Chapter ten - Genetics v3...Microbial Genetics: New Genes for Old Germs Objectives: After reading Chapter Ten, you should understand… • The structure and complexity of the bacterial

Sigler, Microbes and Society Spring 2014

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Chapter 10 Microbial Genetics: New Genes for Old Germs

Objectives: After reading Chapter Ten, you should understand…

• The structure and complexity of the bacterial chromosome and the significance of plasmids.

• How genetic changes occur as a result of spontaneous and induced mutations. • How bacteria can acquire new genes from their environment and incorporate them into

their genomes. • The role of viruses in mutations of bacteria genomes.

1968 – Rash of dysentery broke out in Guatemala (Shigella dysenteriae) - shigellosis Patients received one of four antibiotics to treat the

infection. Treatments became less and less effective until

eventually, none of the four antibiotics would work.

Within three years, 100,000 infected,

and at least 12,000 died.

Shigella dysenteriae cells on a filter membrane.


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Shigella spp. isolates were later the subject of research to investigate antibiotic resistance (tetracycline, sulfonimide, chloramphenicol, and streptomycin).

Patients with antibiotic-resistant Shigella spp. also carried a strain of E. coli that

was resistant to the same four antibiotics. The co-occurrence of resistance to the same drugs in two different bacteria was very

unlikely.

How did this happen then? How did these two different species of bacteria both become resistant to the same antibiotics?

To understand how bacteria change and adapt, you first must understand these changes are due to alterations in genetic material (DNA), called mutations.

3.5 billion years of evolution has allowed bacteria to become the most adapted organisms on the planet. This ability to adapt has allowed bacteria to become increasingly problematic with regard to public health.

The bacterial chromosome Most genetic information in bacteria is stored in the chromosome.

Remember, the chromosome is a loop of DNA, unlike in eukaryotic chromosomes, in which the DNA is linear. It occupies about half of the bacterial volume and would stretch to about 1.5 mm if unpackaged from the cell.

Therefore, the chromosome is about 1500x the length of the bacterium that contains it. How can such a relatively large molecule be packed into such a small space AND remain useful?

It takes on a flower-shaped structure, where loops form about every 50,000 bases.


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This shows that even a simple bacterium is capable of a high level of organization.

An electron micrograph of an E. coli cell immediately after disruption. The tangled mass is the organism’s DNA.


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DNA replication

The bacteria chromosome must copy itself every time the cell divides (once every 20 minutes for E. coli).

The process:

1. The DNA anchors to a point on the cell membrane.

2. An enzyme “nicks” the closed loop at a site known as the origin of replication and the two strands “unzip”.

This establishes a “v”-shaped replication fork.

3. An enzyme called DNA polymerase builds a new strand of DNA using nucleotides with bases complementary to those on the original strand.

Remember: A pairs with T, C pairs with G.


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4. The half-new, half-old strands twist to form a new double helix. This is referred to as semiconservative replication.

In each new double helix, one strand of parent DNA is conserved, while one strand is new.

5. The chromosomes separate.

In some bacteria, this process occurs in as little as 20 min. Can you think of some of the problems with doing something so intricate, so quickly?

Plasmids Plasmids are small loops of DNA (maybe a few thousand bases) that are: 1. Not necessarily mandatory for survival of the bacterium under normal conditions. Might contain <2% of the total genetic information. 2. Independent of the chromosome. They are separate from the chromosome. 3. Replicate independent of the chromosome (but in the same way as the chromosome). With less than 2% of the genetic information contained in the entire genome, why might plasmids be of such interest to us? Conjugation is a process that results in the transfer of certain plasmids from one

bacterium to another. Plasmids can contain genetic information that encodes for the transfer of

themselves and other plasmids from one bacterium to another. These are called fertility plasmids.

Fertility plasmids allow some bacteria to accumulate certain types of

specialized genes also contained on the plasmids, such as… Degradation genes (pollutants, for example) Toxin genes Antibiotic resistance genes (R-plasmids)


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Overall, conjugation represents a significant mechanism for the emergence of antibiotic

resistance in bacteria. Another one is gene mutations…

A mating pair of bacteria initially brought together by means of an F pilus. Electron microscopic image by Charles C. Brinton, Jr.

