Cultivation and characterization of symbiotic bacteria from the gut of Reticulitermes chinensis

Authors

  • Hao Fang
  • Wen Chen
  • Baojun Wang
  • Xiaojuan Li
  • Shuang-Jiang Liu
  • Hong Yang

DOI:

https://doi.org/10.18063/AEB.2016.01.004

Keywords:

nutrient dependent isolation, intestinal symbionts, wood-feeding termite, physiological function

Abstract

The complex symbiotic relationship between wood-feeding termites and microorganisms inhabiting their intestinal tracts is a fascinating phenomenon in nature. To understand the physiological functions of symbiotic micro-organisms, bacteria were isolated from the gut homogenate of Reticulitermes chinensis with different media and culture conditions. Under aerobic conditions, 105 bacterial strains were isolated with 1/5 LB medium, 1/3 TSB medium and a modified basal mineral medium, MM-4. Most dominant isolates were bacteria in the genera Bacillus (27.6%) and Lactococcus (21.9%). Under anaerobic conditions, 60 bacterial strains were isolated with 1/5 LB medium, 1/3 TSB medium and a modified Peptone-Yeast medium. The predominant isolates were bacteria in the genus Enterobacter (41.7%) and Citrobacter (33.3%). Many of these bacterial isolates shared high sequence similarity (>98%) in 16S rRNA genes to bacterial clones obtained from the same termite and the other wood-feeding termites or cockroaches. Several bacterial species such as Deinococcus and Gryllotalpicola were isolated from termite gut for the first time. Characterization of these isolates showed that (i) most of Enterobacteriaceae and Lactococcus strains were able to hydrolyze uric acid; and (ii) many of the Bacillus and Streptomyces strains presented endo-β-1,4-glucanase activity. The preliminary results of this work gave us hints of possible functions of symbiotic bacteria in nitrogen recycling and cellulose degradation in the gut of wood-feeding termites

References

Brune A, 2014, Symbiotic digestion of lignocellulose in termite guts. Nature Reviews Microbiology, vol.12(3): 168–180. http://dx.doi.org/10.1038/nrmicro3182.

Yamin M A, 1981, Cellulose metabolism by the flagellate Trichonympha from a termite is independent of endosymbiotic bacteria. Science, vol.211(2): 58–59. http://dx.doi.org/10.1126/science.211.4477.58.

Inoue T, Moriya S, Ohkuma M, et al. 2005, Molecular cloning and characterization of a cellulase gene from a symbiotic protist of the lower termite Coptotermes formosanus. Gene, vol.349(2): 67–75. http://dx.doi.org/10.1016/j.gene.2004.11.048.

Brune A, 2006, Symbiotic associations between termites and prokaryotes, in The Prokaryotes, 3rd edn. Springer, New York, vol.1: 439–474. http://dx.doi.org/10.1007/0-387-30741-9_17.

Ohkuma M and Kudo T, 1996, Phylogenetic diversity of the intestinal bacterial community in the termite Reticulitermes speratus. Applied and Environmental Microbiology, vol.62(2): 461–8.

Hongoh Y, Ohkuma M and Kudo T, 2003, Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera: Rhinotermitidae), FEMS Microbiology Ecology, vol.44(2): 231–242. http://dx.doi.org/10.1016/S0168-6496(03)00026-6.

Yang H, Schmitt-Wagner D, Stingl U, et al. 2005, Niche heterogeneity determines bacterial community structure in the termite gut (Reticulitermes santonensis). Environmental Microbiology, vol.7(7): 916–32. http://dx.doi.org/10.1111/j.1462-2920.2005.00760.x.

Potrikus C J and Breznak J A, 1977, Nitrogen-fixing Enterobacter agglomerans isolated from guts of wood- eating termites. Applied and Environmental Microbiology, vol.33(2): 392–399.

Schultz J E and Breznak J A, 1978, Heterotrophic bateria present in hindguts of wood-eating termites [Reti-culitermes flavipes (Kollar)]. Applied and Environmental Microbiology, vol.35(5): 930–936.

