococcus hirae ATCC 8043 as bioassay organism, purify and identify secretors by using polyphasic taxonomy, and identify the folate compound secreted by using routine analytical methods; (4) Identify pterins present in termite hindgut fluid and test their inhibitory activity against Methanobrevibacter fliformis in the presence and absence of accompanying spirochetes, which may be necessary to potentiate the inhibitory activity; (5) Examine N2 fixation by spirochetes under in vitro conditions of ambient [N] that mimic those in situ (determined by analysis), as well as the ability of termite hindgut spirochetes to export fixed N; (6) Determine the Ki (for H2) of the nitrogenase of Treponema strain ZAS-9; (7) Determine which of the two homologues of nifHDK genes are expressed by Treponema strains during N2 fixation in vitro and compare this to the genes expressed in situ, and also compare the relative rates of N2 fixation by the "spirochete fraction" of the microbiota physically separated from other gut microbiota; (8) Quantitatively plate, and perform MPN-type liquid enrichment series of, termite hindgut microbiota under microoxic (0.01-0.04 atm O2) conditions and in the presence of reagents known to protect cells from exogenously formed, as well as endogenous, toxic oxygen species.

Progress 08/01/02 to 07/31/07

Outputs
Termites depend on a diverse community of hindgut microbes for nutrition. Acetate, produced by hindgut microbes during the digestion of lignocellulosic plant material (e.g. wood), serves as a major carbon and energy source for the insect. Likewise, nitrogen fixation by hindgut microbes can supply as much as 30-60% of new N needed for colony growth. However, these microbial processes depend on (or are greatly favored by) the maintenance of an anoxia in the hindgut, which is largely attributable to other, O2-consuming microbes located on or near the hindgut wall. Research carried out during the period covered sought to increase our understanding of microbes participating in each of the aforementioned processes, including: acetate-producing, N2-fixing spirochetes (novel species of Treponema representing the first spirochetes ever isolated from termite guts); novel O2-consuming beta-Proteobacteria (Stenoxybacter acetivorans); novel Acidobacteria; and protozoa. Characterization of the physiology, phylogeny, and genomic properties of spirochete isolates supported their recognition as new species. The species name Treponema primitia was applied to the homoacetogenic strains, which produced acetate fermentatively and by CO2 reduction; and the name T. azotonutricium was applied to a carbohydrate-fermenting strain that displayed highest rates of N2-fixing activity of all the spirochete isolates. (Subsequent work by others has supported the notion that spirochetes, which are among the most abundant groups of microbes in termite hindguts, are primarily responsible for acetogenesis that occurs via CO2 reduction). Biochemical studies confirmed that T. primitia possessed all the enzymes of the Wood-Ljungdahl (Acetyl-CoA) pathway for CO2 reduction to acetate, including those enzymes mediating conversion of CO2, via tetrahydrofolate (THF)-linked intermediates, to the methyl group of acetate. However, T. primitia was found to require an exogenous source of folate for growth, and we showed that this requirement could be fulfilled in situ by folinate-secreting strains of Lactococcus lactis and Serratia grimesii, which were isolated from termite guts and characterized. Studies of S. acetivorans revealed that it was a strictly aerobic, obligate microaerophile (optimum O2 conc. = 1-2%; range 0.5-4%) associated primarily with the gut wall, and it could account for ca. 0.2% of the O2 consumption observed with entire extracted guts. Analysis of S. acetivorans-specific mRNA transcripts retrieved from gut homogenates and encoding acetate kinase, phosphotransacetylase, and a cbb3-type cytochrome oxidase, supported the hypothesis that acetate was a major, if not sole, substrate fueling the O2-consuming respiratory activity of S. acetivorans in situ. Novel Acidobacteria were among the O2-consuming bacteria isolated from hindguts, but these present in low numbers and appeared to be allochthonous bacteria of soil origin. Studies of O2 consumption by guts of termites from which major groups of microbes had been eliminated implied that protozoa, long thought to be strict anaerobes, were also responsible for much of the O2 consumption occurring in hindguts.

