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Bacteriology at UW-Madison |
Bacterial Adaptation to the Nutritional and Physical Environment
Unlike plant and animal cells, most bacteria are exposed to a constantly changing physical and chemical environment. Within limits, bacteria can react to changes in their environment through changes in patterns of st ructural proteins, transport proteins, toxins, enzymes, etc., which adapt them to a particular ecological situation. For example, E. coli does not produce fimbriae for colonization purposes when living in a planktonic (free-floating or swimming) environment. Vibrio cholerae does not produce the cholera toxin that causes diarrhea unless it is in the human intestinal tract. Bacillus subtilis does not make the enzymes for tryptophan biosynthesis if it can find preexisting tryptophan in its environment. If E. coli is fed glucose and lactose together, it will use the glucose first because it takes two less enzymes to use glucose than it does to use lactose. The bacterium Neisseria gonorrhoeae will develop a sophisticated iron gathering and transport system if it senses that iron is in short supply in its environment.
Bacteria have developed sophisticated mechanisms for the regulation of both catabolic and anabolic pathways. Generally, bacteria do not synthesize degradative (catabolic) enzymes unless the substrates for these enzymes are present in their environment. For example, synthesis of enzymes that degrade lactose would be wasteful unless the substrate for these enzymes (lactose) is available in the environment. Similarly, bacteria have developed diverse mechanisms for the control of biosynthetic (anabolic) pathways. Bacterial cells shut down biosynthetic pathways when the end product of the pathway is not needed or is readily obtained by uptake from the environment. For example, if a bacterium could find a preformed amino acid like tryptophan in its environment, it would make sense to shut down its own pathway of tryptophan biosynthesis, and thereby conserve energy. However, in real bacterial life, the control mechanisms for all these metabolic pathways must be reversible, since the environment can change quickly and drastically.
Some of the common mechanisms by which bacterial cells regulate and control their metabolic activities are discussed in this chapter It is important for the reader to realize that most of these mechanisms have been observed or described in the bacterium, Escherichia coli, and they are mostly untested and unproved to exist in many other bacteria or procaryotes (although, whenever they are looked for, they are often found). The perceptive reader will appreciate that the origins of the modern science of molecular biology are found in the experiments that explained these regulatory processes in E. coli.
Conditions Affecting Enzyme Formation in Bacteria
As stated above, bacterial cells can change patterns of enzymes, in order to adapt them to their specific environment. Often the concentration of an enzyme in a bacterial cell depends on the presence of the substrate for the enzyme. Constitutive enzymes are always produced by cells independently of the composition of the medium in which the cells are grown. The enzymes that operate during glycolysis and the TCA cycle are generally constitutive: they are present at more or less the same concentration in cells at all times. Inducible enzymes are produced ("turned on") in cells in response to a particular substrate; they are produced only when needed. In the process of induction, the substrate, or a compound structurally similar to the substrate, evokes formation of the enzyme and is sometimes called an inducer. A repressible enzyme is one whose synthesis is downregulated or "turned off" by the presence of (for example) the end product of a pathway that the enzyme normally participates in. In this case, the end product is called a corepressor of the enzyme.
Regulation of Enzyme Reactions
Not all enzymatic reactions occur in a cell to the same extent. Some substances are needed in large amounts and the reactions involved in their synthesis must therefore occur in large amounts. Other substances are needed in small amounts and the corresponding reactions involved in their synthesis need only occur in small amounts.
In bacterial cells, enzymatic reactions may be regulated by two
unrelated
modes: (1) control or regulation of enzyme activity (feedback
inhibition or end product
inhibition), which mainly operates to regulate
biosynthetic pathways; and (2) control or regulation of enzyme
synthesis, including end-product repression, which
functions in the regulation
of biosynthetic pathways, and enzyme induction and catabolite
repression, which regulate mainly degradative pathways. The process
of feedback inhibition regulates the activity of preexisting enzymes in
the cells. The processes of end-product repression, enzyme induction
and
catabolite repression are involved in the control of synthesis of
enzymes.
