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Chapter 4, BIO 3335


Microbial physiology is undergoing a revolution due to the availability of genome sequences.
Organisms that have been difficult to culture are now rapidly studied.
From the genes, we know their biochemical pathways, energy metabolism, and virulence
This is possible because the gene sequence permits determination of the likely protein
Genomic and proteomic tools
Cloning the genome. Chromosomal DNA is extracted, digested with a restriction enzyme to
small pieces, and cloned into a plasmid.
Recently, PCR has been used to isolate DNA from organisms that cannot be cultured.
DNA sequencing.
The dideoxy method of Sanger is used which is based on synthesis from an
oligonucleotide primer, which hybridizes to plasmid DNA.
Each chain terminating nucleotide contains a fluorescent tag. Each of the four nucleotides
has a different tag.
The oligos synthesized are separated by capillary electrophoresis, and a laser detects the
progessively longer oligos.
Computers identify overlaps from the shotgun cloning into contigs.
The final step is annotation, which is to identify ORFs and homologies with other
proteins. Many tools for annotation are available on the Internet.
Information from homology
Orthologs are homologs in two different organisms that have a common ancestoral gene.
Paralogs are homologous proteins that exist in one organism as a result of gene
duplication. They probably have diverged in function also.

The programs can tell which part of a protein is homologous to a characterized protein.
Once tentatively identified, the function of a protein in a pathway might be suggested.
Gene replacement techniques.
The point of homology searches is to provide possible functions. This must be tested, and
a good way to test is to eliminate the gene, and see if there is a phenotype. This is called
reverse genetics, since genetic analysis is to start with a mutant and do what you can to
understand the gene.
One method is shown in Fig 4-2.
A gene of interest is cloned into a plasmid, part of it removed with restriction
enzymes, and replaced with a gene for an antibiotic resistance.
The plasmid contains a gene for a second antibiotic resistance and it is introduced into
cells. If circular, then the plasmid integrates via homologous recombination, and is
resolved by a second recombination event. You then select for cells with the first
antibiotic resistance, but loss of the second.
Another method is to use a linear DNA, which cannot entirely integrate. Selecting for
the antibiotic requires a double crossover.
Gene arrays (Fig 4-3).
This technique allows comparison of the level of all transcripts under two different
A microchip is constructed which contains oligos for each gene. Each oligo occupies a
separate space on the chip. Each chip is about 1 sq inch for 4-5,000 genes.
Next, RNA from each culture is extracted. The RNA is converted to cDNA with reverse
transcriptase. Also each culture uses a different fluorescent nucleotide.
The two cDNA mixtures are combined, and hybridized to the chip.
The chip is read in a chip reader, which reads both flourescent tags with two lasers.
The proteome is defined as the expressed proteins of an organism.
The proteome content varies with the environment, and a variety of stresses.
One way to tell which proteins change is to separate and identify them.

2D gels are often used (Figs 4-4 and 4-5). Separation is first performed by net charge
(isoelectric focusing), and then by size.
Net charge is determined by side chain charges of the charged amino acids.
The proteins migrate in an electric field through a pH gradient. At a certain pH
(the isoelectric point), the protein will be uncharged and will not migrate.
After IEF, the proteins are soaked in detergent (SDS) to denature them and impart a
negative charge on all proteins.
Then they are separated by size.
The proteins are visualized with stain (colored or flourescent) or radioactivity.
Once separated, the proteins must be identified.
They can be cut out and analyzed by mass spec.
Traditional tools
Mutant hunts. Prior to genome sequencing, analysis of biochemical pathways required a
selectable phenotype. Once isolated, the mutated gene could be analyzed, mapped and
isolated (cloned).
It is most desirable to isolate mutants with a positive selection, if possible.
Examples include resistance to an amino acid analog. Only mutants that fail to
transport a compound will grow.
In the absence of a selection, a screen may be necessary. This is labor intensive, and may
require analysis of 10,000 colonies, using replica plating.
This can be done with replica plating or florescent-activated cell sorting (FACS).
FACS can involve green flourescent protein (gfp) fusions. If gfp is fused to a gene
that is expressed at pH 4.5 (non-optimal and stressed condition). Then mutants that do
not express this gene can be separated from those that do.
Gene fusions (reporter genes)
Promoters of interest are fused to genes whose products are easily assayed. There are two
types of fusions (Fig 4-6).

