MICROBIOLOGY LECTURE 2 – Physiology of Bacterial Growth,  utilize oxygen to generate energy by

Genetic Exchange and Variation - Dr. Enrique H. Carandang respiration if it is present, but they
Notes from Lecture can use the fermentation pathway
USTMED ’07 Sec C – AsM to synthesize ATP in the absence of
sufficient oxygen (e.g. E. coli)
GROWTH
c. Obliogate anaerobes
- orderly increase of all chemical constituents of the cell
- entails the replication of all cellular structures,
 cannot tolerate oxygen due to the
absence of catalase and superoxide
organelles and protoplasmic components from the
dismutase to neutralize harmful
nutrients present in the surrounding environment
form of oxygen
A. Increase in cell size – when nuclear division is not  only anaerobic growth (e.g.
accompanied by cell division (coenocytic) Clostridium spp., Bacteroides spp.,
B. Increase in cell number – when microorganism reproduce Fusobacterium)
by processes like budding or binary fission (one parent d. Aerotolerant anaerobes
cell gives rise to two progeny cells)  only anaerobic growth but tolerate
presence of oxygen due to the
Requirements for growth presence of one enzyme, SOD
- source of energy allowing harmful forms of oxygen to
o light – Photo be partially neutralized (e.g.
o Chemical Reaction – Chemo Enterococcus faecalis)
A. Nutrient Requirements e. Microaerophiles
1. Carbon - classification of bacteria according to  requires oxygen in low concentration
carbon source:  exposure to normal atmospheric
a. autotrophic bacteria (litotrophs) oxygen is toxic (e.g. Campylobacter)
 require only water, inorganic salts
and CO2
 synthesize essential organic
metabolites from CO2
b. 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 Light Photolithotroph
CO2 Chemical reaction Chemolithotroph
Organic material Light Photoorganotroph
Organic material Chemical reaction 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 3. Growth Factors
o bacteria needs 2 enzymes to utilize oxygen o classification of Bacteria according to Growth
since the use of oxygen generates 2 toxic factors requirement
molecules: hydrogen peroxide (H2O2) and the a. Prototrophic – can synthesize their own
radical superoxide (O2) growth factor
o Possible targets for damage by H2O2 and O2 b. Auxotrophic – needs exogenous source of
includes: growth factors
 Specific outer membrane proteins  B complex vitamins, amino acids,
 Redox active components of the purines and pyrimidines (from yeast
cytoplasmic membrane extract and whole blood)
 Enzymes in periplasmic space o Inorganic ions
o Two enzymes needed by bacteria to utilize
oxygen: Inorganic Function in metabolisms
a. Superoxide dismutase – to catalyze the ions
reaction: Sulfur Constituent of S-containing amino acids, cysteine,
methionine, thiamine pyrophosphate, coenzyme
 2 O2 + 2 H+ H2O2 + O2
A, biotin, and α-lipoic acid
b. Catalase – to catalyze the reaction
Phosphorus Constituent of nucleic acids, phospholipids,
 2 H2O2 2 H2O + O2 nucleotides
Potassium Major inorganic cation, cofactor (e.g. pyruvate
o Classification of Bacteria according to oxygen kinase)
requirement Magnesium Cofactor of many enzymes (e.g. kinases);
a. Obligate aerobes component of cell walls, membranes, ribosomes,
 require oxygen for growth because and phosphate esters
their ATP- generating system is Calcium Component of exoenzymes (amylases, protease)
dependent on oxygen as the and cell walls; major component of endospores as
hydrogen acceptor Ca-dipicolinate
 presence of enzyme catalase and Iron Present in cytochromes, ferredoxins, and other
superoxide dismutase allows toxic iron-sulfur proteins; cofactor (dehydratases)
forms of oxygen to be neutralized Sodium Transport
(e.g. M. tuberculosis) Chloride Important inorganic anion
b. Facultative anaerobes Zinc Component of the enzymes alcohol
dehydrogenase, alkaline phosphatase aldolase,
 capable of growth under both
RNA and DNA polymerase
aerobic and anaerobic conditions
Manganese Present in superoxide dismutase; cofactor of the
but greater growth in the presence
enzymes PEP carboxykinase isocitrate synthase
of oxygen
Molybdenum Present in nitrate reductase, nitrogenase,
 presence of catalase and superoxide xanthine dehydrogenase and formate
dismutase to neutralize toxic forms dehydrogenase
of oxygen Selenium Component of glycine reductase and formate
 dehydrogenase negative bacteria
Cobalt Required element in coenzyme B12-containing - degrades complex macromolecules of high molecular
enzyme (glutamate mutase, methylmalonyl-CoA weight
mutase) - some are virulence factors of the organism secreting
Copper Present in cytochrome oxidase and nitrite them
reductase
2. Membrane transport
- transfer of solutes from the external environment into
4. Carbon Dioxide the cytoplasm
o in addition to lithotrophs, organotroph also - passage of metabolites is accomplished by specific
have a requirement for adequate supply of transport carrier system except that of water and NH 4
carbon dioxide for heterotrophic CO2 fixation (passive diffusion)
and synthesis of fatty acids TRANSPORT SYSTEMS
o some bacteria (Neisseria and Brucella) require 1. Porin and Maltose Channels
higher concentration (10%) of CO2 for growth Porin Channel Maltose Channel
not energy dependent not energy dependent
5. Nitrogen relatively non-specific lambda receptor
o NH4+ is the source of the nitrogen atom of allow passage of hydrophilic involves with the passage of
amino acids, purines, pyrimidines, and other solutes with a molecular maltose and maltodextrins
biomolecules weight of 600 daltons or less through the outer membrane

