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Nitrogen gas

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  1. Chromosomal DNA in 50% Glycerol Nitrogen gas

  2. 23.13 9.4 6.55 4.4 ~2-4 2.3 2.03 1.35 ~1-2 1.08 0.87

  3. Small-insert library (initial shotgun) Small-Insert clones (1-2 kb) @ 8 PSI Large-Insert clones (2-6 Kb) @ 6 PSI Large-insert library (walking clone) Cloning into pUC18

  4. Forward Reverse

  5. Spanning Clone

  6. Stop codon DNA strands ARTEMIS GC% window

  7. ARTEMIS

  8. Metabolic Schema

  9. COG search

  10. Motif search

  11. Signal search

  12. Excel

  13. Glycolysis Glucose ATP 2.7.1.1 ADP Glucose-6-P 5.3.1.9 Fructose-6-P ATP 2.7.1.11 ADP Fructose-1,6-bisP 4.1.2.13 5.3.1.1 Glyceraldehyde-3-P Dihydroxyacetonephosphate NAD++Pi 1.2.1.12 NADH+H+ 1,3-diphosphoglycerate ADP 2.7.2.3 ATP 3-phosphoglycerate 5.4.2.1 2-phosphoglycerate 4.2.1.11 H2O Phosphoenolpyruvate ADP 2.7.1.40 ATP Pyruvate

  14. Pentosephosphate Pathway 3.1.1.31 1.1.1.49 1.1.1.44 6-phosphoglucolactone 3-keto-6-phosphogluconate Glucose-6-P 6-phosphogluconate NADP+ NADP+ NADPH+H+ NADPH+H+ 1.1.1.44 CO2 5.1.3.1 Sedoheptulose-7-P Xylulose-5-P Erythrose-4-P + + Ribulose-5-P 2.2.1.1 2.2.1.2 Glyceraldehyde-3-P Ribose-5-P Fructose-6-P 5.3.1.6

  15. PEP Pyruvate Acetyl-CoA

  16. PEP 4.1.1.49 PEP PEP PEP PEP PEP PEP NADH+H+ + CO2 Pyruvate 4.1.1.3 Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Acetyl-CoA 1.1.1.40 Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA Oxaloacetate Citrate NAD+ NADH+H+ 4.1.3.7 Aconitate Malate 1.1.1.37 4.2.1.3 4.2.1.2 Fumarate Isocitrate FADH2 1.1.1.42 1.3.99.1 NADPH+H+ Oxalosuccinate Succinate 6.2.1.5 1.2.4.2 CO2 Succinyl-CoA a-ketoglutarate NADH+H+ CO2 Reductive Oxidative

  17. 5.3.1.8 2.7.1.2 Fructose-6-P Mannose-6-P Mannose ADP 2.7.1.11 ATP 2.7.1.56 2.7.1.69 Fructose-1,6-bisphosphate Fructose-1-P Fructose ADP PEP ATP 2.3.1.8 2.7.1.40 2.3.1.54 PEP Pyruvate acetyl-CoA acetyl-P ADP ATP Pi CoA-SH Formic acid 4.1.1.49 ADP NADH, H + 1.2.1.10 NADH H + 2.7.2.1 Oxaloacetate CoA + NAD+ ATP NADH, H + 1.1.1.27 1.1.1.37 1.18.99.1 acetaldehyde NAD+ NAD+ acetate Malate NADH, H + 1.1.1.1 NAD+ 4.2.1.2 H2O Lactate ethanol Fumarate NADH, H + H2 CO2 1.3.99.1 NAD+ Putative mixed-acid fermentation Succinate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate

  18. NADH dehydrogenase Fumarate reductase Fumarate NADH Nitrate reductase Formate dehydrogenase Nitrate Formate Ubiquinone / Menaquinone DMSO reductase Lactate dehydrogenase DMSO Lactate Nitrite reductase Nitrite Succinate dehydrogenase Succinate Cytochrome oxidase Oxygen Glycerol_P DH. Glycerol-3-P