Tholen A, Schink B and Brune A, 1997, The gut microflora of Reticulitermes flavipes, its relation to oxygen, and evidence for oxygen-dependent acetogenesis by the most abundant Enterococcus sp. FEMS Microbiology Ecology, vol.24(2): 137–149. http://dx.doi.org/10.1111/j.1574-6941.1997.tb00430.x.

Leadbetter J R and Breznak J A, 1996, Physiological ecology of Methanobrevibacter cuticularis sp. nov. and Methanobrevibacter curvatus sp. nov., isolated from the hindgut of the termite Reticulitermes flavipes. Applied and Environmental Microbiology, vol.62(10): 3620–3631.

Leadbetter J R, Crosby L D and Breznak J A, 1998, Methanobrevibacter filiformis sp. nov., a filamentous methanogen from termite hindguts. Archives of Microbiology, vol.169(4): 287–292.

Leadbetter J R, Schmidt T M, Graber J R, et al. 1999, Acetogenesis from H2 plus CO2 by spirochetes from termite guts. Science, vol.283: 686–689.

Lilburn T G, Kim K S, Ostrom N E, et al. 2001, Nitrogen fixation by symbiotic and free-living spirochetes. Science, vol.292(5526): 2495–2498. http://dx.doi.org/10.1126/science.1060281.

Wenzel M, Schoenig I, Berchtold M, et al. 2002, Aerobic and facultatively anaerobic cellulolytic bacteria from the gut of the termite Zootermopsis angusticollis. Journal of Applied Microbiology, vol.92(1): 32–40. http://dx.doi.org/10.1046/j.1365-2672.2002.01502.x.

Olsen R A and Bakken L R, 1987, Viability of soil bacteria: optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microbiology Ecology, vol.13(1): 59–74. http://dx.doi.org/10.1007/BF02014963.

Leadbetter J R, 2003, Cultivation of recalcitrant micro-laboratory. Current Opinion in Microbiology, vol.6: 274–281.

http://dx.doi.org/10.1016/S1369-5274(03)00041-9.

Huang Z, Chen X, Shi Y, et al. 2011, Molecular analysis of some Chinese termites based on mitochondrial cytochrome oxidase (CoII) gene. Sociobiology, vol.58(1): 107–118.

Chen W, Wang B, Hong H, et al. 2012, Deinococcus reticulitermitis sp. nov., isolated from a termite gut. International Journal of Systematic and Evolutionary Microbiology, vol.62(1): 78–83. http://dx.doi.org/10.1099/ijs.0.026567-0.

Tschech A and Pfennig N, 1984, Growth yield increase linked to caffeate reduction in Acetobacterium woodii. Archives of Microbiology, vol.137(2): 163–167.

http://dx.doi.org/10.1007/BF00414460.

Widdel F and Pfennig N, 1981, Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. I. Isolation of a new sulfate-reducer enriched with acetate from saline environments. Description of Desulfobacter postgatei gen. nov. sp.nov. Archives of Microbiology, vol.129(5): 395–400.

Edwards U, Rogall T, Blöcker H, et al. 1989, Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Research, vol.17(19): 7843– 7853.

Weisburg W G, Barns S, Pelletier D A, et al. 1991, 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, vol.173(2): 697–703.

Ashelford K E, Chuzhanova N A, Fry J C, et al. 2005, At least 1 in 20 16S rRNA sequence records currently held in public repositories is estimated to contain substantial anomalies. Applied and Environmental Microbiology, vol.71(12): 7724–7736. http://dx.doi.org/10.1128/AEM.71.12.7724-7736.2005.

Potrikus C J and Breznak J A, 1980, Uric acid-degrading bacteria in guts of termites [Reticulitermes flavipes (Kollar)]. Applied and Environmental Microbiology, vol.40(1): 117–124.

Hendricks C W, Doyle J D and Hugley B, 1995, A new solid medium for enumerating cellulose-utilizing bacteria in soil. Applied and Environmental Microbiology, vol.61(5): 2016–2019.

