B
B6
See: C57BL/6
BAC (Bacterial Artificial
Chromosome)
G M Weinstock
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.0098
Bacterial artificial chromosomes (BACs) are plasmids
used for cloning and stably maintaining large seg-
ments of foreign DNA in Escherichia coli. This is
important in various types of analyses of mammalian
and other genomes. A problem in some recombinant
DNA experiments is the stable maintenance of large
(>100 kilobase pairs) inserts in E. coli. Typically, most
plasmid vectors used for cloning foreign DNA into
E. coli can stably carry DNA fragments of 10 kb or
less. When the DNA inserted into a standard cloning
vector exceeds this size, several problems may result:
1. The plasmid clone replicates poorly (due, for
instance, to foreign sequences that adopt structures
that are difficult for the E. coli apparatus to repli-
cate, or do not segregate evenly at cell division).
2. The cell grows slowly (due, for example, to toxic
products being produced by gene expression from
the foreign DNA).
3. The inserted DNA adopts structures, such as cruci-
forms, that are readily deleted in E. coli.
In the first two cases, rare deletions in the insert that
reduce its size and eliminate growth problems will have
a growth advantage over the parental clone and even-
tually overgrow the culture. Thus in all these instances
the large insert is unstable and hard to maintain.
In 1992, Shizuya and Simon at Cal Tech developed
vectors capable of maintaining large inserts without
these problems. These vectors were plasmids that con-
tained the origin of replication from the F factor of
E. coli. The F factor is a large plasmid that is normally
capable of replication of DNA molecules greater than
100 kb in length. It is a low copy number plasmid,
being present in only one to two copies per cell.
These two features aid in preventing problems 1 and
2 above. The mechanism of deletion of structures
such as cruciforms (problem 3) requires that certain
enzymes, such as nucleases, load onto DNA and may
also be influenced by DNA superhelicity, both of
which may be different with a replication fork formed
from the F origin of replication than from other repli-
cons, such as are used in higher copy number vectors.
BAC vectors have been engineered to have other fea-
tures such as selectable antibiotic resistance genes and
restriction enzyme cleavage sites for inserting or
removing foreign DNA.
BAC vectors have been extremely successful for
cloning and maintaining mammalian DNA in E. coli.
In the Human Genome Project, BAC clones from
large libraries (105±106 clones) were first carefully
mapped along each chromosome, and then the DNA
sequences of a subset of these BAC clones were indi-
vidually determined and assembled to provide the
complete human genome sequence.
See also: DNA Cloning; Plasmids; Vectors
Bacillus subtilis
A Danchin
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.0099
Dozens of genomes have been sequenced and many
more will soon be added to the list. Unfortunately,
most genomes have long runs of nucleotides that
encode genes with unknown functions. Achieving an
understanding of model bacteria as a means of organ-
izing our biological knowledge (and more generally
our knowledge about what constitutes life) is there-
fore of particular importance. Only two bacterial
models are available: Escherichia coli, which is the
model for gram-negative bacteria and is the best-
known living organism; and Bacillus subtilis, which is
the model for gram-positive bacteria. The recent con-
troversial proposal of Gupta to classify bacteria into
monoderm and diderm cell types places these two
136 B a c i l l u s s u b tilis
model systems in key positions in investigations of
what constitutes life. In the case of B. subtilis, most
of the studies have been devoted to specific processes
such as sporulation, competence/transformation/re-
combination, or secretion. Until recently, not much
was known about the intermediary metabolism of
B. subtilis. After the elucidation of its genome
sequence, facts and concepts in this area increased
dramatically. Apart from its importance as a model
organism, B. subtilis is also widely employed in bio-
technology, for example in fermentation processes
(secretion of enzymes and processing of plants such
as soybean). Because the sequence of its genome is
now known, it is fast becoming one of the few uni-
versal models for the understanding of the require-
ments for life in unicellular organisms.
Bacillus subtilis and its Biotope
The objective of any living organism is to occupy
a part of the earth's crust. This means, among other
ancillary functions, the exploration, colonization,
maintenance and exploitation of the local resources
dealing with congeners and with other organisms, etc.
As a consequence, one cannot understand an organism
if one does not have knowledge of its habitat. Bacillus
subtilis was first identified in 1872. It is a bacterium that
can be routinely obtained in pure culture by soaking
hay in water for a few hours at 37 8C, then filtering and
boiling for 1 h at neutral pH. Bacillus subtilis has also
been isolated directly from soil-inoculated nutrient
agar, where B. subtilis predominates among the out-
growing cultures. Spores are more readily obtained in
solid media than in liquid media, and they require the
presence of manganese ions. The bacteria produce a
complex lipopeptide, surfactin, that permits them to
glide very efficiently over the surface of certain types of
media. This property is likely to be related to coloni-
zation of the surfaces of leaves (the `phylloplane'),
fruits or sometimes roots. Indeed B. subtilis makes up
the major population of bacteria on flax stems during
the retting process. Vegetative cells of B. subtilis are
responsible for the early stages of breakdown of plants,
and sometimes products of animal origin; some vari-
ants (e.g., B. amyloliquefaciens) cause potato tubers to
rot. When conditions become unfavorable, the onset of
a differentiation process, sporulation, permits the cells
to generate resistant spores that can be easily dispersed
throughout the environment, where they will germin-
ate if conditions are appropriate.
