1. A.J O’Neil, J. A. Lindsay, K. Gould, J. Hinds, and I. Chopra
Transcriptional Signature following Inhibition of Early-Stage Cell Wall Biosynthesis in Staphylococcus aureus
A.J O’Neil, J. A. Lindsay, K. Gould, J. Hinds, and I. Chopra
(2009)Antimicrobial Agents and Chemotherapy :
Angela Garibaldi & Ryan Willhite
BIOL398-01/S10: Bioinformatics Laboratory
April 27, 2010

2. Outline Review of the Experimental Set up Cell Wall Biosynthesis
Review of the State I Biosynthesis Pathway
Results of GenMAPP, using MAPPfinder tool
Top 10 GO Terms
Relation of significant GO Terms to CWB
Conclusions

3. Experimental Design Inhibit the Mur enzymes (A/Z, B, and E)
3 Biological Replicates
2 Technical Replicates
18 hybridizations (6 per condition)
Dye swap design – label orientations are reversed

4. Overview of Cell Wall Biosynthesis
Biosynthesis catalyzed by
Mur A-F and MurG enzymes (1-4 steps)
Filled hexagons
Peptidoglycan
GlcNac: N-acetylglucosamine
MurNac: N-acetyl-muramic acid
Orange circles
Carrier lipids- bactoprenol-phosphate
Transglycosylation and pyrophosphorylation (steps 5&6)
Peptide bond between chains
Those in pink boxes are antibiotics and their ways of inhibiting cell-wall synthesis
Cell-wall biosynthesis: the intracellular steps of murein (peptidoglycan) biosynthesis are catalysed by the enzymes MurA–F and MurG (steps1–4). Peptidoglycan is a polymer of two hexoses (filled hexagons) — N-acetylglucosamine (GlcNac) and N-acetyl-muramic acid (MurNAc). Peptidoglycan units are transferred to a carrier lipid — bactoprenol-phosphate (orange circles) — which transports precursor molecules across the cell membrane, generating Lipids I and II. Sugars and phosphates are added by transglycosylation and pyrophosphorylation (steps 5 and 6), and finally, a peptide bond between the peptide chains is formed (step 7). Antibiotics that inhibit cell-wall synthesis are indicated.

5. Stage I Biosynthesis pathway
Fosfomycin interferes with the function of UDP-GlcNAc and therefore inhibits MurA/Z
Change from lower to higher temperature inhibits stops MurB from being produced
Pspac promoter in the presence of IPTG inhibits the production of MurE 5

6. Stage I Biosynthesis pathway
Fosfomycin interferes with the function of UDP-GlcNAc and therefore inhibits MurA/Z
Change from lower to higher temperature inhibits stops MurB from being produced
Pspac promoter in the presence of IPTG inhibits the production of MurE 6

7. MurB Decrease RNA Modification
Transferase activity- peptidal link formation
RNA processing- capping, polyadenylation and splicing of mRNA
Rna modification, tranferase activity, and RNA processing
Transferase activity- peptidal transferase activity forms the peptidal links during protein biosynthesis
RNA processing- involves the capping, polyadenylation and splicing of mRNA. Trying to generate mature mRNA for translation to create protein.

8. MurB Increase Antioxidant activity Biotin biosynthesis
Breaking chain reaction normally leading to biological damage
Biotin biosynthesis
Plays role in gluconeogenesis
Amide metabolism
Antioxidant activity-Inhibition of the reactions brought about by dioxygen (O2) or peroxides. Usually the antioxidant is effective because it can itself be more easily oxidized than the substance protected. The term is often applied to components that can trap free radicals, thereby breaking the chain reaction that normally leads to extensive biological damage.
Biotin biosynthesis-he chemical reactions and pathways resulting in the formation of biotin. Biotin is a coenzyme in the metabolism of fatty acids and leucine, and it plays a role in gluconeogenesis. Gluconeogenesis (abbreviated GNG) is a metabolic pathway that results in the generation of glucose from non-carbohydrate carbon substrates. Those with gluconeogenesis had thicker cell wall.
Amide metabolic -

9. Stage I Biosynthesis pathway
Fosfomycin interferes with the function of UDP-GlcNAc and therefore inhibits MurA/Z
Change from lower to higher temperature inhibits stops MurB from being produced
Pspac promoter in the presence of IPTG inhibits the production of MurE 9

10. MurE Decrease Translation RNA Binding Non-membrane bound organelle
Translation- process of which mRNA is turned into protein
RNA Binding- a molecular function. Interacting selectively and non-covalently with an RNA molecule or a portion thereof.
Non-membrane bound organelle- Organized structure of distinctive morphology and function, not bounded by a lipid bilayer membrane. Includes ribosomes, the cytoskeleton and chromosomes.

11. MurE Increase Amine biosynthetic process
Results in formation of organic compound with amino group
Carboxylic acid biosynthetic process
Formation of carboxylic acids
Cellular amine biosynthetic process
Naturally occuring biologically active amines
Amine bio process- The chemical reactions and pathways resulting in the formation of any organic compound that is weakly basic in character and contains an amino or a substituted amino group. Amines are called primary, secondary, or tertiary according to whether one, two, or three carbon atoms are attached to the nitrogen atom.
Carboxylic acid-The chemical reactions and pathways resulting in the formation of carboxylic acids, any organic acid containing one or more carboxyl (-COOH) groups.
Cellular amine-The chemical reactions and pathways occurring at the level of individual cells resulting in the formation of any of a group of naturally occurring, biologically active amines, such as norepinephrine, histamine, and serotonin, many of which act as neurotransmitters.

12. Stage I Biosynthesis pathway
Fosfomycin interferes with the function of UDP-GlcNAc and therefore inhibits MurA/Z
Change from lower to higher temperature inhibits stops MurB from being produced
Pspac promoter in the presence of IPTG inhibits the production of MurE 12

13. MurA Decrease

14. MurA Increase Polysacchairide transmebrane transporter activity
polysaccharide localization
Arsenate reductase activity
Poly transmembrane transport- Catalysis of the transfer of a solute or solutes from one side of a membrane to the other according to the reaction: ATP + H2O + capsular polysaccharide(in) = ADP + phosphate + capsular polysaccharide(out).
Poly localization- any process by which a polysaccharide is transported to, or maintained in, a specific location.
Arsenate reductase-Catalysis of the interconversion of arsenate and arsenite. Can be integrated in the cell wall.bacteria reduce this compound for energy. Use as electron donors.

15. Conclusions Not very conclusive results
Terms mostly involved with biological processes of a cell
Those decreased are essential to cell gene expression and functional protein synthesis, so still unclear on specific relationships