Progress 05/15/12 to 05/14/16
Outputs
Target Audience:Research results have been disseminated to our target audience of academics,scientists and industry professionals at national and international meetings across the four years of this granting period. In addition, high profile scientific manuscripts have been published over the same time period of the grant. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?As described in our research papers, twoPostdoctoral Research Associates and two PhDstudents have received an outstanding scientific training in modern molecular as well as traditional cultivation based approaches to tye study of microbial biology. How have the results been disseminated to communities of interest?The results of this researchprogram have been published in high profile, peer reviewed scientific journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have sequenced the genomes of two strains of Selnomonas ruminantium, one strain that utilizes lactate (GA192) and the other a lactate utilizing isolate (HD4). Comparative genomic analyses of Selenomonas ruminantium GA192 and HD4 have revealed many exciting features, including the presence of dissimilatory sulfate reduction genes, a physiology never before described in these organisms. The model rumen Firmicute Ruminococcus albus 8 was grown using ammonia, urea, or peptides as the sole N source; growth was not observed with amino acids as the sole nitrogen source. Growth of R. albus 8 on ammonia and urea showed similar growth rates and maximum cell densities. However, growth on peptides resulted in a nearly identical growth rate and lower maximum cell density. To identify differences in gene expression and enzyme activities, the transcript abundance of ten different genes involved in nitrogen metabolism and specific enzyme activities were analyzed by harvesting mRNA and crude protein from cells at mid- and late-exponential phases of growth on the different N sources. Transcript abundance and enzyme activities varied according to nitrogen source, ammonia concentration, and growth phase. Growth of R. albus 8 on ammonia and urea was similar, with the only observed difference being an increase in urease transcript abundance and enzyme activity in urea-grown cultures. Growth of R. albus 8 on peptides showed a different nitrogen metabolism pattern, with higher gene transcript abundance levels of gdhA, glnA, gltB, amtB, glnK, and ureC as well as higher activities of glutamate dehydrogenase and urease. These results demonstrate that ammonia, urea, and peptides can all serve as nitrogen sources for R. albus, and nitrogen metabolism genes and enzyme activities of R. albus 8 are regulated by nitrogen source and the level of ammonia in the growth medium. Nitrogen utilization and metabolism in commensal gut bacteria is highly important to gut function and health but our understanding of this process is superficial. Using a genomic-based approach, we investigated gene expression and enzyme activities involved in nitrogen metabolism for Ruminococcus albus, a predominant plant cell wall degrading anaerobic bacterium in the rumen and other gut ecosystems. We showed growth on the preferred nitrogen source (ammonium) as well as urea and peptides but not amino acids. Interestingly, several gene transcript abundance patterns on ammonia and urea were contrary to those previously observed in enteric bacteria where GS is a scavenging system highly up-regulated under low environmental ammonia concentrations. Xylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacterium Bacteroides intestinalis DSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. The xyn8A gene encodes an endoxylanase (Xyn8A), and rex8A encodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose (X5) and xylohexaose (X6) to a mixture of xylobiose (X2) and xylotriose (X3), while Rex8A hydrolyzed X3 through X6 to a mixture of xylose (X1) and X2. Moreover, rex8A is located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit beta-xylosidase activity and would be able to further hydrolyze X2 to X1. Mutational analyses of putative active site residues of both Xyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among the Bacteroidetes, indicating that these genes contribute to xylan utilization in the human gut. Ruminococcus albus 7 has played a key role in the development of the concept of interspecies hydrogen transfer. The rumen bacterium ferments glucose to 1.3 acetate, 0.7 ethanol, 2 CO2, and 2.6H2 when growing in batch culture and to 2 acetate, 2CO2 and 4H2 when growing in continuous culture in syntrophic association with H-consuming microorganisms that keep the partial pressure low. The organism uses NAD(+) and ferredoxin for glucose oxidation to acetyl coenzyme A (acetyl-CoA) and CO2, NADH for the reduction of acetyl-CoA to ethanol, and NADH and reduced ferredoxin for the reduction of protons to H2. Of all the enzymes involved, only the enzyme catalyzing the formation of H2 from NADH remained unknown. Here, we report that R. albus 7 grown in batch culture on glucose contained, besides a ferredoxin-dependent [FeFe]- hydrogenase (HydA2), a ferredoxin- and NAD-dependent electron-bifurcating [FeFe]-hydrogenase (HydABC) that couples the endergonic formation of H2 from NADH to the exergonic formation of H2 from reduced ferredoxin. Interestingly, hydA2 is adjacent to the hydS gene, which is predicted to encode an [FeFe]-hydrogenase with a C-terminal PAS domain. We showed that