Isolation of a new phospholipid, phosphatidyl-N-(2-hydroxyethyl)-alanine, from rumen protozoa.
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ABSTRACT:
SUBMITTER: Kemp P
PROVIDER: S-EPMC1184699 | biostudies-other | 1969 Jul
REPOSITORIES: biostudies-other
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Project description:1. A method is presented for identifying and estimating the aminoethylphosphonate (ciliatine)-containing phospholipids in a complex mixture. 2. Evidence was obtained that the phospholipids of a pure culture of Entodinium caudatum and a mixed rumen protozoa sample contain diglyceride ciliatine, and a plasmalogen ciliatine was detected in the latter. 3. A ninhydrin-positive sphingolipid was isolated from rumen protozoa. Although chromatographically homogeneous on silica gel it contains two components, which were provisionally identified as ceramide ciliatine and ceramide phosphorylethanolamine. 4. A detailed phospholipid analysis of E. caudatum and rumen protozoa is presented. They contain no phosphatidylserine or cardiolipin, but an unidentified phosphoglyceride containing a zwitterionic amino acid is present.
| S-EPMC1198385 | biostudies-other
Project description:The Megavirales are a newly described order capable of infecting different types of eukaryotic hosts. For the most part, the natural host is unknown. Several methods have been used to detect these viruses, with large discrepancies between molecular methods and co-cultures. To isolate giant viruses, we propose the use of different species of amoeba as a cellular support. The aim of this work was to isolate new Brazilian giant viruses by comparing the protozoa Acanthamoeba castellanii, A. polyphaga, A. griffini, and Vermamoeba vermiformis (VV) as a platform for cellular isolation using environmental samples. One hundred samples were collected from 3 different areas in September 2014 in the Pampulha lagoon of Belo Horizonte city, Minas Gerais, Brazil. PCR was used to identify the isolated viruses, along with hemacolor staining, labelling fluorescence and electron microscopy. A total of 69 viruses were isolated. The highest ratio of isolation was found in A. polyphaga (46.38%) and the lowest in VV (0%). Mimiviruses were the most frequently isolated. One Marseillevirus and one Pandoravirus were also isolated. With Brazilian environmental samples, we demonstrated the high rate of lineage A mimiviruses. This work demonstrates how these viruses survive and circulate in nature as well the differences between protozoa as a platform for cellular isolation.
| S-EPMC4594340 | biostudies-literature
Project description:This study investigated the associations between abundance of rumen ciliate protozoa and the proportion of the main bioactive fatty acids related to rumen biohydrogenation, as 18:0, t10-18:1, t11-18:1, c9,t11-18:2, 18:3n-3 and 18:2 n-6, in rumen and meat of growing lambs, using data derived from 3 production experiments. A global correlation analysis and a linear regression analysis considering the effect of the experiment were performed. Ten of the 86 lambs involved in the experiments did not present ciliate cells in rumen liquor and the remaining lambs presented an average of 1.35 × 106ciliates / ml rumen liquor. From the nine genera of ciliates identified, Entodinium was the most abundant, averaging 1.17 × 106 cells / ml of rumen liquor. A large variation among lambs was observed for both rumen concentration and community structure of ciliates. Rumen t11-18:1 (P < 0.001) and meat deposition of t11-18:1 (P < 0.001) and of c9,t11-18:2 (P < 0.001) increased linearly with total ciliates, whereas the t10/t11 ratio in rumen (P = 0.002) and in meat (P = 0.036) decreased linearly. Entodiniomorphids seems to be strongly related with meat deposition oft11-18:1 and c9,t11-18:2 and with the reduction of the trans-10 shifted pathway. Completeness of RBH decreased linearly with Holotrichs (P = 0.029), Entodiniomorphids (P = 0.029), Isotricha (P = 0.011) and Epidinium (P = 0.027) abundances. Rumen 18:0 also decreased linearly with increasing counts of total ciliates (P = 0.015), Holotrichs (P = 0.020), Entodiniomorphids (P = 0.010) and Isotricha (P = 0.014). Rumen protozoa were positively linked with the deposition of healthy bioactive FA and simultaneously negatively associated with the occurrence of trans-10 shift.
