Biomedical subjects

H J Flint

Publications and source records attributed to H J Flint.

At least 55 records · Page 3Linked to original sources

PubMed1995

A xylan hydrolase gene cluster in Prevotella ruminicola B(1)4: sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and beta-(1,4)-xylosidase activities.

Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region of 75 nucleotides. xynA is shown to encode a family F endoxylanase of 369 amino acids, including a putative amino-terminal signal peptide. xynB encodes an enzyme of 319 amino acids, with no obvious signal peptide, that shows 68% amino acid identity with the xsa product of Bacteroides ovatus and 31% amino acid identity with a beta-xylosidase from Clostridium stercorarium; together, these three enzymes define a new family of beta-(1,4)-glycosidases. The activity of the cloned P. ruminicola xynB gene product, but not that of the xynA gene product, shows considerable sensitivity to oxygen. Studied under anaerobic conditions, the XynB enzyme was found to act as an exoxylanase, releasing xylose from substrates including xylobiose, xylopentaose, and birch wood xylan, but was relatively inactive against oat spelt xylan. A high degree of synergy (up to 10-fold stimulation) was found with respect to the release of reducing sugars from oat spelt xylan when XynB was combined with the XynA endoxylanase from P. ruminicola B(1)4 or with endoxylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17. Pretreatment with a fungal arabinofuranosidase also stimulated reducing-sugar release from xylans by XynB. In P. ruminicola the XynA and XynB enzymes may act sequentially in the breakdown of xylan.

Amino Acid Sequence↗

PubMed1994

Identification of non-catalytic conserved regions in xylanases encoded by the xynB and xynD genes of the cellulolytic rumen anaerobe Ruminococcus flavefaciens.

xynB is one of at least four genes from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that encode xylanase activity. The xynB gene is predicted to encode a 781-amino acid product starting with a signal peptide, followed by an amino-terminal xylanase domain which is identical at 89% and 78% of residues, respectively, to the amino-terminal xylanase domains of the bifunctional XynD and XynA enzymes from the same organism. Two separate regions within the carboxy-terminal 537 amino acids of XynB also show close similarities with domain B of XynD. These regions show no significant homology with cellulose- or xylan-binding domains from other species, or with any other sequences, and their functions are unknown. In addition a 30 to 32-residue threonine-rich region is present in both XynD and XynB. Codon usage shows a consistent pattern of bias in the three xylanase genes from R. flavefaciens that have been sequenced.

Amino Acid Sequence↗

PubMed1994

Molecular genetics of obligate anaerobes from the rumen.

The rumen is inhabited by a highly specialised microflora consisting of obligately anaerobic bacteria, fungi and protozoa. Rumen bacteria belong to many different phylogenetic groupings and many species exhibit a high degree of rRNA gene sequence diversity, whereas the rumen fungi are monophyletic. At least 21 genes concerned with the degradation and utilisation of plant cell wall polysaccharides, from five species of rumen bacteria and from rumen fungi, have been isolated and sequenced. In general, the catalytic domains of the encoded enzymes belong to enzyme families identified among non-rumen microorganisms, but some show unusual organisation, consisting of multiple catalytic domains. Several bacterial species have been used as recipients for gene transfer by electrotransformation or by conjugation, allowing development of methods for genetic analysis. The rumen is also considered as a potential site for natural gene transfer.

Animals↗

PubMed1994

Genetic diversity and phylogenetic relationships among strains of Prevotella (Bacteroides) ruminicola from the rumen.

A high degree of genetic diversity among 29 strains of Prevotella (Bacteroides) ruminicola from the rumen was revealed by comparing restriction fragment length polymorphisms in 16S rRNA genes, sodium dodecyl sulfate-polyacrylamide gel profiles of total-cell proteins, and G + C contents of chromosomal DNAs. In order to obtain information on phylogenetic relationships, the sequences of a 389-bp region of the 16S rRNA gene, including variable regions 4 and 5, were compared for 10 strains. These 10 strains formed a single group when their sequences were compared with 16S ribosomal DNA sequences from other species, including Bacteroides spp. from the human colon. On the other hand, the great genetic distances between many P. ruminicola strains, including P. ruminicola subsp. brevis B(1)4 and GA33 and P. ruminicola 23T (T = type strain), support the hypothesis that these organisms should be reclassified into new species. We identified signature oligonucleotides based on 16S ribosomal DNA sequences that distinguished strains related to strains 23T, B(1)4, GA33, and M384, as well as an oligonucleotide that specifically recognized all but one of the Bacteroides and Prevotella strains tested. On the basis of the priming activities of these signature oligonucleotides in PCR reactions and on other criteria, we concluded that 12 of the original 29 strains were related to strain 23T, 4 were related to strain B(1)4, and 4 were related to strain GA33. While there are clear grounds for subdividing the species P. ruminicola on the basis of genotypic differences, it is appropriate to delay formal reclassification until further work on the phenotypic differentiation of the new groups is completed.