Unlike most other bacterial species, endospore-
forming bacteria are highly resistant to the lethal
effects of heat, drying, many chemicals, and radiation.
In fact, one fashionable hypothesis of the origin of life
on earth by panspermia by Sven Arrhenius, and more
recently Francis Crick, relies on the notion that bac-
terial spores such as those of B. subtilis could travel
through space and survive for millions of years. Des-
pite its appeal to a wild imagination, this hypothesis
essentially puts the investigation of the origin of life
out of our reach, because exploring the whole Universe
is not possible.
Compartmentalization: Bacillus subtilis
and its Envelopes
Envelope of the Vegetative Cell
Gram-positive bacteria and, in general, monoderms
have complex envelopes comprising one bilayer lipid
membrane separating the cytoplasm from the exterior
of the cell. The membrane is part of a very complex
structure that comprises many layers (up to 40 in the
case of B. subtilis) of murein, or peptidoglycan, a com-
plex of peptides containing d-amino acids (in par-
ticular mesodiaminopimelic acid), and amino sugars.
The cell envelope also has several layers of teichoic
acid (Figure 1).
The possible existence of a periplasm in B. subtilis
in a distinct cell compartment surrounded by the
cytoplasm membrane and the cell wall is a controver-
sial issue. Cytoplasm, membrane, and protoplast
supernatant fractions were prepared from protoplasts
generated from phosphate-limited cells. The proto-
plast supernatant fractions was found to include cell
wall-bound proteins, exoproteins in transit, and con-
taminating cytoplasmic proteins arising through leak-
age from a fraction of protoplasts. By this operational
definition, 10% of the proteins of B. subtilis can be
considered periplasmic.
Sporulation
Upon starvation, B. subtilis stops growing and initi-
ates sporulation. This developmental process involves
differentiation into two cell types (Figure 1). The
process begins with a reorganization of the cell cycle
that leads to the production of cells whose size and
chromosome content is appropriate for the develop-
mental process. The formation of the two cell types,
a forespore and a mother cell twice as large as the
Figure 1 (See Plate 2) Electron micrograph of Bacillus
subtilis in the process of sporulation.

forespore, with differing developmental fates is the
first morphological indication of the early stages of
sporulation in B. subtilis. Endospore formation is a
multistep process that is common among bacilli. This
seemingly simple structure is the product of a very
complex network of interconnected regulatory
pathways that become activated during late growth
in response to unbalanced nutritional shifts and cell
cycle-related signals. Sporulation starts with stage 0
(vegetative growth). Symmetrical cell division, char-
acteristic of vegetative growth, is blocked. Instead, the
cell divides asymmetrically to produce a small polar
prespore cell and a much larger mother cell. During
stage I, asymmetrical preseptation starts. The cellular
DNA takes the shape of an axial filament. At stage II,
septation proceeds and the daughter chromosomes
are separated. Spore development follows at stage III
(engulfment of the forespore and complete separation
of the spore membrane from that of the mother cell).
Stage IVinvolves formation of the spore cortex. In stage
V spore coat proteins are synthesized and assembled.
At stage VI the spore becomes highly refractile under
the microscope, and it acquires heat and stress resist-
ance. Finally, the programmed death of the mother cell
occurs, leading to lysis and release of the mature spore
(stage VII). Pigments are produced that stain the spores
from reddish brown to blackish brown (black in the
presence of tyrosine).
Stage 0 Stage I
Septation
σA σA pro-σ E
σH σH σF
Stage III
Engulfment
pro-σE pro-σ E σE
σF
σE σG pro-σ K
Stage V Stages VI −VII
Coat Maturation, lysis
σK σG
Germination Outgrowth
Figure 2 The stages of sporulation. The various sigma transcription factors that control gene expression processes
during sporulation are indicated within the compartments where they operate.
B a c i l l u s s u b t i l i s 137
Our understanding of sporulation control in
B. subtilis is extensive. The process combines phos-
phorylation cascades mediated by kinases and phos-
phatases with a network of transcription controls
by sigma factors, together with membrane-bound ef-
fector molecules that control compartmentalization
(Figure 2). Despite the intensive work of hundreds of
scientists on many of the signals involved in the onset
and control of sporulation, some still remain unknown.
The spore coat is a complex envelope comprising
several layers of spore coat proteins that protect the
almost entirely desiccated interior of the spore, where
DNA is compacted and protected from the harmful
influence of the environment. Under conditions of
appropriate moisture, in media that contain alanine,
glucose, and minerals, spores are able to germinate.
This process involves swelling and a complex lytic
process that opens and sometimes degrades the coat-
ing envelope, during which time metabolism is
initiated. Cells then resume normal vegetative growth.
Quorum-Sensing and Chemotaxis
It has long been known that bacteria form colonies
on agar plates. If the medium is appropriate these
colonies give rise to bacterial swarming. In the late
1960s, it was observed that cultures of Vibrio fischeri
(a luminescent gram-negative bacterium that colon-
izes squid) remained nonluminescent during the first
Stage II
pro-σ E pro-σ E
σF σF
Stage IV
Cortex
pro-σ K
σG σG
σK
Free spore
138 B a c i l l u s s u b tilis
hours of growth, during which time the number of
cells increased. Luminescence appeared when the
population reached a significant density, at a moment
when the bacteria ran out of nutrients. This collective
behavior meant that a bacterial function was expressed
at a certain cell density: the organisms in the popu-
lation were sensing each other. This, was termed
`quorum sensing.'
