Molecular techniques in rumen biotechnology: A review

Transcription

1 Agricultural Reviews, 37 (1) 2016 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Molecular techniques in rumen biotechnology: A review Manju G. Preedaa* 1, Thulasiraman Parkunan 2, Dhinesh Kumar R. 2, Ramavathi J., Yazhini P., Aasif Ahmad Sheikh 2, Mohammad Rayees Dar 2, Lakshmi Priyadarshini 2, Gunjan Baghel 2 and Chandrasekar T. 2 Veterinary College and Research Institute, Namakkal , India. Received: Accepted: DOI: /ar.v37i ABSTRACT This review analyses the use of molecular techniques in rumen microbial identification. The use of regular methods like roll tube and most probable number resulted in under estimation of rumen microbial growth. So molecular biology acts as an advanced tool for rumen microbial culture and identification of various new species. These techniques give complete descriptions of individual ruminal populations. Use of molecular techniques like PCR, DDGE and FISH, which also pave pathway for genetic manipulation of rumen microbes in the field of rumen manipulation. So the combination of traditional and molecular assays gives accurate and satisfactory results. Key words : DNA, Microbes, Molecular methods, PCR, Rumen, RNA. INTRODUCTION Rumen is the richest habitat of numerous micro organism species, which comprises of prokaryotes and eukaryotes. Predominant organisms are bacteria about viable cells per gram that represent about 200 species, variety of ciliate protozoa per gram about 25 genera and anaerobic fungi of zoospore population per gram, 5 genera (Miron et al., 2001). Rumen microbial composition was analyzed by traditional methods such as roll-tube technique by Hungate or most probable- number (MPN) estimates, but some microbes cannot be cultivated with current techniques and cultured microorganisms represent only a small fraction of natural microbial diversity which grossly under estimated by 10%. The most desirable classification scheme reflects natural evolutionary relationships and evolutionary conservation of 16S/18S ribosomal RNA by modern molecular techniques provides molecular characterization as well as classification scheme without cultivation. New rdna sequences differ from previously known sequences lead to discovery of new phyla. The major advantage is due to lack of microorganism s culture and samples can be collected directly from rumen (Soliva et al., 2004). So the present review attempts to summarize the various techniques involved in the rumen biotechnological study systematically. Sample collection: Time of sample collection depends on time of feeding and place of rumen, because fibrolytic bacteria are seen predominantly in solid fraction, protozoa in liquid fraction and bacteria in liquid or mixed. Samples should be representative and free from bias and this is particularly important in case when objective of the study is quantified. Rumen fluid sample should be frozen or directly preceded for DNA isolation, because of high activities of DNase and RNase. There is also possibility to use frozen bacterial pellet collected after 30 min (approx.) of centrifuging with maximum speed (at least 1000 rpm) at 4 æ% C for DNA isolation. Freezing is an effective way of protecting DNA/RNA from degradation. Repeated thawing of the sample should be avoided prior to the DNA extraction (Tajima et al., 2001). Extraction of DNA can be conducted directly or indirectly. In a direct method the DNA is extracted from the sample collected directly from the rumen environment. This assay is fast, effective and the obtained genomic DNA is more representative. However, in the extracted DNA sample some inhibitors may be present. These can inhibit enzymes that acts during the amplification or cloning. Therefore, an additional step during direct DNA isolation is needed to remove these inhibitors. This step can be done with the help of commercially available DNA purification kits (Leng et al., 2011). The indirect method involves DNA isolation from pure microbial culture that has been isolated with the classic microbial methods from rumen sample. This method is timeconsuming, laborious and reduces the genetic diversity of environmental DNA. Lower genetic diversity within obtained samples is due to lack of an optimized culture system for culturing of many rumen microorganisms. DNA extraction is done by cell lysis. Due to huge diversity and number of microorganisms present in the rumen fluid, and existence of difficult-to-lyse microorganisms, *Corresponding author s preprand@gmail.com. 1 Veterinary College and Research Institute (VC&RI), Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Namakkal, Tamil Nadu ICAR-National Dairy Research Institute (NDRI), Karnal , India.