  19. Assimilation of Nitrate Glutamate Glutamine synthase Nitrite reductase Nitrate reductase NO2- NH3 NO3- ATP H2O H2O Glutamine

  20. O || CH3-(CH2) n-CH2-CH2-C~OH b-oxidation ATP + CoA-SH AcylCoA synthetase (6.2.1.3) O || CH3-(CH3) n-CH2-CH2-C~SCoA AMP + PPi FAD Long-chain-acyl-CoA dehydrogenase (1.3.99.13) O || CH3-(CH2) n-CH=CH-C~SCoA FADH2 H2O Long-chain-enoyl-CoA hydratase (4.2.1.74) O || CH3-(CH2) n-CH-CH2-C~SCoA | OH 3-Hydroxyacyl-CoA dehydrogenase (1.1.1.211 ) NAD+ O || CH3-(CH2) n-CH-CH2-C~SCoA || O NADH + H+ beta-Ketothiolase (2.3.1.16) CoA-SH O || CH3-(CH2) n-1-C~SCoA + CH3-C~SCoA || O

  21. 6.4.1.2 Malonyl-CoA Acetyl-CoA 2.3.1.41 2.3.1.39 2.3.1.41 Malonyl-ACP Acetyl-ACP CO2 +ACP 3-ketoacyl-ACP NADPH, H+ 1.1.1.100 NADP+ 3-hydroxy-acyl-ACP 4.2.1.- H2O enoyl-acyl-ACP NADPH, H+ 1.3.1.9 NADP+ acyl-ACP Fatty acid Biosynthesis

  22. CTP PPi NADH+ H+ NAD+ 2 Fatty acyl-ACP Phosphatidic acid (PA) Dihydroxyacetone phosphate Glycerol 3-phosphate CDP-diacylglycerol 2.3.1.15 / 2.3.1.51 1.1.1.8 2.7.7.41 2.7.1.107 2.7.8.8 Glycerol-P CMP Serine ADP 2.7.8.5 CMP ATP mdoB Phosphatidylglycerol phosphate (PGP) Phosphatidylserine (PS) Diacylglycerol H2O MDO MDO-P-glycerol 3.1.3.27 4.1.1.65 Pi Phosphatidylglycerol (PG) CO2 PG Phosphatidylethanolamine (PE) 2.7.8.- Glycerol Phospholipids Synthesis diphosphatidylglycerol phosphate (Cardiolipin) (CL)

  23. Histidine Biosynthesis in E. coli 3.6.1.31 2.4.2.17 Phosphoribosyl pyrophosphate (PRPP) 2.7.6.1 N’-(5’-phosphoribosyl)-ATP N’-(5’-phosphoribosyl)-AMP Ribose 5-P 3.5.4.19 5.3.1.16 Phosphoribosyl formimino-5-aminoimidazole 4-carboxamide ribonucleotide Pro-phosphoribosyl formimino-5-aminoimidazole 4-carboxamide ribonucleotide HisH Imidazoleglycerol phosphate 4.2.1.19 COG0241 2.6.1.9 3.1.3.15 1.1.1.23 Histidine Imidazoleacetol phosphate histidinol phosphate histidinol

  24. Cysteine, Glycine, and Serine Biosynthesis 1.1.1.95 2.6.1.52 3-phosphoglycerate phosphohydroxypyruvate Phosphoserine NAD + NADH+H+ a-ketoglutarate Glu 3.1.3.3 H2O Pi 2.3.1.30 L-Serine O-acetylserine CoA Acetyl-CoA H2S THF 4.2.99.8 2.1.2.1 Acetic acid Methylene-THF + H2O L-Glycine L-Cysteine

