CH 11 Microbe Coordination of Cell Processes

10.1128/9781555819132.
ch11
11
C h a p t er E lev en
Coordination of Cell
Processes
Key Concepts
This chapter covers the following topics in the ASM Fundamental Statements.
Evolution
1. Cells, organelles (e.g., mitochondria and chloroplasts), and all major metabolic
pathways evolved from early prokaryotic cells.
Metabolic Pathways
2. The survival and growth of any microorganism in a given environment depend
on its metabolic characteristics.
Information Flow and Genetics
3. The regulation of gene expression is influenced by external and internal molecu-
lar cues and/or signals.
Introduction
O
ur earlier chapters on bacterial metabolism and growth noted that some 2,000
chemical reactions drive the assembly and life of a bacterium. We introduced
the notion of phases of metabolism (fueling, biosynthesis, polymerization, and
assembly), discussed the organization of metabolic pathways, and described the
complex overall sequence of biochemical events that generate a living cell from s imple or-
ganic or even completely inorganic substrates.
But that is not all it takes to make a living cell. If each of these 2,000 chemical reactions
acted independently, they would produce not a cell, but chaos! In this chapter, we w ill
examine how independent chemical reactions are linked into a single, coordinated
305
306 par t i F u nda m enta ls o f m icro bia l Life
network—an integrated circuit of enormous complexity and elegance, sur-

passing the highest achievements of human engineers. Without such control
mechanisms, microbes could not respond to changes in the environment
appropriately, and many, perhaps all, environments on Earth would never
have been c olonized.
Case: How can Escherichia coli selectively use sugars from a mixture?
Phase II
1.0
Glucose/lactose concentration
log (optical density, OD600)
Phase I
0.1
0.01
Glucose Lactose
0 3 6 9
Time (hours)
Many heterotrophic bacteria have the remarkable ability to use glucose

preferentially over any other sugar. Student JM demonstrated this by grow-
ing E. coli in mineral medium supplemented with a mix of glucose and
lactose (a disaccharide of glucose and galactose). He monitored growth
using a spectrophotometer to quantify the increase in optical density of
the culture and plotted the data in a semi-log plot. He also measured the
concentration of glucose and lactose in the medium throughout the incu-
bation period and plotted the consumption of these sugars on the second-
ary y axis. He was puzzled when he observed two phases of growth (known
as diauxic growth): the first phase correlated with depletion of glucose and
the second phase with lactose consumption.
Questions arising:
• H ow did E. coli cells selectively use glucose first?
• How did they switch from growing with glucose to growing with lactose?
What evidence shows that metabolic reactions

are coordinated?
Learning Outcomes:
• E xplain the need for coordination of metabolic reactions within a cell.
• Summarize a set of experiments that demonstrates coordination of
biosynthesis in a bacterium.
• Speculate about at least one macromolecule (or group of macromolecules)
that will be in greater abundance for each phase of bacterial culture
growth and undergo coordination of polymerization.
Cha pt er 11 C oordinat ion of Cell Processes 307
As student JM observed experimentally in the case study, microbes are

remarkably attuned to their environment, so much so that they selectively
use one sugar over another to support their growth. To accomplish this
feat, microbes have mechanisms to sense environmental changes and to
respond by rapidly reprogramming their metabolic networks. In the case
study, the cells detected glucose and lactose and responded by using glucose
first, then lactose. How? Below, we describe a few experiments that pro-
vide concrete evidence of the cell’s ability to coordinate its working parts
and become a single integrated system. Coordination can be demon-
strated in each of the major stages of metabolism. For simplicity, we shall
begin with biosynthesis.
Coordination in Biosynthesis
Imagine that you prepare a medium consisting of a mixture of inorganic
salts and radioactive 14C-labeled glycerol as the sole source of carbon and
energy (just enough to support the growth of a bacterial species such as E.
coli to a density of 108 cells per ml). Inoculate the medium with a few cells,
and incubate it aerobically. As the culture grows, the cells take up radioac-
tively labeled glycerol, metabolize it via their oxidative reactions, and pro-
duce carbon dioxide, which is now labeled. You would also find some
radioactive carbon in the cells’ building blocks and macromolecules. In-
deed, the total protein of the cells would be labeled. Furthermore, if you
were to analyze specific amino acid residues in the protein fraction, you
would find that they are all labeled as well. Consider the implications of this
finding: carbon from the substrate (glycerol) flowed through each meta-
bolic pathway in a coordinated fashion to make amino acids and, from them,
proteins. Rather neat and simple.
But h ere is the interesting part. Repeat this experiment, but now in-
clude in the medium not only the labeled glycerol but also an unlabeled
amino acid, for example, histidine. You would discover that, as before, the
radioactive carbon flowed from the glycerol substrate to carbon dioxide and
to the cells’ proteins. However, the histidine residues, alone among all the
amino acids, w ill contain almost no trace of radioactivity. You can deduce that
the cells’ histidine derived almost exclusively from the nonradioactive his-
tidine supplied in the medium instead of the radioactive glycerol substrate.
In fact, if you w ere to analyze any of the intermediates of the histidine
biosynthetic pathway, you would find that they are also unlabeled. Thus,
the cell turned off the entire pathway leading to the synthesis of histidine from
glycerol. This experiment could be repeated with virtually any of the build-
ing blocks, vitamins, or cofactors with the same result. The presence of any
one compound in the medium stops endogenous synthesis of that compound. More-
over, the same pattern would be observed if the experiment was performed
under different temperature, pH, oxygen tension, carbon source, mixture
of nutrients, and so on. In all cases, the cells stop making a particular com-
pound when it is readily available in the medium. Remarkable!
Coordination in Fueling
Does coordination exist in fueling reactions as well? The answer is a big
yes. Fueling reactions provide the precursor metabolites, reducing power
(mainly in the form of NADH), and energy (ATP) needed for the synthesis
of building blocks and macromolecules. The cell adjusts the ratios of the
products of fueling (precursor metabolites, energy, and reducing power) to
satisfy its needs. If precursor metabolites are provided in the medium, the
cell coordinates the fueling reactions to generate only the products of fuel-
ing that it needs. Let us return to our glycerol experiment, but this time
we will grow E. coli in a rich medium that supplies most of the amino ac-
ids. Would you detect radioactive carbon from glycerol in the protein frac-
tion? Only a little, b
ecause the cells no longer need to use the glycerol to
make most of the amino acid building blocks. Instead, the cells reroute the
glycerol through the fueling pathways to generate an adequate supply of
reducing power and ATP to satisfy their biosynthetic needs.
Coordination in Macromolecular Composition

Recall from chapter 4 that, in general, cells maintain their macromolecular
composition despite differences in their growth medium. Cells growing on
a mixture of amino acids are not richer in total protein; those growing on
fatty acids are not richer in lipid content; those growing on nucleosides are
not richer in nucleic acids; t hose growing on sugars are not richer in carbo-
hydrates. On the other hand, the rate of growth does affect a cell’s composi-
tion. Cells growing fast, as when supplied with amino acids, are richer in
RNA than the same cells growing more slowly in a minimal medium.
Therefore, regulatory circuits must sense the cell’s potential to attain a par
ticular growth rate and control the synthesis of each macromolecule.
In summary, all the major fueling and biosynthetic pathways are subject
to powerful controls that bring order out of the potential chaos of a system
with thousands of individual working parts. T hese examples demonstrate
that metabolic reactions are coordinated. The question now becomes, how?
How Do Microbes Regulate Their Metabolism?
Learning Outcomes:
• L ist four reasons why prokaryotes more often control the amounts of
enzymes produced rather than controlling enzymatic activity (as is more
often seen in eukaryotes).
• Define “allostery.”
If you wanted to design a highly coordinated cell, you might first think
about the possible ways the rates of metabolic reactions can be regulated.
Consider the following metabolic reaction (where S1 and S2 are substrates
and P1 and P2 are products) catalyzed by an enzyme:
Enzyme
→ S1 + S2 P1 + P2 →
The rate of the reaction can be controlled by one of three means: (i) vary-
ing the activity of the enzyme, (ii) changing the amount of the enzyme, or
(iii) varying the intracellular concentration of one or both substrates (or
products). Of the three, which would you choose? Microbial cells actually
use all these options. However, as reactions in the cell are coupled, the third
mechanism—varying the concentration of substrate(s) for a reaction—is
Cha p t e r 11 C oordinat ion o f C ell P rocesses 309
A. Protein amount B. Protein activity
Allosteric
change
1 Template
DNA structure
Allosteric
2 Transcription effector
control
mRNA
Reversible
3 Translation modification
control
Protein
Regulation
by sRNA
4 Proteolysis
sRNA
Figure 11.1 Overview of metabolic regulatory devices. Synopsis of the two forms of

control that operate to coordinate metabolism: regulation of the amount of a protein in
the cell (A) and regulation of its activity (B).
brought about by one of the first two mechanisms. Thus, the primary basis
of metabolic coordination is varying either the amounts or the activities of
enzymes (Fig. 11.1).
Controlling Enzyme Amounts