Impacts
Virtually all animals possess a symbiotic gut microbiota that functions to various degrees in the animal's nutrition, in resistance to infection by gut pathogens, in stimulation of immune response, and in general well-being. Research with termites is serving as an excellent model system from which to better understand host interaction with a complex (multispecies) microbiota that works synergistically with the insect to derive nutrition from a nitrogen-poor, relatively refractory food resource (lignocellulosic plant material) in a manner similar, but not identical, to the bovine rumen and its microbial symbionts. Hence, it is not unreasonable to imagine that studies on termite gut microbes will provide information to increase the efficiency of ruminal fermentations to enhance meat, milk and fiber production, as well as provide microbial strains, or enzymes, or genes encoding enzymes, useful in conversion of lignocellulosic plant material to useful products, including nonpolluting fuels and chemical feedstocks.

Publications

  • Wertz, J.T. and Breznak, J.A. 200_. Stenoxybacter acetivorans gen. nov., sp. nov., an acetate-oxidizing obligate microaerophile among diverse O2 consuming bacteria from termite guts. Appl. Environ. Microbiol. (accepted; in revision).
  • Wertz, J.T. and Breznak, J.A. 200_. Physiological ecology of Stenoxybacter acetivorans, an obligate microaerophile in termite guts. Appl. Environ. Microbiol. (accepted; in revision).
  • Eichorst, S. A., Breznak, J.A. and Schmidt, T.M. 2007. Isolation and characterization of soil bacteria that define Terriglobus gen. nov., in the phylum Acidobacteria. Appl. Environ. Microbiol. 73:2708-2717.


Progress 01/01/06 to 12/31/06

Outputs
In termite hindguts, fermentative production of acetate - a major carbon and energy source for the insect - depends on efficient removal of inwardly diffusing oxygen by microbes residing on and near the hindgut wall. However, little is known about the identity of these organisms or the substrate(s) used to support their respiratory activity. A cultivation-based approach was used to isolate O2-consuming organisms from hindguts of Reticulitermes flavipes (ex Dansville, MI). More colonies developed under hypoxia (2% v/v O2) than under air, an increase attributable to colonies of a novel, rod-shaped, obligately microaerophilic beta-proteobacterium (family Neisseriaceae) that was <95% similar (based on 16S rRNA gene sequence) to any other known bacterium. Nearly identical organisms (and/or their 16S rRNA genes) were obtained from geographically separated and genetically distinct populations of R. flavipes, as well as R. santonensis collected in France. Cultivation and PCR-based procedures implied that the novel isolates were autochthonous to the hindgut of R. flavipes, associated with the hindgut wall, present at densities of 1-2 x 10e5/gut, and comprised ca. 2-7% of the hindgut prokaryote community. Representative strain TAM-DN1 utilized acetate and a limited range of other organic and amino acids as energy sources and possessed catalase and superoxide dismutase. On solid medium, the optimal O2 concentration for growth was about 2%, with no growth above 4% or under anoxia. However, cells in liquid medium could grow in higher (up to 16%) headspace O2 concentrations, but only after proportionately extended lag phases and with less efficiency (i.e. less g. biomass yield per mol O2 consumed). The genetic and physiological distinctiveness of TAM-DN1 and related strains support their recognition as a new genus and species, for which the name Stenoxybacter acetivorans gen. nov., sp. nov. is being proposed. Enzymes expressed by S. acetivorans strain TAM-DN1 indicative of growth on acetate included acetate kinase (ACK; EC 2.7.2.1) and phosphotransacetylase (PTA; EC 2.3.1.8), but not acetyl-CoA synthetase (EC 6.2.1.1). TAM-DN1 did not appear to possess typical glyoxylate cycle enzymes, suggesting that it has an alternative pathway to replenish TCA cycle intermediates or can obtain these compounds in situ. All S. acetivorans isolates possessed the ccoN gene, which encodes the oxygen-reducing subunit of the high-affinity cbb3-type cytochrome oxidase. S. acetivorans-specific transcripts of ccoN, ack and pta were detected in hindguts of R. flavipes by RT-PCR, implying that cells are oxidizing acetate and consuming oxygen in situ. The maximum contribution of the S. acetivorans to total hindgut O2 consumption was approximately 0.1 - 2.0%, similar to other hindgut bacterial isolates. During such experiments, it was also found that hindgut protozoa, to be strict anaerobes, may be more important to O2 consumption than previously thought. Work continues on analysis of nif (nitrogenase) genes of spirochetes.