2 56 AGRICULTURAL REVIEWS different DNA extraction protocols were developed. However, all of them are based on enzyme-chemical or mechanic-chemical methods of cell lysis. In the enzymechemical based method, cell lysis is done with lysozyme or proteinase K. The most common lysis method is a mechanicchemical one, involving bead-beating in the presence of the detergent. However the most efficient method is the repeated bead beating plus column (RBB+C) in which proteinase K, detergent and bead-beating is used. Using this technique, the efficiency of DNA isolated can reach 5-6 times more DNA (in ng) per one ml than the amount of DNA obtained by the commercial kits. After the extraction step, the obtained DNA samples should be aliquoted and stored frozen in -20 C (Yu and Morrison, 2004). Molecular techniques, based on the DNA amplification, have been applied to describe the diversity of bacteria present in the rumen (Fig 1). Such techniques are able to provide unique environmental DNA profile, which represents the genetic diversity of microbial community from each sample (Tatsuoka et al., 2007). The structure of microbial communities in environmental rumen samples can be studied without cultivation and further to determine the community dynamics in response to environmental variations. Obtained profiles can be compared with the standard profile and can be sequenced, and then compared with known sequences placed in Genome Base. There are several techniques based on PCR assay which can be used to characterize diversity of rumen microbiota, such as RFLP (Restriction Fragment Length Polymorphism), DGGE (Denaturing Gradient Gel Electrophoresis), TGGE (Temperature Gradient Gel Electrophoresis) and SSCP (Single Strand Conformation Polymorphism) etc of PCR amplicons (up to 500bp) of the same size but different sequences. Different sequences are characterized by different denaturation (melting) profile. PCR DDGE: PCR-DGGE is the culture-independent fingerprinting technique which is based on the separation. Application: In relation to diet, the corn-fed animals have more diverse and rich bacterial populations than hay-fed animals. Mackie et al. (2003) designed DGGE procedures for the detection of Oscillospira spp. Effects of disodium fumarate, monensin on microbial communities and direct identification of microbe & relative abundance of different species was studied. 80% of bacterial population attached to the rumen wall can be defined as uncultured by this method. Protozoal communities in the rumen and duodenum are studied by using specific primers and dominated by Entodinium species. FIG 1: Flow Chart explaining the molecular techniques in rumen microbial identification (Adpated from Weidong et al., 2008).

3 Blot hybridization: Natural microbial populations and dietary effects on the bacterial population in the rumen were studied by this method. Group-specific 16S rrna hybridization probes for determining Butyrivibrio fibrisolvens in the rumen. Ziemer et al. (2000) reported sum of the three cellulolytic bacterial species represented 4.5% of the total bacterial rrna and did not vary with sampling time. Fish: FISH detects nucleic acid sequences by fluorescently labeled probes called fluorochrome which are complementary to the specific region of nucleic acid. Estimation of the methanogens is done by this method as they are very difficult to cultivate in vitro and also to determine microbial mixed population in the rumen. Anaerobic filamentous fugal populations which are difficult to identify are determined by this method by internal transcribed spacers (ITS-1) (Mackie et al. 2003). FISH does not require the pure culture of microorganisms; it can be used in the determination of microbial mixed population in the rumen. Three main steps in the protocol are: (i) cell fixation, (ii) whole-cell hybridization and (iii) analysis in the microscopy, but there are some disadvantages like it is long and laborious, not every oligonucleotide probes can access inside the cell and it is impossible to measure the quantity of analysed microorganisms (Stabnikova et al., 2006). Competitive PCR method: Quantification of the rumen microorganism can be done also with the competitive PCR method. In this technique a known amount of a DNA fragment (a competitor) is added to the sample. An ideal competitor should be (i) amplified by the same primers as the target DNA, (ii) distinguishable from the target DNA (e.g., different size, different restriction fragment pattern). During PCR reaction both templates (the target DNA and competitor) compete for the same set of primers. Due to this