A variety of processes are regulated in a cell density
or growth phase-dependent manner in gram-positive
bacteria. In the early 1990s quorum sensing was dis-
covered in B. subtilis and was certainly linked to spor-
ulation (to swarm or to sporulate, that is the question),
but the functional reason(s) for the existence of the
process are not yet known. Most bacteria that use
quorum sensing systems inhabit an animal or plant.
The microorganisms benefit from the process, but
the host organism may or may not. Each bacterium
produces small diffusible molecules that allow cell-to-
cell communication. As the population of bacteria
increases, so does the concentration of the signaling
molecules. Sensors recognize these molecules. Once
the local concentration in the medium has reached a
threshold value, the sensor proteins transmit a signal
to a transcriptional regulator. Examples of such
quorum-sensing modes are the development of
genetic competence in B. subtilis and Streptococcus
pneumoniae, the virulence response in Staphylococcus
aureus, and the production of antimicrobial peptides
by several species of gram-positive bacteria, including
lactic acid bacteria.
Avariety of ways for bacterial populations to coord-
inate their activities have been discovered. Cell density-
dependent regulation in these systems appears to
follow a common theme. First, the signal molecule (a
posttranslationally processed peptide±pheromone) is
secreted by a dedicated ATP-binding cassette (ABC)
exporter. The role of the secreted peptide pheromone
is to function as the input signal for a specific sensor
component of a two-component signal-transduction
system. Coexpression of the elements involved in
this process results in self-regulation of peptide±
pheromone production. Peptides are secreted and
processed under various conditions that are further
recognized by the cell. Next, in response to phero-
mone, cells swim in a coordinated fashion, thereby
forming a kind of wall surrounding rings of bacteria
having the same exploration behavior (Figure 3).
Bacillus subtilis is a highly motile bacterium, endowed
with a complex flagellar machinery. This permits cells
to swim toward nutrients or away from repellents.
Many genes similar to those known in motile bacteria
are found in B. subtilis, making it likely that the tum-
bling and swimming processes function similarly to
those of E. coli. One can expect that this behavior
permits the cell to invade and colonize the surface of
leaves where they can find nutrients (especially as
carbon and nitrogen sources as well as micronutrients)
secreted by the plant or decaying leaves. The bacteria
secrete antibiotics that permit them to outcompete
other organisms, for example the products of the pks
genes act against Agrobacterium species. This estab-
lishes a cooperation between the plant and the bac-
teria; commensalism rather than symbiosis.
Protein Secretion
Bacillus subtilis is one of the organisms of choice in the
study of protein secretion. At the time of this review,
many fundamental aspects of this process are not yet
understood. Several systems enable proteins to be
inserted into the membrane and/or to be located out-
side of the membrane or secreted into the surrounding
medium. In B. subtilis, the Sec-dependent pathway
(one that recognizes signal peptides) has at least five
different signal peptide peptidases. Proteins that are
periplasmic in gram-negative bacteria are also found
in B. subtilis, presumably as lipoproteins (i.e., posses-
sing a specific signal peptide, cleaved upstream of a
cysteine residue that is covalently coupled to the outer
lipid layer of the cell membrane upon cleavage).
The signal recognition particle (SRP) system is an
oligomeric complex that mediates targeting and inser-
tion of proteins into the cytoplasmic membrane. SRP
consists of a 4.5S RNA and several protein subunits.
One of these subunits, Ffh, interacts with the signal se-
quence of nascent polypeptides. The N-terminal resi-
dues of Ffh include a GTP-binding site (G-domain)
and are evolutionarily related to similar domains in
other proteins. A second protein, the counterpart of
the E. coli FtsY protein, is believed to play a role
similar to that of the docking protein in eukaryotes.
Finally, it appears that some B. subtilis secreted
proteins are made of two parts. The first part remains
inserted in the membrane, presumably as a permease,
and the second part is liberated in the surrounding
medium after cleavage by an unknown protease.
Metabolism
In addition to the need for compartmentalization, liv-
ing cells must chemically transform some molecules
into others. Metabolism is the hallmark of life. Cells
can be in a dormant state, as is the case with spores, for
example, but one cannot be sure that they are living
organisms unless, at some point, they initiate metab-
olism. In general, one distinguishes between primary
metabolism (the transformation of molecules that sup-
port cell growth and energy production) and second-
ary metabolism (transactions involving molecules that
are not necessary for survival and multiplication, but
 B a c i l l u s s u b t i l i s 139

Processing
PEP19
Outside Oligopeptide
permease

Cell membrane

Inside
Modification ?