The simplest and neatest trick to control the rates of a metabolic reaction is
to modulate the amounts of enzymes that are made. How can this be
done? Any step in the flow of information, from DNA to mRNA to protein, can
be controlled to regulate the rates of synthesis (Fig. 11.1A). For example,
the expression of a gene can be controlled by changes in DNA structure
such as supercoiling (chapter 3), or it can be controlled at the level of tran-
scription (a major hot spot of regulation for prokaryotes) or translation
(chapter 8). Interestingly, controlling the amounts of enzymes produced is
much more common in prokaryotes than in cells of plants and animals,
which often control enzyme activity. So, in what ways do prokaryotes differ
from cells of higher organisms? H ere are four key microbial themes.
• F irst, microbes, particularly free-living ones, are often substrate limited.
Microbes cannot afford to make useless or redundant enzymes (re-
member the energy investment required? [chapter 8]), nor can they sur-
vive without producing essential enzymes. To meet both challenges,
they rapidly adjust enzyme synthesis.
• Second, coupled transcription and translation allows prokaryotes to ad-
just quickly the expression of genes encoding an enzyme to control its
availability. Upregulation can start within seconds and can quickly
increase the amount of an enzyme by 1,000-fold or more. Downregula-
310 par t i F u nda m enta ls of m icro bia l Li f e
tion also starts right away. If transcription of an enzyme-encoding gene

is turned off in a growing culture, the amount of that enzyme will
halve e very time the cell divides. If the generation time is 20 minutes,
the enzyme pool can be reduced 8-fold in an hour.
• Third, prokaryotic mRNAs are short-lived. Thus, the pool of mRNA
transcripts available for protein synthesis can be completely renewed
every few minutes—a marvelous opportunity to control enzyme syn-
thesis rapidly.
• Fourth, prokaryotic genomes organize genes of related functions in mul-
ticistronic transcriptional units called operons (chapter 8). This facilitates
the coordinated control of functionally related enzymes (e.g., enzymes
involved in the same metabolic pathway). By adjusting the synthesis of
just one long mRNA, the cell can change the rate of synthesis of an en-
tire pathway.
Controlling Enzyme Activity

Controls on protein activity, though less common, are also used by pro-
karyotes to modulate the rates of a metabolic reaction and to coordinate
some cellular processes (Fig. 11.1B). As we discuss in more detail below,
activity can be positively or negatively modified in a reversible manner by
covalent modification (phosphorylation is most common). In other cases, ac-
tivity is modulated by allostery, a process that involves the reversible asso-
ciation of the protein with another molecule. Compared to covalent modification,
allostery is far more pervasive in microbial regulation.
If reactions can be regulated equally well by controlling the amounts or
the activities of enzymes, why do microbes employ both mechanisms? To
examine this key question of cell physiology, we need more information
about each mode of regulation. Then the benefit of having two modes of
regulation will become clear.
How Is Protein Activity Modulated?
Learning Outcomes:
• D ifferentiate covalent modification and allosteric interactions as ways of
controlling protein activity.
• Use the histidine biosynthetic pathway as an example to explain how
feedback inhibition is an effective means to control biosynthetic
pathways.
• Describe how the ratio of the concentration of ATP to those of ADP and
AMP is maintained almost constant in bacterial cells over a wide range of
energy supply and demand.
Modulation of enzyme activity occurs rapidly, almost instantaneously, ad-

justing metabolic flow in the microbial cell in a fraction of a second. Using
the overview of regulatory devices shown in Fig. 11.1B, we next discuss
the two strategies microbes use to regulate protein activity.
Covalent Modification
Both eukaryotes and prokaryotes control enzyme activity using covalent
modifications. Some of these (phosphorylation, in particu lar) are prev-
alent in bacterial physiology; beautiful examples are motility, chemo-
taxis, and sensory signal transduction, in which cascades of proteins are
activated via phosphorylation (see chapter 12). Other modifications, such
as addition of adenylyl, acetyl, methyl, and other residues, are rarer, but
the cellular processes they regulate are no less import ant (for example,
adenylation regulates glutamine synthetase, a key enzyme in nitrogen
assimilation).
Allosteric Interactions
The most prevalent mode of controlling the activities of enzymes (and
other proteins) occurs via allosteric interactions. Allostery involves a
change in conformation or shape and, therefore, activity upon binding
a molecule called an allosteric effector (Fig. 11.1B). The effector can be a
metabolite (ligand) or another protein or RNA (modulator), but the re-
sult is the same: upon binding the allosteric effector, the protein’s confor-
mation is changed and, consequently, so is its activity. Remember two
features of allosteric changes: (i) the regulatory site at which the allosteric
effector binds is separate from the enzyme’s catalytic site, and (ii) the ef-
fector need bear no steric resemblance to the substrates (or products) of
the enzyme. This is worth emphasizing: allostery provides a means for
modifying the activity of an enzyme by substances not even remotely re-
sembling the substrates or products. Therefore, allostery differs funda-
mentally from competitive inhibition, in which a substance resembling
the substrate competes for binding to the active site of the enzymes.
Can you imagine how allostery works? Allosteric enzymes can exist in at
least two conformations, one with high and another with low activity. The
binding of the effector favors one conformation over the other. In some
cases, what is changed is the velocity (Vmax, the maximum rate of reaction)
of the enzyme reaction; in others, it is affinity for the substrate (Km, the
Michaelis constant). Allosteric effectors may either increase or decrease
the activity of the enzyme, and some enzymes even have both positive
and negative allosteric effectors.
Allostery in biosynthesis. Earlier we noted that, given the choice, a bacte-

rium w ill utilize a building block supplied in the medium rather than
make its own (in our example, histidine). Moreover, recall that by tracing
the radioactively labeled glycerol, we learned that flow through the histi-
dine biosynthetic pathway ceases just seconds after histidine is supplied.
This calls for a rapid mechanism, right? Indeed, the first enzyme in the
histidine biosynthetic pathway is allosteric, and histidine is its negative al-
losteric effector. By this elegant mechanism, exogenous histidine immedi-
ately shuts off the entire biosynthetic pathway at its very first step.
In fact, a more dramatic version of this process takes place continuously
in the cell to regulate histidine biosynthesis. The internal concentration
of histidine, whether made by the cell or imported from the environment,
determines the rate of flow from precursor metabolites to end product.
And realize that histidine is but one example, not an exception: virtually
Enzyme1 Enzyme2 Utilization for

Metabolitea Metaboliteb Metaboliten macromolecule
synthesis
Figure 11.2 Feedback inhibition. The final product of a series of enzymatic reactions,

metaboliten, has the property of binding to the regulatory site of the allosteric protein,
enzyme1, and thereby inhibiting it. A scheme of this sort can guarantee that metaboliten
A Isofunctional enzymes
is produced only as rapidly as it is used in some subsequent process, such as macromol-
ecule synthesis.
d f
a b c
all building blocks control their own synthesis by acting as negative allosteric ef-
e g fectors of the first enzyme in their biosynthetic pathway. This control pro
cess is called feedback inhibition (or end-product inhibition). Since
each building block is consumed by macromolecule synthesis, biosynthetic
B Cumulative feedback inhibition
pathways work by demand feeding: they produce their end products at the
d f
same rate that macromolecular synthesis depletes them. Figure 11.2 sum-
marizes general features of feedback inhibition in biosynthetic pathways.
a b c Feedback inhibition can also be adapted to accommodate the branching of
biosynthetic pathways and to interact with other pathways (Fig. 11.3).
e g
Allostery in fueling. Allosteric inhibition and activation also regulate the