The processes which regulate the synthesis of enzymes may be either
a form of positive control or negative control. End-product repression
and enzyme induction are mechanisms of negative control
because they lead
to a decrease in the rate of transcription of proteins.
Catabolite repression
is considered a form of positive control because it affects an increase
in rates of transcription of proteins.
Allosteric Proteins
Although there are examples of regulatory processes that occur at all stages in molecular biology of bacterial cells (see Table 1 above), the most common points of regulation are at the level of transcription (e.g. enzyme induction and enzyme repression) and changing the activity of preexisting proteins. In turn, these levels of control are usually modulated by proteins with the property of allostery.
An allosteric protein is one which has an active
(catalytic)
site and an allosteric (effector) site. In an allosteric
enzyme,
the active site binds to the substrate of the enzyme and converts it to
a
product. The allosteric site is occupied by some small molecule which
is
not a substrate. However, when the allosteric site is occupied by the
effector
molecule, the configuration of the active site is changed so that it is
now unable to recognize and bind to its substrate (Figure 1). If the
protein
is an enzyme, when the allosteric site is occupied, the enzyme is
inactive,
i.e., the effector molecule decreases the activity of the enzyme. There
is an alternative situation, however. The effector molecule of certain
allosteric enzymes binds to its allosteric site and consequently
transforms
the enzyme from an inactive to an active state (Figure 2). Some
multicomponent
allosteric enzymes have several sites occupied by various effector
molecules
that modulate enzyme activity over a range of conditions.
Figure 1. Example of an
allosteric
enzyme with a negative effector site. When the effector molecule binds
to the allosteric site, substrate binding and catalytic activity of the
enzyme are inactivated. When the effector is detached from the
allosteric
site the enzyme is active.
Figure 2. Example of an
allosteric
enzyme with a positive effector site. The effector molecule binds to
the
allosteric site resulting in alteration of the active site that
stimulates
substrate binding and catalytic activity.
Some allosteric proteins are not enzymes, but nonetheless have an active site and an allosteric site. The regulatory proteins that control metabolic pathways involving end product repression, enzyme induction and catabolite repression are allosteric proteins. In their case, the active site is a DNA binding site, which, when active, binds to a specific sequence of DNA, and which, when inactive, does not bind to DNA. The allosteric or effector molecule is a small molecule which can occupy the allosteric site and affect the active site. In the case of enzyme repression, a positive effector molecule (called a corepressor) binds to the allosteric regulatory protein and activates its ability to bind to DNA. In the case of enzyme induction a negative effector molecule (called an inducer) binds to the allosteric site, causing the active site to change conformation thereby detaching the protein from its DNA binding site.
Feedback Inhibition
Feedback inhibition (or end product inhibition) is a mechanism for the inhibition of preformed enzymes that is seen primarily in the regulation of whole biosynthetic pathways, e.g. pathways involved in the synthesis of the amino acids. Such pathways usually involve many enzymatic steps, and the final (end) product is many steps removed from the starting substrate. By this mechanism, the final product is able to feed back to the first step in the pathway and to regulate its own biosynthesis.
In feedback inhibition, the end product of a biosynthetic pathway inhibits the activity of the first enzyme that is unique to the pathway, thus controlling production of the end product. The first enzyme in the pathway is an allosteric enzyme. Its allosteric site will bind to the end product (e.g. amino acid) of the pathway which alters its active site so that it cannot mediate the enzymatic reaction which initiates the pathway. Other enzymes in the pathway remain active, but they do not see their substrates. The pathway is shut down as long as adequate amounts of the end product are present. If the end product is used up or disappears, the inhibition is relieved, the enzyme regains its activity, and the organism can resume synthesis of the end product. Thus, if a E. coli bacterium swims out of a glucose minimal medium into milk or some other medium rich in growth factors, the bacterium can stop synthesizing any of the essential metabolites that are made available directly from the new environment.