competition, the ratio of the amounts of the two amplified products (T: C ratio) reflects the ratio of the amounts of the target DNA (T) and competitor (C). When the T: C ratio = 1, the initial amount of the target DNA will correspond to the amount of competitor. In the PCR reaction, a series of diluted competitors is added to a known amount of sample to perform competitive PCR. After completion of the reaction, equal aliquots from each sample are analyzed by an agarose gel electrophoresis by visual assessment of band intensities or by digital analysis of the gel image Minimum detection levels for F. succinogenes, Ruminococcus albus and R. flavefaciens were 1 10 cells in pure culture and cells per ml in mixed culture and 1,000 times more sensitive than hybridization Quantitative real-time PCR: Quantitative PCR is a technique enabling the analysis of quantitative microorganism changes in the sample (Zhang and Fang, Volume 37 Issue 1 (2016) ). In contrast to conventional PCR, in which the amplicon detection occurs at the end of each cycle of reactions, in the quantitative PCR product detection occurs only during the phase of exponential growth. At that time, amplified DNA sequence theoretically should be doubled (MacKay, 2007) and detection of amplicons is possible by the use of fluorescent dyes. The cheapest and most widely used dye is SYBR Green. SYBR green binds to every doublestranded structure of DNA, also non-specifically amplified. More specific PCR amplification can be done with the 5 nuclease Taqman assay. Before starting the real-time PCR analysis it has to be remembered, that (i) the size of amplification products should be between bp; (ii) the concentration of used primers should be as low as possible, to reduce primer dimmer amplification; (iii) the concentration of Mg 2+ should be optimal to increase the amplification efficiency and specificity; (iv) the primers should be located in sequence with reduced probability of secondary structure interference; (v) PCR products specificity should be always checked by electrophoresis as well as by melting procedure; (vi) PCR efficiency assay should be performed In real-time PCR the results are interpreted based on the Ct value (cycle threshold). This value describes number of cycles, in which fluorescence is detected above the background during an exponential phase (Cikos and Koppel, 2009). Theoretically, the amount of starting target sample and amount of amplicons within exponential phase of amplification are in quantitative relationship. The change of the Ct value represents a twofold increase of the amount of starting template. Absolute standard curve and the comparative method are frequently used. The standard curve method allows for a direct quantification. The standard curve is constructed by plotting cycles at the Ct against the logarithmic values of known amounts of DNA templates. These curves, as well as amplification efficiency and amount of amplicons can be automatically constructed by software. The reliable standard curve is obtained when the sample and the diluted standard have the same amplification efficiency. Second technique is based on relative quantification of template in the sample. In this method comparison between the target gene and a reference gene ( house-keeping gene, plasmid sequence) is done without the standard curve. The final result expressed the change in the quantity of sample DNA template in comparison to quantity of the reference gene. Ribosomal genes, -actin or glyceraldehyde-3- phosphate dehydrogenase is the most commonly used reference genes Fibrolytic bacterial species, F. succinogenes and R. flavefaciens and the total anaerobic fungal population (Klieve et al., 2003). Accurate quantification of protozoan biomass in the rumen and its passage to the duodenum are performed by this method, as microscopic enumeration does not allow for

4 58 AGRICULTURAL REVIEWS measurement of protozoal passage to the duodenum and protozoal cells are lysed before reaching the duodenum. Gene markers for methanogens: Denman et al. (2005) proposed that methyl-coenzyme M reductase (mcr) gene has been analysed by phylogenetic analysis as Methyl coenzyme- M reductase is involved in crucial step of methane production Metagenomics: A DNA-based approach which has gained rapid adoption as a way of studying community function is called metagenomic analysis. Metagenomics is defined as the study of collected genomes from an ecosystem that can be used to examine the phylogenetic, physical and functional properties of microbial communities. The principle steps in this form of analysis is the