Transcript Differential expression of genes involved in

Differential expression of genes involved in
metabolic and regulatory pathways in
oxygen-limited M. smegmatis
Chris Rhodes, Nicki Harmon
Loyola Marymount University Department of Biology
BIOL 368, 12/07/11
Outline
• Mycobacteria show an extraordinary ability to survive in extreme
environmental conditions
• Studies by Berney et al. (2010) show M. smeg implements three
distinct responses to oxygen limited conditions
• Further study of the Berney et al. (2010) microarray data was
performed using statistical analysis techniques
• Statistical analysis shows:
– Down-regulation of NAD+/NADH dependent pathways
– Up-regulation of regulatory pathways
– Significant up-regulation of ferredoxin enzymes involved in TCA
cycle
• Differential expression data agree partially with previous
conclusions by Berney et al. (2010)
Mycobacteria have shown remarkable adaptability
to oxygen- and energy-limited environments
• Mycobacteria are a group of obligate aerobes which require
oxygen to grow, but are capable of surviving in anaerobic
environments
• Mycobacteria have also been show to survive in conditions of
nutrient deprivation
• This adaptability is indicative of a high degree of metabolic
flexibility within mycobacteria
• The mechanisms behind this flexibility have not been
adequately studied
Berney et al. shows M. smeg adopts 3 different
responses in order to adapt to low oxygen conditions
1. Oxygen scavenging:
– Up-regulation of cytochromes which procure and conserve oxygen in
the cell
2. Up-regulation of NAD+/NADH independent enzymes:
– Ferredoxin reducing and oxidizing enzymes power TCA cycle
independent of NAD+/NADH conserving energy
3. Up-regulation of hydrogenases:
– Hydrogenases carry out metabolic functions while conserving and
recycling energy and oxygen
Statistical analysis was performed using gene
expression data from Berney et al. (2010)
• Microarrays were performed using 5 biological replicates from
2.5% and 50% oxygen level experiment with dye swap chips
– 50% Oxygen Level  Cys3
– 2.5% Oxygen Level  Cys5
• For each microarray the Average log2-value, p-value, and Tstat
were calculated
• This data was run through MAPPFinder in order to find
differentially expressed ontologies
• The most significantly increased and decreased ontologies
were found and compared to the results of Berney et al.
(2010)
Outline
• Mycobacteria show an extraordinary ability to survive in extreme
environmental conditions
• Studies by Berney et al. (2010) show M. smeg implements three
distinct responses to oxygen limited conditions
• Further study of the Berney et al. (2010) microarray data was
performed using statistical analysis techniques
• Statistical analysis shows:
– Down-regulation of NAD+/NADH dependent pathways
– Up-regulation of regulatory pathways
– Significant up-regulation of ferredoxin enzymes involved in TCA
cycle
• Differential expression data partially agree with previous
conclusions by Berney et al. (2010)
Significant genes represent a low
percentage of the entire genome
P<…
Number of Genes Under Threshold
0.05
215
0.01
42
0.001
3
Total Number of Genes
6541
Number of MAPPFinder Errors
221
The ten most differentially expressed genes
show no discernable pattern in function
Protein Name
P-Value
Function
Transcriptional Regulator LysR Family Protein
0.000589
Transcriptional Regulator
Linear Gramicidin Synthetase mbtf
0.000591
Antibiotic Synthesis
Putative Carboxylesterase/Lipase
0.000659
Lipid Hydrolysis
Probable Conserved Transmembrane Protein 0.001346
Transmembrane Protein
MmpL Protein
0.001619
Lipid Transport Protein
Taurine Transport System Permease Protein
TauC
0.002122
Taurine Transport Protein
Hypothetical Protein
0.002569
NA
[2Fe-2S] Binding Domain Protein
0.002674
Ferredoxin enzyme
Conserved Hypothetical Protein
0.003256
NA
Putative Oxygen-Independent
Coproporhyrinogen III Oxidase
0.003371
Oxidase Activity
Up-regulated ontologies are involved in cell
regulation pathways
• RNA Metabolic Process
– Regulation of metabolic pathways involving RNA