2. Facilitated diffusion
B. Physical Requirements - involves a specific membrane protein which facilitates
1. Temperature: the rapid equilibrium of substrate across the cell
o For each bacterium, there is an optimal membrane
temperature at which the organism grows - typified by the process of glycerol uptake
most rapidly and a range of temperatures over
which growth can occur 3. Phosphenolpyruvate: sugar
o Classification of Bacteria based on
• Phosphotransferase System (PTS)
Temperature requirement
- mediates group translocation
Type Temp. Range Optimum Temperature - consist of 4 proteins
Psychrophilic -5oC to 30oC 10oC to 20oC a. Enzyme 1
Mesophilic 10oC to 45oC 20oC to 40oC b. HPr (Histidine-containing Proteins)
Thermophilic 25oC to 80oC 50oC to 60oC Phosphorylate
c. Enzyme II s specific
d. Enzyme III sugars
• Most human pathogens grow best at 37oC. - generally present in facultative anaerobes and anaerobic
organism but not in obligate aerobes
2. Hydrogen Ion Concentration Requirement - protein of PTS acts as chemoreceptor system permitting
o pH of the culture medium affects the growth the organism to recognize sugar sustrates in the
rate extracellular environment
o there is a wider range of pH over which - regulate the utilization of certain non-PTS substrate
growth can occur but there is also an optimal (glycerol, maltose, melibiose and lactose).
pH - has regulatory function (glucose effect or diauxic
o bacteria possess extremely effective growth)
mechanisms for maintaining tight regulatory • Glucose effect or Diauxic Growth:
control over their cytoplasmic pH o In culture media containing both a PTS and a
o In many organism, the pH varies by only 0.1
non-PTS sugar, the PTS sugar is utilized before
units per pH unit change in external pH the induction of the catabolic systems for the
o Classification of Bacteria Based on pH non-PTA sugar.

Type pH 4. Active Transport (Substrate Translocation)

Acidophiles 6.5 – 7 - requires participation of specific membrane associated
Neutrophile 7.5 – 8 proteins
s - requires a source of energy
Alkalophiles 8.4 - 9
5. Iron Uptake and Transport
• most pathogenic bacteria grow between pH 7.2-7.6 a. Siderophores
o flow molecular weight ferric-specific ligands
3. Osmotic Conditions whose function is to supply iron to the cell
o concentration of osmotically active solutes o 2 types: catechols and hydroxamates
inside a bacterial cell is in general, higher b. Specific membrane receptor for siderophores
than the concentration outside the cell.
o majority of bacteria are unusually osmotically MICROBIAL GROWTH
tolerant except for the mycoplasma and other - refers to the number of cells, not the size of the cell
cell-wall defective microorganism - bacteria reproduce by binary fission (undergo
o membrane derived oligosaccharides (MDO) is exponential or logarithmic growth)
the principal source of fixed anion in the
periplasmic space and acts to maintain the Exponential Growth
higher osmotic pressure and Donnan
membrane potential of the periplasmic space. Number of Cells 1 2 4 6 16

4. Oxidative Reduction Potenetials Exponential 20 21 22 23 24

o critical factor whether growth of an inoculum
will occur when transferred to a fresh
Measurement of Bacterial Growth
medium.
A. Direct Method
o for most media in contact with air, the Eh is - plates counts
about + 0.2 to + 0.4 V at pH 7 - filtration method
- most probable number method
Uptake of Nutrients by Bacteria is made possible by 2 - direct microscopic count (Petroff-Hansser cell counter)
mechanisms: B. Indirect Method
- Turbidimetric
1. Secreted enzymes (Exoenzymes) - metabolic Activity determination
- different types of proteins are located outside the - dry wt. measurement
cytoplasmic membrane in both gram + and gram
 Process DNA transferred from one
bacterium to another
Type of Cell involved Prokaryotic
Nature of DNA transferred Chromosomal or plasmic

2. Transduction

Process DNA transferred by a virus from one

cell to another
Type of Cell involved Prokaryotic
Nature of DNA any gene in generalized transduction,
transferred only certain genes in specialized
transduction

3. Transformation
I. Lag Phase
- cell division does not take place Process
- no net increase in mass Type of Cell involved
- cell is synthesizing new components 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
- 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
The Mechanism of Bacterial Conjugation. (a) F+ x F- mating (b) Hfr x F-
b. Nonsense mutation - when the base substitution (the integrated F factor is shown in color).
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 pathogenic properties of bacteria.
B. 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.

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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