Bacterial Metabolism and Growth
미생물학교실
권 형 주
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…...
1) C-source : CO2, organic compounds
Nutritional classification : two parameters
- The nature of energy source, - The nature of principle carbon source
Energy
Light energy
(phototrophs)
Chemical energy
(Chemotrophs)
(Autotrophs)
Photoautotrophs
(higher plants, algae,
photosynthetic bacteria)
Chemoautotrophs
(Nitrifying bacteria, sulfur
oxidizer, Iron bacteria)
Organic carbon
source
(heterotrophos)
Photoheterotrophs
(Purple and green bacteria)
Chemoheterotrophs
(all metazoan animals,
protozoa, fungi, majority of
bacteria)
Carbon
CO2
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.
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
- 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
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)- Helicobacter pylori
- 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
4) Aeration : O2
- Obligate aerobes : aerobic respiration (oxygen : hydrogen acceptor):Mycobacterium tuberculosis
- 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
등
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
그람음성, 양성, 진균 등 다
양한 미생물의 배양 목적으
로 사용하는 배지로서
Soytone, Tryptone,질소원,
Vitamin 등의 영양물질을
함유.
Blood agar plate
미생물 배양과 분리
목적으로 사용하는 배
지로서 혈액을 첨가하
여 균종의 용혈성 확
인
Chocolate agar
Chocolate 한천배지는
기초배지(GC)에
hemoglobin,
Isovitales 등을 첨가한
증균배지로서
Haemophilus
influenzae와 같이 일반
배지에서는 배양이 어려
운 세균의 분리에 사용
MacConkey agar
그람음성간균의 선택배지
이면서 유당발효세균을 확
인하는 감별배지
Lactose fermentor : acid
producer red color
Blood agar plate
미생물을 배양과 분리 목적으로 사용하는배지로서 혈액을 첨
가하여 균종의 용혈성을 알 수 있음. 그러므로 혈액한천평판
배지는 영양강화 배지이면서 용혈성을 구분하는 배지임.
Streptococcus pyogenes의 용혈을 찾기 위해 이 배지를 사용
할 때는 배지 백금이 등으로 배지 바닥까지 여러번 천자한 후
배양. 천자된 부위는 산소압이 낮아져 산소에 불안정한
hemolysin O의 용혈을 증가시킴. S. pyogenes의 선별을 위해
서 혐기성 배양을 시행할 수 있음.
Metabolism and the conversion of energy
Metabolism : 세포내에서 발생되는 모든 생화학반응과 물질변형(transformation)
1) 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
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 potential
Schematic 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-, NO3O2: 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
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
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
: 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
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)