C Sequential feedback inhibition flow of metabolites through fueling pathways (Fig. 11.4). Here, the simple
device of end-product control of the first or an early step in a pathway can-
d f not apply. As we learned in chapter 7, the pathways of formation of the 13
precursor metabolites are so interrelated (some pathways are cyclic) that
a b c
there are no unique early steps. A cell metabolizing glucose may produce ma-
late in the tricarboxylic acid (TCA) cycle, one of the last reactions in glu-
e g
cose fueling (Fig. 11.4). However, if malate is provided as a sole carbon and
energy source, the same cell may reverse the metabolic flow to generate
D Inhibition plus activation
the precursor metabolites from malate (chapter 7). Thus, controls work
e f
internally in each of the main fueling pathways, and both positive and
– negative allosteric interactions abound (Fig. 11.4).
a b The rates of formation of ATP and reducing power (NAD[P]H) must
+ also be regulated during fueling. As an example, consider how tightly ATP
c d g h levels are regulated. B
ecause ATP synthesis and utilization involve a cyclic
Figure 11.3 Patterns of feedback flow through ADP and AMP, it makes sense that all three adenylates play
inhibition found in bacterial regulatory roles in fueling reactions (as well as in biosynthetic pathways)
biosynthetic pathways. (A) Isofunc- (Fig. 11.4). A useful index of a cell’s energy status is the energy charge of
tional enzymes for the regulated
reaction allow differential feedback the cell, defined as follows:
effects by the two pathway end
products. (B) Cumulative feedback [ATP]+ 1/2[ADP])
inhibition involves multiple allosteric Energy charge =
sites on the regulated enzyme, [ATP]+[ADP]+[AMP]
ensuring that there will be some
activity unless all the end products Mathematically, the energy charge of the cell could vary from 0 (all AMP)
are in excess. (C) Sequential feedback
inhibition involves different end
to 1 (all ATP), but in fact, the energy charge of prokaryotes u nder normal
products operating separately on the conditions is held narrowly between 0.87 and 0.95. Starving a cell for a
various branches of the pathway. very long time reduces its energy charge slowly; when it reaches approxi-
(D) Inhibition plus activation uses
mately 0.5, the cell is dead. In general, ATP-replenishing pathways (fuel-
both positive (+) and negative (−)
allosteric effectors to coordinate ing) are inhibited by high levels of energy charge, and ATP-utilizing pathways
complex pathways. (biosynthesis and polymerization) are stimulated.
Pentose phosphate cycle Glycolysis

Pentose-5-phosphate Glucose-6-phosphate
Sedoheptulose-7-
phosphate
Glyceraldehyde-
3-phosphate
Fructose-6-phosphate
Erythrose-4-phosphate –
AMP
–
+
Phosphoenolpyruvate
Fructose-1,6-bisphosphate
ADP
Glyceraldehyde-
Dihydroxyacetone-phosphate
3-phosphate
1,3-Diphosphoglycerate
3-Phosphoglycerate
Acetyl coenzyme A
2-Phosphoglycerate Aspartate
+ –
Phosphoenolpyruvate Oxaloacetate
Fructose-1,6-bisphosphate +
– NADH Malate
Pyruvate Citrate
TCA Fumarate
Acetyl coenzyme A
– Isocitrate cycle
NADH Figure 11.4 Central pathways of
Succinate
Phosphoenolpyruvate fueling reactions showing some of
+ 2-Oxoglutarate
AMP the allosterically controlled steps.
Succinyl coenzyme A −, negative allosteric effector; +,
Acetyl coenzyme A
positive allosteric effector.
Allostery by regulatory RNAs. Prokaryotic cells also use RNA molecules as

allosteric effectors to modulate the activity of numerous proteins. Typi-
cally small in size, these so-called small RNAs (sRNAs) fold into a looped
structure containing binding sites for specific proteins. Protein-R NA pairs
can inhibit, activate, or modify the protein activity or even tether proteins
together to modulate their activities collectively (Fig. 11.5). The 6S RNA of
E. coli, for example, regulates entry in stationary phase by modifying
the activity of the RNA polymerase (RNAP) holoenzyme containing the
housekeeping sigma factor σ70. Once bound to 6S RNA, the RNAP-σ70
holoenzyme can no longer bind its usual promoters, and genes otherwise
activated during exponential phase are repressed. But, as we will discuss
later in the chapter, allosteric regulation is but one of the many ways in
which sRNAs coordinate processes in the cell.
Inhibit Modify A tiva

Act
Activate
Actii ate
ivat
te Tether
Figure 11.5 Control of protein activity by regulatory sRNAs. Upon binding the target
proteins, regulatory sRNAs can affect their activity in various ways. Some are inhibited (as
in the CsrB-CsrA system), some are modified in their activity, and some are activated. In
some cases, the same sRNA may bring (tether) two different proteins together, influencing
their activities through their association.
How Are Protein Amounts Modulated?
Learning Outcomes:
• D escribe two ways in which gene expression can be monitored under
various growth conditions.
• State how the regulation of gene expression in the lac operon is under the
influence of external and internal molecular cues/signals.
• Identify the molecules that serve as the repressor and inducer in the lac
operon.
• List three central conditional properties of operons.
• Speculate on reasons why microbes have such a large diversity of regula-
tory processes that control the synthesis of proteins.
• Describe six ways in which microbes control the initiation of transcription
as a means of regulating gene expression.
• Identify examples of protein-RNA interactions that regulate gene expres-
sion in a cell.
• Explain how the structure of the trp operon mRNA contributes to control-
ling gene expression for enzymes involved in biosynthesis of tryptophan
through attenuation.
• Give two reasons for the evolution of controls that govern sets of genes or
operons together as regulatory units (global regulation).
• Relate operons, regulons, and modulons.
• Use the catabolite repression modulon as an example to identify a reason
why some modulons are considered global regulatory systems.
• Explain how an E. coli cell uses the catabolite repression system to repress
transcription of the lac operon in the presence of both glucose and
lactose.
• Summarize the role of ppGpp in the stringent response modulon.
Even if all the processes described above were functioning optimally, they
would not prevent the synthesis of unneeded proteins. However, such waste
does not occur, b ecause enzyme amounts can be controlled by regulating gene
expression. In the mid-20th century, intensive efforts focused on discovering

how bacteria change the rates of synthesis of their enzymes. The initial
studies were done with E. coli and related enteric bacteria, but all bacteria
(and archaea) are extremely adept at turning genes on and off, thereby
raising and lowering the levels of enzymes by controlling the expression of
the encoding genes.
Evidence that flexibility of gene expression is widespread in both bacteria
and archaea can be quickly obtained using two tools in wide use today:
proteomics and transcriptomics. Monitoring the proteome allows one to survey
the rates of synthesis and the amounts of most proteins of the cell; tran-
scriptional monitoring tells the profile of all or specific mRNA molecules
in the cell. Many different species of prokaryotes radically change their
protein and mRNA profiles in response to different growth conditions. In
many (but not all) instances, the modulation of protein synthesis occurs at
the transcriptional level (that is, by changes in the synthesis of mRNA).
But, as we will discuss below, translational control can also be exerted to
adjust the amount of proteins that are made.
Gene regulation is an epic tale, best told in two parts. In the first, we
ask how individual genes and operons are controlled. In the second part,
we examine how sets of genes and operons are coordinately regulated and
tied into a cellular regulatory network, a regulatory process that integrates
so many gene targets that it is often referred to as global regulation.
Regulation of Gene Expression