Impacts
Virtually all animals possess a symbiotic gut microbiota that functions to various degrees in the animal's nutrition, in resistance to infection by gut pathogens, in stimulation of immune response, and in general well-being. Research with termites is serving as an excellent model system from which to better understand host interaction with a complex (multispecies) microbiota that works synergistically with the insect to derive nutrition from a nitrogen-poor, relatively refractory food resource (lignocellulosic plant material) in a manner similar, but not identical, to the bovine rumen and its microbial symbionts. Hence, it is not unreasonable to imagine that studies on termite gut microbes will provide information to increase the efficiency of ruminal fermentations to enhance meat, milk and fiber production, as well as provide microbial strains, or enzymes, or genes encoding enzymes, useful in conversion of lignocellulosic plant material to useful products, including nonpolluting fuels and chemical feedstocks.

Publications

  • Breznak, J. A. 2006. Termite gut spirochetes pp. 421-443. In J. D. Radolf and S. A. Lukehart (eds.), Pathogenic Treponema: molecular and cellular biology. Horizon Scientific Press, Norfolk, UK.


Progress 01/01/05 to 12/31/05

Outputs
In termite hindguts, removal of inwardly diffusing O2 by microbes residing in the hypoxic zone on and near the hindgut wall is important for continued fermentative production of acetate, an important carbon and energy source for termites. However, little is known about the organisms responsible for O2 consumption or the oxidizable substrate(s) supporting this activity, although acetate seems a likely candidate. Among the most abundant O2-consuming bacteria identified in guts of Reticulitermes flavipes were novel, rod-shaped, obligately microaerophilic Beta-Proteobacteria, whose 16S rRNA gene sequences were 98.5% similar to each other, but < 95% similar to the closest relative (Eikenella corrodens). A PCR-based procedure indicated that these Beta-Proteobacteria were autochthonous to the hindgut, were associated with the hindgut wall, occurred at densities of 1-2 x 10e5 cells/gut, and comprised ca. 5% of the hindgut bacterial community. Nearly identical organisms and/or 16S rRNA gene clones were identified in genetically distinct (as judged by microsatellite DNA analysis) populations of R. flavipes collected from various sites in the USA and in R. santonensis collected in France. Representative strain TAM-1: grew between 1-10% O2 (optimum 2%); possessed both catalase (98.9 U/mg protein) and and superoxide dismutase (31.7 U/mg protein) to combat oxidative stress; used acetate, TCA cycle acids and amino acids, but not sugars, as oxidizable energy sources; and possessed acetate kinase (2.3 U/mg protein) and phosphotransacetylase (0.6 U/mg protein), but not acetyl-CoA synthetase, for activation of acetate. All TAM strains possessed ack and pta (acetate kinase and phosphotransacetlyase genes, respectively), whose deduced protein sequences formed a clade distinct from other known Ack and Pta homologues. Homologues of acs (acetyl-CoA synthetase gene) were not detected. TAM strains also possessed ccoN, which encodes a subunit of the high-affinity cbb3-type cytochrome oxidases. By using RT-PCR, TAM-specific transcripts of ack, pta, and ccoN were detected in hindguts of R. flavipes, implying that the TAM community is oxidizing acetate and consuming oxygen in situ. In vitro rates of acetate-supported O2 consumption by strain TAM-1 [ca. 1x10e-5 pmol O2/(min x cell)] suggest that the TAM strain community can make a significant contribution to O2 consumption in situ. In separate work, dinitrogenase structural genes have been sequenced in the termite gut spirochete, Treponema azotonutricium, and in the free-living spirochete, Spirochaeta aurantia, and are currently being analyzed phylogenetically. In addition, secretion of 5-formyl-tetrahydrofolate (folinate) by termite gut strains of Lactococcus lactis and Serratia grimesii was found to support the folate requirement of the homoacetogenic spirochete, Treponema primitia, an important producer of acetate in hindguts of the dampwood termite, Zootermopsis angusticollis.

Impacts
Results continue to illuminate the role of gut microbes in termite nutrition and global C and N cycling. Findings may also lead to the development of: (i) enhanced processes for bioconversion of lignocellulosic plant materials to food, fuels or chemical feedstocks by using termite gut microbes, or genes and/or enzymes derived from them; and (ii) rational biological control strategies for termites in areas where they are pests.

Publications

  • Graber, J.R. and Breznak, J.A. 2005. Folate cross-feeding supports symbiotic homoacetogenic spirochetes. Appl. Environ. Microbiol. 71:1883-1889.