One of the most intensely studied bacterial pathways is the pathway of tryptophan biosynthesis (Figure 3). The pathway of tryptophan biosynthesis is regulated by feed back inhibition. Tryptophan is the effector molecule for allosteric enzyme a. When the end product of the pathway (tryptophan) attaches to enzyme a, the enzyme is inactive and can no longer join glutamine and chorismic acid into anthranilate. If tryptophan is disjoined from the enzyme the pathway is resumed, and tryptophan synthesis will continue. Tryptophan biosynthesis is also regulated at a genetic level by the processes of enzyme repression (below) and attenuation.
Note: In the case of feedback inhibition (above), the signal molecule, tryptophan, is a negative effector of Enzyme a in the pathway of tryptophan biosynthesis, because when it binds to Enzyme a, it inactivates the enzyme. In enzyme repression (below) tryptophan is a signal molecule that acts as a positive effector of the trp repressor protein because when it binds to the repressor it activates the protein, so that it binds to the trp DNA.
Figure 3. The pathway of
tryptophan
biosynthesis in E. coli. The pathway is regulated by the
process
of feedback inhibition. Tryptophan (trp), the end product of the
pathway,
is the effector molecule that binds to the allosteric site of Enzyme a,
the first enzyme in the pathway. When trp is bound to the enzyme the
catalytic
(active) site of Enzyme a is altered so that it is unable to react with
its substrates and the synthesis of anthranilate is inhibited.
If a metabolic pathway branches, leading to the synthesis of two
amino
acids, each end product (amino acid) can control its own synthesis
without
affecting the other (Figure 4). For example, the amino acids proline
and
arginine are both synthesized from glutamic acid. Each amino acid can
regulate
the first enzyme unique to its own synthesis without affecting the
other,
so that a surplus of arginine will not shut off the synthesis
of proline and vice versa.
Figure 4. Generalized scheme
for regulation of a branched metabolic pathway by the process of
feedback
inhibition.
Enzyme Repression
Enzyme repression is a form of negative control (down-regulation) of bacterial transcription. This process, along with that of enzyme induction, is called negative control because a regulatory protein brings about inhibition of mRNA synthesis which leads to decreased synthesis of enzymes.
Although feedback inhibition shuts off synthesis of the end product of a pathway, it still allows some waste of energy and carbon if the cell continued to manufacture enzymes for which it has no use. It is the process of enzyme repression that prevents the synthesis of the enzymes concerned with the synthesis of that particular end product. In the case of the pathway of tryptophan biosynthesis (Figure 3), the end product of the pathway, tryptophan, serves as an effector molecule that can shutdown the synthesis of the Enzymes a, b, c, d, and e that are concerned with tryptophan biosynthesis. This results in saving of many molecules of ATP which must be expended during protein synthesis, and it conserves amino acid precursors for synthesis of other proteins. The process is slower to act than is feedback inhibition (which acts immediately) because pre-existing enzymes have to be diluted out as a result of cell division before its effects are seen.
The genes for tryptophan biosynthesis in Escherichia coli are
organized on the bacterial chromosome in the tryptophan operon (trp
operon). An operon is a
cluster of genes that are controlled by the
same elements and which are coordinately transcribed and translated.
The
trp operon consists of a Promoter (P) region, an Operator (O) region,
an
Attenuator (A) region, and the five structural genes for the enzymes
involved
in
tryptophan biosynthesis (Trp A-E) The components of the trp operon and
its control elements are described in Figure 5 and Table 2 below.
Figure 5. Genetic organization
of the Trp operon and its control elements.
O = Operator specific nucleotide sequence on DNA to which an active Repressor binds.
P = Promoter specific nucleotide sequence on DNA to which RNA polymerase binds to initiate transcription. If the repressor protein binds to the operator, RNAp is prevented from binding with the promoter and initiating transcription. Therefore, none of the enzymes concerned with tryptophan biosynthesis are synthesized.
A = Attenuator DNA sequence which lies between the operator and the structural genes for trp biosynthesis. The attenuator is a barrier that RNA polymerase must traverse if it is to transcribe the genes for tryptophan biosynthesis. In the presence of trp, most RNAp molecules fall off the DNA before transcribing the trp genes. In the absence of trp, RNAp is able to traverse the attenuator region to successfully transcribe the trp genes.
