This process is experimental and the keywords may be updated as the learning algorithm improves. This is a preview of subscription content, log in to check access. Johanna Laybourn-Parry 1 1. Personalised recommendations. Cite chapter How to cite? ENW EndNote. Per burette, 0. A small portion of the pellet was diluted using ddH 2 O and fixed for 2 h at room temperature with an equal volume of MFS solution [3.
Fractions containing the largest size and concentration of cDNA were pooled then cleaned via ethanol precipitation. LB top agar 0. The packaged reactions were diluted and 2.
The plates were then rocked at room temperature for 1 hour h before the bacteriophage suspension was recovered and pooled. The first screen applied to the library was a functional assay for cellulolytic activity using carboxymethyl cellulose CMC as a substrate and Congo red for post-staining. A layer of 0. The Congo red dye was removed and 1 M NaCl solution added and rocked at rpm for 20 min. The solution was discarded and the process repeated.
Plaques expressing cellulolytic activity would appear yellow on the red background. The second screen applied to the library was a functional assay for lipolytic activity using Tributyrin as a substrate and Spirit Blue as an indicator dye. Plaques expressing lipolytic activity would show a clear zone around their circumference.
The third screen applied to the library was a functional assay for protease activity using egg yolk agar, as described by Fu et al. A positive result would be indicated by off-white precipitation around the plaque. Sequencing data were handled using the Galaxy platform Version The sequences were quality checked using FastQC Version 0. The sliding window size was set at 4 bps and average quality required was set at 20 given as a phred33 value this translates to 1 in probability of an incorrect base call i.
Trinity Version 0. Transdecoder was run using the following parameters: Minimum protein length bps, universal genetic code, retain long ORFs equal to or longer than bps aa and train with the number of top longest ORFs: EggNOG mapper Version 4.
FeatureCounts Version 1. In doing so, multiple results were produced for the same sequence and all sequences returned matches. The same process was used to investigate the few proteases and lipases that were identified. Figure 1. Diagrammatic representation of the workflow employed on the Galaxy platform to analyse the protozoal metatranscriptome.
Phylogenetic trees were constructed using neighbor-joining clustering method Saitou and Nei, The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site Tamura et al.
The analysis for xylanase genes involved 30 nucleotide sequences and there was a total of positions in the final dataset. The analysis for cellulase genes involved 32 nucleotide sequences and there was a total of 70 positions in the final dataset. All positions containing gaps and missing data were eliminated. As samples from three different animals were pooled together, animals may have possessed different types. The holotrichous representatives observed were identified as Isotricha species intestinalis and Dasytricha species ruminantium Supplementary Figures S2J,I Dehority, ; Williams and Coleman, The library produced a titer of 6.
Primary screening of the amplified cDNA library using substrates for cellulase, lipase and protease activities, yielded mixed results. The only putatively positive results were observed in response to CMC substrate, which consistently revealed six positive plaques in addition to the positive control post-staining indicating cellulase activity Figure 2.
No positive results were detected using differential media to screen for lipases and proteases. Viral plaques were observed but showed no zones of clearance or colorimetric change in any instances. Positive controls indicated that the assays were functional.
Figure 2. Amplified cDNA library plated on Escherichia coli lawn with carboxymethyl cellulose overlay and post-staining with Congo red Primary assay. A—E Cellulase positive plaques indicated by yellow coloring; F Positive control cellulase from Aspergillus niger indicated by yellow coloring.
Direct sequencing of the metatranscriptome of the protozoal cDNA resulted in approx. The metatranscriptome sequences were reconstructed using Trinity on Galaxy, which provided 9, contigs Table 1.
Use of BowTie2 resulted in , reads aligned to contigs, which when processed by FeatureCounts resulted in , reads aligning to predicted genes Table 1.
Figure 3. Expression reads per functional subcategory from the protozoal metatranscriptome using Bowtie2, FeatureCounts and EggNOG online mapper. Subcategories are collated into the appropriate main category. After selecting results according to criteria laid out in section 2. Other matches were made to various endo-1, 4-beta-xylanases, xylanases, cellulases and endoglucanases from rumen microorganisms three matches to rumen bacteria and 5 to rumen protozoa; sequence IDs: SCI Table 2.
