Microbio Lec 2 - Physio Of Bacterial Growth

MICROBIOLOGY LECTURE 2 – Physiology of Bacterial Growth, Genetic Exchange and Variation - Dr. Enrique H. Carandang Notes from Lecture USTMED ’07 Sec C – AsM

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GROWTH

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A. B.

c.

orderly increase of all chemical constituents of the cell entails the replication of all cellular structures, organelles and protoplasmic components from the nutrients present in the surrounding environment

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cannot tolerate oxygen due to the absence of catalase and superoxide dismutase to neutralize harmful form of oxygen

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Increase in cell size – when nuclear division is not accompanied by cell division (coenocytic) Increase in cell number – when microorganism reproduce by processes like budding or binary fission (one parent cell gives rise to two progeny cells)

d.

only anaerobic growth (e.g. Clostridium spp., Bacteroides spp., Fusobacterium) Aerotolerant anaerobes

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Requirements for growth source of energy o light – Photo o Chemical Reaction – Chemo A. Nutrient Requirements 1. Carbon - classification of bacteria according to carbon source: a. autotrophic bacteria (litotrophs)

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utilize oxygen to generate energy by respiration if it is present, but they can use the fermentation pathway to synthesize ATP in the absence of sufficient oxygen (e.g. E. coli) Obliogate anaerobes

e.

only anaerobic growth but tolerate presence of oxygen due to the presence of one enzyme, SOD allowing harmful forms of oxygen to be partially neutralized (e.g. Enterococcus faecalis) Microaerophiles  requires oxygen in low concentration

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exposure to normal atmospheric oxygen is toxic (e.g. Campylobacter)

require only water, inorganic salts and CO2

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b.

synthesize essential organic metabolites from CO2 heterotrophic bacteria (organotrophs)  require organic form of carbon for growth  organic materials e.g. proteins, carbohydrates and lipids

Source of Carbon

Energy Source

Classification of Bacteria

CO2 CO2 Organic material Organic material

Light Chemical reaction Light Chemical reaction

Photolithotroph Chemolithotroph Photoorganotroph chemoorganotroph

2.

Oxygen o For most organism, adequate supply of oxygen enhances metabolism and growth o Oxygen acts as hydrogen acceptor in the final steps of energy production catalyzed by flavoproteins and cytochromes o bacteria needs 2 enzymes to utilize oxygen since the use of oxygen generates 2 toxic molecules: hydrogen peroxide (H2O2) and the radical superoxide (O2) o Possible targets for damage by H2O2 and O2 includes:  Specific outer membrane proteins  Redox active components of the cytoplasmic membrane  Enzymes in periplasmic space o Two enzymes needed by bacteria to utilize oxygen: a. Superoxide dismutase – to catalyze the reaction: b.

2 O2 + 2 H+ H2O2 + O2 Catalase – to catalyze the reaction 2 H2O2

2 H2O + O2

Classification of Bacteria according to oxygen requirement a. Obligate aerobes

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require oxygen for growth because their ATP- generating system is dependent on oxygen as the hydrogen acceptor

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b.

Inorganic ions Sulfur

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o

3.

presence of enzyme catalase and superoxide dismutase allows toxic forms of oxygen to be neutralized (e.g. M. tuberculosis) Facultative anaerobes

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capable of growth under both aerobic and anaerobic conditions but greater growth in the presence of oxygen presence of catalase and superoxide dismutase to neutralize toxic forms of oxygen

Phosphorus Potassium Magnesium Calcium Iron Sodium Chloride Zinc Manganese Molybdenum Selenium

Growth Factors o classification of Bacteria according to Growth factors requirement a. Prototrophic – can synthesize their own growth factor b. Auxotrophic – needs exogenous source of growth factors  B complex vitamins, amino acids, purines and pyrimidines (from yeast extract and whole blood) o Inorganic ions Function in metabolisms Constituent of S-containing amino acids, cysteine, methionine, thiamine pyrophosphate, coenzyme A, biotin, and α-lipoic acid Constituent of nucleic acids, phospholipids, nucleotides Major inorganic cation, cofactor (e.g. pyruvate kinase) Cofactor of many enzymes (e.g. kinases); component of cell walls, membranes, ribosomes, and phosphate esters Component of exoenzymes (amylases, protease) and cell walls; major component of endospores as Ca-dipicolinate Present in cytochromes, ferredoxins, and other iron-sulfur proteins; cofactor (dehydratases) Transport Important inorganic anion Component of the enzymes alcohol dehydrogenase, alkaline phosphatase aldolase, RNA and DNA polymerase Present in superoxide dismutase; cofactor of the enzymes PEP carboxykinase isocitrate synthase Present in nitrate reductase, nitrogenase, xanthine dehydrogenase and formate dehydrogenase Component of glycine reductase and formate