  25. PEP + erythrose 4-P 5-enolpyruvylshikimate 3-P 4.1.2.15 4.6.1.4 4.2.1.51 2.6.1.5 5.4.99.5 L-phenylalanine Chroismate Prephenate Phenylpyruvate 3-deoxy-D-arabino-heptulosonate 7-P 2.4.2.18/4.1.3.27 4.6.1.3 Anthralinate 1.3.1.12 3-dehydroquinate 2.4.2.18 4.6.1.3 2.5.1.19 Phosphoribosyl anthralinate 4.2.1.20 3-dehydroshikimate 4-(OH) phenylpyruvate L-tyrosine 4.1.1.48 4.2.1.10 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate 3-dehydroshikimate 1.1.1.25 4.1.1.48 Shikimate Indologlycerol phosphate 2.7.1.71 Aromatic amino acid synthesis 4.2.1.20 Shikimate 3-P L-tryptophan

  26. Glutamate, Glutamine, Aspartate, Asparagine, and Alanine Biosynthesis Glutamate dehydrogenase (EC 1.4.1.4) a-ketoglutarate Glutamate NADPH+H++NH3 NADP++H2O Glutamine sythetase (EC 6.3.1.2) Glutamate Glutamine ATP+NH3 ADP+Pi Aspartate aminotransferase (EC 2.6.1.1) Oxaloacetate Aspartate a-ketoglutarate Glutamate Asparagine synthetase (asnA) (EC 6.3.1.1) Aspartate Asparagine NH3 + ATP AMP + PPi Alanine transaminase (EC 2.6.1.2) Pyruvate Alanine a-ketoglutarate Glutamate

  27. 2.7.2.4 1.2.1.11 1.1.1.3 2.7.1.39 4.2.99.2 Aspartate semialdehyde Threonine Aspartate Aspartyl-P Homoserine-P Homoserine Pyruvate 4.2.1.52 2.3.1.36 H2O 2.3.1.31 O-succinyl homoserine Dihydrodipicolinate NADPH, H + 1.3.1.26 4.2.99.9 NADP+ O-Acetyl-L-homoserine tetrahydrodipicolinate Succinyl-CoA LL-cystathionine 2.3.1.117 CoA 4.2.99.9 4.4.1.8 N-succinyl-a-amino-S-keto-pimelate Glutamate homocysteine 2.6.1.17 a-ketoglutarate 4.2.99.10 N-succinyl diaminopimelate 2.1.1.14 H2O Methionine 3.5.1.18 Succinate LL-Diaminopimelate 5.1.1.7 Lysine, Threonine, and Methionine Biosynthesis Meso-diaminopimelate 4.1.1.20 CO2 L- lysine

  28. Biosynthesis of Branched-Chain Amino Acids Threonine 4.2.1.16 a-isopropylmalate Pyruvate a-ketobutyrate Pyruvate 4.2.1.33 4.1.3.18 H2O CoA CO2 a-aceto-a-hydroxybutyrate dimethylcitraconate a-acetolactate NADPH, H + 4.1.3.12 4.2.1.33 H2O 1.1.1.86 NADP+ b-isopropylmalate a,b-dihydroxy-b-methylvalerate a,b-dihydroxy-isovalerate NAD + Acetyl-CoA 1.1.1.85 NADH, H+ 4.2.1.9 a-ketoisocaproate a-keto-b-methylvalerate a-keto isovalerate Glutamate 2.6.1.42 a-ketoglutarate Glutamate 2.6.1.42 L-leucine a-ketoglutarate L-isoleusine L-valine

  29. a-ketoglutarate L-Orntithine 1.4.1.4 2.1.3.3 2.7.2.11 Glutamate 2.3.1.1 Citrulline Glutamyl-P N-acetylglutamate 6.3.4.5 1.2.1.41 2.7.2.8 Argininosuccinate N-acetyl-g-glutamylphosphate Glutamate-5-semialdehyde 3.5.1.16 4.3.2.1 1.2.1.38 N-acetyl\glutamate-g-semialdehyde L-arginine Pyrroline-5-carboxylate 2.6.1.69 1.5.1.2 N-acetylornithine Arginine and Proline synthesis L-proline

