Progress 10/01/99 to 09/30/04
Outputs
The key enzymes of ammonia assimilation (Glutamate dehydrogenase [GDH], Glutamine synthetase [GS] and Glutamate synthase [GOGAT]) were systematically cloned and characterized. It was established using measurements of gene expression and enzyme activity that ammonia assimilation occurred via the NADP-dependent GDH reaction and that GDH activity was considerably higher ammonia limiting as compared to carbon-limiting growth conditions. In addition, it was shown that GdhA activity was regulated primarily at the level of transcription. Sequence elucidation and cloning of a glutamine synthetase (glnN) from R. flavefaciens FD-1 was performed utilizing linker libraries and genome walking. Upon further examination of the entire sequence, the five regions of homology found in all members of the GS family III proteins were identified. Transcriptional regulation studied with quantitative real-time PCR demonstrated higher levels of glnN transcripts under ammonia than carbon-limited
growth conditions. The GSIII enzyme from R.flavefaciens was characterized at the DNA, RNA and protein level. This has provided vital knowledge concerning the evolution of the GS gene, one of the oldest existing and functioning superfamily of genes. The link between GSIII enzymes and bacteria which reside in the gastrointestinal tract is interesting and may be the result of selection or evolution for that family of enzymes by the nitrogen environment within the intestinal tract. Although initial studies using whole cells or cell free extracts obtained from R. flavefaciens FD-1 grown under different physiological conditions failed to demonstrate GOGAT activity using NAD(P)H as the electron donor, complete gene sequence for both the large (gltB) and small (gltD) subunits of glutamate synthase has been completed. We constructed a genome-walking DNA library and used it to obtain the genes encoding the large (gltB) and small (gltD) subunits of glutamate synthase (GOGAT) by PCR. The gltB
gene codes for a polypeptide of 1,509 amino acid residues with an estimated molecular mass of 165.8 kDa while the gltD gene, located immediately downstream of gltB, codes for a protein of 494 amino acids (54.2 kDa). Transcriptional regulation studied with quantitative RT-PCR demonstrated that the concentration of the gltB mRNA was less responsive to extracellular ammonia concentration than gdhA and glnA. Interestingly, further genome walking showed that immediately downstream of the genes encoding R. flavefaciens FD-1 GOGAT is the gene coding for glnA or Glutamine synthetase I which codes for a protein of 425 amino acid residues (47.8 kDa). The gene next to glnA codes for a putative response regulator of 195 amino acid residues with an estimated molecular mass of 22.2 kDa. Next to the response regulator-encoding gene is a gene coding for an amidophosphoribosyl transferase, the enzyme that controls the rate of de novo biosynthesis of purines. The operonic arrangement of these genes
suggests that in this organism nitrogen regulation and metabolism may be tied to purine biosynthesis.
Impacts
Metabolism and utilization of nitrogen by ruminant tissues during growth and production is dependent on, and regulated by, nitrogen metabolism in the rumen. Ammonia plays a central role as an intermediary in the degradation and assimilation of dietary nitrogen by gastrointestinal microorganisms. However, our understanding of the mechanism of ammonia assimilation in rumen bacteria is superficial despite its overriding importance in ruminant nitrogen metabolism and economy. This research has made major contributions to our understanding of the biochemical and genetic properties of key ammonia assimilating enzymes and their regulation in the Gram-positive, cellulolytic, rumen bacteria, Ruminococcus flavefaciens and Ruminococcus albus, since it is only with a thorough understanding of the biochemical and genetic mechanisms involved that the system can be successfully manipulated and fully exploited. This research has clearly demonstrated that the regulation of ammonia
assimilation and nitrogen metabolism in the Ruminococci differ from the classical enteric paradigm. This research has a major impact on global animal agriculture since some 60-80% of microbial protein synthesized in the rumen uses ammonia as a precursor and thus provides the major source of amino acids required by the host animal. Furthermore, more efficient fixation of nitrogen into bacterial cells will reduce N excretion and assist in developing animal production systems that are sustainable both environmentally and economically.
Publications
- Antonopoulos, D.A., Aminov, R.I., Duncan, P.A., White, B.A. and Mackie, R.I. 2003. Characterization of the gene encoding glutamate dehydrogenase (gdhA) from the ruminal bacterium Ruminococcus flavefaciens FD-1. Arch. Microbiol. 179:184-190.
- Amaya, K.R., Mackie, R.I. and Cann, I.K.O. 2005. Biochemical and mutational analysis of a glutamine synthetase type III (GSIII) from the rumen anaerobe Ruminococcus albus 8. Appl. Environ. Microbiology (Submitted).
- Wallace, S.R., Aminov, R.I., Antonopoulos, D.A., Pfister-Genskow, M.A., Duncan, P.A., White, B.A. and Mackie, R.I. 2005. Glutamine synthetase- Glutamate synthase pathway of ammonia assimilation in Ruminococcus flavefaciens FD-1. Appl. Environ. Microbiology (Submitted).