dehydrogenase Required element in coenzyme B12-containing enzyme (glutamate mutase, methylmalonyl-CoA mutase) Present in cytochrome oxidase and nitrite reductase

Cobalt Copper

2.

4.

5.

B.

Carbon Dioxide o in addition to lithotrophs, organotroph also have a requirement for adequate supply of carbon dioxide for heterotrophic CO2 fixation and synthesis of fatty acids o some bacteria (Neisseria and Brucella) require higher concentration (10%) of CO2 for growth Nitrogen o NH4+ is the source of the nitrogen atom of amino acids, purines, pyrimidines, and other biomolecules

Physical Requirements 1. Temperature: o For each bacterium, there is an optimal temperature at which the organism grows most rapidly and a range of temperatures over which growth can occur o Classification of Bacteria based on Temperature requirement

Type Psychrophilic Mesophilic Thermophilic

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Temp. Range -5oC to 30oC 10oC to 45oC 25oC to 80oC

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TRANSPORT SYSTEMS

1. Porin and Maltose Channels Porin Channel not energy dependent relatively non-specific allow passage of hydrophilic solutes with a molecular weight of 600 daltons or less

Facilitated diffusion - involves a specific membrane protein which facilitates the rapid equilibrium of substrate across the cell membrane typified by the process of glycerol uptake

3.

Phosphenolpyruvate: sugar • Phosphotransferase System (PTS) mediates group translocation - consist of 4 proteins a. Enzyme 1 b. HPr (Histidine-containing Proteins)

Optimum Temperature 10oC to 20oC 20oC to 40oC 50oC to 60oC

Phosphorylate

2.

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Hydrogen Ion Concentration Requirement o pH of the culture medium affects the growth rate o there is a wider range of pH over which growth can occur but there is also an optimal pH o bacteria possess extremely effective mechanisms for maintaining tight regulatory control over their cytoplasmic pH o In many organism, the pH varies by only 0.1 units per pH unit change in external pH o Classification of Bacteria Based on pH pH 6.5 – 7 7.5 – 8

4.

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Osmotic Conditions o concentration of osmotically active solutes inside a bacterial cell is in general, higher than the concentration outside the cell. o majority of bacteria are unusually osmotically tolerant except for the mycoplasma and other cell-wall defective microorganism o membrane derived oligosaccharides (MDO) is the principal source of fixed anion in the periplasmic space and acts to maintain the higher osmotic pressure and Donnan membrane potential of the periplasmic space. Oxidative Reduction Potenetials o critical factor whether growth of an inoculum will occur when transferred to a fresh medium. o for most media in contact with air, the Eh is about + 0.2 to + 0.4 V at pH 7

Uptake of Nutrients by Bacteria is made possible by 2 mechanisms: Secreted enzymes (Exoenzymes) - different types of proteins are located outside the cytoplasmic membrane in both gram + and gram

c. Enzyme II s specific d. Enzyme III sugars generally present in facultative anaerobes and anaerobic organism but not in obligate aerobes protein of PTS acts as chemoreceptor system permitting the organism to recognize sugar sustrates in the extracellular environment regulate the utilization of certain non-PTS substrate (glycerol, maltose, melibiose and lactose). has regulatory function (glucose effect or diauxic growth) Glucose effect or Diauxic Growth: o In culture media containing both a PTS and a non-PTS sugar, the PTS sugar is utilized before the induction of the catabolic systems for the non-PTA sugar.

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Active Transport (Substrate Translocation) requires participation of specific membrane associated proteins requires a source of energy

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Iron Uptake and Transport a. Siderophores o flow molecular weight ferric-specific ligands whose function is to supply iron to the cell o 2 types: catechols and hydroxamates b. Specific membrane receptor for siderophores

8.4 - 9

most pathogenic bacteria grow between pH 7.2-7.6

Maltose Channel not energy dependent lambda receptor involves with the passage of maltose and maltodextrins through the outer membrane

2.