Progress 01/01/04 to 12/31/04

Outputs
In termite hindguts, removal of inwardly diffusing O2 by microbes residing in the hypoxic zone on and near the hindgut wall is important for continued fermentative production of acetate, a major carbon and energy source for termites. However, little is known about the organisms responsible for O2 consumption or the oxidizable substrate(s) supporting this activity. To explore these questions, O2-consuming bacteria were isolated from guts of Reticulitermes flavipes collected in Dansville, MI by using various media and incubation conditions that included hypoxia (2% O2). Highest recoveries were obtained on acetate-containing media under hypoxia (1.1x10e6 CFU/gut), and 7-18% of the colonies consisted of novel, rod-shaped, obligately microaerophilic Beta-Proteobacteria, whose 16S rRNA gene sequences were 98.5% similar to each other, but < 95% similar to those of any other known bacteria. A PCR-based procedure implied that these Beta-Proteobacteria were autochthonous to the hindgut, were associated with the hindgut wall, and comprised ca. 5% of the hindgut bacterial community. Nearly identical organisms and/or 16S rRNA gene clones were also obtained from genetically distinct (as judged by microsatellite DNA analysis) populations of R. flavipes collected in Spring Arbor, MI, Raleigh, NC, and Woods Hole, MA. Representative strain TAM-1: was catalase and oxidase positive; grew only under atmospheres containing 1-4% O2 v/v; used acetate, succinate and amino acids as oxidizable energy sources; and possessed acetate kinase (sp. act 2.3 U/mg. protein), but not acetyl-CoA synthetase, for activation of acetate. All TAM strains possessed ack and pta (acetate kinase and phosphotransacetlyase genes, respectively), whose deduced protein sequences formed a distinct clade separate from other known Ack and Pta homologues. Homologues of acs (acetyl-CoA synthetase gene) were not detected. TAM strains also possessed ccoN, which encodes a subunit of the high-affinity cbb3-type cytochrome oxidases. By using RT-PCR, TAM-specific transcripts of ack, pta, and ccoN were detected in hindguts of R. flavipes, implying that the TAM community is oxidizing acetate and consuming oxygen in situ. Ongoing studies continue to test the hypothesis that TAM strains are quantitatively important to O2 consumption in the hypoxic zone of R. flavipes hindguts, and that acetate oxidation supports much of this O2 consumption. In separate work on nitrogen fixation by spirochetes, the nifHDK genes (encoding the nitrogenase complex) have been sequenced in the termite gut spirochete, Treponema azotonutricium, and in the free-living spirochete, Spirochaeta aurantia. N2-fixing cells of S. aurantia exhibit a rapid shut-off of nitrogenase activity on addition of ammonium to growth media, as well as an immediate attenuation of nitrogenase gene expression, as judged by RT-PCR analysis of nitrogenase-specific mRNA. Similar experiments are underway with T. azotonutricium. Results will increase our understanding of N2 fixation in spirochetes and the importance of spirochetal N2 fixation to termite nutrition and global N cycling.

Impacts
This project contributes to a better understanding of how gut microbes assist animals in digestion and nutrition. Results may also reveal rational targets for controlling the destructive activity of termites, or provide novel microbes, genes, or gene products of commercial value.

Publications

  • Breznak, J.A. 2004. Invertebrates - Insects, pp. 191-203. In A. T. Bull (ed.), Microbial Diversity and Bioprospecting. ASM Press, Washingtion, DC.
  • Graber, J.R. and Breznak, J.A. 2004. Physiology and nutrition of Treponema primitia, an H2/CO2-acetogenic spirochete from termite hindguts. Appl. Environ. Microbiol. 70:1307-1314.
  • Graber, J.R., Leadbetter, J.R. and Breznak, J.A. 2004. Description of Treponema azotonutricium sp. nov. and Treponema primitia sp. nov., the first spirochetes isolated from termite guts. Appl. Environ. Microbiol. 70:1315-1320.
  • Stevenson, B.S., Eichorst, S.A., Wertz, J.T., Schmidt, T.M. and Breznak, J.A. 2004. New strategies for the cultivation and detection of yet-uncultured microbes. Appl. Environ. Microbiol. 70:4748-4755.