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Progress 01/01/03 to 12/31/03
Outputs
Genome sequencing and annotation of the ruminal cellulolytic bacterium Ruminococcus albus 8 has enabled the search and identification of genes involved in ammonia assimilation and regulation. Using this information we have identified and constructed a hypothetical model of the nitrogen assimilation and regulation for R. albus 8 based on regulatory scheme of the well-studied low G+C gram positive bacterium Bacillus subtilis. In the R. albus 8 gene list after 8x coverage the key ammonia assimilation enzymes GDH, GOGAT, and GS type I enzymes were identified. In addition, a novel glutamine synthetase type III (GSIII) encoded by the glnN gene, not found in B. subtilis, was identified. The novel GSIII was further confirmed by the identification of conserved domains from other hypothetical and biochemically characterized GSIII enzymes. The novel glnN gene was amplified by PCR from genomic DNA isolated from R. albus 8, cloned and expressed in a pET28 vector under a T7
promoter. The recombinant GSIII was purified to >90% by affinity (cobalt) column and anion exchange. Kinetic studies indicate a biologically functional GSIII, and an increase in glnN mRNA transcription under nitrogen limiting conditions further supports the importance of the GSIII enzyme in ammonia assimilation in R. albus 8. Mutational studies that change the conserved glutamic acid amino acids to alanine in the four GSIII motifs resulted in a reduction of activity using both the transferase and biosynthetic assays, excluding mutation E380A which resulted in a 20% increase in biosynthetic activity over the wild type GSIII. Reduced GSIII activity was also exhibited by mutating the lysine to alanine at position 308, which is located in the identified ATP binding site. The data presented here suggests an importance of the GSIII to Ruminococcus albus 8 during growth under nitrogen limiting conditions, while the mutagenic studies of the conserved amino acids in the identified motifs
indicate their functional importance for GSIII activity.
Impacts
This research is of fundamental importance in understanding the enzymes involved in the assimilation of ammonia, an essential nutrient for the growth of the cell.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Sequence elucidation and cloning of glnA from R. flavefaciens FD-1 was performed utilizing linker libraries and genome walking. A 2.6 kb composite sequence was deduced from three overlapping clones and primers were designed to amplify and then clone the entire region. The clone contains the 2 kb GS open reading frame (2,085 bp ORF) which is flanked upstream by a possible -35 and -10 promoter region and putative 54s-dependent RNA polymerase binding site, while downstream it is flanked by a termination loop. Upon further examination, the four regions of homology found in all members of the GS family III genes were identified in the sequence: Region I, the proposed site where the adjacent subunits of the enzyme meet to form the active site; Regions II and V, b-strands closely associated with two Mn2+ cations of one of the enzyme's subunits; Region III, the ATP binding site; and Region IV, the possible glutamate binding site (Hill et al. 1989). These regions in concert
form the GS active site (Almassy et al. 1986). In vitro transcription and translation analyses confirmed a 69.5 kDa protein (ca 600 amino acids), the estimated molecular weight of the R. flavefaciens FD-1 GlnA protein. Transcriptional regulation studied with quantitative real-time PCR demonstrated higher levels of glnA transcripts (2.04 vs 0.04 pg/100ng total RNA) under ammonia and carbon-limited growth conditions respectively. Until recently it appeared that all GS enzymes could be placed into two Families, GSI and GSII (Kumada et al. 1993) regardless of their origin in the domains Eubacteria, Archaea or Eucarya. However,a new Family, GSIII, has been reported in B.fragilis (Southern et al. 1987), Butyrivibrio fibrisolvens (Goodman and Woods 1993) and a few other bacteria. The characterization of the GSIII enzyme from R.flavefaciens at the DNA, RNA and protein level has provided vital knowledge concerning the evolution of the GS gene, one of the oldest existing and functioning genes
(Kumada et al. 1993). The closest match was to the glnA gene of Butyrivibrio fibrisolvens (Goodman and Woods 1993), also a rumen inhabitant. This grouping is not congruent with the 16S rDNA phylogeny of these bacteria. The link between GSIII enzymes and bacteria which reside in the gastrointestinal tract is interesting and may be the result of selection or evolution for that family of enzymes by the nitrogen environment within the intestinal tract. A surprising discovery was that the C-terminal portion (345 bp) of the pyrimidine biosynthetic pathway enzyme CTP synthase (pyrG) lies directly upstream (15 bp) from glnA. This sequence information will be utilized to investigate whether there is a transcriptional link between GS and CTP synthase, which would be a novel connection between these two pathways. Transcript size determination via Northern blots will also help verify the putative transcriptional link between GS and CTP synthase.