Trp A, B, C, D, E = Structural genes for enzymes involved in tryptophan biosynthesis.
Trp = tryptophan end product of the biosynthetic pathway. When combined with the repressor protein the Repressor is active. Trp is called a corepressor.
The trp operon is regulated by a regulatory gene (Trp L) associated
with
the trp promoter. The product of the Trp L gene is the trp Repressor,
an allosteric protein which is regulated by tryptophan. The Repressor
is
produced constitutively in small amounts in an inactive form. When the
Repressor combines with tryptophan it becomes activated and binds to
the
DNA of the trp operon in such a way that it blocks the transcription of
the structural genes for tryptophan. Thus, in the presence of
tryptophan,
transcription of the genes for tryptophan biosynthesis are repressed
(tryptophan
is not produced), while in the absence of tryptophan, the genes for
tryptophan
biosynthesis can be transcribed (tryptophan is produced); See Figure 6
below.
Figure 6a. Derepression of the
trp operon. In the absence of trp the inactive repressor cannot bind to
the operator to block transcription. The cell must synthesize the amino
acid.
Figure 6b. Repression of the
trp operon. In the presence of tryptophan the trp operon is repressed
because
trp activates the repressor. Transcription is blocked because the
active
repressor binds to the DNA and prevents binding of RNA polymerase.
Enzyme Induction
In some cases, metabolites or substrates can turn on inactive genes so that they are transcribed. In the process of enzyme induction, the substrate, or a compound structurally similar to the substrate, evokes the formation of enzyme(s) which are usually involved in the degradation of the substrate. Enzymes that are synthesized as a result of genes being turned on are called inducible enzymes and the substance that activates gene transcription is called the inducer. Inducible enzymes are produced only in response to the presence of a their substrate and, in a sense, are produced only when needed. In this way the cell does not waste energy synthesizing unneeded enzymes.
The best known and best studied case of enzyme induction involves
the
enzymes of lactose degradation in E. coli. Only in the presence
of lactose does the bacterium synthesize the enzymes that are necessary
to utilize lactose as a carbon and energy source for growth. Two
enzymes
are required for the initial breakdown of lactose: lactose permease,
which actively transports the sugar into the cell, and beta
galactosidase,
which splits lactose into glucose plus galactose. The genes for these
enzymes
are contained within the lactose operon (lac operon) in the
bacterial
chromosome (Figure 7).
Figure 7. The Lac operon and
its control elements
The mechanism of enzyme induction is similar to end product
repression
in that a regulatory gene, a promoter, and an operator are involved,
but
a major difference is that the lac Repressor is active only in the
absence
of the inducer molecule (lactose). In the presence of lactose, the
Repressor cannot bind to the operator region, so that the genes for
lactose
transport and cleavage are transcribed. In the absence of lactose, the
Repressor is active and will bind to the operator with the result that
the genes for lactose metabolism are not transcribed. The induction
(presence
of lactose) and the repression (absence of lactose) of the lactose
operon
is represented in Figure 8. The functions of the components and control
elements of the lac operon are shown in Table 3.
Figure 8. Enzyme Induction.
Induction (or derepression) of the lac operon.
O = Operator specific nucleotide sequence on DNA to which an active Repressor binds.
P = Promoter specific nucleotide sequence on DNA to which RNA polymerase binds to initiate transcription. (The promoter site of the lac operon is further divided into two regions, an upstream region called the CAP site, and a downstream region consisting of the RNAp interaction site. The CAP site is involved in catabolite repression of the lac operon.). If the Repressor protein binds to the operator, RNAp is prevented from binding with the promoter and initiating transcription. Under these conditions the enzymes concerned with lactose utilization are not synthesized.
Lac Z, Y and A = Structural Genes in the lac operon. Lac Z encodes for Beta-galactosidase; Lac Y encodes the lactose permease; Lac A encodes a transacetylase whose function is not known.
lac = lactose, the inducer molecule. When lactose binds to the Repressor protein, the Repressor is inactivated; the operon is derepressed; the transcription of the genes for lactose utilization occurs.
