Hostname: page-component-5c6d5d7d68-wbk2r Total loading time: 0 Render date: 2024-08-23T17:36:45.304Z Has data issue: false hasContentIssue false
  • English
  • Français

DGGE and 16S rDNA analysis reveals a highly diverse and rapidly colonising bacterial community on different substrates in the rumen of goats

Published online by Cambridge University Press:  01 March 2008

Y.-Z. Sun ,
S.-Y. Mao ,
W. Yao  and
W.-Y. Zhu
Y.-Z. Sun
Affiliation:
Laboratory of Gastrointestinal Microbiology, College of Animal Science and Technology, Nanjing Agricultural University, 210095 Nanjing, PRChina
S.-Y. Mao
Affiliation:
Laboratory of Gastrointestinal Microbiology, College of Animal Science and Technology, Nanjing Agricultural University, 210095 Nanjing, PRChina
W. Yao
Affiliation:
Laboratory of Gastrointestinal Microbiology, College of Animal Science and Technology, Nanjing Agricultural University, 210095 Nanjing, PRChina
W.-Y. Zhu*
Affiliation:
Laboratory of Gastrointestinal Microbiology, College of Animal Science and Technology, Nanjing Agricultural University, 210095 Nanjing, PRChina
*
E-mail: zhuweiyunnjau@hotmail.com
Get access

Abstract

In the rumen, plant particles are colonised and degraded by the rumen micro-organisms. Although numerous important findings about fibre-associated bacterial community were obtained using traditional or molecular techniques, little information is available on the dynamics of bacteria associated with feed particles during incubation in the rumen. In the present study, ryegrass leaf, ryegrass stem and rice straw, representing different carbohydrate compositions, were used as substrates and placed in the rumen of goats by using nylon bags, and PCR/DGGE (denaturing gradient gel electrophoresis) with subsequent sequence analysis were used to monitor the dynamics of and identify bacteria associated with the substrates during 24 h of incubation. DGGE results showed that substrate samples collected from 10 min to 6 h had similar DGGE patterns, with up to 24 predominant bands to each sample, including 14 common bands to all samples, suggesting a rapid and stable colonisation by a highly diverse bacterial community. Substrate samples collected at 12 and 24 h showed similar DGGE patterns but had great difference in DGGE patterns from those collected at 10 min to 6 h, suggesting an apparent shift in bacterial community. Sequence analysis indicated that most substrate-associated bacteria were closely related to fibrolytic bacteria. In conclusion, a highly diverse and similar rumen bacterial community could immediately colonise to different substrates and remained stable during the initial 6 h of incubation, but experienced a marked change after 12 h of incubation. Italian ryegrass leaf, Italian ryegrass stem and rice straw were colonised with a similar bacterial community.

Keywords

16S rDNA analysisDGGEgoatssubstrate-associated bacterial community
Type
Full Paper
Information
animal , Volume 2 , Issue 3 , March 2008 , pp. 391 - 398
Copyright
Copyright © The Animal Consortium 2008