hydS and hydA2 are part of a larger transcriptional unit also harboring putative genes for a bifunctional acetaldehyde/ethanol dehydrogenase (Aad), serine/threonine protein kinase, serine/threonine protein phosphatase, and a redox-sensing transcriptional repressor. Since HydA2 and Aad are required only when R. albus grows at high H2 partial pressures, HydS could be a H-sensing [FeFe]-hydrogenase involved in the regulation of their biosynthesis. We are continuing this work by examining the role of the putative hydrogen sensing hydrogenase Hyd S in interspecies hydrogen transfer. This involves growth of R. albus 7 under both low and high external H concentrations followed by paired growth with a series of H utilizing partners including Wolinella succinogenes (fumarate reducer), Methanobrevibacter smithii (methanogen) and Acetitomaculum ruminis (acetogen). The mutually-beneficial interdependence of hydrogen producing and hydrogen utilizing bacteria was discovered by M.P. Bryant, M. J. Wolin and R.S. Wolfe at the University of Illinois in 1967. This discovery led to the realization that metabolically distinct microorganisms in the absence of air, act not as individual species but as tightly linked multispecies units coupled by the energetic need to maintain the exchanged hydrogen at very low concentration. This multispecies interaction, called syntrophy, allows biodegradation to proceed uni-directionally in the rumen colon waste digesters and aquatic sediments. This is critical to efficient functioning of the rumen and hence production efficiency on beef and dairy cows. This research carried out using high throughput sequencing approaches demonstrates the sequence and importance of bacterial interactions and communication in rumen fermentation and consequently efficient degradation of forages and other fiber sources in the diets of beef and dairy cattle.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Cann, I., Bernardi, R. and Mackie, R.I. 2016. Cellulose degradation in the human gut: Ruminococcus champanellensis expends the cellulosome paradigm. Environ. Microbiol. 18:307-310.
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Progress 05/15/14 to 05/14/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Postdoctoral training for one scientist (Dr. Amy Biddle) and PhD training for two gradutae students (Mr. Inhyuk Kwon and Mr. Michael Lakiviak). How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Continue with research experimentation and complete manuscripts for submission to high impact scientific journals.
Impacts
What was accomplished under these goals?
1. Ruminococcus albus 7 has played a key role in the development of the concept of interspecies hydrogen transfer. The rumen bacterium ferments glucose to 1.3 acetate, 0.7 ethanol, 2 CO2, and 2.6 H2 when growing in batch culture and to 2 acetate, 2 CO2, and 4 H2 when growing in continuous culture in syntrophic association with H2-consuming microorganisms that keep the H2 partial pressure low. The organism uses NAD(+) and ferredoxin for glucose oxidation to acetyl coenzyme A (acetyl-CoA) and CO2, NADH for the reduction of acetyl-CoA to ethanol, and NADH and reduced ferredoxin for the reduction of protons to H2. Of all the enzymes involved, only the enzyme catalyzing the formation of H2 from NADH remained unknown. Here, we report that R. albus 7 grown in batch culture on glucose contained, besides a ferredoxin-dependent [FeFe]-hydrogenase (HydA2), a ferredoxin- and NAD-dependent electron-bifurcating [FeFe]-hydrogenase (HydABC) that couples the endergonic formation of H2 from NADH to the exergonic formation of H2 from reduced ferredoxin. Interestingly, hydA2 is adjacent to the hydS gene, which is predicted to encode an [FeFe]-hydrogenase with a C-terminal PAS domain. We showed that hydS and hydA2 are part of a larger transcriptional unit also harboring putative genes for a bifunctional acetaldehyde/ethanol dehydrogenase (Aad), serine/threonine protein kinase, serine/threonine protein phosphatase, and a redox-sensing transcriptional repressor. Since HydA2 and Aad are required only when R. albus grows at high H2 partial pressures, HydS could be a H2-sensing [FeFe]-hydrogenase involved in the regulation of their biosynthesis. We are continuing this work by examining the role of the putative hydrogen sensing hydrogenase Hyd S in interspecies hydrogen transfer. This involves growth of R. albus 7 under both low and high external H2 concentrations followed by paired growth with a series of H2 utilizing partners including Wolinella succinogenes (fumarate reducer), Methanobrevibacter smithii (methanogen) and Acetitomaculum ruminis (acetogen). The mutually-beneficial interdependence of hydrogen producing and hydrogen utilizing bacteria was discovered by M.P. Bryant, M. J. Wolin and R.S. Wolfe at the University of Illinois in 1967. This discovery led to the realization that metabolically distinct microorganisms in the absence of air, act not as individual species but as tightly linked multispecies units coupled by the energetic need to maintain the exchanged hydrogen at very low concentration. This multispecies interaction, called syntrophy, allows biodegradation to proceed uni-directionally in the rumen colon waste digesters and aquatic sediments. This is critical to efficient functioning of the rumen and hence production efficiency on beef and dairy cows. 2. The rumen microbial ecosystem is known for its biomass-degrading and methane-producing