Selenium uptake by Butyrivibrio fibrisolvens and Bacteroides ruminicola

Abstract The uptake and incorporation of ⁷⁵[Se]selenite by Butyrivibrio fibrisolvens and Bacteroides ruminicola were by constitutive systems. Rates of uptake were higher in chemostat culture than in batch culture and there may be some inducible component. Uptake of [⁷⁵Se]selenite was distinct from sulphate or selenate transport, since sulphate and selenate did not inhibit selenite uptake, nor could sulphate or selenate uptake be demonstrated in these organisms. Selenite uptake in B. fibrisolvens had and apparent K _m of 1.74 mM and a V _max of 109 ng Se · min⁻¹· (mg protein)⁻¹. An apparent K _m of 1.76 mM and V _max of 1.5 μg Se · min⁻¹· (mg protein)⁻¹ was obtained for B. ruminicola . [⁷⁵Se]Selenite uptake by both organisms was partially sensitive to inhibition by 2,4-DNP. Uptake by B. fibrisolvens was also partially inhibited by azide and arsenate and in B. ruminicola it was partially inhibited by fluoride. CCCP, CPZ, DCCD or quinine did not inhibit uptake in either B. fibrisolvens or B. ruminicola . Selenite transport by both organisms was sensitive to IAA and NEM and was strongly inhibited by sulphite and nitrite. [⁷⁵Se]Selenite was converted to selenocystine, selenohomocystine and selenomethionine by B. fibrisolvens. B. ruminicola did not incorporate [⁷⁵Se]selenite into organic compounds, but did reduce it to red elemental selenium.     

Increased expression of a molecular chaperone GroEL in response to unsaturated fatty acids by the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens

Butyrivibrio fibrisolvens is the most active bacterial species in the biohydrogenation of polyunsaturated fatty acids (PUFA) in the rumen. It needs to remove the unsaturated bonds in order to detoxify the PUFA to enable the growth of the bacterium. Here, we investigated the response of cell membrane-associated proteins in B. fibrisolvens to growth in the presence of PUFA. Numerous changes were observed in the cell membrane-associated proteome. One of the main modifications occurring when the 18:2 fatty acids, linoleic acid and conjugated linoleic acid, were added, was an increased expression of the molecular chaperone GroEL.     

Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: Comparison with other Butyrivibrio plasmids and application in the development of a cloning vector

Abstract A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79 bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM β1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens .     

Cloning and sequencing of an endoglucanase (end1) gene from Butyrivibrio fibrisolvens H17c

Summary The nucleotide sequence of a 2.8 kb DNA segment containing an endoglucanase gene (end1) from Butyrivibrio fibrisolvens H17c was determined. The B. fibrisolvens H17c gene was expressed from its own regulatory region in Escherichia coli and three putative consensus promoter sequences were identified upstream of a ribosome binding site and an ATG start codon. The complete amino acid sequence (547 residues) was deduced and homology with the Clostridium thermocellum ME gene product (EGE) was demonstrated. The endoglucanase contained a typical amino-terminal signal sequence and five repeated sequences (PDPTPVD) between amino acids 412–447. The endoglucanase showed relatively high endoglucanase activity against endoglucanase-specific substrates with 1-4 linkages but low activity against xylan and an exoglucanasespecific substrate, p-nitrophenyl--d-cellobioside.Abbreviations CMCase carboxymethylcellulase - DNS dinitrosalicylic acid - end1 gene coding for End1 - End1 endo-1,4--glucanase - nt nucleotide - ORF open reading frame     

Isolation of a novel strain of Butyrivibrio fibrisolvens that isomerizes linoleic acid to conjugated linoleic acid without hydrogenation, and its utilization as a probiotic for animals

AIM: Isolation of a new strain of Butyrivibrio fibrisolvens possessing great capacity to produce conjugated linoleic acid (CLA) in order to utilize as a probiotic for animals. METHODS AND RESULTS: A novel strain (MDT-5) was isolated from the goat rumen, which exclusively converted linoleic acid (LA) to CLA, because of its high LA isomerase activity with virtually no CLA reductase activity. MDT-5 also converted linolenic acid to conjugated linolenic acid that may be more bioactive than CLA. The oral administration of MDT-5 every other day to mice for 2 weeks resulted in increased amounts of CLA in the contents of the large intestine (2.5-fold), as well as in adipose tissue (threefold). Feeding a high-LA diet, as well as prolonging the period of MDT-5 administration, further increased the CLA content in body fat. CONCLUSIONS: MDT-5 has by far greater ability to produce CLA than any other known bacteria. Administration of MDT-5 to mice increases CLA production in the large intestine, which results in increased CLA absorption. SIGNIFICANCE AND IMPACT OF THE STUDY: MDT-5 may be useful in pet animals as a probiotic to provide CLA continuously.     