| S-EPMC6730912 | biostudies-literature
Project description:Buffalo, Jersey cow Raw sequence reads
| PRJNA376856 | ENA
Project description:Ciliate protozoa are an integral part of the rumen microbiome and were found to exert a large effect on the rumen ecosystem itself as well as their host animal physiology. Part of these effects have been attributed to their ability to harbor a diverse ecto- and endo-symbiotic community of prokaryotic cells. Studies on the relationship between the protozoa population and their associated prokaryotic community in the rumen mainly focused on the methanogens, revealing that protozoa play a major role in enhancing methanogenesis potential. In contrast, little is known about the composition and function of the bacteria associated with rumen protozoa and the extent of this association. In this study, we characterize the prokaryotic communities associated with different protozoa populations and compare their structure to the free-living prokaryotic population residing in the cow rumen. We show that the overall protozoa associated prokaryotic community structure differs significantly compared to the free-living community in terms of richness and composition. The methanogens proportion was significantly higher in all protozoa populations compared to the free-living fraction, while the Lachnospiraceae was the most prevalent bacterial family in the protozoa associated bacterial communities. Several taxa not detected or detected in extremely low abundance in the free-living community were enriched in the protozoa associated bacterial community. These include members of the Endomicrobia class, previously identified as protozoa symbionts in the termite gut. Our results show that rumen protozoa harbor prokaryotic communities that are compositionally different from their surroundings, which may be the result of specific tropism between the prokaryotic community and protozoa.
| S-EPMC6217230 | biostudies-literature
Project description:Dr. Burk Dehority was an international expert on the classification and monoculture of ruminal ciliated protozoa. We have summarized many of the advancements in knowledge from his work but also in his scientific way of thinking about interactions of ruminal ciliates with the entire rumen microbial community and animal host. As a dedication to his legacy, an electronic library of high-resolution images and video footage catalogs numerous species and techniques involved in taxonomy, isolation, culture, and ecological assessment of ruminal ciliate species and communities. Considerable promise remains to adapt these landmark approaches to harness eukaryotic cell signaling technology with genomics and transcriptomics to assess cellular mechanisms regulating growth and responsiveness to ruminal environmental conditions. These technologies can be adapted to study how protozoa interact (both antagonism and mutualism) within the entire ruminal microbiota. Thus, advancements and limitations in approaches used are highlighted such that future research questions can be posed to study rumen protozoal contribution to ruminant nutrition and productivity.
| S-EPMC7058926 | biostudies-literature
Project description:The rumen protozoa, alongside fungi, comprise the eukaryotic portion of the rumen microbiome. Rumen protozoa may account for up to 50% of biomass, yet their role in this ecosystem remains unclear. Early experiments inferred a role in carbohydrate and protein metabolism, but due to their close association with bacteria, definitively attributing these functions to the protozoa was challenging. The advent of 'omic technologies has created opportunities to broaden our understanding of the rumen protozoa. This study aimed to utilize these methods to further our understanding of the role that protozoa play in the rumen in terms of their metabolic capacities, and in doing so, contribute valuable sequence data to reduce the chance of mis or under-representation of the rumen protozoa in meta'omic datasets. Rumen protozoa were isolated and purified using glucose-based sedimentation and differential centrifugation, extracted RNA was Poly(A) fraction enriched and DNase treated before use in a phage-based, cDNA metatranscriptomic library. Biochemical activity testing of the phage library showed 6 putatively positive plaques in response to carboxymethyl cellulose agar (indicative of cellulose activity), and no positive results for tributyrin (indicative of esterase/lipase activity) or egg yolk agar (indicative of proteolysis). Direct sequencing of the cDNA was also conducted using the Illumina HiSeq 2500. The metatranscriptome identified a wealth of carbohydrate-active enzymes which accounted for 8% of total reads. The most highly expressed carbohydrate-active enzymes were glycosyl hydrolases 5 and 11, polysaccharide lyases and deacetylases, xylanases and enzymes active against pectin, mannan and chitin; the latter likely used to digest rumen fungi which contain a chitin-rich cell membrane. Codon usage analysis of expressed genes also showed evidence of horizontal gene transfer, suggesting that many of these enzymes were acquired from the rumen bacteria in an evolutionary response to the carbohydrate-rich environment of the rumen. This study provides evidence of the significant contribution that the protozoa make to carbohydrate breakdown in the rumen, potentially using horizontally acquired genes, and highlights their predatory capacity.