Expression number of reads and percentage of total reads per type and family of carbohydrate active enzyme. Figure 4. Using the sequences as queries for a BLASTp alignment against dbCAN helped to give a more comprehensive overview of the glycosyl hydrolases found in this dataset. Only four enzymes were commonly identified by all three tools used by dbCAN, only two of which fell within the top most expressed genes in the dataset and were of some interest.
This protein is produced by Ruminococcus sp. Other carbohydrate-active enzymes within the top most expressed enzymes were from glycosyl hydrolase GH family 11 xylanases and 45 endo-glucanases. Finally, phylogenetic trees constructed from cellulases and xylanases identified from this dataset target sequences and sequences retrieved from GenBank GH5, cellulase, xylanase, GH10 and GH11 sequences from the rumen protozoa, bacteria and fungi to visualize their similarity.
From the phylogenetic tree constructed using cellulase sequences it can be observed that several 7 target sequences cluster together with some similarity shown to AF Piromyces sp. Cel9A Figure 5. One target sequence shows some similarity to other fungal enzymes produced by Anaeromyces sp.
AF and Neocallimastix sp. HQ Figure 5. The remaining 6 target sequences showed similarity to those produced by E. The phylogenetic tree constructed from xylanase sequences shows clustering of fungal genes GU, EU, and AF and of a fungal and bacterial gene M and MH Murphy et al.
Four target xylanase genes show some, limited similarity to xynD11 and xylanases produced by P. Five target xylanases showed close similarity to those produced by E. Figure 5. Comparative phylogenetic tree of all putative cellulase or GH5 genes detected within the protozoal metatranscriptome. Twelve representative protozoal sequences from Epidinium ecaudatum 6 , Epidinium caudatum 4 , Polyplastron multivesiculatum , 1 and an uncultured bovine rumen ciliate 1 , three fungal cellulases produced by Neocallimastix sp.
Target sequences from the metatranscriptome are shown in bold with a diamond shape node marker, the most highly expressed enzyme is marked with a square, protozoal sequences are given in black, bacterial in red and fungal in blue text. Figure 6. Comparative phylogenetic tree of all putative xylanase GH10 or GH11 genes detected within the protozoal metatranscriptome.
Fourteen representative protozoal sequences from Epidinium ecaudatum 4 , Polyplastron multivesiculatum 5 , uncultured bovine rumen ciliates 3 Epidinium caudatum 1 and Eudiplodinium maggii 1 , three fungal cellulases produced by Neocallimastix sp. Target sequences from the metatranscriptome are shown in bold with a diamond shape node marker, the most highly expressed enzyme is marked with a square, protozoal sequences are given.
With the view to developing the current understanding of the role and functions of the rumen protozoa, this study created a phage-based metatranscriptomic library from purified protozoal cDNA that was functionally screened for lipase, protease and cellulase activity. The sequencing data also revealed high expression levels of chitinases, which are likely utilized by rumen protozoa to digest rumen fungi which possess chitin-rich cell walls Morgavi et al.
This study suggests that the primary roles of the rumen ciliates are predation and carbohydrate metabolism. The majority of studies concerning the carbohydrate-active enzymes of the rumen protozoa span the period between and , pre-dating the genomic era Guttmacher and Collins, ; Konstantinidis et al.
As such, very little sequence data are available and most research uses faunated vs. The only other published metagenomic library to be constructed exclusively from rumen protozoa was that by Findley et al. Major differences between the studies include the kits and protocols used to construct the metagenomic library and the use of in vivo excision, which was not possible in the current study.
Excising the phagemid vector using in vivo excision allows for transformation into eukaryotic cells which would allow functional screening followed by PCR and Sanger sequencing. The use of phage in functional assays is more complex when compared to other vector systems and is not widely used, as any one plaque may contain up to 1 million individual phages, of which only one or two may be expressing the desired activity.