Progress 01/01/03 to 12/31/03

Outputs
Characterization of the first pure cultures of termite gut spirochetes has been sufficient to support the application of new species names: Treponema primitia, for the H2/CO2-consuming homoacetogenic strains (ZAS-1 and ZAS-2); and Treponema azotonutricium, for the sugar-fermenting, H2-producing, N2-fixing strain (ZAS-9). Two papers documenting the characterization and naming are currently in press (Applied & Environmental Microbiology). Genomic DNA fosmid libraries have been prepared in from T. azotonutricium ZAS-9 and T. primitia ZAS-2, as well as from the free-living N2-fixing spirochete, Spirochaeta aurantia. Libraries are being screened for clones containing genes encoding the nitrogenase enzyme complex, i.e. nifHD(G)K. Each of the above spirochetes contains two different nifH genes, and we now seek to answer: (i) whether each nifH is also associated with nifD(G)K homologues; and (ii) which of these nif genes is expressed during N2 fixation, and under what conditions. In addition, cellobiose-dependent co-cultures have been established with T. azotonutricium (which ferments cellobiose with H2 production) and T. primitia ZAS-2 (which consumes H2, but does not utilize cellobiose) to test the hypothesis that interspecies transfer of H2 occurs between spirochetes and results in increased acetate and biomass production per mol cellobiose than in monocultures. As the medium is N-limited, experiments will also examine interspecies transfer of fixed N between the strains (ZAS-9 exhibits 100-fold greater rates of N2 fixation than does ZAS-2). Other work is focusing on the novel microaerophilic beta-Proteobacterium TAM-1 isolated from guts of Reticulitermes flavipes. Twelve additional TAM-strains have been isolated, and these form a phylogenetically coherent cluster with no known close relatives (the nearest relative is Eikenella corrodens at only 94% 16S rRNA sequence similarity). Quantitative PCR with specific primers imply an in situ population of about 3 x 105 TAM cells per gut, which is equivalent to approx. 5% of the total prokaryotic community. Cells are true microaerophiles, only growing at O2 concentrations between 0-4% v/v. We hypothesize that TAM strains occur in the peripheral, hypoxic region of termite hindguts, where they are important to O2 consumption and help keep the luminal region anoxic for fermentative production of acetate (the major energy source of termites). We further hypothesize that acetate supports the respiratory consumption of O2 by TAM strains in situ. We have sequenced the acetate kinase gene of TAM isolates and found that the deduced proteins form a novel phylogenetically coherent cluster. We have begun to examine the regulation acetate kinase synthesis in vitro, including quantification of transcripts by using RT-PCR, and will use this approach to examine expression of TAM-specific acetate kinase transcripts in situ.

Impacts
This project contributes to a better understanding of how gut microbes assist animals in digestion and nutrition. Results may also reveal rational targets for controlling the destructive activity of termites, or provide novel microbes, genes, or gene products of commercial value.

Publications

  • No publications reported this period


Progress 01/01/02 to 12/31/02

Outputs
Continued studies of termite gut symbiotic spirochetes revealed that, unlike Treponema strains ZAS-1 and ZAS-2 (which are H2-consuming homoacetogens), strain ZAS-9 produces H2 during fermentation of mono- and disaccharides, yielding (mmol/100 mmol maltose): acetate, 280; ethanol, 80; H2, 520; and CO2, 360. The mol% G+C content of genomic DNAs were: ZAS-1, 51.04; ZAS-2, 50.87; and ZAS-9, 50.02 (HPLC method). Genome sizes, estimated by pulsed field gel electrophoresis of macro-restricted genomic DNA, were (restriction enzyme): ZAS-1, 3.61 Mb (Pme I); ZAS-2, 3.82 Mb (Xba I); and ZAS-9, 3.80 Mb (Pme I). For comparison, the genome size of the free-living spirochete, Spirochaeta aurantia, was determined to be 3.72 Mb (Ase I). Estimates of rrn copy number, based on Southern hybridizations of restricted genomic DNA with a 16s rDNA probe, were two for each ZAS strain, as well as for S. aurantia. Results suggest that Treponema ZAS-1 and ZAS-2 be regarded as two strains of a single species distinct from that represented by ZAS-9, and several Latinate epithets are under consideration for application to these important termite gut symbionts. A separate effort was initiated to isolate and identify O2-consuming microbes present in hindguts of Reticulitermes flavipes. Maintenance of an anoxic lumen is important to the fermentative production of acetate - a major carbon and energy source for the insect. Microbes residing in the hypoxic peripheral region, on and within ca. 100 um from the hindgut wall, are critical to the maintenance of anoxia by consuming inwardly diffusing O2. However, little is known about these microbes. We used acetate-containing media and incubation conditions that included hypoxia. Based on CFUs, 3x10e4 bacteria per hindgut were capable of growth on plates incubated in 95% air/5% CO2. Prevalent among these were acetate-oxidizing strains closely related to known species of Enterobacteriaceae and Burkholderia. A "Plate Wash PCR" technique developed in our laboratory facilitated the recognition and isolation of novel Verrucomicrobia and Acidobacteria, whose colonies might otherwise have been overlooked owing to their tiny size. The Verrucomicrobia were not acetate oxidizers and presumably grew on other nutrients included in the isolation media. A semi-quantitative PCR method suggested that the Verrucomicrobia were authochthonous and occurred at a cell density of about 4x10e3 cells per gut. By contrast, ca. 3x10e3 CFU per gut consisted of novel acetate-oxidizing, microaerophilic Proteobacteria that only grew on plates incubated under 2% O2 and whose closest relative was Eikenella corrodens (94% 16S rRNA sequence similarity). Cells resembling the Verrucomicrobia and microaerophilic Proteobacteria were observed among the hindgut wall-associated microbiota by transmission electron microscopy. The Acidobacteria appeared to be allochthonous transients presumably acquired from soil. Given the small size of hindguts (ca. 1 ul total volume) and the narrow zone in which O2 depletion occurs, we believe that many of the isolates obtained are important to O2 consumption in situ and merit continued study.