extraction of DNA from the microbial community followed by cloning of the DNA fragments in a suitable host (eg E. coli) using a plasmid or bacterial artificial chromosome vector which results in a library of many thousands of clones. The clone library can then be screened using specific DNA sequences in a PCR or hybridization based approach for genes encoding discrete steps in known metabolic pathways or enzymatic activities (Ferrer et al., 2007). This approach has been employed to screen rumen-dna metagenomic libraries for enzymes involved in depolymerisation of lignocellulose and starch with the discovery of a range of novel enzymes. A bacterial artificial chromosome library has also been constructed from the rumen of a dairy cow and is being used to screen for novel enzyme activities. Genetically engineered bacterial species: One area for manipulation of rumen fermentation via use of genetically engineered micro-organisms would be to focus on changing the rumen digestion of specific dietary components. Numerous animal digestion studies have shown that rumen starch digestion is somewhat incomplete, particularly with high-grain-level-fed beef animals having high feed intakes. In this case, introduction of a genetically engineered bacterial species having high expression of amylases for starch degradation and possibly, high xylanase activities for branhemicellulose degradation, would seem feasible. In contrast, rumen digestion of dietary proteins often occurs at a greater rate than that for polysaccharides. This often results in rumen ammonia levels that are in excess of microbial and animal needs and loss of dietary N from the animal through urinary excretion. Based on current knowledge, protease production by rumen bacterial species appears to be constitutive. Genetically then, the problem of excessive proteolysis might be approached by linking the protease gene(s) of one or more species to other Nitrogen (N) metabolism genes whose expression is regulated by ammonia levels. Such a gene would be urease or glutamine synthetase which has been studied in rumen micro-organism species such as Selenomonas ruminantium and Succinivibrio dextrinosolvens. Overall plant fibre digestion in the rumen is commonly slow and incomplete with most animals, particularly when feed intake levels are high. Fibre digestion might be improved by amplifying the cellulolytic and related activities of bacterial species such as Ruminococcus albus, R. flavefaciens, Bacteroides succinogenes or Butyrivibrio vibrisolvens (Hespell et al., 1997). The regulation of fermentation products made by the rumen microbial population would be another target area for use of genetically engineered micro-organisms. For steers, this would be to increase propionate usually at the expense of acetate. This might be done by amplifying the genes for selected enzymes involved with propionate formation or increasing the rumen numbers of propionate-forming bacteria such as S. ruminantium, Bacteroides ruminicola, Megasphera elsdenii or Veionella species. Lactate acidosis could be reduced or brought under control by increasing the abilities of certain bacteria to utilize lactate. S. ruminantium has a rather poor affinity for lactate and genetic modifications of the appropriate binding proteins or transport mechanisms for lactate might be the only genetic engineering needed to turn this species into a super-lactate using organism. A third area for manipulation of rumen fermentation would be control of the numbers of specific microbial species in the rumen population. An approach to this problem would be to genetically engineer a bacterial species to produce and secrete a compound that would inhibit the growth or be lethal to another micro-organism. The control of the rumen protozoal population by inhibition compounds would seem attractive because of their eukaryotic cell nature and it would allow them to be susceptible to a number of compounds that would have little or no effect on the prokaryotic bacterial cells the rumen methanogenic micro-organisms could also be sensitive because of their archaebacterial cell nature and loss of these hydrogen-gas-utilizing methanogenic organisms would drastically disrupt the entire rumen fermentation system. The metabolism of other bacterial species would also have to be genetically engineered to provide a hydrogen sink. One possibility would be to engineer Eubacterium limosum, a relatively numerically minor species in the rumen, preferentially forms acetate and butyrate. Elimination of rumen methane production is attractive since it represents a loss of 6-10% of the digestible feed energy. Finally, by controlling the numbers of Streptococcus bovis and other rapidly growing, lactate producing bacterial species, the problem of lactate acidosis may also be eliminated or reduced. Genetic markers: In the area of genetics research, three aspects have to be noted. First, finding genes that are suitable for selecting and identifying strains that have acquired or lost genetic material. These marker strains might be developed by mutagenic techniques using U.V. light or chemical agents (nitrous acid, nitrosoguanidine) with rumen species to generate mutants for specific amino acids or sugars. A second aspect of genetic research has to include