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Akin, DE 1980. Evaluation by electron microscopy and anaerobic culture of types of rumen bacteria associated with digestion of forage cell walls. Applied and Environmental Microbiology 39, 242–252.CrossRefGoogle ScholarPubMed
Braune, A, Gütschow, M, Engst, W, Blaut, M 2001. Degradation of quercetin and luteolin by Eubacterium ramulus. Applied and Environmental Microbiology 67, 5558–5567.CrossRefGoogle ScholarPubMed
Cheng, KJ, McAllister, TA 1997. Compartmentation in the rumen. In The rumen microbial ecosystem, 2nd edition (ed. PN Hobson and CS Stewart), pp. 492–522. Blackie Academic and Professional, London.CrossRefGoogle Scholar
Cheng, KJ, Fay, JP, Coleman, RN, Milligan, LP, Costerton, JW 1981. Formation of bacterial microcolonies on feed particles in the rumen. Applied and Environmental Microbiology 41, 298–305.CrossRefGoogle ScholarPubMed
Cheng, KJ, Stewart, CS, Dinsdale, D, Costerton, JW 1984. Electron microscopy of bacteria involved in the digestion of plant cell walls. Animal Feed Science and Technology 10, 93–120.CrossRefGoogle Scholar
Craig, WM, Broderick, GA, Ricker, DB 1987. Quantitation of microorganisms associated with the particulate phase of ruminal ingesta. Journal of Nutrition 117, 56–62.CrossRefGoogle ScholarPubMed
Dehority, BA, Orpin, CG 1997. Development of, and natural fluctuations in, rumen microbial populations. In The rumen microbial ecosystem, 2nd edition (ed. PN Hobson and CS Stewart), pp. 196–245. Blackie Academic and Professional, London.CrossRefGoogle Scholar
Gomes, NCM, Fagbola, O, Costa, R, Rumjanek, NG, Buchner, A, Mendona-Hagler, L, Smalla, K 2003. Dynamics of fungal communities in bulk and maize rhizosphere soil in the tropics. Applied and Environmental Microbiology 69, 3758–3766.CrossRefGoogle ScholarPubMed
Ho, YW, Abdullah, N, Jalaludin, S 1996. Microbial colonisation and degradation of some fibrous crop residues in the rumen of goats. Asian-Australian Journal of Animal Science 9, 519–524.CrossRefGoogle Scholar
Jackson, CR, Roden, EE, Perry, F 1998. Changes in bacterial species composition in enrichment cultures with various dilutions of inoculum as monitored by denaturing gradient gel electrophoresis. Applied and Environmental Microbiology 64, 5046–5048.CrossRefGoogle ScholarPubMed
Kocherginskaya, SA, Aminov, RI, White, BA 2001. Analysis of the rumen bacterial diversity under two different diet conditions using denaturing gradient gel electrophoresis, random sequencing, and statistical ecology approaches. Anaerobe 7, 119–134.CrossRefGoogle Scholar
Koike, S, Kobayashi, Y 2001. Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. FEMS Microbiology Letters 204, 361–366.CrossRefGoogle ScholarPubMed
Koike, S, Pan, J, Kobayashi, Y, Tanaka, K 2003a. Kinetics of in sacco fiber-attachment of representative ruminal cellulolytic bacteria monitored by competitive PCR. Journal of Dairy Science 86, 1429–1435.CrossRefGoogle ScholarPubMed
Koike, S, Yoshitani, S, Kobayashi, Y, Yanaka, K 2003b. Phylogenetic analysis of fibre-associated rumen bacterial community and PCR detection of uncultured bacteria. FEMS Microbiology Letters 229, 23–30.CrossRefGoogle Scholar
Konstantinov, SR, Zhu, WY, Williams, BA, Tamminga, S, de Vos, WM, Akkermans, ADL 2003. Effect of fermentable carbohydrates on piglet faecal bacterial communities as revealed by denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA. FEMS Microbiology Ecology 43, 225–235.CrossRefGoogle ScholarPubMed
Krumholz, LR, Bryant, MP 1986. Eubacterium oxidoreducens sp. nov. requiring H2 or formate to degrade gallate, pyrogallol, phloroglucinol and quercitin. Archives of Microbiology 144, 8–14.CrossRefGoogle Scholar
Lane, DJ 1991. 16S/23S rRNA sequencing. In Nucleic acids techniques in bacterial systematics (ed. E Stackebrandt and M Goodfellow), pp. 115–175. John Wiley and Sons, Chichester, UK.Google Scholar
Maidak, BL, Cole, JR, JrParker, CT, Garrity, GM, Larsen, N, Li, B, Lilburn, TG, McCaughey, MJ, Olsen, GJ, Overbeek, R, Pramanik, S, Schmidt, TM, Tiedje, JM, Woese, CR 1999. A new version of the RDP (Ribosomal Database Project). Nucleic Acids Research 27, 171–173.CrossRefGoogle ScholarPubMed