Impacts
This project contributes to a better understanding of how gut microbes assist animals in digestion and nutrition. Additionally, this research may suggest targets for controlling the destructive activity of termites, or enhancing their ability to decompose (and hence assist turnover of) earth's most abundant form of biomass, i.e. lignocellulosic plant material.

Publications

  • Breznak, J.A. 2002. Phylogenetic diversity and physiology of termite gut spirochetes. Integr. Comp. Biol. 42:313-318.
  • Breznak, J.A. and Leadbetter, J.R. 2002. Termite gut spirochetes. In M. Dworkin, S. Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackelbrandt (eds.), The prokaryotes, an evolving electronic resource for the microbiological community. On line. Springer Verlag, New York.


Progress 01/01/01 to 12/31/01

Outputs
Spirochetes are a major component of the termite hindgut microbiota, accounting for one-half of all prokaryotes in some termite species. Recently, the first pure cultures of such forms were isolated in our laboratory and found to members of the genus Treponema and to possess two metabolic activities previously unknown in the Spirochaetes phylum of the Bacteria: (i) acetogenesis from H2 + CO2; and (ii) nitrogen fixation. These processes appear to contribute significantly to termite carbon and energy requirements and to termite nitrogen nutrition, respectively. Based on enzyme activities present in cell extracts, H2/CO2 acetogenesis appears to proceed via the Wood-Ljungdahl (AcetylCoA) pathway, which includes reduction of one molecule of CO2 to the methyl group of acetate via tetrahydrofolate (THF) as the C1 carrier. However, the spirochetes are unable to synthesize folate compounds and require folate (or THF, or related compounds) in media for growth. We hypothesized that their growth in situ was supported by other hindgut microbes that secrete folate compounds, and we screened for folate-secretors by using a bioassy. Major cultivable folate-secretors were most closely related Serratia grimesii and Lactococcus lactis subsp. lactis on the basis of 16S rRNA sequence analyses. Cell-free culture supernatant fluids from both isolates replaced folate and permitted growth of the spirochetes in vitro. Identification of the specific folate compound(s) present in such fluids is currently underway. Separate studies of oxygen tolerance revealed that the termite gut treponemes cannot tolerate the presence of more than 0.5 percent O2 in the gas phase. Ongoing research seeks to determine the genome size and rrn copy number of termite gut treponemes, as well as of certain free-living spirochetes, and to establish genomic libraries of such strains. The latter will be used to clarify the nature and organization of N2 fixation and acetogenesis genes which, in turn, will facilitate a study of the regulation of their expression.

Impacts
Termites are one of earth's most abundant and important terrestrial insects; in some locations they are also one of the our most destructive pests. A clearer understanding of their symbiotic gut microbiota (on which they depend for nutrition and vitality): enhances our appreciation of their positive roles in our biosphere; may suggest ways to manage them for increasing N2 fixation and, ultimately, soil fertility; and could suggest rational, environmentally-safe methods for controlling them and their destructive activities.

Publications

  • Lilburn, T. G., K. S. Kim, N. E. Ostrom, K. R. Byzek, J. R. Leadbetter and J. A. Breznak. 2001. Nitrogen fixation by symbiotic and free-living spirochetes. Science 292:2495-2498.
  • Breznak, J. 2001. The genus Sporomusa. In M. Dworkin, S. Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackelbrandt (ed.), The prokaryotes, an evolving electronic resource for the microbiological community,.On line. Springer Verlag, New York.


