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 01309-08: 1701-1704 Angela Garibaldi & Ryan Willhite BIOL398-01/S10: Bioinformatics Laboratory April 27, 2010

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

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

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. http://images.google.com/imgres?imgurl=http://www.nature.com/nrmicro/journal/v1/n1/images/nrmicro727-f2.jpg&imgrefurl=http://www.nature.com/nrmicro/journal/v1/n1/fig_tabnrmicro727_F2.html

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

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

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.

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 -

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

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.

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.

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

MurA Decrease

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.

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