Bacterial Metabolism and Growth

미생물학교실권 형 주

Metabolic requirementsCultivation ( 배양 ) : 적당한 영양 및 환경 조건을 제공하여 미생물을 증식하는 과정 : Essential components - 1) Nutrients 2) Metabolic energy 3) Environmental factorsGrowth requirements 1) energy 2) Nutrition 3) IonsMetabolic energy 1) Fermentation 2) Respiration 3) Photosynthesis

Nutrition

Organic matter ( 유기물 ) 구성성분

inorganic ions ( 무기물 ): enzymatic catalysis, chemical gradients…...

Nutritional classification : two parameters - The nature of energy source, - The nature of principle carbon source

1) C-source : CO2, organic compounds

Light energy (phototrophs)

Chemical energy(Chemotrophs)

CO2

(Autotrophs)

Photoautotrophs(higher plants, algae, photosynthetic bacte-

ria)

Chemoautotrophs(Nitrifying bacteria, sulfur oxidizer, Iron

bacteria)Organic carbon

source (heterotrophos

)

Photoheterotrophs(Purple and green bac-

teria)

Chemoheterotrophs(all metazoan animals, protozoa, fungi, major-

ity of bacteria)

EnergyCarbon

2) O – source : O2, H2O, organic compounds3) H – source : H2, H2O, organic compounds4) N- source : protein, nucleic acids, NH3 Assimilation, dissimilation5) S – source : for biosynthesis of cysteine, Methionine, CoA, biotin, a-lipoic acid etc. SO4

2-, HS-, So, organic sulfur compounds6) P – source : for Nucleic acids, phospholipids, nucleotides, coenzymes, etc.

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, hydroxamatesGrowth factors : 미생물이 합성할 수 없어서 외부에서 영양분으로 공급해주어야 하는 물질 미생물이 스스로 합성할 수는 없으나 증식에 반드시 필요한 유기물 Amino acids, Purines, pyrimidines, Vitamins

- Prototrophs : glucose 와 같은 하나의 영양소로부터 증식에 필요한 탄수화물을 합성할 수 있는 미생물-Auxotrophs : 하나 또는 그 이상의 유기물을 스스로 합성하지 못하여 growth factor 를 필요로 하는 미생물

Use of Biochemical mutantsAuxotrophic mutants: require as growth factors biosynthetic intermediates thatthe parental strain can synthesize de novo

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/L2) Hydrogen ion concentration (pH) - Acidophils : pH 3.0 에서 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)- Helicobacter pylori - Alkaliphiles : pH 10.5 에서 최적성장 internal pH ~9.5 (external range, 9.0-11.0)

3) Temperature - Psychrophilic forms: 15-20oC - Mesophilic forms: 30-37oC - Thermophilics: 50-60oC

4) Aeration : O2- Obligate aerobes : aerobic respiration (oxygen : hydrogen acceptor):Mycobacterium tuberculosis 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: catalase 및 superoxide dismutase 가 없음 O2 is not reduced, No H2O2, O2

-

- Peptococcus anaerobius; 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, Vibrio parahaemolyticus- Osmophilic: high osmotic pressure

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 species선택배지 (selective media) : 분리하고자 하는 세균 이외의 다른 세균들의 성장을 억제하는 물질을 첨가하여 원하는 균만을 자라게 하는 배지

그람음성 , 양성 , 진균 등 다양한 미생물의 배양 목적으로 사용하는 배지로서 Soytone, Tryptone,질소원 , Vitamin 등의 영양물질을 함유 .

Nutrient agar Blood agar plate Chocolate agar MacConkey agar

그람음성간균의 선택배지이면서 유당발효세균을 확인하는 감별배지 Lactose fermentor : acid producer red color

미생물 배양과 분리 목적으로 사용하는 배지로서 혈액을 첨가하여 균종의 용혈성 확인

Chocolate 한천배지는 기초배지(GC) 에 hemoglo-bin, Isovitales 등을 첨가한 증균배지로서 Haemophilus in-fluenzae 와 같이 일반배지에서는 배양이 어려운 세균의 분리에 사용

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

Blood agar plate

Metabolism and the conversion of energyMetabolism : 세포내에서 발생되는 모든 생화학반응과 물질변형 (transformation)

1) Catabolism ( 이화작용 ) : Organic substrates 를 분해하면서 세포구성성분의 생합성에 필요한 에너지와 소단위 분자들을 생산하는 과정2) Anabolism ( 동화작용 ) : 이화작용에 의해 생산된 에너지와 소단위 분자들을 이용하여 새로운 세포 구성분자들을 (cellular constituents: cell walls, proteins, fatty acids, nucleic acids etc) 생합성하는 과정

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) Phosphoribosyl transferase purine/pyrimidine bases + PEP nocleoside-P + PPi

Metabolism of glucose

Generation of energy 1) substrate phosphorylation : 기질의 인산화

2) Oxidative phosphorylation (via electron transport chain) : oxidation-reduction potentialSchematic representation of an electron transport chain

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

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, SO4

2-, 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 accep-tors.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

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. 4-4Fig. 4-7

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

Biosynthesis -Peptidoglycan, lipopolysaccharide, protein, nucleic acids…….

Nucleic acid synthesis - ribose-5-phosphate - Purine nucleotide : inosine phosphate - guanosine, adenosine - Pyrimidine nucleotide : orotate - orotidine monophosphate - cytidine or uridine monophosphate

Transcription - DNA-dependent RNA polymerase : rifamycinTranslation 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

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)