Catabolite Repression
Enzyme Induction is still considered a form of negative control because the effect of the regulatory molecule (the active repressor) is to decrease or downregulate the rate of transcription. Catabolite repression is a type of positive control of transcription, since a regulatory protein affects an increase (upregulation) in the rate of transcription of an operon. The process was discovered in E. coli and was originally referred to as the glucose effect because it was found that glucose repressed the synthesis of certain inducible enzymes, even though the inducer of the pathway was present in the environment. The discovery was made during study of the regulation of lac operon in E. coli. Since glucose is degraded by constitutive enzymes and lactose is initially degraded by inducible enzymes, what would happen if the bacterium was grown in limiting amounts of glucose and lactose? A plot of the bacterial growth rate resulted in a diauxic growth curve which showed two distinct phases of active growth (Figure 9). During the first phase of exponential growth, the bacteria utilize glucose as a source of energy until all the glucose is exhausted. Then, after a secondary lag phase, the lactose is utilized during a second stage of exponential growth.
Figure 9. The Diauxic Growth
Curve of E. coli grown in limiting concentrations of a mixture
of
glucose and lactose
During the period of glucose utilization, lactose is not utilized because the cells are unable to transport and cleave the disaccharide lactose. Glucose is always metabolized first in preference to other sugars. Only after glucose is completely utilized is lactose degraded. The lactose operon is repressed even though lactose (the inducer) is present. The ecological rationale is that glucose is a better source of energy than lactose since its utilization requires two less enzymes.
Only after glucose is exhausted are the enzymes for lactose utilization synthesized. The secondary lag during diauxic growth represents the time required for the complete induction of the lac operon and synthesis of the enzymes necessary for lactose utilization (lactose permease and beta-galactosidase). Only then does bacterial growth occur at the expense of lactose. Since the availability of glucose represses the enzymes for lactose utilization, this type of repression became known as catabolite repression or the glucose effect.
Glucose is known to repress a large number of inducible enzymes in many different bacteria. Glucose represses the induction of inducible operons by inhibiting the synthesis of cyclic AMP (cAMP), a nucleotide that is required for the initiation of transcription of a large number of inducible enzyme systems including the lac operon.
The role of cyclic a cAMP is complicated. cAMP is required to activate an allosteric protein called CAP (catabolite activator protein) which binds to the promoter CAP site and stimulates the binding of RNAp polymerase to the promoter for the initiation of transcription. Thus, to efficiently promote gene transcription of the lac operon, not only must lactose be present to inactivate the lac repressor, but cAMP must be available to bind to CAP which binds to DNA to facilitate transcription. In the presence of glucose, adenylate cyclase (AC) activity is blocked. AC is required to synthesize cAMP from ATP. Therefore, if cAMP levels are low, CAP is inactive and transcription does not occur. In the absence of glucose, cAMP levels are high, CAP is activated by cAMP, and transcription occurs (in the presence of lactose).
Many positively controlled promoters, such as the lac promoter, are not fully functional in the presence of RNAp alone and require activation by CAP. CAP is encoded by a separate Regulatory gene, and is present in constitutive levels. CAP is active only in the presence of cAMP. The binding of cAMP to CAP causes a conformational change in the protein allowing it to bind to the promoter near the RNAp binding site. CAP can apparently interact with RNAp to increase the rate of operon transcription about 50-fold. Positive control of the lac operon is illustrated in Figure 10.
Figure 10. Catabolite repression is positive control of the lac operon. The effect is an increase in the rate of transcription. In this case, the CAP protein is activated by cAMP to bind to the lac operon and facilitate the binding of RNA polymerase to the promoter to transcribe the genes for lactose utilization.
As a form of catabolite repression, the glucose effect serves a useful
function in bacteria: it requires the cells to use the best available
source
of energy. For many bacteria, glucose is the most common and readily
utilizable
substrate for growth. Thus, it inhibits indirectly the synthesis of
enzymes
that metabolize poorer sources of energy.