Impacts
This research is important because it documents a unique form of Glutamine synthetase belonging to the GSIII Family. This research utilizes real-time quantitative PCR to quantitate transcript levels of these genes in the cell of this key fiber degrading bacterium. This research will lead to increased understanding of how rumen, and other gut, bacteria procure an essential nutrient making this process more efficient leading to reduced nitrogen excretion and less environmental polution.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
The Glutamine synthetase - Glutamate synthase (GS/GOGAT) pathway of ammonia assimilation is assumed to play a role in scavenging a limiting nutrient, ammonia (representing the total ionized and unionized species), when environmental concentrations are low. After detecting the presence of Glutamine synthetase activity in cell-free extracts of Ruminococcus flavefaciens FD-1 our further experiments focussed on cloning and sequencing of enzymes of ammonia assimilation (GS and GOGAT). Information on the molecular features of these two genes and their phylogenetic analysis was reported previously. Transcriptional regulation was studied with quantitative real-time PCR to detect mRNA transcripts using the GeneAmp 5700 Sequence Detection System. The glnA transcripts were shown to have higher levels of expression under nitrogen-limitation (2.90pg/100ng RNA) than under carbon-limitation (0.44pg/100ng RNA). Levels of the gltB transcripts were less responsive to the extracellular
level of ammonia, 1.14 and 0.78pg/100ng RNA for nitrogen-limitation and carbon-excess respectively.
Impacts
This research has importance in a number of different areas. It documents a unique form of Glutamine synthetase (GS) that has an evolutionary relationship to a unique family of these genes termed the GS III family. It also documents the presence of a Gluatamate synthase (GOGAT) gene that uses ferrodoxin as an electron carrier. This research also uses real-time PCR to quantitate transcript levels of these genes in cells of an important fiber-degrading bacterium. This research will lead to increased understanding of how rumen, and other gut, bacteria procure an essential nutrient making this process more efficient leading to decreased nitrogen excretion and less environmental pollution.
Publications
- KRAUSE, D.O., SMITH, W.J., RYAN, F.M.E., MACKIE, R.I. and MCSWEENEY, C.S. 2000. Use of 16S rRNA based techniques to investigate the ecological succession of microbial populations in the immature lamb rumen: Tracking of specific strains of inoculated Ruminococcus and interactions with other microbial populations in vivo. Microb. Ecol. 38:365-376.
- KRAUSE, D.O., BUNCH, R.J., CONLAN, L.L., KENNEDY, P., SMITH, W.J., MACKIE, R.I. and MCSWEENEY, C.S. 2001. Molecular ecology of fiber digestion: Functional evaluation and tracking of introduced Ruminococcus strains. Microbiology 147:1719-1729.
- WALLACE, S.R., AMINOV, R.I., ANTONOPOULOS, D.A., PFISTER-GENSKOW, M.A., DUNCAN, P.A., WHITE, B.A. and MACKIE, R.I. 2002. Gluatamine synthetase- Glatamate synthase pathway of Ruminococcus flavefaciens FD-1. Appl. Environ. Microbiol. (Submitted).
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Progress 01/01/00 to 12/31/00
Outputs
The protocol for in situ PCR for amplification and detection of specific target nucleic acid sequences within bacterial cells has been the focus of research during the review period. Ruminococcus flavefaciens FD-1 was grown under conditions of carbon and nitrogen limitation, harvested by centrifugation, washed and fived in 4% fresh paraformaldehyde. Fixed cells are applied to silane-coated slides, followed by enzymatic treatment with lysozyme, proteinase K and DNase. RT-PCR was performed using the Titan One Tube RT-PCR kit (Boehringer) using a GeneAmp in situ PCR System (Perkin Elmer) and oligonucleotide primers (21-mer) targeting the GDH, GS and GOGAT genes. Forward primers have been synthesized with the flourochrome CY3 incorpoarted at the 5' end to allow in situ visualization. Cell heterogeneity in signal intensity is a problem.
Impacts
Whole cell hybridization using in situ techniques which allow direct detection and identification as well as measurement of activity are powerful tools. This allows ecologists to link the presence of specific genes and their activity in the natural ecosystem of the intestinal tract.
Publications
- Mackie,R.I., Aminov,R.I., White,B.A. and McSweeney,C.S. 2000. Molecular ecology and diversity in gut microbial ecosystems. In: Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction pp.61-77 (P.B. Cronje, Editor) CABI Publishing, New York.
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Progress 01/01/99 to 12/31/99
Outputs
Progress for this project has been limited based on the short duration (2 months) from start date to report date. Research has continued on the validation of the in situ RT-PCR technique for detection of glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT). Cell fixation and permeabilization have been optimized with inclusion of mutanolysin to improve probe penetration. A wide range of treatments has been attempted in order to reduce the non-specific signals obtained using the flourescent primers targeting each of the genes of interest. This signal is apparently related to non-specific priming of DNA which is diifficult to elimuinate in whole cells with DNA's treatment. Visualization of signal at the individual cell level using epiflourescence microscopy has been successful and in situ expression of genes encoding enzymes of ammonia assimalation can be detected and imaged. Further research will make use of this technique to examine
level of expression in R. flavefaciens FD-1 grown under conditions of ammonia limitation and ammonia excess (carbon limited).
Impacts
More efficient fixation of nitrogen, and specifically ammonia, in bacterial cells will reduce N excretion and assist in developing animal production systems which are sustainable both environmentally and economically.
Publications
- No publications reported this period
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