Cole J R, Wang Q, Fish J A, et al. 2014, Ribosomal database project: data and tools for high throughput rRNA analysis. Nucleic Acids Research, vol.41(Database issue): D633–D642. http://dx.doi.org/10.1093/nar/gkt1244.

Tamura K, Peterson D, Peterson N, et al. 2011, MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, vol.28: 2731–2739. http://dx.doi.org/10.1093/molbev/msr121.

Thong-On A, Suzuki K, Noda S, et al. 2012, Isolation and characterization of anaerobic bacteria for symbiotic recycling of uric acid nitrogen in the gut of various termites, Microbes and Environments, vol.27(2): 186–192. http://dx.doi.org/10.1264/jsme2.ME11325.

Cho M J, Kim Y, Shin K, et al. 2010, Symbiotic adaptation of bacteria in the gut of Reticulitermes speratus: low endo-beta-1,4-glucanase activity. Biochemical and Biophysical Research Communications, vol.395: 432– 435. http://dx.doi.org/10.1016/j.bbrc.2010.04.048.

Chou J H, Chen W M, Arun A B, et al. 2007, Trabulsiella odontotermitis sp. nov., isolated from the gut of the termite Odontotermes formosanus Shiraki. International Journal of Systematic and Evolutionary Microbiology, vol.57(4): 696–700. http://dx.doi.org/10.1099/ijs.0.64632-0.

Pace N R, Stahl D A and Lane D J, 1986, The analysis of natural microbial populations by ribosomal RNA sequences. Advances in Microbial Ecology, vol.9: 1–55. http://dx.doi.org/10.1007/978-1-4757-0611-61.

Fang H, Lv W, Huang Z, et al. 2015. Gryllotalpicola reticulitermitis sp. nov. isolated from a termite gut. International Journal of Systematic and Evolutionary Microbiology, vol.65(1): 85–89. http://dx.doi.org/10.1099/ijs.0.062984-0.

Pasti M B and Belli M L, 1985, Cellulolytic activity of actinomycetes isolated from termite (Termitidae) gut. FEMS Microbiology Letter, vol.26(1): 107–112. http://dx.doi.org/10.1111/j.1574-6968.1985.tb01574.x.

Roes-Hill M L, Rohland J and Burton S, 2011, Actinobacteria isolated from termite guts as a source of novel oxidative enzymes. Antonie Leeuwenhoek, vol.100(4): 589–605. http://dx.doi.org/10.1007/s10482-011-9614-x.

Stevenson B S, Eichorst S A, Wertz J T, et al. 2004, New strategies for cultivation and detection of previously uncultured microbes. Applied and Environmental Microbiology, vol.70(8): 4748–4755.

http://dx.doi.org/10.1128/AEM.70.8.4748-4755.2004.

Brune A and Ohkuma M, 2011, Role of the termite gut microbiota in symbiotic digestion, in Biology of Termites: A Modern Synthesis. Springer, Dordrecht: 439–475.

Ohkuma M and Brune A, 2011, Diversity, structure, and evolution of the termite gut microbial community, in Biology of Termites: A Modern Synthesis. Springer, Dordrecht: 413–438.

Ohkuma M, Noda S and Kudo T, 1999, Phylogenetic diversity of nitrogen fixation genes in the symbiotic microbial community in the gut of diverse termites. Applied and Environmental Microbiology, vol.65(11): 4926–4934.

Du X, Li X, Wang Y, et al. 2012, Phylogenetic diversity of nitrogen fixation genes in the intestinal tract of Reticulitermes chinensis Snyder. Current Microbiology, vol.65(5): 547–551.

Eutick M L, O’Brien R W and Slaytor M, 1978, Bacteria from the gut of Australian termites. Applied and Environmental Microbiology, vol.35(5): 823–828.

Pasti M B, Pometto A L, Nuti M P, et al. 1990, Lignin-solubilizing ability of actinomycetes isolated from termite (Termitidae) gut. Applied and Environmental Microbiology, vol.56(7): 2213–2218.

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2016-04-01

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RESEARCH ARTICLES