From: Grohmann et al., Microbiology and Molecular Biology Reviews, June 2003, p. 277–301.

Plasmid name Host Size Antibiotics Organism the plasmid can be transferred to.


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Gene mutations Changes in genetic information can impact the success of pathogenic bacteria. Example: tetracycline resistance Tetracycline is an antibiotic used to combat a variety of bacterial infections.

In susceptible bacteria, tetracycline acts by affecting the ribosomes in the target bacteria.

Tetracycline prevents the ribosomes from joining amino acids together in

a growing polypeptide chain (i.e. tetracycline prevents protein synthesis in bacteria).

In tetracycline-resistant bacteria, ribosomes are altered so that they do not bind tetracycline.

Mutations in the DNA that encodes for the structure of ribosomes can cause this change.

From: Ross et al., Antimicrobial Agents and Chemotherapy (1998) 42:1702-1705.


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Mutations are “permanent” (long-term) changes in the sequence of the DNA.

These changes can represent a gain or loss of genetic material, or an altered nucleotide base. AACCGGTT vs. AACCGGTA Mutations can occur during replication (error) or after replication has

occurred (mutagen). What cellular components will ultimately be affected by DNA mutations?

Causes of mutations

1. They can occur spontaneously.

At least one spontaneous mutation occurs in every 10 billion bases replicated.

Is this a lot or a little? …I wish my mistakes only occurred once in every 10 billion tries.

However, think about this in terms of a bacteria colony on a Petri dish that

contains millions of bacteria…

A single Shigella contains ~4,600,000 base pairs of DNA. This means that in one-billion Shigella, there are 4 x 1015 base pairs of DNA.


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If a mutation spontaneously occurs once in every 10 billion bases replicated, than a single, large colony theoretically contains 800,000 mutations. The math: 4 x 1015 base pairs x 2 bases = 8 x 1015 bases 8 x 1015 bases / 10 billion = 800,000 mutations So, what if the “one-in-ten-billion” mutation is the one that alters the ribosome structure, as we discussed earlier in the case of tetracycline resistance? …then 999,200,000 of the bacteria in the colony are susceptible to

tetracycline, while 800,000 are resistant and can grow and divide to produce an ever expanding population of resistant bacteria.

So, is one in ten billion a lot or a little?

2. The second way mutations can arise is as a result of exposure to identifiable factors known as mutagens.

Remember, the spontaneous mutation rate is 0.0000000001 (one in ten billion

bases) The presence of a mutagen can increase this rate by 1000-fold. A. UV light – can create a thymine dimer.

C T C T T T C T

G A G A A A G A


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Adjacent thymine bases bind together, leading to replication errors and mutations in the resulting DNA.

B. Chemicals in the environment can also act as mutagens. These fall into several groups. We will discuss two of these.

i) Base-altering mutagens – chemicals that cause changes to the chemical

structure of bases in the DNA.

Many base-altering mutagens are complex organic molecules such as those found in smoke (leads to mutated respiratory cells and lung cancer)

ii) Base analogs – molecules whose chemical structure is similar to one of the four DNA bases (adenine, cytosine, guanine thymine).

Because of this similarity, they can be incorporated into the DNA helix during replication. The problem with this is that the analogs can often form base pairs with more than one other base, which decreases the specificity of base pairing and leads to mispairing during the next round of replication.

Example: Assume that a base analog “X” can pair with both A and G.

Some analogs can have beneficial uses – e.g. AZT, the antiviral treatment for HIV infections.

AZT prevents the replication of the DNA made from HIV RNA.


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Remember we discussed that the deoxyribose sugars formed the DNA backbone. The N3 group in AZT keeps that backbone from forming.


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Gene Recombinations Remember the Guatemala story… patients with drug-resistant Shigella spp. also carried a strain of E. coli that was resistant to the same four antibiotics.