phenotype. Fermentation of recalcitrant plant material, comprised of a multitude of interwoven fibers, necessitates the synergistic activity of diverse microbial taxonomic groups that inhabit the anaerobic rumen ecosystem. Although interspecies hydrogen (H2) transfer, a process during which bacterially generated H2 is transferred to methanogenic Archaea, has obtained significant attention over the few last decades, the temporal variation of the different taxa involved in in situ biomass-degradation, H2 transfer and the methanogenesis process remains to be established. Here we investigated the temporal succession of microbial taxa and its effect on fiber composition during rumen incubation using 16S rRNA amplicon sequencing. Switchgrass filled nylon bags were placed in the rumen of a cannulated cow and collected at nine time points for DNA extraction and 16S pyrotag profiling. The microbial community colonizing the air-dried and non-incubated (0 hours) switchgrass was dominated by members of the Bacilli (recruiting 63% of the pyrotag reads). During in situ incubation of the switchgrass, two major shifts in the community composition were observed: Bacilli were replaced within 30 min by members belonging to the Bacteroidia and Clostridia, which recruited 34 and 25% of the 16S rRNA reads generated, respectively. A second significant shift was observed after 16 h of rumen incubation, when members of the Spirochaetes and Fibrobacteria classes became more abundant in the fiber-adherent community. During the first 30 minutes of rumen incubation ~13% of the switchgrass dry matter was degraded, whereas little biomass degradation appeared to have occurred between 30 minutes and 4 hours after the switchgrass was placed in the rumen. Interestingly, methanogenic members of the Euryarchaeota (i.e., Methanobacteria) increased up to 3-fold during this period of reduced biomass-degradation, with peak abundance just before rates of dry matter degradation increased again. We hypothesize that during this period microbial-mediated fibrolysis was temporarily inhibited until H2 was metabolized into CH4 by methanogens. Collectively, our results demonstrate the importance of inter-species interactions for the biomass-degrading and methane-producing phenotype of the rumen microbiome-both microbially facilitated processes with global significance. This research carried out using high throughput sequencing approaches demonstrates the sequence and importance of bacterial interactions and communication in rumen fermentation and consequently efficient degradation of forages and other fiber sources in the diets of beef and dairy cattle.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Zheng, Y., Kahnt, J., Kwon I.H., Mackie R.I. and Thauer R.K. Hydrogen formation and its regulation in Ruminococcus albus: Involvement of an electron-bifurcating [FeFe]-hydrogenase, of a non-electron-bifurcating [FeFe]-hydrogenase, and of a putative hydrogen-sensing [FeFe]-hydrogenase. J. Bacteriology 196:3840-3852.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Piao, H., Lachman, M., Malfatti, S., Sczyrba, A., Knierem, B., Auer, M., Tringe, S.G., Mackie, R.I., Yeoman, C.J. and Hess, M. Temporal dynamics of fibrolytic and methanogenic rumen microorganisms during in situ incubation of switchgrass determined by 16S rRNA gene profiling. Front. Microbiology 2014 Jul 22;5:307. doi: 10.3389/fmicb.2014.00307.
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Progress 05/15/13 to 05/14/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Postdoctoral training for one scientist (Dr Emily Henriksen) and PhD training for two gradutae students (Mr Inhyuk Kwon and Mr Michael Iakiviak). How have the results been disseminated to communities of interest? Through peer-reviewed scientific publications in high-impact journals and throughinvited presentations at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. The model rumen Firmicute Ruminococcus albus 8 was grown using ammonia, urea, or peptides as the sole N source; growth was not observed with amino acids as the sole nitrogen source. Growth of R. albus 8 on ammonia and urea showed similar growth rates (0.08 h-1) and maximum cell densities (ammonia, OD600 1.01; urea, OD600 0.99). However, growth on peptides resulted in a nearly identical growth rate (0.09 h-1) and lower maximum cell density (OD600 0.58). To identify differences in gene expression and enzyme activities, the transcript abundance of ten different genes involved in nitrogen metabolism and specific enzyme activities were analyzed by harvesting mRNA and crude protein from cells at mid- and late-exponential phases of growth on the different N sources. Transcript abundance and enzyme activities varied according to nitrogen source, ammonia concentration, and growth phase. Growth of R. albus 8 on ammonia and urea was similar, with the only observed difference being an increase in urease transcript abundance and enzyme activity in urea-grown cultures. Growth of R. albus 8 on peptides showed a different nitrogen metabolism pattern, with higher gene transcript abundance levels of gdhA, glnA, gltB, amtB, glnK, and ureC as well as higher activities of glutamate dehydrogenase and urease. These results demonstrate that ammonia, urea, and peptides can all serve as nitrogen sources for R. albus, and nitrogen metabolism genes and enzyme activities of R. albus 8 are regulated by nitrogen source and the level of ammonia in the growth medium. 