  30. Pyrimidine Biosynthesis Pathway 1.3.3.1 6.3.4.16 2.1.3.2 3.5.2.3 Carbamoyl-P Carbamoyl-aspartate dihydroorotate orotate HCO3 ADP 2.7.2.2 2.7.6.1 5-phosphoribosyl-pyrophosphate (PRPP) Ribose 5-phosphate ATP + NH3+CO2 2.4.2.10 6.3.4.2 2.7.4.6 2.7.4.- 4.1.1.23 2.7.1.48 Orotidine monophosphate CDP CTP UTP UDP UMP 1.17.4.1 1.17.4.2 dUDP 1.17.4.2 2.7.1.48 dCTP dTDP dUTP dUMP dTMP dCDP 3.6.1.23 3.5.4.13 2.1.1.45 2.7.1.48 2.1.1.45 2.7.1.48 dTTP

  31. Pyrimidines Salvage pathway 2.7.1.48 Uridine UMP 3.5.4.5 2.4.2.3 2.4.2.9 Cytidine CMP cytosine uracil 3.5.4.1 2.7.1.48 Nucleotide transport systems C (nupC) uraA Cytidine/uridine Uracil

  32. Organization of the upp Operon uraA upp Hypothetical

  33. Purine Biosynthesis Pathway 5-phosphoribosyl-1-pyrophosphate 5-phosphoribosyl-amine 5-phosphoribosyl-glycineamide 5-phosphoribosyl-N-formylglycineamide 2.4.2.14 6.3.4.13 6.3.5.3 2.1.2.2 5-phosphoribosyl-4-(N-succinocarboxyamide)- 5-aminoimidazole 5-phosphoribosyl-5-aminoimidazole-4-carboxylic acid 5-phosphoribosyl-5-aminoimidazole 5-phosphoribosyl-N-formylglycineamidine 6.3.2.6 4.1.1.21 6.3.3.1 4.3.2.2 5-phosphoribosyl-4-carboxyamide-5-aminoimidazole (AICAR) 5-phosphoribosyl-4-carboxyamide-5-formaminoimidazole AMP IMP dAMP 2.1.2.3 3.5.4.10 1.17.4.1 4.3.2.2 1.1.1.205 GMP XMP dGMP 1.17.4.1 6.3.5.2

  34. Purine Salvage Pathway Succinyil- AMP AMP GMP IMP XMP 4.3.2.2 6.3.4.4 1.1.1.205 6.3.5.2 2.4.2.8 2.4.2.7 Deoxydenylate Adenine Adenylate Guanylate Guanine deoxyguanylate 2.4.2.1 2.4.2.1 2.4.2.1 2.4.2.1 Nucleotide transport systems C (nupC)

  35. Pantothenate, and CoA Valine 2.6.1.42 a-ketoisovalerate Holo-Acyl carrier protein Folate 2.1.2.11 COG3697 Tetrahydrofolate Apo-ACP Coenzyme A a-ketopantoate NADPH+H + ADP 1.1.1.86 2.7.1.24 ATP NADP+ pantoate Dephosphocoenzyme A PPi 6.3.2.1 2.7.7.3 ATP lpxC 2.7.1.33 pantothenate Pantetheine 4’-Phosphopantetheine PanF ATP 2.7.1.33 4.1.1.36 ADP 6.3.2.5 4’-Phosphopantothenate 4’-Phosphopantethenoylcysteine CMP + PPi CTP