RapA
phosphatase Phr
PEP6

Spo0F + Pi

+ −

ATP KinA Spo0F −P Spo0B Spo0A −P

Spo0E
ADP KinA −P Spo0F Spo0B −P Spo0A Pi

Figure 3 Competence is triggered when Bacillus subtilis encounters a signal from the environment and when an
appropriate quorum is reached, monitored by phenomones synthesized by the bacteria. The chain of events is
depicted. A sensor controls a regulator which, though a phosphorylation cascade, controls transcription. The onset
of sporulation negatively controls competence under appropriate conditions through the action of the protein
SpoOK.

assist in the exploration and occupation of biotopes,
e.g., antibiotic synthesis).
Transport of Basal Cell Atoms
Carbon, oxygen, nitrogen, hydrogen, sulfur, and
phosphorus are the core atoms of life. Electron trans-
fers and catalytic processes, as well as the generation of
electrochemical gradients, require many other atoms
in the form of ions. Metabolic processes allow the cell
to concentrate, modify, and excrete ions and molecules
that are necessary in energy management, growth and
cell division.
Nutrients and ions are transported into cells by
a number of more or less specific permeases, most
of which belong to the ABC permease category. In
B. subtilis these permeases generally comprise a bind-
ing lipoprotein responsible for part of the specificity,
located at the external surface of the membrane, an
integral membrane channel made of proteins of two
different types, and a dimeric, membrane-bound cyto-
plasmic complex, which binds and hydrolyzes ATP as
the energy source. For positively charged ions, select-
ivity is the most important feature of the permease,
because the electrochemical gradient is oriented
toward the interior of the cell (negative inside). Ions
must be concentrated from the environment until they
reach the concentration required for proper activity,
but must not reach inhibitory levels. Apart from iron,
which is scavenged from the environment with highly
selective siderophores synthesized in response to iron
limitation, manganese is the most important transition
metal ion for B. subtilis. It is required for many en-
zyme activities, such as superoxide dismutase, agmatin-
ase, phosphoglycerate mutase, pyrophosphatase, etc.
Copper is important for electron transfer and cobalt is
required by the important recycling protein methio-
nine aminopeptidase. Nickel is required by urease, zinc
is needed as a cofactor of polymerases and dehydro-
genases, and magnesium is involved in catalytic com-
plexes with substrate in about one-third of enzyme
reactions. Potassium is needed to construct the elec-
trochemical gradient of the cell's cytoplasm, and is a
likely cofactor in many reactions. Calcium is probably
needed in major reactions during the division cycle,
but the importance of this ion still remains a mystery.
Anions are also important, and they need to be
imported against a strong electrochemical gradient.
Phosphate in particular requires a set of highly
involved transport systems. For B. subtilis a main
source of phosphate is probably phytic acid, a slowly
degraded phosphate-rich molecule. Sulfate is the pre-
cursor of many important coenzymes in addition
to cysteine and methionine, but not much is yet
known about its transport and metabolism, except
140 B a c i l l u s s u b tilis
by comparison with the counterparts known to be
present in E. coli.
Intermediary Metabolism
Carbon and nitrogen metabolism in B. subtilis follow
the general rules of intermediary metabolism in aerobic
bacteria, with a complete glycolytic pathway and
a tricarboxylic acid cycle. Electron transfer to oxygen
is mediated by a set of cytochromes and cytochrome
oxidases, allowing efficient respiration in B. subtilis.
This organism is generally said to be a strict aerobe,
and indeed it respires very efficiently. However, it can
grow in the absence of molecular oxygen, provided
that appropriate electron acceptors such as nitrate
are present in the environment. Coupled to electron
transfer, a proton addition between NAD(P) and
NAD(P)H occurs. Bacillus subtilis does not possess
a transhydrogenase that could equilibrate the pools
of NADH and NADPH. Therefore, because the
enzymes using NAD and NADP often differ, there
must be a means of equilibrating the corresponding
pools of reduced molecules.
As expected from its vegetal biotope, B. subtilis can
grow on many of the carbohydrates synthesized by
plants. In particular, sucrose can function as a major
carbon source in this organism, via a very compli-
cated set of highly regulated pathways. As in many
other eubacteria, the phosphoenolpyruvate-dependent
(PTS) system plays a major role in carbohydrate trans-
port and regulation. Catabolite repression control,
mediated by a unique system involving specific factors
(and no cyclic AMP), exists in this organism. Some
knowledge about nitrogen metabolism in B. subtilis
has accumulated, but significantly less than in its E. coli
counterpart. Many nitrogenous compounds, such as
arginine or histidine, can be transported and used by
B. subtilis. A specific transcription factor controls
nitrogen availability. Amino acid biosynthesis is not
yet well documented, but purine and pyrimidine
metabolism is well understood. In B. subtilis, in con-
trast to E. coli, there are two carbamoylphosphate
synthases: one specific for arginine synthesis and the
other for pyrimidine synthesis. As in other living
organisms, the ubiquitous polyamines putrescine and
spermidine play a fundamental, yet enigmatic, role.
They arise via the decarboxylation of arginine to
agmatine, coupled to a manganese-containing agmatin-
ase, and not from decarboxylation of ornithine, as in
higher eukaryotes.