Animals↗

PubMed1994

Multiple lactate dehydrogenase activities of the rumen bacterium Selenomonas ruminantium.

The lactate utilizing strain of Selenomonas ruminantium 5934e was found to contain three lactate dehydrogenase (LDH) activities in sonicated cell extracts. One activity, an NAD dependent L-LDH (L-nLDH) was measured at 15-fold greater levels in extracts of cells grown to mid-exponential phase on glucose compared to cells grown to the equivalent growth stage on DL-lactate. A second nLDH activity specific for D-lactate (D-nLDH) was detected at similar levels in both lactate-grown cell extracts and glucose-grown cell extracts. The third activity, an NAD independent DLDH (D-iLDH) was very low in cells grown on glucose but was induced more than 10-fold when DL-lactate was used as the carbon source. The three LDH activities could be separated by gel filtration. Recovery of the activities was low due to the apparent instability of the enzymes at 4 degrees C, which was most pronounced in the case of the D-iLDH. A Km for lactate of 0.5 mM was estimated for the D-iLDH and this was considerably lower than the values of 45 mM and 70 mM measured for L-nLDH and D-nLDH respectively. It is proposed that the D-iLDH may be largely responsible for the formation of pyruvate in lactate-grown cells of S. ruminantium strain 5934e. Three other lactate utilizing strains of S. ruminantium, HD4, 5521C1 and JW13 exhibited a similar profile of LDH activities to strain 5934e when grown on glucose and DL-lactate.

Animals↗

PubMed1993

Mode of action, kinetic properties and physicochemical characterization of two different domains of a bifunctional (1-->4)-beta-D-xylanase from Ruminococcus flavefaciens expressed separately in Escherichia coli.

Two catalytic domains, A and C, of xylanase A (XYLA) from Ruminococcus flavefaciens were expressed separately as truncated gene products from lacZ fusions in Escherichia coli. The fusion products, referred to respectively as XYLA-A1 and XYLA-C2, were purified to homogeneity by anion-exchange chromatography and chromatofocusing. XYLA-A1 was isoelectric at pH 5.0 and had a molecular mass of 30 kDa, whereas XYLA-C2 had a pI of 5.4 and a molecular mass of 44 kDa. The catalytic activity shown by both domains was optimal at 50 degrees C, but XYLA-A1 was more sensitive than XYLA-C2 to temperatures higher than the optimum. XYLA-A1 showed a higher sensitivity to pH than XYLA-C2. The enzyme activity of both domains was completely inactivated in the presence of copper or silver ions and partially inactivated by iron or zinc ions. Neither domain was active on xylo-oligosaccharides shorter than xylopentaose: the rate of degradation of longer xylo-oligosaccharides (degree of polymerization 5-10) increased as the chain length increased. Analysis of the products of hydrolysis of xylo-oligosaccharides and xylan (arabinoxylan) polysaccharide showed that the two domains differed in their modes of action: xylobiose was the shortest product of the hydrolysis. With oat spelt xylan as substrate, XYLA-A1 activity was apparently restricted to regions where xylopyranosyl residues did not carry arabinofuranosyl substituents, whereas XYLA-C2 was able to release hetero-oligosaccharides carrying arabinofuranosyl residues. Neither domain was able to release arabinose from oat spelt xylan.

Animals↗

PubMed1993

Probiotics and manipulation of rumen development and function.

There is good evidence that the bacterial and fungal probiotics are effective in the manipulation of rumen development and function. The effects of adherent Streptococcus bovis preparations were manifested by significant increase of adherent S. bovis bacteria and alpha amylase activity in the rumen wall of young ruminants. Fungal probiotics based on Saccharomyces cerevisiae and Aspergillus oryzae are rapidly gaining acceptance as a means of improving productivity in adult ruminants. However, fungal probiotics may also have a complementary role to bacterial probiotics in young animals. Dietary yeast improved performance of calves and lambs in terms of feed intake and live weight gain and Aspergillus foetidus was effective in altering rumen fermentation in newly weaned lambs.

Animals↗

PubMed1993

A bifunctional enzyme, with separate xylanase and beta(1,3-1,4)-glucanase domains, encoded by the xynD gene of Ruminococcus flavefaciens.