Much of what we know about the regulation of gene expression comes from
pioneering studies of bacterial operons. As we have noted repeatedly, the
operon is a hallmark of the prokaryotic cell. It is a unit of transcription: a
DNA segment containing a promoter, two or more (typically more) genes
encoding polypeptides (cistrons), and a transcription terminator. Thus,
all of the genes in an operon are transcribed simultaneously in a single mRNA
molecule. A historic picture of the regulation of an operon, as first proposed
by F. Jacob and J. Monod in 1961 for the genes encoding the enzymes
metabolizing lactose in E. coli, designated lac, is shown in Fig. 11.6. The lac
operon contains the three genes (lacZ, lacY, and lacA) encoding all of the
proteins needed to metabolize lactose: LacZ (β-galactosidase, the enzyme
that hydrolyzes lactose), LacY (a membrane protein, or permease, that trans-
ports lactose into the cell), and LacA (a transacetylase of uncertain func-
tion). Upstream of the operon is the promoter, which is the binding site for
RNAP. The promoter overlaps with a control region, or operator, which
binds an allosteric protein called LacI (the product of the nearby gene lacI).
LacI is produced constitutively and thus is always available for binding the
operator. In the absence of lactose (Fig. 11.6A), LacI binds the operator and
prevents RNAP from transcribing the operon (either by preventing it from
binding or by blocking its movement). Thus, LacI is a negative regulator, or
repressor, of the lac operon: when bound to its operator, none of the three
enzymes encoded by the operon is made. Occasionally, the repressor disso-
ciates from the operator and a few transcript molecules are made, ensuring
that the cell will have a low basal level of the three products of the operon.
Why does the cell maintain a small amount of the Lac proteins? Once
lactose becomes available, t hese basal protein levels equip the cell to take
A
lac operon structural genes
Regulatory Promoter Operator
gene (lacI) (P) (O) lacZ lacY lacA
DNA
mRNA
Binding of repressor to operator

prevents transcription of lac genes
by RNAP.
Active
repressor
B
lacI P O lacZ lacY lacA
DNA
Transcription
Inactive
repressor
Translation
Galactosidase Permease Transacetylase

Inducer
Inducer binds to repressor and
inactivates it, allowing RNAP to
transcribe the lac genes.
Figure 11.6 Original operon model of Jacob and Monod, proposed in 1961 for the
regulation of the lac genes of E. coli. (A) Uninduced state of the lac operon in cells
growing on substrates other than lactose; (B) induced state during growth on lactose.
See the text for an explanation.
up and metabolize some of this sugar, an event that is sufficient to acti-

vate the lac operon (Fig. 11.6B). Lactose is brought into the cells by
LacY permease and is converted into a metabolite called allolactose by the
β-galactosidase LacZ. This derivative of lactose is the a ctual inducer of the
lac operon: it binds the LacI repressor, triggering a conformational change that
releases this allosteric regulatory protein from its promoter. RNAP is now free to
make lacZYA transcripts. More of the operon products are made, and the
cells bring more lactose in. But, you may be wondering, if lactose is all
the cells need to make the inducer that removes the repressor of the lac
operon, why did E. coli grow with lactose only after glucose was consumed in
the case study? Later in the chapter, we will learn the answer: catabolite
repression.
Research on the lac operon of enteric bacteria generated a number of

fundamental concepts of the operon, including the following.
1. Initiation of transcription from the promoter of an operon can be a site
of regulation.
2. Initiation of transcription can be controlled by allosteric proteins (i.e.,
the protein’s activity is governed by the binding of specific ligands).
3. Increase in the expression of an operon can be triggered by relief of a
negative control.
The lac operon is one elegant example of how microbes regulate a meta-
bolic adaptation, but it is not the only mechanism. Microbes exhibit a
dazzling diversity of solutions to metabolic and physiologic challenges. In
general, operons differ as to (i) the site at which control is exerted, (ii) the
mode of control (positive or negative), and (iii) the particular molecular
device used to bring about the regulation. Some operons even employ
more than one promoter; positioning a second regulatory site within the
operon provides greater flexibility and sensitivity of regulation. E very con-
ceivable step that leads from a gene to its finished protein product has been
singled out for control in some gene or operon or in some bacterium
(Fig. 11.7). Furthermore, some of these regulated steps may involve mul-
tiple molecular mechanisms. To examine some tricks that have evolved,
we will use a few well-studied examples. To guide the discussion, we w ill
σS
σH 2 Promoter recognition
70
σ
Inactive Inactive
activator Inducer repressor
RNA
4 Transcriptional polymerase 3 Transcriptional
Ligand Corepressor
activation repression
Active Active
activator repressor 6 Transcription
termination
DNA
P
A R
mRNA
DNA-bending
protein 5 Transcriptional 7 Regulatory sRNA 8 Messenger
enhancement E stability
9 Translational
control
10 Proteolysis
Ribosome
1 DNA topology
Figure 11.7 Some of the many regulatory processes that can control the synthesis of proteins and, thus, affect their
amounts in the cell. The boxes show various regulatory processes: those acting on the DNA (1, blue) and during (2 to 6, green) or
after (8 to 10, red) transcription. Regulatory sRNAs (7, tan) can modulate transcription or translation.
refer to the various regulatory mechanisms by the numbers (1 to 10) fol-

lowing the order of regulatory events shown in Fig. 11.7.
Transcriptional control. Transcription is a major regulatory hub in pro-

karyotes and transcription initiation, the most commonly controlled step.
By preventing the synthesis of the mRNA transcript, neither the encoding
RNA nor the protein is made; thus resources are not wasted. How is this
accomplished? Recall that transcription initiation involves the binding of
the σ-R NAP holoenzyme to a promoter and the subsequent opening of
the DNA in this region (chapter 8). Thus, any regulatory mechanism that
controls any of t hese early events in transcription can be targeted to control
the frequency of initiation. And though not as prevalent, transcription ter-
mination can also be regulated. Here we examine some of the most relevant
steps of control of transcription initiation (steps 1 to 5 in Fig. 11.7) and regu-
lated transcription termination (step 6 in Fig. 11.7). We will also describe
regulation of both by sRNAs (step 7 in Fig. 11.7).
1. DNA topology: An effective way to control the initiation of transcrip-

tion is by changing the DNA topology to either prevent or promote
σ-R NAP binding to the promoter. Most promoters are, for example,
sensitive to the degree to which the DNA is supercoiled or packed (step 1 in
Fig. 11.7). In fact, cells devote many proteins to the control of the topol-
ogy of their DNA.
2. Promoter recognition: A second way to control the initiation of tran-
scription from certain promoters is by interfering with promoter recog-
nition (step 2 in Fig. 11.7). Recall that binding to the σ factor allows
RNAP to recognize and bind to the –10 and –35 promoter sequences
and locally melt the promoter to initiate transcription (chapter 8). Bac-
teria control promoter recognition, producing alternative σ factors, which
when available compete with the housekeeping sigma factor σ70 and
redirect the σ-R NAP holoenzyme to specific genes in the chromosome.
This is an especially effective mechanism to coordinate the expression
of numerous genes scattered across the genome in a single step. We will
see other examples of global reprogramming later in the chapter and in
the next chapters, when discussing the activation of the heat shock re-
sponse and entry into stationary phase (chapter 12) and spore forma-
tion (chapter 13).
3, 4, and 5. Transcriptional repression, activation, and enhance-
ment: Initiation of transcription can also be activated, repressed, or
enhanced (steps 3, 4, and 5 in Fig. 11.7) in regulatory events involving
sequences called DNA control regions. Some of these control regions
are adjacent to or overlap the –10 and –35 promoter sequences (as the
operator in the lac operon), but others are dozens of nucleotides up-
stream of the promoter or downstream of the operon terminator.
In most cases, these control regions are the binding sites for allosteric
regulatory proteins. Each binding site is called a box, and many are
named after the protein they bind (such as the CRP box) or the process
they regulate (such as the nitrogen box). As we saw for the lac operon,
some regulatory proteins called repressors bind to DNA control regions
(operators) and decrease the frequency of transcription initiation