Special Environmental Conditions
Another aspect of the B. subtilis life cycle is that it can
grow over a wide range of different temperatures up to
54±55 8C. This indicates that its biosynthetic machinery
comprises control elements and molecular chaperones
that permit this versatility. Specific transcription con-
trol processes allow the cell to adapt to changes of
temperature by transiently synthesizing heat shock or
cold shock proteins, according to the environmental
conditions. In addition, gene duplication may permit
adaptation to high temperature, with isozymes having
low and high temperature optima. As a case in point,
B. subtilis has two thymidylate synthases. The one
coded by the thyA gene is thermostable (and more
related to the archebacterial type) and the other, ThyB,
is thermosensitive. Bacillus subtilis is also able to adapt
to strong osmotic stresses, such as the one that occurs
during dehydration and can adapt to high oxygen
concentrations and changes in pH. Not much is yet
known about the corresponding genes and regulation.
Because the ecological niche of B. subtilis is linked
to the plant kingdom it is subjected to rapid alternat-
ing drying and wetting. Accordingly, this organism is
very resistant to osmotic stress, and can grow well in
media containing 1 m NaCl, and indeed B. subtilis has
been recovered from sea water.
Secondary Metabolism
In Bacillus species, starvation leads to the activation of
a number of processes that affect the ability to survive
during periods of nutritional stress. Capabilities that
are induced include competence and sporulation, the
synthesis of degradative enzymes, motility, and anti-
biotic production. Some genes in these systems are
activated during the transition from exponential to
stationary growth. They are controlled by mechan-
isms that operate primarily at the level of transcription
initiation. One class of genes functions in the synthesis
of special metabolites such as peptide antibiotics, as
well as the cyclic lipopeptide surfactin. These genes
include the srfA operon that codes for the enzymes of
the surfactin synthetase complex or the pks operon,
presumably controlling synthesis of polyketides. Sev-
eral antifungal antibotics, some of which are used in
agriculture, are produced by B. subtilis strains, indi-
cating that competition with fungi is probably a major
feature of the B. subtilis biotope. Peptide or polyketide
antibiotic biosynthesis genes are regulated by factors as
diverse as the early sporulation gene product Spo0A,
the transition-state regulator AbrB, and gene products
such as ComA, ComP, and ComQ, required for the
initiation of the competence developmental pathway.
Information Transfer: B. subtilis Genome
and its Organization
The complete sequence (4 214 820 bp) of the B. subtilis
genome (strain 168) was published in November 1997,
and further corrected after several rounds of se-
quence verification. The reference specialized database,

SubtiList, updates the genome sequence and annota-
tion as work on B. subtilis progresses throughout the
world. Of the more than 4100 protein-coding genes,
53% are represented once. A quarter of the genome
corresponds to several gene families that have been
greatly expanded by gene duplication, the largest of
which is a family containing 77 putative ATP-binding
cassette permeases.
Features of Genome Sequence
Analysis for repeated sequences in the genome
demonstrated that strain 168 does not contain inser-
tion sequences. A strict constraint on the spatial dis-
tribution of repeats longer than 25 bp was found in the
genome, in contrast to the situation in E. coli. This was
interpreted as a hallmark of selective processes leading
to the insertion of new genetic information into the
genome. Such insertion appears to rest on the uptake
of nonspecific DNA by the competent cell and its
subsequent integration in the chromosome in a circu-
lar form through a Campbell-like mechanism. Similar
patterns are found in other competent genomes of
gram-negative bacteria as well as Archaea, suggesting
a similar evolutionary mechanism. The correlation of
the spatial distribution of repeats and the absence of
insertion sequences in the genome suggests that
mechanisms aiming at their avoidance and/or elimin-
ation have been developed.
Knowledge of whole genome sequences allows one
to investigate the relationships between gene and gene
products at a global level. Although there is generally
no predictable link between the structure and function
of biological objects, the pressure of natural selection
has created some fitness among gene, gene products,
and survival. Biases in features of predictably unbiased
processes is evidence for prior selective pressure. In
the case of B. subtilis one observes a strong bias in the
polarity of transcription with respect to replication:
70% of the genes are transcribed in the direction of the
replicating fork movement. Global analysis of oligo-
nucleotides in the genome demonstrated that there is a
significant bias, not only in the base or codon compos-
ition of one DNA strand with respect to the other, but,
quite surprisingly, also at the level of the amino acid
content of the proteins. The proteins coded by the
leading strand are valine-rich, and those coded by
the lagging strand are threonine ‡ isoleucine-rich.
This first law of genomics seems to extend to most
bacterial genomes. It must result from a strong selec-
tion pressure of a yet unknown nature.
Codon Usage and Organization of the Cell's
Cytoplasm
Because the genetic code is redundant, coding
sequences exhibit highly variable patterns of codon
B a c i l l u s s u b t i l i s 141
usage. If there were no bias, all codons for a given
amino acid should be used more or less equally. The
genes of B. subtilis have been split into three classes on
the basis of their codon usage bias. One class comprises
the bulk of the proteins, another is made up of genes
that are expressed at a high level during exponential
growth, and a third class, with A‡T-rich codons, cor-
responds to portions of the genome that have been
horizontally exchanged. What is the source of such
biases? Random mutations would be expected to have
smoothed out any differences, but this is not the case.
There are also systematic effects of context, with some
DNA sequences being favored or selected against.
