Bacterial Metabolism and Growth

1. Bacterial Metabolism and Growth 미생물학교실 권 형 주

2. Metabolic requirements Cultivation (배양) : 적당한 영양 및 환경 조건을 제공하여 미생물을 증식하는 과정 : Essential components - 1) Nutrients 2) Metabolic energy 3) Environmental factors Growth requirements 1) energy 2) Nutrition 3) Ions Metabolic energy 1) Fermentation 2) Respiration 3) Photosynthesis Nutrition Organic matter (유기물) 구성성분 inorganic ions (무기물): enzymatic catalysis, chemical gradients…...

3. 1) C-source : CO2, organic compounds Nutritional classification : two parameters - The nature of energy source, - The nature of principle carbon source Energy Carbon 2) O – source : O2, H2O, organic compounds 3) H – source : H2, H2O, organic compounds 4) N- source : protein, nucleic acids, NH3 Assimilation, dissimilation 5) S – source : for biosynthesis of cysteine, Methionine, CoA, biotin, a-lipoic acid etc. SO42-, HS-, So, organic sulfur compounds 6) P – source : for Nucleic acids, phospholipids, nucleotides, coenzymes, etc.

4. 7) Minerals Enzyme cofactors, cell wall, membrane, ribosome, RNA & DNA polymerase, etc. Iron : 10-8 M 이하의 농도에서 bacteria grow poorly, Most human tissue : less than 10-18M Most pathogens possess special mechanism for acquiring iron Siderophores : bind free iron tightly, : Fe2+와 친화성을 가진 작은 단백질로 세균이 감염숙주에서 Fe2+를 이용하는데 중요한 역할을 함 : Catechols, hydroxamates Growth factors : 미생물이 합성할 수 없어서 외부에서 영양분으로 공급해주어야 하는 물질 미생물이 스스로 합성할 수는 없으나 증식에 반드시 필요한 유기물 Amino acids, Purines, pyrimidines, Vitamins

5. - Prototrophs : glucose 와 같은 하나의 영양소로부터 증식에 필요한 탄수화물을 • 합성할 수 있는 미생물 • Auxotrophs : 하나 또는 그 이상의 유기물을 스스로 합성하지 못하여 • growth factor를 필요로 하는 미생물 Use of Biochemical mutants Auxotrophic mutants: require as growth factors biosynthetic intermediates that the parental strain can synthesize de novo

6. Environmental factors affecting growth - Nutrients, pH, Temperature, Aeration 1) Nutrients - Hydrogen donor acceptors : ~2g/L - Carbon source : ~ 1g/L - Nitrogen source : ~ 1g/L - Minerals: sulfur and phosphorus, ~ 50 mg/L Trace elements, 0.1-1 mg/L - Growth factors: amino acids, purines, pyrimidines , 50 mg/L Vitamins, 0.1-1mg/L 2) Hydrogen ion concentration (pH) - Acidophils : pH 3.0에서 최적 성장 internal pH ~6.5 (external range, 1.0-5.0) - Neutralophiles : pH 6.0-8.0 internal pH ~7.5 (external range, 5.5-8.5)-Helicobacterpylori - Alkaliphiles : pH 10.5에서 최적성장 internal pH ~9.5 (external range, 9.0-11.0) 3) Temperature - Psychrophilic forms: 15-20oC - Mesophilic forms: 30-37oC - Thermophilics: 50-60oC

7. 4) Aeration : O2 • Obligate aerobes : aerobic respiration (oxygen : hydrogen acceptor):Mycobacteriumtuberculosis 등 • Obligate anaerobes: Oxygen is a toxic substance : Clostridium, Bacteriods…… • Facultative aerobes (or anaerobes) : 대장균, 이질균등 • Aerobic metabolism 에서 Hydrogen peroxide(H2O2) 및 superoxide(O2-) 생산 • Hydrogen radical 생산: • Superoxide dismutase: • Catalase: • Obligate anaerobe: catalase및superoxide dismutase 가 없음 • O2 is not reduced, No H2O2, O2- • Peptococcusanaerobius; • NADH oxidase • Supply of O2 : shaking, pressure • Exclusion of O2 : reducing agent, 예) Thyoglycolate, cysteine, H2S • CO2 :몇 종류의 세균에서는 대사과정을 원활하게 하기 위하여 5-10% CO2 를 필요로 함 • 예) Haemophilus, Neisseria, Brucella, B. anthrax, etc. 5) Ionic strength & Osmotic pressure - Halophilic: high salt conc. Vibrio cholera, Vibrioparahaemolyticus - Osmophilic: high osmotic pressure

8. Cultivation methods • Growing cells of a given species • : simulate the natural environment, pH, Temperature, aeration…… • e.g) host tissues, brain heart infusion broth Growing cells of a given species 선택배지(selective media) :분리하고자 하는 세균 이외의 다른 세균들의 성장을 억제하는 물질을 첨가하여 원하는 균만을 자라게 하는 배지 Nutrient agar Blood agar plate Chocolate agar MacConkey agar 그람음성, 양성, 진균 등 다양한 미생물의 배양 목적으로 사용하는 배지로서 Soytone, Tryptone,질소원, Vitamin 등의 영양물질을 함유. Chocolate 한천배지는 기초배지(GC)에 hemoglobin, Isovitales 등을 첨가한 증균배지로서 Haemophilus influenzae와 같이 일반배지에서는 배양이 어려운 세균의 분리에 사용 미생물 배양과 분리 목적으로 사용하는 배지로서 혈액을 첨가하여 균종의 용혈성 확인 그람음성간균의 선택배지이면서 유당발효세균을 확인하는 감별배지 Lactose fermentor : acid producer red color

