Microbiology and Immunology

WORLD OF MICROBIOLOGY AND IMMUNOLOGY Porins

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process, two strands of the targeted DNA are separated from

each other. Each strand is capable of being a template. The

second step is carried out around 122°F (50°C). At this low-

ered temperature, the two primers anneal to their complemen-

tary sequence on each template. The DNA polymerase then

extends the primer using the provided nucleotides. As a result,

at the end of each cycle, the numbers of DNA molecules dou-

ble.

PCR was carried out manually in incubators of different

temperatures for each step until the discovery of DNA poly-

merase from thermophilic bacteria. The bacterium Thermus

aquaticuswas found in Yellow Stone National Park. This bac-

terium lives in the hot springs at 203°F (95°C). The DNA

polymerase from T. aquaticuskeeps its activity at above

203°F (95°C) for many hours. Several additional heat-resist-

ant DNA polymerases have also now been identified.

Genetic engineered heat resistant DNA polymerases,

that have proofreading functions and make fewer mutationsin

the amplified DNA products, are available commercially. PCR

reactions are now carried out in different thermocyclers.

Thermocyclers are designed to change temperatures automat-

ically. Researchers set the temperatures and the time, and at

the end of the procedure take the test tube out of the machine.

The invention of PCR was revolutionary to molecular

biology. PCR is valuable to researchers because it allows them

to multiply the quantity of a unique DNA sequence to a large

and workable amount in a very short time. Researchers in the

Human Genome Project are using PCR to look for markers in

cloned DNA segments and to order DNA fragments in

libraries. Molecular biologists use PCR to cloningDNA. PCR

is also used to produce biotin or other chemical-labeled

probes. These probes are used in nucleic acid hybridization, in

situhybridization and other molecular biology procedures.

PCR, coupled with fluorescence techniques and com-

puter technology, allows the real time amplification of DNA.

This enables quantitative detection of DNA molecules that

exist in minute amounts. PCR is also used widely in clinical

tests. Today, it has become routine to use PCR in the diagno-

sis of infectious diseases such AIDS.

See alsoChromosomes, eukaryotic; Chromosomes, prokary-

otic; DNA (Deoxyribonucleic acid); DNA chips and micro

arrays; DNA hybridization; Immunogenetics; Laboratory

techniques in immunology; Laboratory techniques in microbi-

ology; Molecular biology and molecular genetics

PPorinsORINS

Porins are proteins that are located in the outer membrane of

Gram-negative bacteria. They function to form a water-filled

pore through the membrane, from the exterior to the

periplasm, which is a region located between the outer and

inner membranes. The porin channel allows the diffusion of

small hydrophilic (water-loving) molecules through to the

periplasm. The size of the diffusing molecule depends on the

size of the channel.

A porin protein associates with two other porin proteins

of the same type in the outer membrane. This may act to sta-

bilize the three-dimensional structure of each porin molecule.

Each porin contains a pore, so that there are three pores in the

triad of porins.

The size of the water-filled channel that is created by a

porin depends on the particular porin protein. For example, in

the bacterium Escherichia coli, the so-called maltoporin and

phosphoporin have different specificities (for the sugar malt-

ose and phosphorus, respectively).

Since the discovery of porins in the 1970s in

Escherichia coli, these proteins have been shown to be a gen-

eral feature of the Gram-negative outer membrane. Much of

the early work on porins came from the laboratories of Hiroshi

Nikaido and Robert Hancock. Some examples of the bacteria

known to possess porins are Pseudomonas aeruginosa, many

other species of Pseudomonas, Aeromonas salmonicida,

Treponema pallidum, and Helicobacter pylori.

A bacterium typically contains a variety of porins.

Possession of porins of different sizes and chemistries is very

advantageous for a bacterium. The various channels allow for

the inward diffusion of a variety of nutrients required by the

bacterium for survival and growth. Moreover, the diffusional

nature of the molecule’s entry means that a bacterium is able to

acquire some needed nutrients without having to expend energy.

Another example of porin importance is found in

Escherichia coli. In this bacterium, a duo of porins, which are

designated OmpF and OmpC, function in response to changes

in osmolarity. The production of these porins is under the con-

trol of a protein that senses the osmotic character of the envi-

ronment. Depending on the ionic conditions, the amounts of

OmpF and OmpC in the outer membrane can be altered so as

to control the types of ions that enter the bacterium.

Porins share the same function in these bacteria from

various habitats. This similar function is mirrored by the sim-

ilarity in the three-dimensional structure of the proteins. Each

porin is visually reminiscent of a barrel that is open to both

ends. The slats of the barrel are arrangements of the con-

stituent amino acids of the protein (beta sheets). The sequence

of amino acids that makes up a beta sheet region allows the

region to assume a zigzag configuration of the amino acids in

one plane. The result is a narrow, flat strip of amino acids.

When such strips are linked together, the barrel shape can be

created. The outer surface of the porin barrel is more

hydrophobic(water-hating) and so the partitioning of this sur-

face into the hydrophobic interior of the membrane will be

favored. The inner surface of the porin barrel contains side

groups of the amino acids that prefer to interact with water.

The function of porin proteins was discovered by isolat-

ing the particular protein and then inserting the protein into

model systems, that consisted either of lipid membranes float-

ing in solution (liposomes) or floating as a sheet on the surface

of a liquid (black lipid bilayer membranes). The passage of

radioactive sugars of various sizes out of the liposomes or

across the black lipid bilayer membranes could be measured,

and the various so-called exclusion limits for each porin could

be determined.

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