Unit 2: Bacteriology
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Lecture 3 - The Physiology of
Metabolism and Growth in Bacteria
Fine structure of Both: The Cell Wall of Gram-Positive Bacteria
(left) and the
Cell Wall
of Gram-Negative Bacteria (right)
Unit 2: Bacteriology
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Types of Metabolism
Metabolism is the totality of chemical reactions occurring
in bacterial cells. They can be subdivided into anabolic
(synthetic) reactions that consume energy and catabolic
reactions that supply energy. In the anabolic, endergonic
reactions, the energy requirement is consumed in the form
of light or chemical energy—by photosynthetic or
chemosynthetic bacteria, respectively. Catabolic reactions
supply both energy and the basic structural elements for
synthesis of specific bacterial molecules. Bacteria that
feed on inorganic nutrients are said to be lithotrophic,
those that feed on organic nutrients are organotrophic.
Human pathogenic bacteria are always chemosynthetic,
organotrophic bacteria (or chemo-organotrophs).
Catabolic Reactions
Organic nutrient substrates are catabolized in a wide
variety of enzymatic processes that can be schematically
divided into four phases:
Digestion
. Bacterial exoenzymes split up the nutrient
substrates into smaller molecules outside the cell. The
exoenzymes represent important pathogenicity factors in
some cases.
Uptake
. Nutrients can be taken up by means of passive
diffusion or, more frequently, specifically by active
transport through the membrane (s). Cytoplasmic
membrane permeases play an important role in these
processes.
Preparation for oxidation
. Splitting off of carboxyl
and amino groups, phosphorylation, etc.
Oxidation.
This process is defined as the removal of
electrons and H+ ions. The substance to which the H2
atoms are transferred is called the hydrogen acceptor.
The two basic forms of oxidation are defined by
the final hydrogen acceptor.
o Respiration. Here oxygen is the hydrogen acceptor. In
anaerobic respiration, the O2 that serves as the hydrogen
acceptor is a component of an inorganic salt.
o Fermentation. Here an organic compound serves as the
hydrogen acceptor. The main difference between
fermentation and respiration is the energy yield, which
can be greater from respiration than from fermentation for
a given nutrient substrate by as much as a factor of 10.
Fermentation processes involving microorganisms are
designated by the final product, e.g., alcoholic
fermentation, butyric acid fermentation, etc.
The energy released by oxidation is stored as chemical
energy in the form of a thioester (e.g., acetyl-CoA) or
organic phosphates (e.g., ATP).
The role of oxygen. Oxygen is activated in one of
three ways:
o Transfer of 4e
–
to O
2
, resulting in 2 oxygen ions (2 O2
–
).
o Transfer of 2e
–
to O
2
resulting in 1 peroxide anion(1 O2
2–
o Transfer of 1e– to O2, resulting in one superoxide anion
(1 O2 –).
Hydrogen peroxide and the highly reactive superoxide
anion are toxic and therefore must undergo further
conversion immediately.
Bacteria are categorized as the following
according to their O2-related behavior:
o Facultative anaerobes. These bacteria can oxidize
nutrient substrates by means of both respiration and
fermentation.
o Obligate aerobes. These bacteria can only reproduce in
the presence of O2.
o Obligate anaerobes. These bacteria die in the presence of
O2. Their metabolism is adapted to a low redox potential
and vital enzymes are inhibited by O2.
o Aerotolerant anaerobes. These bacteria oxidize nutrient
substrates without using elemental oxygen although,
unlike obligate anaerobes, they can tolerate it.
Basic mechanisms of catabolic metabolism.
The principle of the biochemical unity of life asserts that
all life on earth is, in essence, the same. Thus, the
catabolic intermediary metabolism of bacteria is, for the
most part, equivalent to what takes place in eukaryotic
cells. The reader is referred to textbooks of general
microbiology for exhaustive treatment of the pathways of
intermediary bacterial metabolism.
Anabolic Reactions
It is not possible to go into all of the biosynthetic feats of
bacteria here. Suffice it to say that they are, on the whole,
quite astounding. Some bacteria (E. coli) are capable of
synthesizing all of the complex organic molecules that
they are comprised of, from the simplest nutrients in a
very short time. These capacities are utilized in the field
of microbiological engineering. Antibiotics, amino acids,
and vitamins are produced with the help of bacteria. Some
bacteria are even capable of using aliphatic hydrocarbon
compounds as an energy source. Such bacteria can “feed”
on paraffin or even raw petroleum. Culturing of bacteria
Unit 2: Bacteriology
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in nutrient mediums based on methanol is an approach
being used to produce biomas in addition to many other
ones.
Metabolic Regulation: Bacteria are highly efficient
metabolic regulators, coordinating each individual
reaction with other cell activities and with the available
nutrients as economically and rationally as possible.