(step 3 in Fig. 11.7). By contrast, activators are positive regulators; that
is, they increase the frequency of transcription initiation (step 4 in
Fig. 11.7). Regulatory proteins can also bind control regions, called en-
hancers, that are distant from the gene or operon they regulate. To
exert their control, the enhancer-binding proteins often loop the DNA,
sometimes with help from DNA-bending proteins, to get closer to the
promoter region and promote initiation (step 5 in Fig. 11.7).
6. Transcription termination: Bacteria also regulate the termination of
transcription to control the expression of specific genes. Some bacteria
do so by a mechanism called attenuation, whereby the majority of
transcripts are prematurely terminated shortly after initiation. The best-
studied example of attenuation (and the system in which it was first
discovered) is the E. coli trp operon, encoding the enzymes for trypto-
phan biosynthesis. A 5′ segment of the trp mRNA (or leader sequence)
that precedes the trp operon structural genes can adopt two configura-
tions in the nascent mRNA: one that c auses premature transcription
termination and another that does not (Fig. 11.8). The speed of transla-
tion dictates which configuration this leader mRNA assumes. This, in
turn, depends on how fast the ribosomes are at translating two con-
secutive tryptophan codons in the leader sequence, which serve as
“sensing codons” of tryptophan availability.
• W
hen tryptophan is scarce, the ribosomes stall at these tryptophan
codons, waiting for tryptophanyl-tRNAs. The mRNA is then elon-
gated and acquires the antitermination configuration (Fig. 11.8) so
the enzymes for making tryptophan are produced.
• When tryptophan is abundant, the ribosomes translate the sensing co-
dons without delay and prevent the antitermination configuration from
forming. The mRNA adopts instead a conformation that pauses the
RNAP and then prematurely terminates transcription of the operon.
7. Regulatory RNAs (sRNAs): Transcription initiation and termination
can also be regulated by sRNA molecules. Recall, for example, the al-
losteric regulation exerted by the 6S RNA of E. coli, which we described
earlier. The 6S RNA transcript has a secondary structure that mimics
the open conformation of DNA when transcription is initiated (the so-
called open complex; see chapter 8). As stationary-phase cells produce
6S RNA transcripts, they bind σ70-R NAP holoenzymes, effectively pre-
venting them from interacting with their promoters. As a result, the
frequency of initiation of exponential-phase genes is reduced (step 7 in
Fig. 11.7). In a few instances, the sRNA molecule base pairs directly
with a strand of DNA in the promoter region to prevent transcription
from initiating. A more prevalent mode of transcriptional regulation by
sRNAs is, however, regulated transcription termination, whereby the
sRNA base pairs with a complementary region in the nascent mRNA
and acquires a conformation such that a termination loop forms that
stops RNAP elongation. As we will discuss later, sRNAs are versatile ef-
fector molecules and are also implicated in the control of mRNA stabil-
ity and translation (steps 8 and 9 in Fig. 11.7).
A Pause loop
1:2
AA A
Stop G C
U G
A AGUUCACG C G
U Ser C G
A U C Terminator loop
A U A 3:4
A Thr C G 80
A A U AAA
A Arg C G U G
G G U C A
A
G 60 C 120 C G
U
G G U G C
U Trp G C C G
20 A U A C G
U Trp G C C G
C 40 G C G C 140
GA C A A UGA A AGC A A UUUUCGU A CUGA A AGGUU A AUCAGAUACCCA UUUUUUU
Met Lys Ala Ile Phe Val Leu Lys Gly U A
G C
C G 100
G A
U A
A
B Antiterminator loop
2:3
A
U A
G A
AAGU C G 100
U G C
C U A
A A A
C C U
G C C
U A A
A C G
A U A
U
A A U
A U A
A G C
G U C
G 80 G C
G A A
U C G
20 A G C
U G C
C Met Lys Ala Ile Phe Val Leu Lys Gly Trp Trp Arg Thr Ser Stop G C
GA C A A UGA A AGC A A UUUUCGU A CUGA A AGGUUGGUGGCGC A CUUCCUGA A A C GCCU A A UGAGCGGGCUUUUUUU
40 60 120 140
Figure 11.8 Alternative secondary structures of the trp leader region of E. coli. The numbers 1, 2, 3, and 4 indicate the RNA
segments that form the secondary structures pictured. (A) Termination configuration. The arrowhead indicates the site of transcrip-
tion pausing. (B) Antitermination configuration.
Posttranscriptional control. Prokaryotic cells also use posttranscriptional

mechanisms to control gene expression (steps 8 to 10 in Fig. 11.7), which
we highlight next.
8. mRNA stability: On average, prokaryotic transcripts are short-lived
(chapter 8). By altering the stability of mRNA, a cell can regulate the
amount of its protein products (step 8 in Fig. 11.7). For example, upon
binding, some sRNAs trigger the degradation of their target mRNA molecules.
9. Translational control: Initiation of translation can be repressed or ac-
tivated (step 9 in Fig. 11.7). Noteworthy examples are the operons that
encode ribosomal proteins (r-proteins). Some of the mRNAs encoding
r-proteins contain nucleotide sequences that are recognized and bound
A B
ON OFF
Translation
SD
5’ SD AUG 5’ AUG
Translational Translational
repression activation
OFF ON
Translation
SD
5’ AUG 5’ SD AUG
Figure 11.9 Antisense mechanism of translational control by sRNAs. (A) Repression;

(B) activation.
to their own r-protein products when in free form (that is, not yet
assembled into ribosomes). Binding of r- proteins to their own
mRNAs prevents their translation, resulting in translational re-
pression. Consider how clever this mechanism is: the cell balances
the rate of r-protein synthesis with the rate of ribosome assembly
because the pool of free r-proteins can control the translation of their
own mRNAs.
As we mentioned above, translational control can also involve reg-
ulatory sRNA molecules. The same base-pairing interactions that direct
some sRNAs to bind a nascent mRNA and terminate its transcription
also allow them to interfere with mRNAs and affect their translation.
For example, binding of an sRNA to its mRNA target can block or
expose the Shine-Dalgarno sequence in the mRNA to ribosomes,
thus preventing or promoting ribosome binding and initiation of
translation, respectively (Fig. 11.9). Recognition of target mRNA is often
facilitated by RNA chaperones, which bind the sRNA molecules, es-
cort them to their targets, and stabilize the sRNA-m RNA interaction.
In chapter 12, we w ill learn more about sRNAs and one well-k nown
chaperone called Hfq.
Regulatory sRNAs are not the only effector molecules that bind
mRNAs and control their translation. Metabolites, for example, can
also bind target mRNAs with great specificity. Upon binding, the ef-
fector molecule changes the structure of the target mRNA to control
whether or not they get translated. The binding sequences in the
mRNAs, collectively known as riboswitches, are commonly found
in the 5′ untranslated region of the mRNAs. Translational control is,
along with transcriptional termination, the most prevalent regulatory
mechanism used by riboswitches to control gene expression. However,
some riboswitches can have dual functions, regulating both transcrip-
tion and translation.
10. Proteolysis: Though not high on most prokaryotic cells’ lists of regu-
latory devices, proteolysis plays a definite role (step 10 in Fig. 11.7).
Enzyme degradation provides, for example, a means to control a met-
abolic reaction. More commonly, however, proteolysis is employed to
remove a protein that regulates other proteins. For example, alternative σ
factors used to reprogram the promoter specificity of the core RNAP

under specific conditions are constantly being made and degraded.
Proteolysis keeps their cellular level below that of the major σ factor
(σ70 in E. coli) when not needed. When an environmental change calls
for a particular σ factor to come into play, its degradation is stopped, rap-
idly raising its concentration. A σ factor can be protected from prote-
olysis in part by its increased binding to core RNAP. This process is
significant in controlling the σ factor that triggers the heat shock re-
sponse, a topic that we will discuss in the next chapter.
Note that all forms of posttranscriptional regulation we have consid-
ered seem wasteful in the sense that mRNA is made but not used. True,
but this cost may be offset by other considerations that we know little
about. For several reasons, the overall energy budget of using one or an-
other regulatory device is hard to assess. So, the book is not closed on this
amazing panoply of regulatory devices. In fact, the broadest question—
why there are so many—is wide open (and for you to explore).
Global Regulation
Operons are a sleek way to coordinate the expression of multiple genes in
a biosynthetic pathway. But some processes require the coordinate expres-
sion of many genes scattered on the genome. We call this global regulation.
In the next chapter, we will learn just how important global regulatory
networks are for cells to succeed in a fluctuating environment. Here, we
will introduce the topic and describe, as an example, two well-known
global regulators.
Regulatory units above the operon. Prokaryotes have devised multiple

ways to integrate individual genes and operons into coordinated units. One
simple strategy is to control more than one transcriptional unit with the same
regulatory protein. How? Place the regulator’s DNA control region, or box,
in front of each of these genes and operons! Enteric bacteria use this
regulatory device in their SOS, oxidation damage, and anaerobic electron
transport systems. A second strategy to coordinate multiple genes and op-
erons, which we discussed earlier, is to reprogram RNAP using an alterna-
tive σ factor that recognizes each of the promoters of member operons. The
heat shock (chapter 12) and sporulation systems (chapter 13) of various
species employ this regulatory design. But σ f actors are not the only mod-
ulators of RNAP activity. For one of the largest networks, the stringent
control system, the member operons are regulated by the nucleotide
guanosine tetraphosphate (ppGpp), which binds and reprograms
RNAP to regulate multiple cellular pathways. We discuss this interesting
control mechanism below.
How do we name regulatory units above the operon? The group of in
dependent genes and operons governed by the same regulator comprise a reg-
ulon (Fig. 11.10). For example, the arg regulon of E. coli includes operons
scattered around the chromosome that together encode all the enzymes of
the arginine biosynthetic pathway. We also use “regulon” to refer to the
genes and operons regulated by a particular σ factor (e.g., the RpoS or σS
regulon). Above regulons are modulons (Fig. 11.10). A modulon is a set
of different regulons that display similar regulatory responses to a partic
A Regulon Rr R1
Operon A
P Gene Gene Gene