| S-EPMC7200989 | biostudies-literature
Project description:The aim of this study was to determine if rumen protozoa could form large amounts of reserve carbohydrate compared to the amounts formed by bacteria when competing for glucose in batch cultures. We separated large protozoa and small bacteria from rumen fluid by filtration and centrifugation, recombined equal protein masses of each group into one mixture, and subsequently harvested (reseparated) these groups at intervals after glucose dosing. This method allowed us to monitor reserve carbohydrate accumulation of protozoa and bacteria individually. When mixtures were dosed with a moderate concentration of glucose (4.62 or 5 mM) (n = 2 each), protozoa accumulated large amounts of reserve carbohydrate; 58.7% (standard error of the mean [SEM], 2.2%) glucose carbon was recovered from protozoal reserve carbohydrate at time of peak reserve carbohydrate concentrations. Only 1.7% (SEM, 2.2%) was recovered in bacterial reserve carbohydrate, which was less than that for protozoa (P < 0.001). When provided a high concentration of glucose (20 mM) (n = 4 each), 24.1% (SEM, 2.2%) of glucose carbon was recovered from protozoal reserve carbohydrate, which was still higher (P = 0.001) than the 5.0% (SEM, 2.2%) glucose carbon recovered from bacterial reserve carbohydrate. Our novel competition experiments directly demonstrate that mixed protozoa can sequester sugar away from bacteria by accumulating reserve carbohydrate, giving protozoa a competitive advantage and stabilizing fermentation in the rumen. Similar experiments could be used to investigate the importance of starch sequestration.
| S-EPMC4325151 | biostudies-literature
Project description:Defaunation studies have shown that rumen protozoa are one of the main causes of low nitrogen utilization efficiency due to their bacterivory and subsequent intraruminal cycling of microbial protein in ruminants. In genomic and transcriptomic studies, we found that rumen protozoa expressed lysozymes and peptidases at high levels. We hypothesized that specific inhibition of lysozyme and peptidases could reduce the activity and growth of rumen protozoa, which can decrease their predation of microbes and proteolysis and subsequent ammoniagenesis by rumen microbiota. To test the above hypothesis, we evaluated three specific inhibitors: imidazole (IMI), a lysozyme inhibitor; phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor; and iodoacetamide (IOD), a cysteine protease inhibitor; both individually and in combinations, with sodium dodecyl sulfate (SDS) as a positive control. Rumen fluid was collected from two Jersey dairy cows fed either a concentrate-based dairy ration or only alfalfa hay. Each protozoa-enriched rumen fluid was incubated for 24 h with or without the aforementioned inhibitors and fed a mixture of ground wheat grain, alfalfa, and grass hays to support microbial growth. Live protozoa cells were morphologically identified and counted simultaneously at 3, 6, 12, and 24 h of incubation. Fermentation characteristics and prokaryotic composition were determined and compared at the end of the incubation. Except for IOD, all the inhibitors reduced all the nine protozoal genera identified, but to different extents, in a time-dependent manner. IOD was the least inhibitory to protozoa, but it lowered ammoniagenesis the most while not decreasing feed digestibility or concentration of volatile fatty acids (VFA). ANCOM analysis identified loss of Fibrobacter and overgrowth of Treponema, Streptococcus, and Succinivibrio in several inhibitor treatments. Functional prediction (from 16S rRNA gene amplicon sequences) using the CowPI database showed that the inhibitors decreased the relative abundance of the genes encoding amino acid metabolism, especially peptidases, and lysosome in the rumen microbiota. Overall, inhibition of protozoa resulted in alteration of prokaryotic microbiota and in vitro fermentation, and peptidases, especially cysteine-peptidase, may be targeted to improve nitrogen utilization in ruminants.
| S-EPMC6908469 | biostudies-literature