Biohydrogenation of C18 unsaturated fatty acids to stearic acid by a strain of Butyrivibrio hungatei from the bovine rumen

AIMS: To identify a ruminal isolate which transforms oleic, linoleic and linolenic acids to stearic acid and to identify transient intermediates formed during biohydrogenation. METHODS AND RESULTS: The stearic acid-forming bacterium, isolated from the rumen of a grazing cow, was a Gram-negative motile rod which utilized a range of growth substrates including starch and pectin but not cellulose or xylan. From its 16S rRNA gene sequence, the isolate was identified as a strain of Butyrivibrio hungatei. During conversion of linoleic acid, 9,11-conjugated linoleic acid formed as a transient intermediate before trans-vaccenic acid accumulated together with stearic acid. Unlike previously studied ruminal biohydrogenating bacteria, B. hungatei Su6 was able to convert alpha-linolenic acid to stearic acid. Linolenic acid was converted to stearic via conjugated linolenic acid, linoleic acid and trans-vaccenic acid as intermediates. Oleic acid and cis-vaccenic acid were converted to a series of trans monounsaturated isomers as well as stearic acid. An investigation of these isomers indicated that mixed trans positional isomers are intermediate in the biohydrogenation of cis monounsaturated fatty acids to stearic acid. CONCLUSION: This, the first rigorous identification and characterization of a ruminal bacterium which forms stearic acid, shows that B. hungatei plays an important role in unsaturated fatty acid transformations in the rumen. SIGNIFICANCE AND IMPACT OF THE STUDY: Biohydrogenating bacteria which convert C18 unsaturated fatty acids to stearic acid have not been available for study for many years. Access to B. hungatei Su6 now provides a fresh opportunity for understanding biohydrogenation mechanisms and rumen processes which lead to saturated fat in ruminant products.     

一株瘤胃纤维素降解菌的分离鉴定及其纤维素降解特性

从蒙古绵羊瘤胃内容物中分离到一株纤维素降解细菌WH-1, 通过形态、生理生化特征、G+C mol%含量和16S rRNA序列分析对分离菌株进行鉴定, 鉴定为溶纤维丁酸弧菌属(Butyrivibrio fibrisolvens)的溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)。同时, 用Mega 4.1软件构建的系统发育树显示分离菌株WH-1与多株溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)的亲缘关系最近。对该菌株纤维素降解特性的初步研究表明：当温度为37°C、     

Extracellular metabolomics: a metabolic footprinting approach to assess fiber degradation in complex media

This work reports the implementation and optimization of a method for high-throughput analysis of metabolites produced by the breakdown of natural polysaccharides by microorganisms. Our simple protocol enables simultaneous separation and quantification of more than 40 different sugars and sugar derivatives, in addition to several organic acids in complex media, using 50-mul samples and a standard gas chromatography-mass spectrometry platform that was fully optimized for this purpose. As an implementation proof-of-concept, we assayed extracellular metabolite levels of three bacterial strains cultivated on complex medium rich in polysaccharides and under identical growth conditions. We demonstrate that the metabolic footprinting profile data distinguish among sample types such as typical metabolomics data. Moreover, we demonstrate that the differential metabolite-level data provide insight on specific fibrolytic activity of the different microbial strains and lay the groundwork for integrated proteome-metabolome studies of fiber-degrading microorganisms.     

Identification of L-altrose in the extracellular polysaccharide from Butyrivibrio fibrisolvens strain CF3

Abstract Butyrivibrio fibrisolvens strain CF3, a stricyly anaerobic bacterial isolate from the ovine cecum, produces extracellular polysaccharides (EPS) when grown on a defined medium containing glucose as the carbon source. EPS were purified from culture supernatants and their monosaccharide composition was determined by two different procedures. Analysis of EPS hydrolysates by thin-layer chromatography (TLC) yielded spots coincident with standard glucose, altrose, and 1,6-anhydroaltrose. These results were corroborated by both gas-liquid chromatography (GLC) and GLC-mass spectroscopy (GLC-MS) of alditol acetates prepared from EPS hydrolysates. Purification of the altrose from EPS hydrolysates was accomplished by preparative paper and column chromatography. Polarimetry demonstrated the isolated altrose to have the L -configuration. The occurrence of this hexose in nature has not yet been reported.