Rr R2
Operon B
P
Rr R3
Operon C
P
Regulon regulator
B Modulon Rr
Operon D
Rm Rr
Operon E
Regulon 1
Rm Rr
Operon F
Rm Rr
Operon G
Modulon Rm Rr
Operon H
Regulon 2
Rm Rr
Operon I
Rm
Operon J
Operon
Modulon regulator
Figure 11.10 Patterns of operon organization into higher (global) regulatory units.

(A) Regulon organization. Each operon (A, B, or C) is depicted as a segment of DNA
consisting of a control site at which a common protein regulon regulator (Rr) and operon-
specific protein regulators (R1, R2, and R3) act to control transcription of the genes of the
operon. The promoter (P) of each operon is also indicated. (B) Modulon organization. A
modulon consisting of six operons (E to J), controlled by a single modulon regulator (Rm) or
global regulator, is depicted. Operons E and F are part of one regulon (regulon 1), along
with operon D (which is not in the modulon). All three operons (G, H, and I) of regulon 2
are included in the modulon, along with the independent operon J. The two individual
regulon regulators are shown, as are the individual regulators of each operon.
ular physiological condition. Hence, modulons coordinate the expression

of regulons. Below, we describe two examples of modulons.
Examples of global regulatory systems. Some modulons are so pervasive in

their physiological effects that they help us understand the “global” nature
of global regulatory systems. Two modulons that fit this category are the
so-called catabolite repression system (which, as you will learn below,
does not r eally involve a “repressor”) and the stringent response system.
Together, these two systems directly or indirectly control probably three-
fourths of the protein-synthesizing capacity of the E. coli cell.
Catabolite repression. The ability of enteric bacteria to ensure that a pre-

mium substrate, glucose, is utilized to the exclusion of lesser substrates is
controlled (in part) by a global regulator called the cyclic AMP receptor
protein (CRP or CAP). When this allosteric regulatory protein binds the
small nucleotide ligand, cyclic AMP (cAMP), it recognizes and binds CRP-
specific boxes located upstream of the transcriptional start site of several
operons. Upon binding to the CRP box, cAMP-CRP increases the fre-
quency of transcription initiation, provided the operon has already been
induced by the operon’s own substrate. (Note that the name “catabolite
repression” refers to the cell’s response to glucose, which is controlled by a
transcriptional activator, CRP.)
Catabolite repression has extraordinary importance in the physiology
of many bacteria. The operons in its network can be expressed at very high
levels when induced by their substrates. Preventing such unnecessary ex-
pression of these operons is so critical to bacterial economy that their con-
trol within a modulon is widespread in the bacterial world, even though the
molecular mechanisms of control differ. For example, Gram-positive bac-
teria lack cAMP yet possess an effective catabolite repression system.
To illustrate how catabolite repression can override other regulatory
switches, let’s return to student JM, who grew E. coli with a mixture of glu-
cose and lactose (case study). Glucose uptake by the cells used the phos-
photransferase (Pts) system, a complex transport system that includes
proteins that regulate the phosphorylation state and, therefore, activity of
the enzyme (adenylate cyclase) that makes cAMP from ATP (Fig. 11.11).
Figure 11.11 Glucose catabolite Cell membrane

repression in E. coli. AC, adenylate
cyclase; CRP, cyclic AMP receptor
P
protein. The EIIGlc transporter of the
phospotransferase system (orange) is HPr EIIAGlc
regulated by phosphates transferred
from enzyme I (pink) to histidine P P
Glucose 6P
protein (green) to enzyme II (light
green). EP EI
HPr EIIAGlc P EIIGlc
P B
Glucose
C
ATP
CRP AC
cAMP
cAMP CRP
Activation of catabolic genes

So in JM’s culture, when glucose was transported, adenylate cyclase was

inhibited; consequently, the cellular pool of cAMP declined, and the
amount of cAMP-CRP decreased. Without this allosteric activator, RNAP
could not transcribe the lac operon to high levels even though lactose was
also present in the medium to induce the operon. Thus, the cells could not
use lactose until the glucose was consumed. Once this happened, the ad-
enylate cyclase was activated and cAMP levels increased, providing the
ligand for CRP activation.
Stringent response. The stringent response modulon is a gigantic regulatory

network geared to respond to starvation, for either energy or building blocks.
The response is extensive and affects a large number of operons and regu-
lons with diverse functions (Fig. 11.12). For example, limitation of amino
acids restricts the formation of one or more species of aminoacyl-tRNA, and this,
in turn, stalls ribosomes on the corresponding codons on the mRNA being
translated. A sensor on the stalled ribosomes (the enzyme RelA) is then
activated to convert GTP into ppGpp, a small molecule appropriately called
an “allarmone” for its critical role in signaling the cell that it is starving.
The ppGpp alarmone cooperates with other components to alter RNAP’s
affinity for many regulatory sites on DNA and reprogram the cell to respond to
starvation (Fig. 11.12). By activating some metabolic processes and re-
pressing others, the stringent response equips the cell to balance the avail-
able resources and cope with nutrient restriction. So powerful is the stringent
response that bacteria encode multiple elaborate biochemical paths for
making, degrading, and responding to ppGpp.
Amino acid depletion
Inhibition of
Blocked protein synthesis DNA initiation
(p)ppGpp
Inhibition Inhibition Inhibition Inhibition of Direct stimulation

of other of tRNA of rRNA protein chain of biosynthetic and
operons synthesis synthesis elongation catabolic operons
Inhibition Increased fidelity

of r-protein of protein
synthesis synthesis
Increased capacity to
produce necessary biosynthetic
and catabolic enzymes
Figure 11.12 Stringent response. The series of events ensuing after amino acid
restriction of bacteria are shown. The solid arrows indicate processes shown experimen-
tally to result from ppGpp accumulation. The dashed arrows depict more speculative
relations.
Why Regulate Both Protein Activity And Amounts?
Learning Outcomes:
• E
xplain why evolution has primarily favored two modes of metabolic
regulation in microbes (controlling either the amount or activity of
enzymes) over a single mode, such as allostery.
Why should a cell regulate its metabolism by changing both enzyme ac-
tivity and enzyme amount? Controlling the activity of an allosteric enzyme is a
swifter and more precise mechanism to adjust flow through the many meta-
bolic pathways. For example, recall the experiment where E. coli was
grown with glycerol only or with glycerol and histidine; histidine biosyn-
thesis was active in the first culture but repressed in the other. This is a
case of end-product inhibition by allosteric enzymes (Fig. 11.2). As soon as
histidine was added to the medium, it inhibited the first reaction of the
biosynthetic pathway. In fact, a cell could accomplish the task of coordinat-
ing metabolic flow in an orderly fashion using allosteric enzymes alone.
If allosteric enzymes can be so effective, why do prokaryotes also go to
great lengths to regulate the synthesis of their proteins (Fig. 11.7)? Evi-
dently, in a given situation evolution has pressured microbes to be highly
sensitive about what proteins they make and in what quantity. A reason-
able theory about the force b ehind this evolution is the need for economy and
efficiency. Over half the cell’s dry mass is protein, and making proteins is ex-
pensive (75% of the energy budget is devoted to protein synthesis!). Opti-
mizing the rate of growth in each environment requires that cells not make
redundant or irrelevant proteins. By this argument, metabolic coordination—
making order out of chaos—would seem to be the domain of allosteric
control of enzyme activity. On the other hand, competing successfully for
food and space would seem to demand the elegant and effective controls on
transcription and translation. From these considerations, one might specu-
late that allostery developed at an earlier stage of evolution than did con-
trols on gene expression. What do you t hink?
Case Revisited: How can E. coli selectively use sugars

