Microbiology and Immunology

WORLD OF MICROBIOLOGY AND IMMUNOLOGY Bacterial growth and division

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sequence of the DNA. BACs proved useful in the sequencing

of the human genome.

The BAC is based on a plasmid in Escherichia colithat

is termed the F (for fertility) plasmid. The F plasmid (or F fac-

tor) contains information that makes possible the process called

conjugation. In conjugation, two Escherichia colibacteriacan

physically connect and an exchange of DNA can occur.

A BAC contains the conjugation promoting genetic infor-

mation as well as stretch of DNA that is destined for incorpora-

tion into the bacterium. The foreign DNA (e.g., portion of

human genome) is flanked by sequences that mark the bound-

aries of the insert. The sequences are referred to as sequence tag

connectors. When the BAC becomes incorporated into the

genome of Escherichia colithe sequence tag connectors act as

markers to identify the inserted foreign DNA.

Using a BAC, large stretches of DNA can be incorpo-

rated into the bacterial genome and subsequently replicated

along with the bacterial DNA. In molecular biologyterminol-

ogy, pieces of DNA that contain hundreds of thousands of

nucleotides (the building blocks of DNA) can be inserted into

a bacterium at one time. As the process is done using different

sections of the foreign DNA, the amount of DNA that can be

analyzed can be very large.

BACs were developed in 1992. Since then, their useful-

ness has grown immensely. The primary reason for this popu-

larity is the stability of the inserted DNA in the bacterial

genome. Because the inserted DNA remains in the bacterial

genome during repeated cycles of replication, the information

is not lost. As well, the BAC can be sequenced using the nor-

mal tools of molecular biology.

The most dramatic recent example of the power of

BACs is their use by The Institute for Genomic Research

(TIGR) in the technique of shotgun cloningthat was employed

in the sequence determination of the human genome. Many

fragments of the human genome could be incorporated into

BACs. The resulting “library” could be expressed in

Escherichia coliand the sequences determined. Subsequently,

these sequences could be reconstructed to produce the orderly

sequence of the actual genome. This approach proved to be

less expensive and quicker than the method known as directed

sequencing, where a genome was sequenced in a linear fash-

ion starting at one end of the genome.

The total number of fragments of the DNA from the

human genome that have been expressed in Escherichia coli

by the use of BACs is now close to one million. In addition to

the human genome, BACs have also been used to sequence the

genome of agriculturally important plants such as corn and

rice, and of animals such as the mouse.

With the realization of the sequence of the human

genome, the use of BACs is becoming important in the screen-

ing of the genome for genetic abnormalities. Indeed, BAC

cloning kits are now available commercially for what is

termed genomic profiling.

See alsoBiotechnology; Plasmid and plastid

BACTERIAL EPIDEMICS•seeEPIDEMICS, BACTERIAL

BACTERIAL FOSSILIZATION •seeFOSSILIZATION

OF BACTERIA

BACTERIAL GENETICS•seeMICROBIAL GENETICS

BBacterial growth and divisionACTERIAL GROWTH AND DIVISION

The growth and division of bacteriais the basis of the increase

of bacterial colonies in the laboratory, such as colony forma-

tion on agarin a liquid growth medium, in natural settings,

and in infections.

A population of bacteria in a liquid medium is referred

to as a culture. In the laboratory, where growth conditions of

temperature, light intensity, and nutrients can be made ideal

for the bacteria, measurements of the number of living bacte-

ria typically reveals four stages, or phases, of growth, with

respect to time. Initially, the number of bacteria in the popula-

tion is low. Often the bacteria are also adapting to the envi-

ronment. This represents the lag phase of growth. Depending

on the health of the bacteria, the lag phase may be short or

long. The latter occurs if the bacteria are damaged or have just

been recovered from deep-freeze storage.

After the lag phase, the numbers of living bacteria rap-

idly increases. Typically, the increase is exponential. That is,

the population keeps doubling in number at the same rate. This

is called the log or logarithmic phase of culture growth, and is

the time when the bacteria are growing and dividing at their

maximum speed. For Escherichia coli, for example, the rate

of growth and division of a single bacterium (also called the

generation time) during the log phase is 15 to 20 minutes. In

the log phase, most of the bacteria in a population are growing

and dividing.

The explosive growth of bacteria cannot continue for-

ever in the closed conditions of a flask of growth medium.

Nutrients begin to become depleted, the amount of oxygen

becomes reduced, and the pHchanges, and toxic waste prod-

ucts of metabolic activity begin to accumulate. The bacteria

respond to these changes in a variety of ways to do with their

structure and activity of genes. With respect to bacteria num-

bers, the increase in the population stops and the number of

living bacteria plateaus. This plateau period is called the sta-

tionary phase. Here, the number of bacteria growing and

dividing is equaled by the number of bacteria that are dying.

Finally, as conditions in the culture continue to deterio-

rate, the proportion of the population that is dying becomes

dominant. The number of living bacteria declines sharply over

time in what is called the death or decline phase.

Bacteria growing as colonies on a solid growth medium

also exhibit these growth phases in different regions of a

colony. For example, the bacteria buried in the oldest part of

the colony are often in the stationary or death phase, while the

bacteria at the periphery of the colony are in the actively divid-

ing log phase of growth.

Culturing of bacteria is possible such that fresh growth

medium can be added at rate equal to the rate at which cul-

ture is removed. The rate at which the bacteria grow is

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