On the other hand, using a bacterial or eukaryotic vector allows for more straightforward functional screening in terms of both working conditions and identification of positive colonies. Other studies have utilized cDNA libraries to characterize single species of protozoa, which have produced mostly partial and one complete gene encoding carbohydrate-active enzymes Wereszka et al. More recently, NGS has allowed some partial mRNA to be deposited in Genbank for GH 5 and 9 as well as uncategorized cellulases and xylanases produced by ruminant protozoa, but no characterization or further work has been done e.
The study by Wang et al. The use of NGS provides many advantages over traditional culture-based methods, the primary benefit being the bypassing of establishing and maintaining a culture of protozoa. Rumen protozoa are notoriously difficult to maintain in culture and although decades of optimization have resulted in improved methods, it remains impossible to maintain them in axenic culture Newbold et al. The use of direct sequencing also negates issues with contamination; the rumen protozoa are well-known for engulfing bacterial and fungal cells which can significantly skew assays, rendering results inaccurate and unreliable Belanche et al.
After sequencing, there is also the option to align data to various genomes host, plant, fungal, bacterial etc. Utilizing these techniques, this study approached the rumen protozoa from both a functional and sequence-based perspective to give a well-rounded picture of their role in the rumen.
Illumina sequencing revealed a wealth of carbohydrate-active enzymes in addition to those that specifically target the rumen fungi and in some cases bacteria. Such a result highlights the lack of data available for the rumen protozoa but also adds value to that presented here, which could contribute significantly to our current understanding and future sequence-based work. Two of these enzymes: glycoside hydrolase family 11 and polysaccharide deacetylase are ranked in the top 20 most expressed genes in the whole metatranscriptome.
Polysaccharide deacetylases accounted for 1. Polysaccharide deacetylases fall into carbohydrate esterase family 4 and catalyze the hydrolysis of N- or O-linked acetyl groups from polysaccharide residues Arnaouteli et al. These enzymes are active against plant polysaccharides but are also used by bacteria to modify peptidoglycan in their cell walls to adjust to varying environmental conditions Kobayashi et al.
Some of the most abundant enzymes were the cellulases, or GH5s. When aligned to known protozoal sequences, several 7 of the sequences grouped away, showing no similarity, instead the phylogenetic tree indicated a relationship with cel9A from Piromyces sp.
AF and cellulases from R. The Amoeba moves by extending part of its cell. This extruding part is the pseudopod, and allows the Amoeba to drag itself from one place to another see Fig. Its movement is slow, and changing directions is just a matter of extending a pseudopod in a new direction.
Amoebas do not seem to have a particular shape, with the exception of the pseudopodia that consistently protrude from the cell. Paramecia are smaller than Amoebas. They move with the help of microscopic hair-like structures called cilia, which act like oars to push them through the water.
They swim by rotating slowly and changing directions often. If the Paramecium comes upon an obstacle, it stops, swims backwards, and then angles itself forward on a slightly different course. Cilia help the Paramecium move as well as feed.
When the Paramecia feed, it does so by drawing its food into a funnel-shaped opening called the oral groove that is lined with cilia see Fig. The oral groove is like a mouth, taking food in with the help of cilia, which direct and move the food inward. The Euglena moves rapidly, using its flagellum to propel itself through the water rather quickly, shifting directions with whip-like movements.
Unlike the Amoeba and the Paramecium , the Euglena has plant-like characteristics. The organelle that gives it this plant-like quality is the chloroplast see Fig. Since it can undergo photosynthesis, Euglena is able to make its own food just like plants. The three protists examined in this lab are examples of protists that use specialized structures for locomotion. These protists exemplify the animal-like and motile types of protozoans. As compared to other protists, the animal-like features of the protists we observed allow them to be motile.
Their motility comes in handy for moving about their environment and finding food. They may be contrasted to another class of protist, the sporozoans. Sporozoans have no form of locomotion and are primarily parasitic, ingesting their food by absorption through their cell membranes.
No matter what type of locomotion a protist uses, all protists must be able to carry out the metabolic functions of multicellular organisms. Based on the observations in this lab, protists are very small yet highly complex. They have all the organelles necessary for a variety of functions such as digestion, excretion, reproduction, respiration, and movement.
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