from a mixture?
Even though lactose was present in the medium and the inducer had re-
moved the LacI repressor, something e lse was needed to activate the RNAP
holoenzyme and stimulate transcription of the lac operon. Clearly, the “stim-
ulating factor” was only available when glucose was no longer present.
What was it?
As you now know, RNAP needs stimulation by cAMP-CRP. Once
bound by cAMP, CRP binds to sequences upstream of the lac promoter and
interacts with RNAP to help it clear the promoter, allowing the operon to
be transcribed. Why was cAMP-CRP not available until all the glucose
was exhausted from the medium? Because the protein system that trans-
ports glucose (Pts) antagonizes the enzyme (adenylate cyclase) that makes
cAMP. As long as glucose was present, the adenylate cyclase was inacti-
vated and the levels of cAMP decreased. But once glucose was used up,
cAMP was made, and cAMP-CRP became available. The RNAP received
its “stimulating factor” and the lac operon was transcribed. The cells were
now able to grow on lactose, and the second phase of diauxic growth
started. Note that there was a lag phase between the two phases. Clearly,
it takes time for adenylate cyclase to be activated, for cAMP to be made
and bind CRP, and for the cAMP-CRP complex to bind and stimulate the
RNAP at the lac operon.
Lactose (no Lacl repressor)
lac operon
Glucose RNAP Glucose
Plac Z Y A
(low cAMP) growth
cAMP
lac operon
No glucose CRP RNAP Lactose
Z Y A
(high cAMP) growth
Conclusions
To avoid chaos, the approximately 2,000 individual chemical reactions a

typical prokaryotic cell requires to live and grow must be integrated into a
single complex system, with controls on the rate of each reaction. We have
seen that specific control devices have evolved to govern enzyme activity and
enzyme synthesis. We have also had a glimpse into the world of feedback
control and complex circuitry that integrates the activity of all the cell’s
parts and processes. As we considered how prokaryotic cells coordinate
their metabolism, the question of how they cope with their environment
has gradually come to the surface.
In real life, nothing is constant. All niches in the biosphere of Earth are
subject to changes in temperature, chemical composition, and all the other
parameters important for life. The environment of microbial cells is often
subject to catastrophic changes of a sort not usually met by the individual
cells of higher plants and animals. Microbes must cope with an environ-
ment that continually presents new challenges: changes in nutritive value,
temperature, pH, redox potential, barometric pressure, and the presence
of toxic factors. Such fluctuations exert an extraordinary burden on the
regulatory devices that coordinate the internal processes of the bacterial
cell. The story of how metabolic reactions are coordinated cannot be sepa-
rated from the intricate, clever ways in which microbial cells successfully
cope with environmental stresses. Therefore, the next chapter is a contin-
uation of this one.
Representative ASM Fundamental Statements

and Learning Outcomes for the Chapter
Evolution
1. Cells, organelles (e.g., mitochondria and chloroplasts), and all major metabolic pathways
evolved from early prokaryotic cells.
a. Give two reasons for the evolution of controls that govern sets of genes or operons
together as regulatory units (global regulation).
b. Explain why evolution has primarily favored two modes of metabolic regulation in
microbes (controlling either the amount or activity of enzymes) over a single mode,
such as allostery.
Metabolic Pathways
2. The survival and growth of any microorganism in a given environment depend on its
metabolic characteristics.
a. Explain the need for coordination of metabolic reactions within a cell.
b. Summarize a set of experiments that demonstrates coordination of biosynthesis in a
bacterium.
c. Speculate about at least one macromolecule (or group of macromolecules) that will
be in greater abundance for each phase of bacterial culture growth and undergo
coordination of polymerization.
d. List four reasons why prokaryotes more often control the amounts of enzymes pro-
duced rather than controlling enzymatic activity (as is more often seen in eukaryotes).
e. Define allostery.
f. Differentiate covalent modification and allosteric interactions as ways of controlling
protein activity.
g. Use the histidine biosynthetic pathway as an example to explain how feedback inhi-
bition is an effective means to control biosynthetic pathways.
h. Describe how the ratio of the concentration of ATP to those of ADP and AMP is main-
tained almost constant in bacterial cells over a wide range of energy supply and
demand.
Genetics and Information Flow

3. The regulation of gene expression is influenced by external and internal molecular cues
and/or signals.
a. Describe two ways in which gene expression can be monitored under various growth
conditions.
b. State how the regulation of gene expression in the lac operon is under the influence
of external and internal molecular cues/signals.
c. Identify the molecules that serve as the repressor and inducer in the lac operon.
d. List three central conditional properties of operons.
e. Speculate on reasons why microbes have such a large diversity of regulatory pro
cesses that control the synthesis of proteins.
f. Describe six ways in which microbes control the initiation of transcription as a means
of regulating gene expression.
g. Identify examples of protein-RNA interactions that regulate gene expression in a
cell.
h. Explain how the structure of the trp operon mRNA contributes to controlling gene
expression for enzymes involved in biosynthesis of tryptophan through attenuation.
i. Relate operons, regulons, and modulons.
j. Use the catabolite repression modulon as an example to identify a reason why some
modulons are considered a global regulatory system.
k. Explain how an E. coli cell uses the catabolite repression system to repress transcrip-
tion of the lac operon in the presence of both glucose and lactose.
l. Summarize the role of ppGpp in the stringent response modulon.
SUPPLEMENTAL MATERIAL
References
• Görke B, Stülke J. 2008. Carbon catabolite repression in bacteria: many ways to make
the most out of nutrients. Nat Rev Microbiol 6:613–624. doi:10.1038/nrmicro1932.
• Hauryliuk V, Atkinson GC, Murakami KS, Tenson T, Gerdes K. 2015. Recent functional
insights into the role of (p)ppGpp in bacterial physiology. Nat Rev Microbiol 13:298–
309. doi:10.1038/nrmicro3448.
• Lewis M. 2013. Allostery and the lac operon. J Mol Biol 425:2309–2316. d oi:10.1016/
j.jmb.2013.03.003.
• Storz G, Vogel J, Wassarman KM. 2011. Regulation by small RNAs in bacteria: expand-
ing frontiers. Mol Cell 43:880–891. doi:10.1016/j.molcel.2011.08.022.
Supplemental Activities
Check your understanding