Adjacent regions of a Ruminococcus flavefaciens 17 DNA fragment were found to encode xylanase and beta(1,3-1,4)-glucanase activities. Sequencing of this fragment showed that both activities are encoded by a single 2,406-bp open reading frame corresponding to the xynD gene. The predicted product has a characteristic signal sequence that is followed by an amino-terminal domain related to family G xylanases, while the carboxyterminal domain is related to beta(1,3-1,4)-glucanases from several other bacterial species. These two domains are connected by a region of unknown function that consists of 309 amino acids and includes a 30-amino-acid threonine-rich sequence. A polypeptide having a molecular weight of approximately 90,000 and exhibiting xylanase and beta(1,3-1,4)-glucanase activities was detected in Escherichia coli cells carrying the cloned xynD gene. This is one of the first cases in which a microbial polysaccharidase has been shown to carry separate catalytic domains active against different plant cell wall polysaccharides within the same polypeptide. xynD is one of a family of related genes in R. flavefaciens that encode enzymes having multiple catalytic domains, and the amino terminus of XYLD exhibits a high degree of similarity with the corresponding regions of another xylanase, XYLA, which carries two different xylanase catalytic domains.

Amino Acid Sequence↗

PubMed1993

Transfer of hybrid plasmids based on the replicon pRRI7 from Escherichia coli to Bacteroides and Prevotella strains.

New shuttle vectors based on a Prevotella ruminicola 9.5 kb cryptic plasmid (pRRI7) inserted within the Escherichia coli vector pKC71, carrying the Ccr/Emr Bacteroides marker, were constructed. These constructs (pKBR23-1 and pKBR23-2) were transferred into Bacteriodes distasonis, Bacteroides thetaiotaomicron, Bacteroides uniformis and into P. ruminicola NCFB 2202 either by conjugal mobilization or by electroporation. Another pRRI7 derivative based on pKC72, pKBR23-3, was smaller (13.1 kb) and non-mobilizable. By electroporation, it was transferred to Bact. distasonis and P. ruminicola. Being derived from pRRI7 which is compatible with the shuttle plasmid pRRI207, the host/vector combination involving P. ruminicola NCFB 2202 and pKBR23-3 offers new possibilities for genetic investigations in rumen anaerobic bacteria after further introduction of a second readily selectable marker within pRRI207 or pKBR23-3.

Bacteroides↗

PubMed1992

Distribution of xylanase genes and enzymes among strains of Prevotella (Bacteroides) ruminicola from the rumen.

The distribution of two xylanase genes was examined by Southern hybridization among 26 strains of the rumen anaerobic bacterium Prevotella (Bacteroides) ruminicola. Hybridization with a xylanase/endoglucanase gene from the type strain 23 was found in six strains while hybridization with a xylanase gene from strain D31d was found in 14 strains. Sequences related to both genes were present, on different restriction fragments, in six strains, whereas no hybridization to either gene was detected in five other strains capable of hydrolysing xylan, or in seven strains that showed little or no xylanase activity. Zymogram analyses of seven xylanolytic strains of P. ruminicola demonstrated interstrain variation in the apparent molecular masses of the major xylanases and carboxymethylcellulases that could be renatured following SDS polyacrylamide gel electrophoresis.

Animals↗

PubMed1992

Expression of Escherichia coli homoserine kinase in mouse 3T3 cells.

The Escherichia coli gene for homoserine kinase (thrB) has been cloned into a simian-virus-40-based eukaryotic expression vector which also includes a neomycin-resistance gene. Mouse 3T3 cells transfected with this plasmid were selected for resistance and screened for homoserine kinase activity. It has thus been possible to isolate clones which are capable of accumulating homoserine O-phosphate when supplied with homoserine. In broken-cell preparations the kinetic constants for the production of homoserine O-phosphate were similar to those of the wild-type E. coli enzyme. These experiments demonstrate that E. coli homoserine kinase can be expressed in an animal cell and that it can successfully phosphorylate L-homoserine in the intact cell utilizing endogenous ATP.

3T3 Cells↗

PubMed1992

A bifunctional xylanase encoded by the xynA gene of the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 comprises two dissimilar domains linked by an asparagine/glutamine-rich sequence.

The nucleotide sequence of the xynA gene of Ruminococcus flavefaciens 17 was determined and found to consist of a 2862bp open reading frame beginning with a TTG start codon. The predicted product, XYLA, consisted of distinct amino-terminal (A) and carboxy terminal (C) domains (248 amino acids, including a putative signal sequence, and 332 amino acids, respectively) linked by a repetitive sequence (B, 374 amino acids) extraordinarily rich in asparagine (45%) and glutamine (26%) residues. Domains A and C were shown to be capable of expressing xylanase activity independently of each other when suitably truncated derivatives of the xynA coding region were expressed as lacZ fusions. The activities associated with the two domains were shown to differ with respect to the average size of hydrolysis products formed from oat-spelt xylan, with domain C releasing relatively more xylose and domain A more xylo-oligosaccharides. The amino acid sequence of domain A of XYLA closely resembled that of the Bacillus pumilus xynA enzyme (45% identical residues). On the other hand domain C showed significant similarity (33% to 40% identical residues) to a different group of bacterial xylanases and exoglucanases exemplified by the Caldocellum saccharolyticum xynA and celB products. The xynA product is, therefore, a bifunctional enzyme having two dissimilar catalytic domains capable of acting on xylan.

