Recent developments in nucleic acid based techniques for use in rumen manipulation

Abstract

-Nucleic acid-based techniques which can be used to characterise complex microbial communities without incubation are now being employed regularly in ruminant nutrition studies. Conventional culture-based methods for enumerating rumen microorganisms (bacteria, archaea, protozoa, and fungi) have been superseded and are now used mainly to obtain pure isolates of novel organisms and reference strains that are required for the development and validation of the nucleic acid approaches. These reference strains are also essential for physiological studies of the lifestyle of the organisms as well as sources of genomic DNA and RNA that can be analysed for functional gene activity. The foundation of the molecular ecology techniques is 16S/18S rDNA sequence analysis which has provided a phylogenetically based classification scheme for enumeration and identification of microbial community members. The use of this marker gene in assays involving the use of single nucleic acid probes or primer sets is rapidly evolving to high throughput approaches such as microarray analysis and new generation sequencing technologies. While these analyses are very informative for determining the composition of the microbial community and monitoring changes in population size, they can only infer function based on these observations. The focus of nucleic acid research is now shifting to the functional analysis of the ecosystem which involves the measurement of functional genes and their expression in the predominant or specific members of the rumen microbial community. Functional gene studies are less developed than 16S rDNA-based analysis of community structure. Also for gene expression studies there are inherent problems involved in extracting high quality RNA from digesta, and priming cDNA synthesis from bacterial mRNA. This paper reviews nucleic acid based molecular methods which have recently been developed for studying the structure and function of rumen microbial communities.Key Words: ecology, functional analysis, microbial, molecular, ribosomal, rumen Recent developments in nucleic acid based techniques for use in rumen manipulationRESUMO -Metodologias baseadas na análise de ácidos nucléicos, que possam ser utilizadas para caracterizar comunidades microbianas complexas, sem a necessidade da incubação de amostras, têm sido empregadas atualmente, de forma rotineira, em estudos de nutrição de ruminantes. Os métodos convencionais, dependentes do cultivo microrganismos ruminais (bactérias, archaea, protozoarios e fungos) para sua enumeração foram superados e atualmente passaram a ser utilizados especialmente para o isolamento de cepas puras de organismos ainda desconhecidos e para o cultivo de cepas de referência, necessários para o desenvolvimento e a validação das metodologias de análise de ácidos nucléicos. Tais cepas de referência são também essenciais para estudos fisiológicos e ainda, funcionam como fontes de DNA e RNA para avaliação da expressão gênica. O fundamento das metodologias de ecologia molecular é a análise de seq...

Integrated metagenomic analysis of the rumen microbiome of cattle reveals key biological mechanisms associated with methane traits

“…Examples include the description of well characterized rumen bacteria based on the isolation of the functionally significant bacterial groups [9], [10]. While successfully identifying more than 200 microbial species including bacteria and protozoa from the rumen [1], [8], culture-dependent techniques requiring a careful design of protocol for growth of organism exhibit several significant limitations [11]. They are not only time consuming and cumbersome [8] but more importantly, culture-based studies are usually unable to reveal the full extent of microbial diversity due to the nature of protocol design and constraints due to culture conditions [11], [12].…”

Acetobase: database update and reanalysis of formyltetrahydrofolate synthetase amplicon sequencing data from the anaerobic digesters

“…As it is well known and mentioned earlier, FTHFS, although a marker for acetogens, is also present is some syntrophic acid degrading bacteria and sulphate reducing bacteria (Lovell, 1994;Singh et al, 2019. In several previous studies on FTHFS gene amplicon-based community profiling, for instance studies by Leaphart and Lovell, 2001;Leaphart et al, 2003;Lovell and Leaphart, 2005;McSweeney et al, 2009;Singh et al, 2021bSingh et al, , 2021a etc. syntrophic acid degrading bacteria and sulphate reducing bacteria have been reported together with acetogenic bacteria.…”

AcetoBase Version 2: a database update and re-analysis of formyltetrahydrofolate synthetase amplicon sequencing data from anaerobic digesters

“…Although FTHFS is commonly used as a marker for acetogens, as mentioned earlier it is present in some syntrophic acid-degrading and sulphate-reducing bacteria ( 1 , 3 , 4 ). In several previous studies on FTHFS gene amplicon-based community profiling ( 2 , 7 , 8 , 11 , 23 , 24 ), syntrophic acid-degrading bacteria and sulphate-reducing bacteria have been reported together with acetogenic bacteria. These groups of bacteria use the FTHFS gene in e.g., biosynthesis of folate or short-chain fatty-acid oxidation but not in reductive acetogenesis, unlike the acetogens.…”