9. Blood agar plate 미생물을 배양과 분리 목적으로 사용하는배지로서 혈액을 첨가하여 균종의 용혈성을 알 수 있음. 그러므로 혈액한천평판배지는 영양강화 배지이면서 용혈성을 구분하는 배지임. Streptococcus pyogenes의 용혈을 찾기 위해 이 배지를 사용할 때는 배지 백금이 등으로 배지 바닥까지 여러번 천자한 후 배양. 천자된 부위는 산소압이 낮아져 산소에 불안정한 hemolysin O의 용혈을 증가시킴. S. pyogenes의 선별을 위해서 혐기성 배양을 시행할 수 있음.

10. Metabolism and the conversion of energy • Metabolism : 세포내에서 발생되는 모든 생화학반응과 물질변형(transformation) • Catabolism (이화작용) : Organic substrates 를 분해하면서 세포구성성분의 생합성에 필요한 • 에너지와 소단위 분자들을 생산하는 과정 • 2) Anabolism (동화작용) : 이화작용에 의해 생산된 에너지와 소단위 분자들을 이용하여 새로운 • 세포 구성분자들을 (cellular constituents: cell walls, proteins, fatty acids, nucleic acids • etc) 생합성하는 과정

11. Membrane transport • Passive transport : concentration gradient • Facilitated diffusion • : transfer mediated by specific membrane proteins : permeases(=carrier proteins) • : sugars…. • Active transport • : transfer a solute to enter the cell against a thermodynamically • unfavorable gradient of concentration • : Need a source of metabolic energy • 1) Binding proteins • : located in periplasm of gram-negative bacteria • : roles of binding proteins – binding to the substrate with high affinity • - interacting with carrier protein • 2) Secondary active transport • : transfer of a molecule across the membrane at the expense of a previously • established gradient of another molecule species • : Uniport, Symport, Antiport • 3) Group translocation • : substrate 가 세포막을 투과할 때 변화되어 (phosphorylation) 세포질내에 기질이 • 계속해서 축정 되게 함 • : Phosphotransferase system (PTS) ; phosphate bond 의 high energy 이용 • sugar (out) + PEP (in)  sugar-P (in) + pyruvate (in) • Phosphoribosyltransferase • purine/pyrimidine bases + PEP  nocleoside-P + PPi

12. Metabolism of glucose Generation of energy 1) substrate phosphorylation : 기질의 인산화 2) Oxidative phosphorylation (via electron transport chain) : oxidation-reduction potential Schematic representation of an electron transport chain

13. Heterofermentative lactic acid bacteria CO2 production by lactic acid bacteria in tubes of a sugar rich-medium with agar seals a) Streptococcus lactic, b) Leuconostoc mesenterroides

14. 2) Respiration (호흡) : 산화적 인산화에 의한 ATP 생산 - require a closed membrane (Fig. 4-5) • Oxidant (electron acceptor) : O2, CO2, SO42-, NO3- • O2: aerobic respiration (호기적호흡) • CO2, SO42-, NO3-: anaerobic respiration (혐기적호흡) • - Reductant (electron donor) : lactic acid, NADH…. The Eo’ values of various half reactions of primary electron donors, electron carriers, and terminal electron acceptors. H2 = 2H+ + 2e- (Eo’ = -0.42V) H2O = 1/2O2 + 2H+ + 2e- (Eo’ = +0.82V) Net reaction: H2 + 1/2O2 H2O DGo’ = -nFDEo’ DGo’= -2 x 23,000 [0.82-(-0.42)] = -57,040 cal

15. TCA (tricarboxylic acid) Cycle • Major mechanism for the generation of ATP • Serves as the final common pathway for the complete oxidation of amino acids, • fatty acids, and carbohydrates • Supplies key intermediates (a-ketoglutarate, pyruvate, oxaloacetate) for the ultimate • synthesis of amono acids, lipids, purines, and pyrimidines • : Amphibolic cycle • Fig. 4-4 • Fig. 4-7

16. Pentose phosphate pathway (Hexose monophosphate shunt) • - NADPH 생산 • metabolic building 의 전구체 생산: • ribose-5-phosphate, erythrose-4-phosphate • -Transketolase, transaldolase : Biosynthetic precursor, glycolytic pathway 에 이용되는 • 여러 종류의 sugar 생산

17. Biosynthesis • Peptidoglycan, lipopolysaccharide, protein, nucleic acids……. • Nucleic acid synthesis • - ribose-5-phosphate • - Purine nucleotide : inosine phosphate - guanosine, adenosine • - Pyrimidine nucleotide : orotate - orotidinemonophosphate • - cytidine or uridinemonophosphate • Transcription • - DNA-dependent RNA polymerase • : rifamycin • Translation • Fig. 4-9 • 70S ribosome : protein synthesis • Aminoglycosides(streptomycin, gentamicin), Tetracyclines • : binding to the small ribosomal subunits • Macrolide (erythromycin), lincosamide (clindamycin) • : binding to large ribosomal subunit

18. Bacterial growth • - Bacterial replication : Two equivalent daughter cells 생산 • (Fig. 4-10) • Alarmones production : Depletion of metabolites (starvation), toxic by-products (e.g. EtOH) • Population dynamics • (Fig. 4-11)