Progress 01/01/00 to 12/31/00

Outputs
Spirochetes are a major component of the termite gut microbiota, accounting for one-half of all the prokaryotes in some termites. However, their role in the gut and their relevance to termite nutrition have long been obscure, owing to the lack of pure cultures of these forms for in vitro study. Research during 2000 was entirely focussed on a continuing characterization of the first-ever isolates of these symbionts (Treponema strains ZAS-1, -2 and -9) originally obtained in our laboratory. Biochemical studies on homoacetogenic strains ZAS-1 and ZAS-2 revealed that cell-free extracts possessed virtually all the enzymes of the Wood-Ljungdahl (acetylCoA) pathway of acetogenesis from CO2, including hydrogenase (methyl transferase was not asayed). Cell extracts also possessed NADH peroxidase (but not catalase, SOD, or SOR) as the major protective enzyme against peroxides, which are likely to be formed in the gut from O2 inwardly diffusing into the peripheral region near the epithelium. Nutritional studies revealed that ZAS-1 and -2 each requires a folate-compound for growth and each can also ferment mono- and disaccharides as energy sources (ZAS-2 also grew slowly by demethylation of methoxylated aromatics). ZAS-9 ferments mono- and disaccharides, but is not a homoacetogen and cannot grow with H2+CO2. All three strains contained two homologues of nifH and were capable of N2 fixation. Conservative estimates suggest that spirochetes can account for a significant amount (in some cases, all) of the N2 fixation activity of the live insects. Homologues of nifH were also found in human oral (T. denticola and T. pectinovorum), bovine ruminal (T. bryantii), and free-living spirochetes (Spirochaeta aurantia, S. zuelzerae, S. stenostrepta). However, of the latter only the free-living forms fixed N2 in vitro. Fixation of 15N2 was confirmed with ZAS-9 and S. aurantia. This is the first demonstration of N2 fixation in the "Spirochaetes" phylum of Bacteria. Results reveal a new dimension to the physiological versatility of spirochetes and a heretofore unrecognized role for them in global N cycling and termite nutrition. These findings also help reconcile the old observation that elimination of spirochetes from termite guts results in decreased survival of termites.

Impacts
Termites are one of earth's most abundant and important terrestrial insects; in some locations they are also one of the our most destructive pests. A clearer understanding of their symbiotic gut microbiota (on which they depend for nutrition and vitality): enhances our appreciation of their positive roles in our biosphere; may suggest ways to manage them for increasing N2 fixation and, ultimately, soil fertility; and could suggest rational, environmentally-safe methods for controlling them and their destructive activities.

Publications

  • Breznak, J.A. 2000. Ecology of prokaryotic microbes in guts of wood- and litter-feeding termites. In Abe, T., Bignell, D.E., Higashi, M. (ed.), Termites: evolution, sociality, symbiosis, ecology. Kluwer Academic Pub., Dordrecht, NL, 209-231pp.
  • Brauman, A., Dore, J., Eggleton, P., Bignell, D., Breznak, J.A., Kane, M.D. 2001. Molecular phylogenetic profiling of prokaryotic communities in guts of termites with different feeding habits. FEMS Microbiol. Ecol. In press.