  36. A B

  37. NA-PR transferase (NadV) PPi ATP NMN 2.7.7.1 PPi PRPP Anaerobic Aerobic Aspartate+dihydroxyacetone phosphate Aspartate+Formate Tryptophan Quinolinate 2.4.2.19 Nicotinic acid mononucleotide 2.7.7.18 Nicotinic acid adenine dinucleotide 6.3.5.1 PnuC Nicotinamide Nicotinamide NAD+ 3.1.3.- ATP PPi NADP+ NAD & NADP Biosythesis

  38. NadR domains A Nicotinamide mononucleotide-binding domain Nicotinamide mononucleotide adenylyltransferase B

  39. Alignment of NadV protein from H. ducreyi and A. actinomycetemcomitans

  40. http://www.ultranet.com/~jkimball/BiologyPages/

  41. A PEP EI HPr-P Pyruvate EI-P HPr PTS systems in A. actinomycetemcomitans Sugar C D C C a C B B P P A a P sugar-P A B P P P

  42. Biotin Biosynthesis Pimeloyl CoA Alanin 2.3.1.47 CO2+CoA 7-keto-8-aminopelargonate SAM 2.6.1.62 S-adenosyl-2-oxo-4-thiomethylbutyrate 7,8-diaminopelargonate ATP + CO2 6.3.3.3 ADP + Pi Dethiobiotin Cystein 2.8.1.6 Biotin

  43. Actinobacillus actinomycetemcomitans Haemophilus influenzae Escherichia coli Biotin synthesis operon Hypothetical bioA bioC bioF bioB bioD

  44. P- P- P- -P-NAM-NAG pep -P-P NAM-P- pep -P -P -P-NAM-NAG pep NAG-NAM-P- pep n+1 Peptidoglycan Biosynthesis Fructose 6-P 2.6.1.16 3.6.1.27 Glucoseamine 6-P 5.4.2.2 Acetyl-CoA 2.7.8.13 Glucosamine 1-phosphate 3.5.1.25 PBP CoASH UMP N-acetylglucoseamine 6 -P murG msbB 2.7.8.13 5.4.2.3 UDP N-acetylglucoseamine 1 -P murG UTP transglycosylase 2.7.7.23 PPi UDP-muramyl-pentapeptide (UDP-NAM-pep) UDP-N-acetylglucoseamine (UDP-NAG) murC murD murE murF PEP 5.1.1.3 5.1.1.1 6.3.2.4 2.5.1.7 Pi NADPH, H+ NADP+ UDP-N-acetylglucoseamine 3-enoylpyruvylether UDP-N-acetylmuramic acid 1.1.1.158

  45. 2.3.1.129 UDP-3-monoacyl-UDP-glucoseamine UDP-2,3-diacyl-UDP-glucoseamine 3.5.1.- 2.3.1.- UDP-3-monoacyl-UDP-NAG Acetate Acyl-ACP ACP Acyl-ACP ACP UDP UMP ADP ATP 2.4.1.182 UDP-2,3-diacyl-glucoseamine-1-P 2.4.99.- KDO2-Lipid IVA Disaccharide-1-P (Lipid X) Lipid IVA 2 CMP-KDO 2.3.1.- Lauroyl-ACP ACP 4.1.2.16 PPi 4.1.2.16 KDO KDO-8-P Arabinose-5-P Myristoyl-ACP CTP Pi PEP 2.3.1.- ACP KDO2-Lipid A Lipid A Biosynthesis UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG) UDP-N-acetylglucoseamine (UDP-NAG)

  46. Initiation DnaA HU FIS Chromosomal Segregation MukB ParA ParB MinD Priming Complex DnaB ssb GyrA GyrB DnaG DnaC Termination DNA repair ParC ParE XerC XerD 2.1.1.72 MutH MutL MutS MutU Tus RecJ ExoI A B

  47. O || B A O-CH3 | O-CH3 | 4.2.99.18 O6-methylguanine O4-methylthymine 2.1.1.63 3.1.11.1 O || 2.7.7.7 + 6.5.1.2

  48. DNA polymerase I + DNA Ligase UvrABC UvrD +