This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Herlemann, D. P. R.
Right arrow Articles by Brune, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Herlemann, D. P. R.
Right arrow Articles by Brune, A.
Agricola
Right arrow Articles by Herlemann, D. P. R.
Right arrow Articles by Brune, A.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, October 2007, p. 6682-6685, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.00712-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Termite Group I Phylum Is Highly Diverse and Widespread in the Environment{triangledown}

Daniel P. R. Herlemann, Oliver Geissinger, and Andreas Brune*

Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany

Received 29 March 2007/ Accepted 9 August 2007


arrow
ABSTRACT
 
The bacterial candidate phylum Termite Group I (TG-1) presently consists mostly of "Endomicrobia," which are endosymbionts of flagellate protists occurring exclusively in the hindguts of termites and wood-feeding cockroaches. Here, we show that public databases contain many, mostly undocumented 16S rRNA gene sequences from other habitats that are affiliated with the TG-1 phylum but are only distantly related to "Endomicrobia." Phylogenetic analysis of the expanded data set revealed several diverse and deeply branching lineages comprising clones from many different habitats. In addition, we designed specific primers to explore the diversity and environmental distribution of bacteria in the TG-1 phylum.


arrow
INTRODUCTION
 
Termite Group I (TG-1) represents a deep branch in the tree of bacterial 16S rRNA gene sequences (18) and has been recognized as a candidate phylum (10). TG-1 comprises a large number of the bacteria in the hindguts of Reticulitermes species (6, 25), where they occur as intracellular symbionts of flagellate protists (22). These symbionts, for which the name "Endomicrobia" has been proposed, form a monophyletic lineage occurring exclusively in the hindguts of termites and wood-feeding cockroaches (11, 22).

However, a few sequences only distantly related to the "Endomicrobia" but clearly affiliated with the TG-1 phylum have been reported to also occur in habitats other than termite guts (3, 19, 20, 24). Moreover, Nakajima et al. (17) obtained two sequences from the gut of Reticulitermes speratus that fall outside the "Endomicrobia" lineage. At present, public databases contain a growing number of sequences from various habitats that are phylogenetically unassigned but seem to be affiliated with the TG-1 phylum.

In this study, we screened public databases for hitherto unrecognized TG-1 sequences and conducted a comprehensive phylogenetic analysis of the expanded data set. In addition, we designed specific PCR primers to investigate the diversity and environmental distribution of major lineages of TG-1 bacteria in soils, sediments, and intestinal tracts.


arrow
Data mining.
 
Sequences affiliated with the TG-1 phylum were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/) using various characteristic oligonucleotide signatures deduced from the originally available 16S rRNA gene sequences, and they were added to the database of the ARB program suite (15). By continuously adapting the signatures to the growing data set (data not shown), we obtained approximately 50 previously unassigned phylotypes that fell into the radiation of the TG-1 phylum. Most of these phylotypes were from large-scale diversity studies of various environments, including soils, sediments, and intestinal tracts. Sequences were aligned with the ARB Fast Aligner tool. The alignment was manually corrected, and highly variable regions and ambiguous positions were excluded from the analysis. Rigorous chimera checking with Bellerophon (9) and fractional treeing (14) identified only one sequence as a putative chimera (accession no. DQ830579), which was removed from the data set. All shorter sequences (500 to 1,300 bp) were added to the core tree using the parsimony tool implemented in ARB.


arrow
Primer design and PCR.
 
Phylogenetic analysis of the nearly full-length sequences (>1,300 bp) fully supported the conclusion that the TG-1 phylum forms a separate line of descent in the bacterial tree, consisting of several diverse and deeply branching lineages (Fig. 1). Designing a single phylum-specific primer that excluded all representatives of other phyla proved to be impossible. A primer set for specific amplification of "Endomicrobia" (primer set 1) (Table 1) was designed in a previous study (22). Based on the expanded data set, we designed three additional primer sets (primer sets 2 to 4) that covered most of the other lineages in the TG-1 phylum. When applicable, information from shorter sequences was included to improve primer design.


Figure 1
View larger version (30K):
[in this window]
[in a new window]

  		FIG. 1. Phylogenetic tree of all sequences in the TG-1 phylum obtained in this and previous studies. A core tree was constructed with 1,262 unambiguously aligned sequence positions of all nearly full-length sequences retrieved from GenBank (in bold type), using maximum-likelihood analysis (fastDNAml). Short sequences (>500 bp), positionally filtered by base frequency (50%), were added without changing the global tree topology using the ARB parsimony tool. The scale bar is only approximate because the procedure distorts branch length. Representatives of 10 other phyla were used as the outgroup. Clones obtained in this study are indicated by filled squares. The topology of the core tree and individual clusters was tested separately by neighbor-joining and parsimony analysis (DNAPARS) with bootstrapping (seqboot; 1,000 bootstraps). Only nodes supported by high bootstrap values are marked (filled circles, >95%; open circles, >75%); nodes not supported by all analyses are shown as multifurcations. Original sequence definitions in the GenBank database were replaced with a consistent nomenclature including sequence length (in base pairs), habitat, geographic origin, and accession number. Roman numerals indicate the lineages referred to in the text and in Table 1.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Primer sets used for amplification of major lineages in the TG-1 phylum and annealing temperatures and MgCl2 concentrations used in the PCR assaya