5 Volume 37 Issue 1 (2016) 59 TABLE 1: Shows the various techniques in Rumen microbial identification with its application, advantage and disadvantages (Adapted from McSweeney et al., 2007) Method Application Advantages Disadvantages Roll-tube Isolation and enumeration - Not representative; slow and laborious; identification acquires additional 16S/18S rrna/rdna-based approaches; low sensitivity MPN Enumeration - Not representative; slow and laborious; low sensitivity Blot hybridization - Detection; quantify relative abundance FISH Denaturing Gradient Gel Electrophoresis (DGGE) Quantitative real time reverse transcriptase Polymeriase Chain Reaction (qrt-pcr) Enumeration of microrganisms in situ within environment Fingerprint pattern analysis of changes mixed microbial community composition Quantitative estimates of microbial gene expression in complex microbial populations Visualise culturable and unculturable microrganisms spatially in relation to substrate and other community members Culturable and unculturable organisms identified Rapid quantitative method for estimating expression levels of single or select number of important genes in a mixed environmental sample Requires sequence information; little probes; laborious at species Laborious Sometimes, only predominant organisms identified Global analysis of gene expression in complex ecosystem not possible Clone library Phylogenetic identification - Laborious; subject to PCR biases; expensive Stable isotope probing DNA expression array Identification of a metabolically active microbial group Semi-quantitative estimates of microbial gene expression in complex microbial populations Culturable and unculturable organisms that are involved in a particular metabolic process can be distinguished from the general microbial community Rapid method for monitoring gross changes in gene expression of large numbers of genes from multiple organisms in a complex ecosystem Not quantitative. Culture conditions may bias for growth of organisms that are not the main group responsible for the metabolism under normal conditions in the ecosystem Not quantitative Lower sensitivity and specificity than qpcr finding or making suitable vehicles to carry the genes of interest and to allow for gene replication or expression independent of chromosomally located genes, or both. It would be highly preferable for this vehicle to be a shuttlevector plasmid capable of replication in the rumen species and in a well-studied bacterium (e.g. Escherichia coli or Bacillus subtilis). The genes or DNA fragments of the rumen species cloned into such a vector can then be subjected to the wide range of genetic tools and analyses available with these well-studied organisms. Finally, the most important aspect is to develop a system for introducing DNA into the cells of the rumen bacterium under study. Probably the best system may be transformation as there are a number of available techniques used with other bacteria (Smith and Hespell, 1983). In the above review, the biotechnological intervention is highly recommended in the rumen study with respect to different feeds digestion and absorption. Each biotechnological intervention has its own ups and downs (Table 1). So, according to your experiment design you will choose the optimal techniques for the betterment of animal survival. REFERENCES Cikos, S. and Koppel, J. (2009). Transformation of real-time PCR fluorescence data to target gene quantity. Anal. Biochem. 384:1-10. Denman, S.E. and McSweeney, C. S. (2005). PCR-based methods for analysis of populations and gene expression quantitative Real time PCR. In Methods in Gut Microbial Ecology for Ruminants. (Ed. H. Makkar and C. S. McSweeney), Springer, The Netherlands. Ferrer, M., Beloqui, A. and Golyyshina, O.V. (2007). Biochemical and structural features of a novel cyclodextrinase from cow rumen metagenome. Biotech. J. 2:

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