• Regulation of Translation
– Modulation of DNA dependent transcription
• Regulation of Nitrogen Compound Metabolic Processes
– Modulation of chemical pathways involving nitrogen compounds
• Regulation of Nucleic Acid Metabolism
– Modulation of chemical reactions involving nucleic acids
• Regulation of Macromolecule Biosynthetic Process
– Modulation of the processes of formation of macromolecules
Up-regulated ontologies are involved in cell
regulation pathways
• Regulation of Gene Expression
– Modulation of processes involved in gene expression
• Regulation of Cellular Macromolecule Biosynthetic Process
– Modulation of the cellular macromolecule biosynthetic process
• Regulation of Cellular Biosynthetic Process
– Modulation of the formation of cellular substances
• Regulation of Biosynthetic Process
– Modulation of the formation of substances
• Regulation of Macromolecule Metabolic Process
– Modulation of the macromolecule metabolic process
Down-regulated ontologies are involved in NAD+/NADH
dependent pathways
• Fructuronate Reductase Activity
– Catalyses: D-mannonate + NAD+ = D-fructuronate + H+ + NADH
• Precorrin-3B C17-methyltransferase Activity
– Catalyses: S-adenosyl-L-methionine + precorrin-3B = S-adenosyl-Lhomocysteine + precorrin
• Precorrin-2 C20-methyltransferase Activity
– Catalyses: S-adenosyl-L-methionine + precorrin-2 = S-adenosyl-Lhomocysteine + H+ + precorrin-3A
• Coniferyl-aldehyde Dehydrogenase Activity
– Catlyses coniferyl aldehyde + H2O + NADP+ = ferulate + NADPH + H+
• Inositol-Monophosphatase Activity
– Catalyses: myo-inositol phosphate + H2O = myo-inositol + phosphate
Down-regulated ontologies are involved in NAD+/NADH
dependent pathways
• Trans-2-enoyl-CoA Reductase NADPH Activity
– Catalyses: acyl-CoA + NADP+ = trans-2,3-dehydroacyl-CoA + NADPH + H+
• Aldehyde Dehydrogenase [NADP+] Activity
– Catalyses: aldehyde + NADP+ + H2O = an acid + NADPH + H+
• Oxidoreductase Activity
– Catalysis of redox reactions relating to energy functions such as
NAD+/NADH
• Inositol Phosphatase Activity
– Inositol phosphate(n) + H2O = inositol phosphate(n-1) + phosphate
• Cellular Aldehyde Metabolic Process
– Cellular chemical reactions and pathways involving aldehydes
Outline
• Mycobacteria show an extraordinary ability to survive in extreme
environmental conditions
• Studies by Berney et al. (2010) show M. smeg implements three
distinct responses to oxygen limited conditions
• Further study of the Berney et al. (2010) microarray data was
performed using statistical analysis techniques
• Statistical analysis shows:
– Down-regulation of NAD+/NADH dependent pathways
– Up-regulation of regulatory pathways
– Significant up-regulation of ferredoxin enzymes involved in TCA
cycle
• Differential expression data partially agrees with previous
conclusions of Berney et al. (2010)
Analysis of gene expression data agrees with
certain findings of Berney et al. (2010)
• Berney et al. (2010) shows up-regulation of NAD+/NADH
independent enzymes coupled with down regulation of
NAD+/NADH dependent enzymes
• Berney et al. (2010) shows differential regulation of ferredoxin
usage and synthesis
• Berney et al. (2010) shows up-regulation of numerous regulatory
pathways
• Analysis shows no up-regulation of hydrogenases or oxygen
scavengers which conflicts with conclusions made by Berney et al.
(2010)
Summary
• Mycobacteria show a high degree of metabolic plasticity under
environmental stress
• Studies by Berney et al. (2010) show differential gene expression
in different oxygen-limited conditions
• Statistical analysis was performed to further study the Berney et
al. data
• Analysis showed up-regulation of regulatory pathway genes and
down-regulation of NAD+/NADH dependent enzymes
• Results correlate to similar conclusions made Bernie et al. (2010)
Acknowledgments
Kam D. Dahlquist, Ph. D
Berney, Michael, and Gregory M. Cook. "Unique Flexibility in
Energy Metabolism Allows Mycobacteria to Combat
Starvation and Hypoxia." Ed. David M. Ojcius. PLoS ONE 5.1
(2010): E8614. Print.