1. In the case study, when JM cultured E. coli in a medium supplemented with both
glucose and lactose, E. coli initially utilized glucose rather than lactose. Why?
a. A lactose derivative relieves inhibition of the lac operon.
b. The lac operon is activated in the presence of lactose and glucose.
c. In the presence of glucose, cells have reduced amounts of the cAMP-CRP allosteric
r egulator.
d. Binding of cAMP to CRP increases transcription of the lac operon.
e. All of the above
2. Enzyme activity can be modulated by __________.
a. allostery by an sRNA molecule
b. increased expression of the gene encoding the enzyme
c. phosphorylation
d. methylation
e. all of the above
3. What fitness advantage do bacteria gain by controlling enzyme amount instead of
enzyme activity?
a. The enzyme is essential for viability.
b. Cells do not invest energy-synthesizing proteins that are not needed.
c. The enzyme is available for immediate use.
d. Less energy is required to synthesize protein and store it.
4. Of the dozen or so different mechanisms for regulating the synthesis of specific
proteins, name two that involve direct participation of r ibosomes.
5. Explain how control regions located hundreds of nucleotides away from a
promoter can control gene expression from that promoter. What name is given to
such control r egions?
Dig deeper
1. Prokaryotic cells coordinate metabolic pathways by controlling the amount of enzymes
or modulating their activity by allosteric interactions.
a. Which type of control is generally faster?
b. What advantage would be sacrificed if all metabolic coordination were achieved
solely by modulating protein activity?
2. The transcription of bacterial operons is often controlled by regulatory proteins, which
bind the operator region and either decrease (repressors) or increase (activators) the
frequency of initiation. In the late 1990s, Savageau proposed the “demand theory” of
gene regulation (M. A. Savageau, Genetics, 149:1665–1676, 1998), which stated that neg-
ative or positive modes of regulation are selected for genes encoding functions in low or
high demand, respectively.
330 par t i F u nda m enta ls o f m icro b ia l Life
a. Based on your knowledge of the regulation of the lac operon, would you predict
lactose to be often found in environments inhabited by E. coli?
b. Arabinose utilization in E. coli requires the expression of the araBAD genes, which
are encoded in an operon that is positively regulated by a regulatory protein (AraC)
but only when bound to arabinose. Would you predict arabinose to be abundant in
the environment(s) inhabited by E. coli?
c. Would you predict operons involved in the synthesis of building blocks to be
positively or negatively regulated?
d. Operons encoding biosynthetic enzymes (e.g., synthesis of amino acids) are
generally turned on unless their building block end product is present in the
environment. In these cases, the end-product feedback inhibits an allosteric
enzyme early in the biosynthetic pathway. The end product can sometimes perform
double duty and activate an allosteric repressor protein to shut off the transcription
of the operon. Bacteria in the human gut are constantly exposed to a hefty supply
of amino acids derived from dietary protein. Based on the “demand theory,” do you
expect the human intestinal environment to select for positive or negative modes of
control of operons involved in the synthesis of amino acids?
e. Consider now the economic cost if a cell acquires a null mutation in a positive
regulator versus a negative regulator. How might this contribute to the pattern that
operons encoding products in high demand tend to be positively regulated,
whereas those with intermittent or low demand are negatively controlled?
3. Riboswitches are sequence elements typically located in the 5′ untranslated region of
mRNA that bind effector molecules with great specificity. Upon binding the effector
molecule, the structure of the nascent mRNA changes and its transcription and/or trans-
lation are promoted or prevented. Search the Web for examples of riboswitches that
a. terminate transcription of the mRNA prematurely.
b. prevent the translation of the mRNA.
c. use an antisense sRNA as the effector molecule.
d. use a metabolite as an effector molecule.
4. Team work: Read the following research article on the Staphylococcus aureus sRNA
SprD, which regulates bacterial virulence: S. Chabelskaya et al., PLoS Pathog, 6:e1000927,
2010; doi:10.1371/journal.ppat.1000927.
a. What experiments did the authors perform to determine that the sRNA SprD
regulates the expression of the Sbi protein at the translational level but not
transcriptional level? (Hint: see Fig. 2.)
b. Draw a schematic to illustrate how SprD regulates the expression of the sbi mRNA.
c. How did the authors show that SprD RNA enhanced the virulence of S. aureus in
mice?
d. Discuss possible ways to control virulence of S. aureus through SprD.
Supplemental Resources
Visual
• Walter Gilbert describes how they designed the experiment that allowed his group to
isolate the LacI repressor (1:09 min): https://w ww.dnalc.org/view/15254-The-experimental
-design-for-the-lac-operon-Walter-Gilbert-.html.
• Animation of attenuation at the trp operon: http://w ww.microbelibrary.org/library
/biology/2825-regulation-of-biosynthesis-attenuation-of-the-trp-operon.
• Beatrix Suess discusses how synthetic biologists are engineering riboswitches in bacte-
ria to manipulate the expression of genes of interest (22:15 min): https://w ww.youtube
.com/watch? v=Ptsv5ZtnnXw.
Written
• S. Marvin Friedman tells us how some plant pathogens inject double-stranded sRNAs in
their host during an infection to suppress their immune system in the Small Things Con-
sidered blog post “Plant Pathogen Silences Host’s Immune Genes” (http://schaechter
.asmblog.org/schaechter/2014/07/plant-pathogen-silences-hosts-immune-genes.html).
• Fred Neidhardt gives a personal account of how capturing cells’ protein profiles by two-
dimensional gel electrophoresis opened the door to the proteomics field of today in the
Small T
hings Considered blog post “How Proteomics Got Started” (http://schaechter
.asmblog.org/schaechter/2009/12/how.html).
• Fred Neidhardt discusses how the stringent response coordinates not only bacterial
growth but also virulence of a number of pathogens, including Legionella pneumophila, in
the Small T
hings Considered blog post “Bacterial Physiology and Virulence: The Cultures
Converge” (http://schaechter.asmblog.org/schaechter/2010/09/bacterial-physiology-and
-virulence-the-cultures-converge.html).

CH 11 Microbe Coordination of Cell Processes

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CH 11 Microbe Coordination of Cell Processes

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10.1128/9781555819132.

network—an integrated cir­cuit of enormous complexity and elegance, sur-

Case: How can Escherichia coli selectively use sugars from a mixture?

Many heterotrophic bacteria have the remarkable ability to use glucose

What evidence shows that metabolic reactions

As student JM observed experimentally in the case study, microbes are

Coordination in Macromolecular Composition

How Do Microbes Regulate Their Metabolism?

A. Protein amount B. Protein activity

Figure 11.1 ​Overview of metabolic regulatory devices. Synopsis of the two forms of

Controlling Enzyme Amounts

tion also starts right away. If transcription of an enzyme-­encoding gene

Controlling Enzyme Activity

How Is Protein Activity Modulated?

Modulation of enzyme activity occurs rapidly, almost instantaneously, ad-

Allostery in biosynthesis. Earlier we noted that, given the choice, a bacte-

Enzyme1 Enzyme2 Utilization for

Figure 11.2 ​Feedback inhibition. The final product of a series of enzymatic reactions,

Allostery in fueling. Allosteric inhibition and activation also regulate the

Pentose phosphate cycle Glycolysis

Allostery by regulatory RNAs. Prokaryotic cells also use RNA molecules as

Inhibit Modify A tiva

How Are Protein Amounts Modulated?

expression. In the mid-20th ­century, intensive efforts focused on discovering

Regulation of Gene Expression

Binding of repressor to operator

Galactosidase Permease Transacetylase

up and metabolize some of this sugar, an event that is sufficient to acti-

Research on the lac operon of enteric bacteria generated a number of

refer to the vari­ous regulatory mechanisms by the numbers (1 to 10) fol-

Transcriptional control. Transcription is a major regulatory hub in pro-

1. DNA topology: An effective way to control the initiation of transcrip-

(operators) and decrease the frequency of transcription initiation

Posttranscriptional control. Prokaryotic cells also use posttranscriptional

Figure 11.9 ​Antisense mechanism of translational control by sRNAs. (A) Repression;

f­actors used to reprogram the promoter specificity of the core RNAP

Regulatory units above the operon. Prokaryotes have devised multiple

P Gene Gene Gene

Figure 11.10 ​Patterns of operon organ­ization into higher (global) regulatory units.

u­lar physiological condition. Hence, modulons coordinate the expression

Examples of global regulatory systems. Some modulons are so pervasive in

Catabolite repression. The ability of enteric bacteria to ensure that a pre-

Figure 11.11 ​Glucose catabolite Cell membrane

Activation of catabolic genes

So in JM’s culture, when glucose was transported, adenylate cyclase was

Stringent response. The stringent response modulon is a gigantic regulatory

Amino acid depletion

Inhibition Inhibition Inhibition Inhibition of Direct stimulation

Inhibition Increased fidelity

Why Regulate Both Protein Activity And Amounts?

Case Revisited: How can E. coli selectively use sugars

Lactose (no Lacl repressor)

To avoid chaos, the approximately 2,000 individual chemical reactions a

Representative ASM Fundamental Statements

Genetics and Information Flow

Check your understanding

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network—an integrated circuit of enormous complexity and elegance, sur-

Figure 11.1 Overview of metabolic regulatory devices. Synopsis of the two forms of

tion also starts right away. If transcription of an enzyme-encoding gene

Figure 11.2 Feedback inhibition. The final product of a series of enzymatic reactions,

expression. In the mid-20th century, intensive efforts focused on discovering

refer to the various regulatory mechanisms by the numbers (1 to 10) fol-

Figure 11.9 Antisense mechanism of translational control by sRNAs. (A) Repression;

factors used to reprogram the promoter specificity of the core RNAP

Figure 11.10 Patterns of operon organization into higher (global) regulatory units.

ular physiological condition. Hence, modulons coordinate the expression

Figure 11.11 Glucose catabolite Cell membrane