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3.

1.

Membrane transport transfer of solutes from the external environment into the cytoplasm - passage of metabolites is accomplished by specific transport carrier system except that of water and NH 4 (passive diffusion)

Most human pathogens grow best at 37oC.

Type Acidophiles Neutrophile s Alkalophiles

negative bacteria degrades complex macromolecules of high molecular weight some are virulence factors of the organism secreting them

MICROBIAL GROWTH

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refers to the number of cells, not the size of the cell bacteria reproduce by binary fission (undergo exponential or logarithmic growth)

Exponential Growth Number of Cells

1

2

4

6

16

Exponential

20

21

22

23

24

Measurement of Bacterial Growth A. Direct Method plates counts filtration method most probable number method direct microscopic count (Petroff-Hansser cell counter) B. Indirect Method Turbidimetric metabolic Activity determination dry wt. measurement

Process Type of Cell involved Nature of DNA transferred 2.

Transduction

Process Type of Cell involved Nature of DNA transferred

3. I. Lag Phase cell division does not take place no net increase in mass cell is synthesizing new components

DNA transferred from one bacterium to another Prokaryotic Chromosomal or plasmic

DNA transferred by a virus from one cell to another Prokaryotic any gene in generalized transduction, only certain genes in specialized transduction

Transformation

Process Type of Cell involved Nature of DNA transferred

Purified DNA taken up by a cell Prokaryotic or eukaryotic (human) Any DNA

II. Exponential (log) Phase microorganisms are growing and dividing at maximal rate - rate of growth is constant (balance growth) - bacterial population is most uniform in terms of chemical and physiologic properties (culture used in biochemical and physiologic studies). III. Stationary Phase - total number of viable microorganisms remain constant due to: o nutrient limitation o limited O2 availability o accumulation of toxic waste products o change in pH attained by bacteria at a population level of around 109 cells per ml. IV. Death Phase - death of microbial population is usually logarithmic (constant proportion of cell dies every hour) - brought about by total depletion of nutrients and building of toxic waste. GENETICS, VARIATION AND EXCHANGE

Mutation

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a change in the base sequence of DNA that usually results in insertion of a different amino acid into a protein and the appearance of an altered phenotype.

Three types of Molecular Changes in Mutation 1. Base substitution – when 1 base is inserted in the place of another a. Missence mutation - when the base substitution results in a codon that simply causes a different amino acid to be inserted b. Nonsense mutation - when the base substitution generates a termination codon that stops protein synthesis prematurely 2. Frame shift mutation - occurs when one or more base pairs are added or deleted, which shifts the reading frame on the ribosome and result in incorporation of the wrong amino acids “downstream” from the mutation and in the production of an inactive protein. 3. DNA integration of transposons or insertion sequences Mutations can be caused by: 1. Chemicals (nitrous acid, alkylating agents, benzpyrene) 2. Radiation (X-rays, UV light) 3. Viruses (mutation bacteriophage) Transfer of DNA within Bacterial Cells 1. Transposons - transfer DNA from one site on the bacterial chromosome to another site or to a plasmid 2. Programmed rearrangements – movement of a gene from a silent storage site where the gene is not expressed to an active site where transcription and translation occur. Transfer of DNA between Bacterial Cells 1.

Conjugation

The Mechanism of Bacterial Conjugation. (a) F+ x F- mating (b) Hfr x F(the integrated F factor is shown in color).

B.

the pathogenic properties of bacteria. Compatibility Two plasmids are said to be compatible if they can stably coexist in the same bacterial cell and incompatible if they cannot. Plasmids that are not related to each other in any way are usually compatible.

Barriers Reducing the transfer of Genes between different Species 1. Conjugal transfer between different species are presumably quite inefficient 2. Existence of restriction - modification system - destroy incoming DNA unless it has the fingerprint of a closely related bacteria. [email protected] [email protected]

Classification of Plasmids A. Genetic Content 1. Drug-resistance plasmid 2. Colicinogenic (col) plasmid - encode small proteins (colicins) that kill a variety of enteric bacteria related to the producing organism 3. Virulence plasmid - encode various proteins involved in