Amino Acid Sequence↗

PubMed1991

Sequence of a cellulase gene from the rumen anaerobe Ruminococcus flavefaciens 17.

A cellulase gene (endA) was isolated from a library of Ruminococcus flavefaciens strain 17 DNA fragments inserted in pUC13. The endA product showed activity against acid-swollen cellulose, carboxymethyl-cellulose, lichenan, cellopentaose and cellotetraose, but showed no activity against cellotriose or binding to avicel. Nucleotide sequencing indicated an encoded product of 455 amino acids which showed significant sequence similarity (ranging from 56% to 61%) with three endoglucanases from Ruminococcus albus, and with Clostridium thermocellum endoglucanase E. Little relatedness was found with a cellodextrinase previously isolated from R. flavefaciens FD1.

Amino Acid Sequence↗

PubMed1991

Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 cloned in pUC13.

Two distinct xylanase genes (designated xynA and xynB) were subcloned in pUC13 from non-homologous restriction fragments of Ruminococcus flavefaciens 17 DNA originally isolated in lambda EMBL3. The products of the two genes showed similar pH optima for hydrolysis of oat spelt xylan (around 5.5) and had little or no activity against carboxymethylcellulose. Trace activities against p-nitrophenyl (pNP) cellobioside and pNP-xyloside were detected in clones containing xynA, but not in one harbouring xynB. The xylanase associated with clones carrying xynA produced mainly xylobiose and xylose from xylan and did not give hydrolysis of xylobiose, while that encoded by xynB produced mainly xylobiose and higher xylo-oligosaccharides from xylan. There was evidence of increased expression, at the RNA level, of these two genes, and of another cloned region encoding multiple activities including xylanase, in R. flavefaciens 17 grown with xylan, as compared with cellobiose, as energy source. Total cell-associated xylanase and beta-xylosidase activities, and supernatant xylanase activity, were shown to be similarly induced in xylan-grown R. flavefaciens, 17.

Animals↗

PubMed1990

Restricted activation of general amino acid control under conditions of glutamine limitation in Neurospora crassa.

In Neurospora crassa limitation for single amino acids normally results in increased formation of enzymes required for amino acid synthesis via 'general amino acid control'. Glutamine limitation, however, led to comparatively low and delayed derepression of enzyme synthesis. Nitrate reductase activity increased steeply under these conditions confirming that de novo protein synthesis could occur. Derepression levels were unaffected by addition of glutamine-derived metabolites. Only small and delayed increases in mRNA levels occurred for the anabolic enzyme genes arg-12, his-3 and trp-1 under conditions of glutamine limitation in contrast to the immediate and far larger increase found on histidine limitation. The trans-acting regulatory gene of general amino acid control in Neurospora, cpc-1, responded with a significant increase in mRNA level to histidine and to glutamine limitation. The restricted response of the amino acid synthesis genes could imply a post-transcriptional block to the positive regulatory function of cpc-1 under condition of glutamine limitation. The results suggest that the expression of general amino acid control is restricted under conditions of inadequate nitrogen supply.

Alcohol Oxidoreductases↗

PubMed1990

The properties of forms of Ruminococcus flavefaciens which differ in their ability to degrade cotton cellulose.

Two mutant strains of Ruminococcus flavefaciens strain 007 that differ in their ability to hydrolyse cotton fibres have been shown also to differ in their cell-surface topology, in that the cotton degrading form possessed larger and more protuberant cell surface structures. The strains had similar CMCase, cellobiosidase and beta-glucosidase activities. The results indicate the importance of cell-surface properties in cotton degradation by R. flavefaciens.

Animals↗

PubMed1990

A molecular biological approach to reducing dietary amino acid needs.

Rapid developments in transgenic animal technology make it possible to consider introducing new metabolic capabilities into animals, using genes from other species. Lysine and threonine are both essential amino acids in mammals, and are commonly the first and second limiting amino acids, respectively, for protein accretion in pigs and poultry fed cereal based diets. Here we consider the potential for transgenic animals with microbial biosynthetic pathways for these amino acids.

Amino Acids, Essential↗

PubMed1990

Deoxyribonuclease activity in rumen bacteria.

Deoxyribonuclease activity was surveyed in 22 strains belonging to 12 species of rumen bacteria, with lambda bacteriophage DNA as substrate. Activity was readily detected in broken cell preparations from 15 of these strains. Particularly high levels of activity were present in cells and culture supernatant of all 5 strains of Bacteroides succinogenes, and 2 out of 6 strains of Bacteroides ruminicola, examined.

Animals↗