The cytoplasm of a cell is not a tiny test tube. One
of the most puzzling features of the organization of
the cytoplasm is that it accommodates the presence of
a very long thread-like molecule, DNA, which is tran-
scribed to generate a multitude of RNA threads that
usually are as long as the length of the whole cell. If
mRNA molecules were left free in the cytoplasm, all
kinds of knotted structures would arise. There must
exist therefore, some organizational principles that
prevent mRNA molecules and DNA from becoming
entangled. Several models, supported by experiments,
postulate an arrangement where transcribed regions
are present at the surface of a chromoid, in such a way
that RNA polymerase does not have to circumscribe
the double helix during transcription. Compartmen-
talization is important even for small molecules,
despite the fact that they can diffuse quickly. In a
B. subtilis cell growing exponentially in rich medium,
the ribosomes occupy more than 15% of the cell's
volume. The cytoplasm is therefore a ribosome lattice,
in which the local diffusion rates of small molecules,
as well as macromolecules, is relatively slow. Along
the same lines, the calculated protein concentration
of the cell is ca. 100±200 mg ml 1, a very high concen-
tration.
The translational machinery requires an appro-
priate pool of elongation factors, aminoacyl-tRNA
synthetases, and tRNAs. Counting the number of
tRNA molecules adjacent to a given ribosome, one
conceptualizes a small, finite number of molecules. As
a consequence, a translating ribosome is an attractor
that acts upon a limited pool of tRNA molecules. This
situation provides a form of selective pressure, whose
outcome would be adaptation of the codon usage bias
of the translated message as a function of its position
within the cytoplasm. If codon usage bias were to
change from mRNA to mRNA, these different mole-
cules would not see the same ribosomes during the life
cycle. In particular, if two genes had very different
codon usage patterns, this would predict that the cor-
responding mRNAs are not formed within the same
sector of the cytoplasm.
142 B a c i l l u s s u b tilis
When mRNA threads are emerging from DNA
they become engaged by the lattice of ribosomes,
and they ratchet from one ribosome to the next, like
a thread in a wiredrawing machine (note that this is
exactly opposite to the view of translation presented in
textbooks, where ribosomes are supposed to travel
along fixed mRNA molecules). In this process, nas-
cent proteins are synthesized on each ribosome, and
spread throughout the cytoplasm by the linear diffu-
sion of the mRNA molecule from one ribosome to the
next. However, when mRNA disengages from DNA,
the transcription complex must sometimes break up.
Broken mRNA is likely to be a dangerous molecule
because, if translated, it would produce a truncated
protein. Such protein fragments are often toxic, because
they can disrupt the architecture of multisubunit com-
plexes (this explains why many nonsense mutants are
negative dominant, rather than recessive). There exists
a process that copes with this kind of accident in B.
subtilis. When a prematurely terminated mRNA mol-
ecule reaches its end, the ribosome stops translating,
does not dissociate, and waits. A specialized RNA,
tmRNA, which is folded and processed at its 30 end
like a tRNA and charged with alanine, comes in, inserts
its alanine at the C-terminus of the nascent polypep-
tide, then replaces the mRNAwithin a ribosome, where
it is translated as ASFNQNVALAA. This tail is a
protein tag that is then used to direct it to a proteolytic
complex (ClpA, ClpX), where it is degraded.
The organization of the ribosome lattice, coupled
to the organization of the transcribing surface of the
chromoid, ensures that mRNA molecules are trans-
lated parallel to each other, in such a way that they do
not make knots. Polycistronic operons ensure that
proteins having related functions are coexpressed
locally, permitting channeling of the corresponding
pathway intermediates. In this way, the structure of
mRNA molecules is coupled to their fate in the cell,
and to their function in compartmentalization. Genes
translated sequentially in operons are physiologically
and structurally connected. This is also true for
mRNAs that are translated parallel to each other,
suggesting that several RNA polymerases are engaged
in the transcription process simultaneously, yoked
as draft animals. Indeed, if there is correlation of
function and/or localization in one dimension, there
exists a similar constraint in the orthogonal directions.
Because ribosomes attract tRNA molecules, they
bring about a local coupling between these molecules
and the codons being translated. This predicts that a
given ribosome would preferentially translate mRNAs
having similar patterns of codon usage. As a conse-
quence, as one moves away from a strongly biased ribo-
some, there would be less and less availability of the
most biased tRNAs. This creates a selection pressure
for a gradient of codon usage as one goes away from
the most biased messages and ribosomes, nesting tran-
scripts around central core(s), formed of transcripts
for highly biased genes. Finally, ribosome synthesis
creates a repulsive force that pushes DNA strands
away from each other, in particular from regions
near the origin of replication. Together these processes
result in a gene gradient along the chromosome, which
is an important element of the architecture of the cell.
Information Transfer
The DNA polymerase complex of B. subtilis is
attached to the membrane. During replication, the
DNA template moves through the polymerase. This
might be caused in part by the formation of planar
hexagonal layers of DnaC, the homolog of E. coli
DnaB helicase. The B. subtilis chromosome starts
replicating at a well-defined Ori site, and terminates
in a symmetrical region, probably using a recombin-
ational process to resolve the knotted structure at the
terminus. This may account for the presence of hori-
zontally exchanged genetic material (prophages in
particular) near the terminus.