2. Spotlight: Articles of significant interest selected from this issue by the Editors. Nitrogen metabolism in predominant rumen bacteria differs from the enteric paradigm. Nitrogen utilization and metabolism in commensal gut bacteria is highly important to gut function and health but our understanding of this process is superficial. Using a genomic-based approach, we investigated gene expression and enzyme activities involved in nitrogen metabolism for Ruminococcus albus (AEM00029-14), a predominant plant cell wall degrading anaerobic bacterium in the rumen and other gut ecosystems. We showed growth on the preferred nitrogen source (ammonium) as well as urea and peptides but not amino acids. Interestingly, several gene transcript abundance patterns on ammonia and urea were contrary to those previously observed in enteric bacteria where GS is a scavenging system highly up-regulated under low environmental ammonia concentrations. 3. Xylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacterium Bacteroides intestinalis DSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. The xyn8A gene encodes an endoxylanase (Xyn8A), and rex8A encodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose(X5) and xylohexaose (X6) to a mixture of xylobiose (X2) and xylotriose (X3), while Rex8A hydrolyzed X3 through X6 to a mixture of xylose (X1) and X2. Moreover, rex8A is located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit beta-xylosidase activity and would be able to further hydrolyze X2 to X1. Mutational analyses of putative active site residues of bothXyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among the Bacteroidetes, indicating that these genes contribute to xylan utilization in the human gut.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Kim, J.N., Henriksen, E.D., Cann, I.K.O. and Mackie, R.I Nitrogen Utilization and Metabolism in Ruminococcus albus 8. Appl. Environ. Microbiol. 80:3095-3102.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Articles of significant interest selected from this issue by the Editors:
Nitrogen metabolism in predominant rumen bacteria differs from the enteric paradigm. Appl. Environ. Microbiol. 80:2965.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Hong, P.Y., Iakiviak, M., Dodd, D., Zhang, M., Mackie, R.I. and Cann, I.K.O. Two new xylanases with different substrate specificities from the human gut bacterium Bacteroides intestinalis DSM 17393. Appl. Environ. Microbiol. 80:2084-2093.
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Progress 05/15/12 to 05/14/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Early results of this work were communicated at the 2013 Congress on Gastrointestinal Function in Chicago, Illinois. A manuscript is in preparation that includes these genomic analyses as well as physiological studies to confirm genetic findings. In the course of this experimentation, the postdoctoral researcher working on this project received significant training in culturing of anaerobic microorganisms and maintenance of anaerobic chambers and gassing stations. We will continue the project, simultaneously addressing Specific Objectives 1 and 2 by establishing mono-, bi-, and tri-cultures with P. ruminicola, S. ruminantium, and our newly isolated Methanobrevibacter and employing RNA-Seq to study the individual and meta-transcriptomes. Following these experiments, Specific Objective 3 will addressed by performing metabolomic and enzymatic assessments of the cultures and integrating this data with that gleaned in the transcriptomic studies. How have the results been disseminated to communities of interest? Early results of this work were communicated at the 2013 Congress on Gastrointestinal Function in Chicago, Illinois. A manuscript is in preparation that includes these genomic analyses as well as physiological studies to confirm genetic findings. What do you plan to do during the next reporting period to accomplish the goals? We will continue the project, simultaneously addressing Specific Objectives 1 and 2 by establishing mono-, bi-, and tri-cultures withP. ruminicola,S. ruminantium, and our newly isolatedMethanobrevibacterand employing RNA-Seq to study the individual and meta-transcriptomes. Following these experiments, Specific Objective 3 will addressed by performing metabolomic and enzymatic assessments of the cultures and integrating this data with that gleaned in the transcriptomic studies.
Impacts
What was accomplished under these goals?
Early studies revealed that laboratory evolution of Methanobrevibacter ruminantium M1 has dramatically changed its growth rate, precluding use of this strain in further experimentation. We have isolated four new Methanobrevibacter strains from ruminal fluid and will sequence their genomes. The Methanobrevibacter isolate with a growth rate most similar to Prevotella ruminicola and Selenomonas ruminantium will be used for further mono-, bi-, and tri-culture transcriptomic and metatranscriptomic experiments. Comparative genomic analyses of Selenomonas ruminantium GA192 and HD4 have revealed many exciting features, including the presence of dissimilatory sulfate reduction genes, a physiology never before described in these organisms.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
Kim, J.N., E.D. Henriksen, I.K.O. Cann, J.J. Loor, and R. I. Mackie. 2013. Ammonia Utilization and Metabolism in Ruminococcus albus 8. Appl. Environ. Microbiology.
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