Progress 01/01/99 to 12/31/99

Outputs
This project seeks to clarify the role of gut microbes in termite nutrition and vitality. We have recently isolated the first pure cultures of termite gut spirochetes, which constitute a dominant component of the termite hindgut microbiota. Some of the strains (Treponema sp. strains ZAS-1 and ZAS-2) were capable of H2-CO2 acetogenesis. Another strain (ZAS-9) formed acetate and H2 as major products of sugar fermentation, suggesting that H2-based spirochete-spirochete trophic interactions are an important aspect of the biology of termite gut spirochetes in situ. The production of acetate by all strains implies a major role for spirochetes in the C and energy nutrition of termites. Ongoing studies revealed that termite gut spirochetes require folic acid (or a folate derivative) for growth, suggesting that growth and acetate production in situ may depend on folate-compound secretion by other members of the gut microbiota. We recently discovered N2 fixation in termite gut spirochetes as NH4Cl-repressible acetylene-reduction activity. Likewise, NifH-encoding sequences were found in genomic DNAs of strains ZAS-1, 2 and 9, as well as in the human oral treponemes (T. denticola and T. maltophilum) and in the free-living spirochetes, Spirochaeta stenostrepta, S. zuelzerae, and S. aurantia. The NifH-encoding sequences from these spirochetes were similar to as-yet-unaffiliated sequences obtained by PCR from termite gut contents, from zooplankton samples, and from rice root-associated microbes, suggesting a possible spirochete origin for these genes. In fact, the NifH-encoding sequence from S. aurantia was identical to one cloned by other investigators from guts of the Formosan termite, Coptotermes formosanus. This is the first demonstration of diazotrophy in spirochetes, and it implies a significant role for spirochetes in termite N nutrition, as well as a heretofore unappreciated role for spirochetes in global N cycling. Research planned for the year 2000 includes: (i) a search for folate compound secretors among the termite gut microbiota as a prelude to studies of their interaction with termite gut spirochetes in vitro; (ii) an estimate of the contribution of termite gut spirochetes to H2-CO2 acetogenesis in situ by using a combined rRNA probe-14CO2 autoradiography approach; and (iii) further studies to clarify the nature and nutritional control of N2 fixation in spirochetes.

Impacts
This research reveals: a new dimension to the physiological versatility of spirochetes; the heretofore unrecognized importance of spirochetes in termite nutrition; and a new role for spirochetes in global carbon and nitrogen cycling.

Publications

  • Lilburn, T.G., Schmidt, T.M. and Breznak, J.A. 1999. Phylogenetic diversity of termite gut spirochaetes. Environ. Microbiol. 1:331-345.
  • Leadbetter, J.R., Schmidt, T.M., Graber, J.R. and Breznak, J.A. 1999. Acetogenesis from H2 plus CO2 by spirochetes from termite guts. Science 283:686-689.


Progress 01/01/98 to 12/31/98

Outputs
This project seeks to clarify the role of gut microbes in termite nutrition and vitality. In wood-feeding termites, H2 produced by hindgut microbes is used primarily for CO2 reduction to acetate, thereby providing up to 1/3 of the insect's respiratory substrate. By contrast methanogenesis, the energetically-favored process of CO2 reduction in most anoxic (dark) habitats, is a minor sink for H2. The basis for this unexpected, but nutritionally-advantageous outcome, appears to be the in situ location of the H2-oxidizing microbes. In Reticulitermes flavipes, methanogens reside primarily on the hindgut wall. Hence they are furthest downstream in the outwardly diffusing H2 gradient and closest to the inwardly diffusing O2 gradient, which undoubtedly compromises their full methanogenic potential. By contrast spirochetes, recently isolated by us in pure culture and shown to be CO2-reducing acetogens (paper in press), reside in the luminal region of termite hindguts where H2 concentrations are as high as 50,000 ppmv (ca. 10 to 100-fold greater than typical H2 thresholds for acetogenesis). Hence, they are not at the mercy of an otherwise inferior (with respect to methanogens) H2 threshold. In termites whose hindgut protozoa harbor methanogens, CO2-reductive methanogenesis becomes more competitive with acetogenesis as an H2 sink. Ours has been the first isolation of termite gut spirochetes in pure culture and the first demonstration of H2-CO2 acetogenesis in the spirochete phylum of bacteria. Our analysis of the 16S rRNA-based phylogeny of recently-cultured and also not-yet-cultured termite gut spirochetes imply that the earth's termites constitute an enormous reservoir of novel spirochetal diversity -- all affiliated with the treponemes, but none closely related to any known species of Treponema. As many as 21 different species of Treponema are present in guts of single termite species. Most of them fall into a distinct subgroup of Treponema - referred to by us as the 'termite cluster' - whose 16S rRNA has the following signature nucleotides (E. coli numbering): 812A; and an inserted nucleotide between 1273 and 1274 (submitted for publication). Continuing research includes: further studies on the physiology and biochemistry of the recently-isolated termite gut spirochetes; an evaluation of the extent to which O2-dependent, acetate-oxidizing gut bacteria compete with termites for the same substrate (acetate); and a survey gut microbes capable of secreting quorum-sensing compounds.

Impacts
(N/A)

Publications

  • Leadbetter, J.R., Crosby, L.D. and Breznak, J.A. 1998. Methanobrevibacter filiformis sp. nov., a filamentous methanogen from termite hindguts. Arch. Microbiol. 169:287-292.