Michener, CD, Sokal, RR 1957. A quantitative approach to a problem in classification. Evolution 11, 130–162.CrossRefGoogle Scholar
Minato, H, Endo, A, Ootomo, Y, Uemura, T 1966. Ecological treatise on the rumen fermentation. II. The amylolytic and cellulolytic activities of the fractionated bacterial portions attached to the rumen solids. Journal of General and Applied Microbiology 12, 53–69.CrossRefGoogle Scholar
Mosoni, P, Fonty, G, Gouet, P 1997. Competition between ruminal cellulolytic bacteria for adhesion to cellulose. Current microbiology 35, 44–47.CrossRefGoogle ScholarPubMed
Muyzer, G, de Waal, EC, Uitterlinden, AG 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology 59, 695–700.CrossRefGoogle Scholar
Nübel, U, Engelen, B, Felske, A, Snaidr, J, Wieshuber, A, Amann, RI, Ludwig, W, Backhaus, H 1996. Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymixa detected by temperature gradient gel electrophoresis. Journal of Bacteriology 178, 5636–5643.CrossRefGoogle Scholar
Odenyo, AA, Mackie, RI, Stahl, DA, White, BA 1994. The use of 16S rRNA- targeted oligonucleotide probes to study competition between ruminal fibrolytic bacteria: pure-culture studies with cellulose and alkaline peroxide-treated wheat straw. Applied and Environmental Microbiology 60, 3697–3703.CrossRefGoogle ScholarPubMed
Rölleke, S, Gurtner, C, Drewello, U, Drewello, R, Lubitz, W, Weissmann, R 1999. Analysis of bacterial communities on historical glass by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Journal of Microbiological Methods 36, 107–114.CrossRefGoogle Scholar
Sanguinetti, CJ, Dias Neto, E, Simpson, AJG 1994. Rapid silver staining and recovery of PCR products separated on polyacrylamide gels. Biotechniques 17, 914–921.Google ScholarPubMed
Sekiguchi, H, Tomioka, N, Nakahara, T, Uchiyama, H 2001. A single band does not always represent single bacterial strains in denaturing gradient gel electrophoresis analysis. Biotechnology Letters 23, 1205–1208.CrossRefGoogle Scholar
Shi, Y, Odt, CL, Weimer, PJ 1997. Competition for cellulose among three predominant ruminal cellulolytic bacteria under substrate-excess and substrate-limited conditions. Applied and Environmental Microbiology 63, 734–742.CrossRefGoogle ScholarPubMed
Sigler, WV, Turco, RF 2002. The impact of chlorothalonil application on soil bacterial and fungal populations as assessed by denaturing gradient gel electrophoresis. Applied Soil Ecology 21, 107–118.CrossRefGoogle Scholar
Sinha, RN, Ranganathan, B 1983. Cellulolytic bacteria in buffalo rumen. Journal of Applied Bacteriology 54, 1–6.CrossRefGoogle Scholar
Stackebrandt, E, Goebel, BM 1994. Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. International Journal of Systematic Bacteriology 44, 846–849.Google Scholar
Stewart, CS, Flint, HJ, Bryant, MP 1997. The rumen bacteria. In The rumen microbial ecosystem, 2nd edition (ed. PN Hobson and CS Stewart), pp. 10–72. Blackie Academic and Professional, London.CrossRefGoogle Scholar
Tajima, K, Aminov, RI, Nagamine, T, Ogate, K, Nakamura, M, Matsui, H, Benno, Y 1999. Rumen bacterial diversity as determined by sequence analysis of 16S rDNA libraries. FEMS Microbiology Ecology 29, 159–169.CrossRefGoogle Scholar
Tajima, K, Arai, S, Ogata, K, Nagamine, T, Matsui, H, Nakamura, M, Aminov, RI, Benno, Y 2000. Rumen bacterial community transition during adaptation to high-grain diet. Anaerobe 6, 273–284.CrossRefGoogle Scholar
Zhu, WY, Williams, BA, Konstantinov, SR, Tamminga, S, De Vos, WM, Akkermans, ADL 2003. Analysis of 16S rRNA reveals bacterial shift during in vitro fermentation of fermentable carbohydrate using piglet faeces as inoculum. Anaerobe 9, 175–180.CrossRefGoogle Scholar
Zoetendal, EG, Akkermans, ADL, De Vos, WM 1998. Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria. Applied and Environmental Microbiology 64, 3854–3859.CrossRefGoogle ScholarPubMed
Zoetendal, EG, Akkermans, ADL, De Vos, WM 2001. Molecular characterization of microbial communities based on 16S rRNA sequence diversity. In New approaches for the generation and analysis of microbial typing data (ed. L Dijkshoorn, KJ Towner and M Struelens), pp. 267–298. Elsevier Science, Amsterdam, The Netherlands.CrossRefGoogle Scholar