This seemed very strange, i.e., what are the odds that two different bacteria from the same people were resistant to the same four antibiotics?

Could the resistance genes have been transferred between the bacteria, thereby

making them resistant to the same antibiotics? Yes…gene recombination can occur as a result of genes moving between bacterial cells. Can be significant, as it is thought that some bacteria (e.g. E. coli), have acquired

20% of their genes from other microbes. Three mechanisms of gene transfer: 1. Transduction – gene transfer is mediated by a virus.

2. Transformation – gene transfer occurs as bacteria “pick up” naked DNA.

3. Conjugation - gene transfer mediated by physical exchange between bacteria 1. Transduction – gene transfer occurs with the assistance of a bacteriophage, a virus that infects bacteria. Like all viruses, bacteriophages incorporate their own genetic material into the bacteria chromosome. …however, what would happen if, during the replication of the viral genome, parts

of the bacteria chromosome or bacteria plasmid were incorporated into the virus DNA?


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When the phages are released from the bacterium, they might carry bacterial genes and transfer them to other bacteria = transduction.


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Although infrequent in the environment, the consequences of transduction can be significant and long-lived.

The infamous E. coli 0157:H7 toxin genes are the result of transduction events. This strain of E. coli was first isolated in 1982 in contaminated hamburger

from a Jack-in-the-Box restaurant. E. coli 0157:H7 has been implicated in several food contamination

incidents including the fall 2006 spinach E. coli outbreak, the Topps meat recall in 2007, and many others.

2. Transformation – DNA is acquired from the surrounding environment. Involves the direct uptake of foreign DNA.


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Example: A patient is infected with E. coli that is resistant to the antibiotic chloramphenicol.

Knowing that chloramphenicol will not work, the doctor treats the patient using the antibiotic gentamycin, which kills the bacteria.

DNA fragments from the killed bacterial are distributed throughout the

intestinal tract of the patient. Then the patient comes in contact with a reservoir of Shigella spp., which

subsequently infects the gut. The Shigella could become resistant through transformation by taking up

the resistance genes left behind by the dead, chloramphenicol-resistant E. coli.

A chloramphenicol-resistant Shigella population can now enter the environment. 3. Conjugation – two bacteria cells are joined by a pilus (remember attachment?) through which

genetic information can be exchanged. Conjugation is mediated by a plasmid called an F plasmid (F = fertility).

Two types of bacteria are involved; F+ and F-.

The fertility plasmid contains ~20 genes that encode for conjugation events, like…

…replication of DNA movement from donor to recipient cell pilus construction. …and sometimes other genes of interest like resistance and degradation genes.

How could transduction, transformation and conjugation provide the means for

“multiple antibiotic resistance” as was demonstrated in the Guatemala example?

F+

F-

F+

F+

F-factor is now in both bacteria.


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Genetic engineering All of the genetic processes we have discussed are real and work together to alter bacterial genomes and subsequently, the performance of the bacterium. However, the natural occurrence of these processes is relatively infrequent and uncontrolled. To take advantage of the pathways and products produced by beneficial bacteria, scientists have developed the ability to artificially alter the genetics of microbes. This is called genetic engineering.

Genetic engineering is only possible if you can move pieces of DNA from one organism to another.

Genetic engineering began with the discovery of enzymes called endonucleases (also

called restriction enzymes). These enzymes are used naturally by all types of organisms to keep foreign DNA

from integrating into the host genome. They cut the sugar-phosphate backbone of the DNA.

Most importantly, endonucleases cut DNA at very specific locations (specific sequences), so they are like a pair of biochemical scissors.


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The first recombinant DNA molecules. Endonucleases can be used to cut pieces of DNA from two different organisms, and then another enzyme can be used to splice them together. Engineering is most often performed using plasmids. This was first done in 1971 when viral DNA was spliced into the genome of E. coli.

Resulted in the first recombinant DNA molecule.


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The implications of genetic engineering

Documents

Chapter ten - Genetics v3...Microbial Genetics: New Genes for Old Germs Objectives: After reading Chapter Ten, you should understand… • The structure and complexity of the bacterial