The primers were used for PCR-based screening of various habitats for the presence of TG-1 bacteria. DNA was extracted by bead beating (16), and humic substances were removed by passing aqueous extracts over an Autoseq G-50 column (Amersham Bioscience). For PCR amplification a standard protocol optimized for the primer pairs was used (Table 1). The products of two identical reactions were combined, cleaned with a MinElute PCR purification kit (QIAGEN), and cloned with an pGEM-T Easy vector kit (Promega). Positive clones were amplified with M13 vector primers and checked for inserts on a 1% agarose gel. Clones with inserts that were the correct lengths were sorted by restriction fragment length polymorphism analysis as previously described (21). Inserts were sequenced on both strands.


arrow
Phylogenetic analysis.
 
Phylogenetic analysis of the resulting clone libraries documented that all primer pairs were highly specific and amplified only 16S rRNA genes of bacteria in the TG-1 phylum.

Primer set 1 gave a PCR product only with the termite hindgut samples (Table 2). This corroborates the specificity of this primer set for its target group, the "Endomicrobia" (lineage I in Fig. 1), which seem to be restricted to termites and wood-feeding cockroaches harboring gut flagellates (1, 22). "Endomicrobia" sequences from termite guts were not further investigated since they are the subject of a separate study (11).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Detection of TG-1 bacteria in various habitats using the new designed primer sets for specific amplification of major lineages in the phyluma

Primer set 2 matched the sequences of three deeply branching lineages (lineages IIa, IIb, and IIc) comprising clones from soil or sediment samples. An amplification product of the expected size was obtained only with Italian rice soil. The resulting clone library contained several phylotypes falling into lineage IIa and a single phylotype falling into lineage IIb (Fig. 1). Generally, the large number of sequences from soils and sediments retrieved from public databases suggests that bacteria in lineage II are widely distributed in these habitats.

Primer set 3 was designed to target the sequences in lineage III, which consists of sequences from intestinal habitats and also includes strain Pei191, the first isolate from the TG-1 phylum obtained from the gut of a beetle larva (O. Geissinger and A. Brune, unpublished data). Several new phylotypes were also obtained from the hindguts of Reticulitermes santonensis and Zootermopsis nevadensis (Table 2). All were distantly related to the clones previously retrieved from an R. speratus gut wall sample (17), underlining the finding that termite guts harbor a second lineage of TG-1 bacteria besides the "Endomicrobia." Cow rumen amplification products yielded a diverse but monophyletic group of sequences clustering with a single TG-1 sequence (accession no. AB034017) in a bacterial clone library from this habitat and erroneously assigned to the Proteobacteria (23).

Primer set 2 also yielded PCR products with the termite gut samples, but cloning analysis revealed that the products consisted exclusively of "Endomicrobia" sequences, which indicated that this primer set insufficiently discriminates against this group. Also, the termite clone libraries obtained with primer set 3 contained sequences belonging to the "Endomicrobia" (Table 2). Although both primer sets had two or more mismatches with all nontarget sequences within the TG-1 phylum, they apparently lack differentiating power for the corresponding subgroups if too many "Endomicrobia" are present in a sample, a problem encountered only with lower termites.

None of the DNA samples used in this study gave a PCR product with primer set 4, designed to detect sequences from lineage IV, which comprises clones from many different habitats (Fig. 1). Lahn River sediment yielded PCR products only with primer set T (Table 1), which was designed to detect most TG-1 sequences other than those of lineage I. This primer set yielded PCR products for all habitats tested but turned out to be nonspecific. Sequence analysis revealed that only about 10% of the clones in each clone library fell into the TG-1 phylum (lineage III in the Pachnoda ephippiata gut; lineages IIb and IIc in Lahn River sediment). Other clones were mostly representatives of the Bacteroidetes and the Acidobacteria, which was in agreement with the lack of discrimination of this primer set with a few representatives of these phyla (data not shown).


arrow
Abundance in the environment.
 