Growth and Culturing of Bacteria
Nutrients
The term bacterial culture refers to proliferation of
bacteria with a suitable nutrient substrate. A nutrient
medium in which chemoorganotrophs are to be
cultivated must have organic energy sources (H2
donors) and H2 acceptors. Other necessities include
sources of carbon and nitrogen for synthesis of specific
bacterial compounds as well as minerals such as
sulfur, phosphorus, calcium, magnesium, and trace
elements as enzyme activators. Some bacteria also
require “growth factors,” i.e., organic compounds they
are unable to synthesize themselves. Depending on the
bacterial species involved, the nutrient medium must
contain certain amounts of O2 and CO2 and have
certain pH and osmotic pressure levels. Growth
factors are required in small amounts by cells because
they fulfill specific roles in biosynthesis. The need for a
growth factor results from either a blocked or missing
metabolic pathway in the cells. Growth factors are
organized into three categories:
1. Purines and pyrimidines: required for synthesis of
nucleic acids (DNA and RNA).
2. Amino acids: required for the synthesis of proteins.
3. Vitamins: needed as coenzymes and functional groups
of certain enzymes.
Some bacteria (e.g E. coli) do not require any growth
factors: they can synthesize all essential purines,
pyrimidines, amino acids and vitamins, starting with their
carbon source, as part of their own intermediary
metabolism. Certain other bacteria (e.g. Lactobacillus)
require purines, pyrimidines, vitamins and several amino
acids in order to grow. These compounds must be added
in advance to culture media that are used to grow these
bacteria. The growth factors are not metabolized directly
as sources of carbon or energy, rather they are assimilated
by cells to fulfill their specific role in metabolism. Mutant
strains of bacteria that require some growth factor not
needed by the wild type (parent) strain are referred to as
auxotrophs. Thus, a strain of E. coli that requires the
amino acid tryptophan in order to grow would be called a
tryptophan auxotroph and would be designated E. colitrp-.
The function(s) of these vitamins in essential enzymatic
reactions gives a clue why, if the cell cannot make the
vitamin, it must be provided exogenously in order for
growth to occur.
Growth and Cell Death
Bacteria reproduce asexually by means of simple
transverse binary fission. Their numbers (n) increase
logarithmically (n = 2G). The time required for a
reproduction cycle (G) is called the generation time (g)
and can vary greatly from species to species. Fast-
growing bacteria cultivated in vitro have a generation
time of 15–30
minutes. The same bacteria may take
hours to reproduce in vivo. Obligate anaerobes grow
much more slowly than aerobes; this is true in vitro as
well. Tuberculosis bacteria have an in-vitro generation
time of 12–24 hours. Of course the generation time also
depends on the nutrient content of the medium.
The so-called normal growth curve for bacteria is
obtained by inoculating a nutrient broth with bacteria the
metabolism of which is initially quiescent, counting them
at intervals and entering the results in a semilog
coordinate system . The lag phase is characterized by an
increase in bacterial mass per unit of volume, but no
increase in cell count. During this phase, the metabolism
of the bacteria adapts to the conditions of the nutrient
medium. In the following log (or exponential) phase , the
cell count increases logarithmically up to about 109/ml.
This is followed by growth deceleration and transition to
the stationary phase due to exhaustion of the nutrients and
the increasing concentration of toxic metabolites. Finally,
death phase processes begin. The generation time can
only be determined during log phase, either graphically or
by determining the cell count (n) at two different times.
Normal Growth Curve of a Bacterial Culture
Unit 2: Bacteriology
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The summary:
Human pathogenic bacteria are
chemosynthetic and organotrophic (chemo-
organotrophic). They derive energy from the breakdown
of organic nutrients and use this chemical energy both for
resynthesis and secondary activities. Bacteria oxidize
nutrient substrates by means of either respiration or
fermentation. In respiration, O2 is the electron and proton
acceptor, in fermentation an organic molecule performs
this function. Human pathogenic bacteria are classified in
terms of their O2 requirements and tolerance as
facultative anaerobes, obligate aerobes, obligate
anaerobes, or aerotolerant anaerobes. Nutrient broth or
agar is used to cultivate bacteria. Nutrient agar contains
the inert substrate agarose, which liquefies at 100°C and
gels at 45°C. Selective and indicator mediums are used
frequently in diagnostic bacteriology. Bacteria reproduce
by means of simple transverse binary fission. The time
required for complete cell division is called generation
time. The in-vitro generation time of rapidly proliferating
species is 15–30 minutes. This time is much longer in
vivo. The growth curve for proliferation in nutrient broth
is normally characterized by the phases lag, log (or
exponential) growth, stationary growth, and death.