Operon or transcriptional fusions fuse a promoterless lacZ (or another gene) to a

promoter of interest. The factors that control the promoter of interest now control
Translational fusions unite a lacZ without a promoter or ribosome binding site to a
promoter and rbs of a gene of interest. Such a fusion has the ability to detect
translational control, if any.
Some fusions can be randomly inserted, and those that are regulated in a certain way can
be screened. Alternately, a fusion can be constructed on a plasmid, and transferred to the
chromosome by homologous recombination.
Once constructed, the effects of various environments on gene expression can be
Mutants hunts can also be devised.
This is shown well in Fig 4-7.
The point is to isolate large amounts of a specific DNA fragment.
The basic procedure of 20-30 cycles can amplify DNA over a million fold.
Thermostable DNP is used in order to survive the heat step in the cycling.
Many variations of the procedure have been developed.
(So many have been developed that the inventor does not understand all of them.)
A powerful tool of this procedure is to engineer mutations by site-specific mutagenesis.
The purpose of this procedure is to test predictions from homology analysis. For
example, if a homology search suggests a specific function for a protein, this can be
tested by generating an alteration in a critical amino acid, and see if it has an effect on
the function of the gene product.
Fig 4-8 gives a complex example of how this is done. It will not be discussed.
DNA mobility shift assays (Fig 4-9).
If a homology search indicates that a protein is a regulatory protein, one way to test this is
to see if it binds to DNA.

This is tested by adding the protein to DNA and analyzing whether the protein changes
the molecular weight of the DNA.
The size is determined by gel electrophoresis.
A further test of binding can include adding an antibody to the regulatory protein, and
seeing if the size increases still more (a supershift).
The binding site of the protein can also be mutated, and presumably the mobility change
will not be seen.
Primer extension to determine the start site of transcription (Fig 4-10).
Genes can have more than one promoter, and each might be used in a different
environmnent. This is tested by primer extension.
A primer is used that is complementary to the mRNA. RTase is added, and the primer is
extended until it reaches the 5 end of the mRNA. Sizing or sequencing the DNA product
then determines the start site of transcription.
Detecting DNA (Southerns), RNA (Northerns), Proteins (Westerns), and DNA-binding
proteins (Southwesterns)
E. M. Southern developed a method to detect DNA.
DNA is cut with one or more restriction enzyme, separated by electrophoresis,
transferred to a nitrocellulose membrane (either by diffusion of electrophoresis), and
attached to the membrane.
The membrane is then probed with a labeled DNA. Those that hybridized are
Northerns (a play on words) is when RNA is bound to the membrane and probed with a
labeled DNA. This can be used to determine the size of the transcript.
Westerns (Fig 4-11) are used to detect whether a protein is present and how much is
In the first step, proteins are bound to a membrane, after the proteins have been
separated by size.
The membrane is then probed with an rabbit antibody to detect if the protein is
This is then probed with a secondary antibody (e.g., a mouse antibody) against the
rabbit antibody, which is coupled to something detectable.

Southwesterns are used to detect proteins that bind specific DNA sequences.
The proteins are transferred to a membrane as for Westerns.
It is not mentioned, but there must be some method to renature the proteins after
adding SDS to denature the proteins.
The membrane is then probed with labeled DNA.
Two-hybrid analysis (Fig 4-12)
The purpose of this method is to detect protein-protein interactions.
The classic example is the yeast two-hybrid system.
Two parts of the GAL4 protein are used. The DNA-binding domain binds DNA, and
the activator region binds to RNP and activates transcription. An interaction is
required to stabilize the activation domain near RNP.
The GAL4 promoter controls lacZ expression.
The two domains do not interact (normally they are part of one protein, but they have
been separated.)
Instead, the DNA coding for the DNA binding region is fused to a gene coding for
the protein of interest.
Then the activation domain is fused to another protein (at the DNA level). If the
two regions or interest interact, lacZ will be expressed.
The strength of the procedure is that any DNA can be cloned to the activation
domain DNA, and screens can be performed to isolate any gene that interacts with
the protein fused to the DNA-binding domain.
Other two-hybrid systems have been developed.
Conclusion. As the authors note, these and other methods have revolutionized the study of
microbial physiology. The methods of 15-20 years ago might take decades to solve a problem
that now can be solved in months.