Transcription in B. subtilis is similar to that in other
eubacteria. The major RNA polymerase is a holoen-
zyme made up of four subunits (two as, b, b0 ) and a
sigma factor. Eighteen sigma factors have been identi-
fied within the genomic sequence. Apart from s54,
which is specialized for the control of nitrogen meta-
bolism, the other ss specifically control specialized
processes such as sporulation, stress response, or che-
motaxis and motility.
Translation in B. subtilis is typical of eubacterial
translation. A new type of control of the synthesis
of aminoacyl-tRNA synthetase was discovered in
B. subtilis. Most aminoacyl-tRNA synthetase genes
belong to the so-called T-box family of genes. They
are regulated by a common mechanism of transcrip-
tional antitermination. Each gene is induced by spe-
cific amino acid limitation; the uncharged cognate
tRNA is the effector that induces transcription of the
full-length message. The mRNA leader regions of
the genes in this family share a number of conserved
primary sequence and secondary structural elements,
some of which are involved in binding the charged
tRNA molecule.
Horizontal Gene Transfer and Phylogeny
Three principal modes of transfer of genetic material,
namely transformation, conjugation, and transduction
occur naturally in prokaryotes. In B. subtilis there
is not much evidence for conjugation processes
(although DNA can be conjugated into the organism),
but transformation is an efficient process (at least in

some B. subtilis species such as the Marburg strain
168) and transduction with the appropriate carrier
phages is well understood.
Bacillus subtilis Phages
An unexpected result that emerged from an analysis of
the B. subtilis genome sequence was that it harbors at
least 10 prophages or prophage-like elements. While
the lysogenic SPbeta phage, as well as the defective
PBSX and skin elements, was known to be present, no
other phage had been identified. Many phages how-
ever can utilize B. subtilis as a host, in particular phi-
29, phi-105, SPO1, SPP1, beta 22 or SF6, but the
details of their biology are generally not well docu-
mented. Bacteriophage PBS1, or the phages IG1, IG3,
and IG4 can perform specialized transduction.
Among the remarkable features of the phage genomes
are the presence of introns or inteins, especially in
genes involved in modulating DNA synthesis by the
host. A three-dimensional reconstruction of phage
phi-29 and its empty prohead precursor has been per-
formed using cryoelectron microscopy. The head±tail
connector, which is the central component of the
DNA packaging machine, has been visualized in situ.
The connector, with 12- or 13-fold symmetry, appears
to fit loosely into a pentameric vertex of the head, a
symmetry mismatch that may be required to rotate
the connector to package DNA. An RNA molecule,
pRNA, is required in the form of an hexamer to
package DNA in the capsid.
Competence and Transformation
In addition to sporulation, B. subtilis enjoys another
developmental process, i.e., competence, which can
lead to genetic transformation. Interconnected regu-
latory networks control the initiation of sporulation
and the development of genetic competence. These
two developmental pathways have both common
and unique features and make use of similar regulatory
strategies. This explains why, before the genome of
B. subtilis was sequenced, the vast majority of ex-
periments using this organism were dealing with
these processes. Quorum-sensing, used by cells to
monitor local cell density, controls the transformation-
competence of B. subtilis. This control system is part
of the 11 phosphorylation cascades comprising a
regulatory aspartate phosphatase that have been dis-
covered in the strain 168 genome.
Recombination
The presence in the B. subtilis genome of local repeats,
suggesting Campbell-like integration of foreign
DNA, is consistent with a strong involvement
of recombination processes in its evolution. In add-
ition, recombination must be involved in mutation
B a c i l l u s s u b t i l i s 143
correction. It is therefore interesting to analyze the
proofreading systems at the level of replication. In B.
subtilis MutS and MutL homologs exist, presumably
for the purpose of recognizing mimatched base pairs.
But no MutH activity could be identified that would
allow the daughter strand to be distinguished from its
parent. It is therefore not known yet how long-patch
mismatch repair corrects mutations in the proper
strand. Excision of misincorporated uracil instead of
thymine might be a general process that would not
require extra information.
Restriction±Modification Systems
Bacillus subtilis strains contain many restriction±
modification systems, mostly of type II, many of
which were probably transferred from phages. The se-
quence specificities of several restriction±modification
systems are known: BsuM (CTCGAG); BsuE
(CGCG); BsuF (CCGG); BsuRI (GGCC); and
BsuBI, which is similar to the PstI system. BsuC is a
type I system, which is very similar to the ones found
in enterobacteria.
Phylogeny
Bacillus subtilis is a typical gram-positive eubacter-
ium. As such it is significantly more similar to Archaea
than is E. coli. Many metabolic genes have a distinct
archaeal flavour, in particular genes involved in the
synthesis of polyamines, but it is rare to find genes in
B. subtilis that are similar to eukaryotic genes. This led
Gupta to propose that ancestral bacteria comprised
a monoderm organism that diverged into gram-
positive bacteria and Archaea, and that gram-positive
bacteria further led to gram-negative bacteria with
their typical double membrane (diderms). This
hypothesis stirred a very heated, but interesting, debate
about the origin of the first cell(s). As such, bacilli
form a heterogenous family of bacteria that can be
split into at least five distinct groups. Bacillus subtilis
is part of group 1 and is strongly linked to B. licheni-
formis (which is often found on the cuticle of insects),
and to the group of animal pathogens formed by B.
thuringiensis, B. cereus, and B. anthracis. In this clas-
sification B. sphaericus is typical of group 2, B. poly-
myxa of group 3, and B. stearothermophilus of group
5. The pathogen Listeria monocytogenes (in between
groups 2 and 5) is related to B. subtilis, and, indeed, its
genome has many features in common with that of the
genome of B. subtilis. Accordingly, B. subtilis is an
excellent model for these groups of bacteria.