TG-1 bacteria are highly abundant only in bacterial clone libraries from the hindguts of lower termites (6, 7, 8, 18, 25). Fluorescence in situ hybridization corroborated the conclusion that "Endomicrobia" constitute a significant portion of the gut microbiota (11, 22). In contrast, bacterial clone libraries from other habitats generally contain only small numbers of sequences from members of the TG-1 phylum, if they contain any at all. In rumen fluid clone libraries (150 clones) and farm soil clone libraries (1,700 clones), only a single clone each fell into the TG-1 phylum (23, 24). Clone libraries of P. ephippiata larva gut homogenates (113 clones) (4) did not contain any clones affiliated with the TG-1 phylum, although a representative of the "intestinal cluster" was obtained from this species with primer set T (this study). Possible explanations for these phenomena may lie in either a low relative abundance of such bacteria in the respective communities or a mismatch in the "universal" Bacteria primers used in these studies.


arrow
Conclusion.
 
The results of this study document that bacteria affiliated with the TG-1 phylum form a separate line of descent, as proposed previously on the basis of a much smaller data set (10, 18). This phylum consists of numerous diverse and deeply branching lineages comprising bacteria from a wide range of chemically and geographically distinct habitats, including soils, sediments, and intestinal tracts. Although TG-1 bacteria seem to be numerically abundant only in the hindguts of lower termites (lineage I; "Endomicrobia"), the great diversity and wide environmental distribution of other lineages suggest a hitherto unrecognized role in the environment.


arrow
Nucleotide sequence accession numbers.
 
Sequences determined in this study have been deposited in the EMBL database under accession numbers AM491071 to AM491086, AM491098, AM491123, and AM491125 (http://www.ebi.ac.uk/).


arrow
ACKNOWLEDGMENTS
 
This study was funded by the Max Planck Society. D.P.R.H. received a fellowship from the International Max Planck Research School for Environmental, Cellular, and Molecular Microbiology.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany. Phone: 49-6421-178701. Fax: 49-6421-178709. E-mail: brune{at}staff.uni-marburg.de Back