Industrial Processes
As a model organism, B. subtilis possesses most of the
functions that one would expect to find in bacteria.
144 Bac kcross
It is an organism Generally Recognized As Safe
(GRAS). This explains why it is a source of many
products synthesized by the agro-food industry.
Bacillus subtilis has often been thought to be a desir-
able host for foreign gene expression or fermentation
and it is commonly used at the industrial level for both
enzyme production (amylase, proteases, etc.) and food
supply fermentation (Bacillus natto, a close parent of
B. subtilis, is used in Japan to ferment soybean, produ-
cing the popular `natto'). Riboflavin is derived from
genetically modified B. subtilis using fermentation
techniques. For some time, high levels of heterologous
gene expression in B. subtilis was difficult to achieve.
Knowledge of the genome allowed identification of
one of the major bottlenecks in this process: Although
it has a counterpart of the rpsA gene, this organism
lacks the function of the corresponding ribosomal S1
protein that permits recognition of the ribosome bind-
ing site upstream of the translation start codons. In
general gram-positive bacteria have transcription and
translation signals that must comply with rules much
more stringent than do gram-negative bacteria. Trad-
itional techniques (e.g., random mutagenesis followed
by screening; ad hoc optimization of poorly defined
culture media) are important and will continue to be
utilized in the food industry. But modern biotechnol-
ogy now includes genomics, which adds the possi-
bility to target genes constructed in vitro at precise
position, as well as to modify intermediary metabol-
ism. As a complement to standard genetic engineering
and transgenic technology, this has opened up a whole
new range of possibilities in food product develop-
ment, in particular allowing `humanization' (i.e., adapt-
ation to the human metabolism and even adaptation to
sickness or health) of the content of food products.
These techniques provide an attractive means of pro-
ducing healthier food ingredients and products that are
presently not available or are very expensive. Bacillus
subtilis will remain a tool of choice in this respect.
Conclusion: Open Questions
The complete genome sequence of B. subtilis contains
information that remains underutilized in the current
prediction methods applied to gene functions, most of
which are based on similarity searches of individual
genes. Methods that utilize higher level information
on molecular pathways to reconstruct a complete
functional unit from a set of genes have been devel-
oped. The reconstruction of selected portions of the
metabolic pathways using the existing biochemical
knowledge of similar gene products has been under-
taken. But it often remains necessary to validate such
in silico (using computers) reconstruction by in vivo
and in vitro experiments. The completeness of a
reconstructed pathway (i.e., no missing reaction) is
an indicator of the correctness of the initial functional
assignment. The core biosynthetic pathways of all
20 amino acids have been completely reconstructed
in B. subtilis. However, many satellite or recycling
pathways have not been identified yet. Finally, there
remains at least 800 completely unknown genes in the
genome of strain 168. Functional genomics is aimed at
identifying their role.
Further Reading
Bron S, Bolhuis A, Tjalsma H et al. (1998) Protein secretion and
possible roles for multiple signal peptidases for precursor
processing in bacilli. Journal of Biotechnology 64: 3±13.
Gupta RS (1998) Protein phylogenies and signature sequences: a
reappraisal of evolutionary relationships among archaebac-
teria, eubacteria, and eukaryotes. Microbiology and Molecular
Biology Reviews 62: 1435±1491.
Kunst F, Ogasawara N, Moszer I et al. (1997) The complete
genome sequence of the gram-positive bacterium Bacillus
subtilis. Nature 390: 249±256.
Perego M (1998) Kinase±phosphatase competition regulates
Bacillus subtilis development. Trends in Microbiology 6: 366±370.
Sonenshein AL, Hoch JA and Losick R (eds) (1993) Bacillus
subtilis and Other Gram-Positive Bacteria: Biochemistry, Physi-
ology and Molecular Genetics. Washington, DC: ASM Press.
Reference
Subti List Database. http://genolist.pasteur.fr/SubtiList/
See also: Archaea, Genetics of; Bacterial Genetics;
Bacterial Transcription Factors; Codon Usage
Bias; Escherichia coli
Backcross
L Silver
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.0100
Backcross is the term for a cross between a class of
organisms that is heterozygous for alternative alleles
at a particular locus under investigation and a second
class that is homozygous for one of these alleles.
The term is often used by itself to describe a two-
generation breeding protocol that begins with a cross
between two inbred strains to produce F1 hybrid off-
spring (see F1 Hybrid). These F1 hybrid offspring are
heterozygous atnumerousloci throughoutthe genome.
The F1 organisms are `backcrossed' to organisms from
one of the original parental strains to obtain a second-
generation population of organisms in which segrega-
tion and assortment of alleles occurs independently