{triangledown} Published ahead of print on 17 August 2007. Back


arrow
REFERENCES
 
    1
  1. Brune, A., and U. Stingl. 2005. Prokaryotic symbionts of termite gut flagellates: phylogenetic and metabolic implications of a tripartite symbiosis, p. 39-60. In J. Overmann (ed.), Molecular basis of symbiosis. Springer, Berlin, Germany.
    2. 2
  2. DeSantis, T. Z., P. Hugenholtz, N. Larsen, M. Rojas, E. L. Brodie, K. Keller, T. Huber, D. Dalevi, P. Hu, and G. L. Andersen. 2006. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 72:5069-5072.[Abstract/Free Full Text]
    3. 3
  3. Dojka, M. A., P. Hugenholtz, S. K. Haack, and N. R. Pace. 1998. Microbial diversity in a hydrocarbon- and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Appl. Environ. Microbiol. 64:3869-3877.[Abstract/Free Full Text]
    4. 4
  4. Egert, M., B. Wagner, T. Lemke, A. Brune, and M. W. Friedrich. 2003. Microbial community structure in midgut and hindgut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Appl. Environ. Microbiol. 69:6659-6668.[Abstract/Free Full Text]
    5. 5
  5. Frenzel, P., F. Rothfuss, and R. Conrad. 1992. Oxygen profiles and methane turnover in a flooded rice microcosm. Biol. Fertil. Soils 14:84-89.[CrossRef]
    6. 6
  6. Hongoh, Y., M. Ohkuma, and T. Kudo. 2003. Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera, Rhinotermitidae). FEMS Microbiol. Ecol. 44:231-242.[Medline]
    7. 7
  7. Hongoh, Y., H. Yuzawa, M. Ohkuma, and T. Kudo. 2003. Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment. FEMS Microbiol. Lett. 221:299-304.[CrossRef][Medline]
    8. 8
  8. Hongoh, Y., P. Deevong, T. Inoue, S. Moriya, S. Trakulnaleamsai, M. Ohkuma, C. Vongkaluang, N. Noparatnaraporn, and T. Kudo. 2005. Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host. Appl. Environ. Microbiol. 71:6590-6599.[Abstract/Free Full Text]
    9. 9
  9. Huber, T., G. Faulkner, and P. Hugenholtz. 2004. Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317-2319.[Abstract/Free Full Text]
    10. 10
  10. Hugenholtz, P., B. M. Goebel, and N. R. Pace. 1998. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180:4765-4774.[Free Full Text]
    11. 11
  11. Ikeda-Ohtsubo, W., M. Desai, U. Stingl, and A. Brune. Phylogenetic diversity of "Endomicrobia" and their specific affiliation with termite gut flagellates. Microbiology, in press.
    12. 12
  12. Lane, D. J. 1991. 16S/23S rRNA sequencing, p. 115-175. In E. Stackebrandt and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. John Wiley & Sons, New York, NY.
    13. 13
  13. Lemke, T., U. Stingl, M. Egert, M. W. Friedrich, and A. Brune. 2003. Physicochemical conditions and microbial activities in the highly alkaline gut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Appl. Environ. Microbiol. 69:6650-6658.[Abstract/Free Full Text]
    14. 14
  14. Ludwig, W., S. H. Bauer, M. Bauer, I. Held, G. Kirchhof, R. Schulze, I. Huber, S. Spring, A. Hartmann, and K. H. Schleifer. 1997. Detection and in situ identification of representatives of a widely distributed new bacterial phylum. FEMS Microbiol. Lett. 153:181-190.[CrossRef][Medline]
    15. 15
  15. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, et al. 2004. ARB: a software environment for sequence data. Nucleic Acids Res. 32:1363-1371.[Abstract/Free Full Text]
    16. 16
  16. Lueders, T., and M. W. Friedrich. 2003. Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts. Appl. Environ. Microbiol. 69:320-326.[Abstract/Free Full Text]
    17. 17
  17. Nakajima, H., Y. Hongoh, R. Usamib, T. Kudo, and M. Ohkuma. 2005. Spatial distribution of bacterial phylotypes in the gut of the termite Reticulitermes speratus and the bacterial community colonizing the gut epithelium. FEMS Microbiol. Ecol. 54:247-255.[CrossRef][Medline]
    18. 18
  18. Ohkuma, M., and T. Kudo. 1996. Phylogenetic diversity of the intestinal bacterial community in the termite Reticulitermes speratus. Appl. Environ. Microbiol. 62:461-468.[Abstract]
    19. 19
  19. Reardon, C. L., D. E. Cummings, L. M. Petzke, B. L. Kinsall, D. B. Watson, B. M. Peyton, and G. G. Geesey. 2004. Composition and diversity of microbial communities recovered from surrogate minerals incubated in an acidic uranium-contaminated aquifer. Appl. Environ. Microbiol. 70:6037-6046.[Abstract/Free Full Text]
    20. 20
  20. Spear, J. R., J. J. Walker, T. M. McCollom, and N. R. Pace. 2005. Hydrogen and bioenergetics in the Yellowstone geothermal ecosystem. Proc. Natl. Acad. Sci. USA 102:2555-2560.[Abstract/Free Full Text]
    21. 21
  21. Stingl, U., and A. Brune. 2003. Phylogenetic diversity and whole-cell hybridization of oxymonad flagellates from the hindgut of the wood-feeding lower termite Reticulitermes flavipes. Protist 154:147-155.[Medline]
    22. 22
  22. Stingl, U., R. Radek, H. Yang, and A. Brune. 2005. "Endomicrobia": cytoplasmic symbionts of termite gut protozoa form a separate phylum of prokaryotes. Appl. Environ. Microbiol. 71:1473-1479.[Abstract/Free Full Text]
    23. 23
  23. Tajima, K., S. Arai, K. Ogata, T. Nagamine, H. Matsui, M. Nakamura, R. I. Aminov, and Y. Benno. 2000. Rumen bacterial community transition during adaptation to high-grain diet. Anaerobe 6:273-284.[CrossRef]
    24. 24
  24. Tringe, S. G., C. von Mering, A. Kobayashi, A. A. Salamov, K. Chen, H. W. Chang, M. Podar, J. M. Short, E. J. Mathur, J. C. Detter, P. Bork, P. Hugenholtz, and E. M. Rubin. 2005. Comparative metagenomics of microbial communities. Science 308:554-557.[Abstract/Free Full Text]
    25. 25
  25. Yang, H., D. Schmitt-Wagner, U. Stingl, and A. Brune. 2005. Niche heterogeneity determines bacterial community structure in the termite gut (Reticulitermes santonensis). Environ. Microbiol. 7:916-932.[CrossRef][Medline]

Applied and Environmental Microbiology, October 2007, p. 6682-6685, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.00712-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Geissinger, O., Herlemann, D. P. R., Morschel, E., Maier, U. G., Brune, A. (2009). The Ultramicrobacterium "Elusimicrobium minutum" gen. nov., sp. nov., the First Cultivated Representative of the Termite Group 1 Phylum. Appl. Environ. Microbiol. 75: 2831-2840 [Abstract] [Full Text]  
  • Herlemann, D. P. R., Geissinger, O., Ikeda-Ohtsubo, W., Kunin, V., Sun, H., Lapidus, A., Hugenholtz, P., Brune, A. (2009). Genomic Analysis of "Elusimicrobium minutum," the First Cultivated Representative of the Phylum "Elusimicrobia" (Formerly Termite Group 1). Appl. Environ. Microbiol. 75: 2841-2849 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Herlemann, D. P. R.
Right arrow Articles by Brune, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Herlemann, D. P. R.
Right arrow Articles by Brune, A.
Agricola
Right arrow Articles by Herlemann, D. P. R.
Right arrow Articles by Brune, A.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»