BROCK BIOLOGY OF MICROORGANISMS Chapter 9 -- Introduction

Chapter 9: Microbial Genetics
Introduction

OVERVIEW

Chapter 9 (pages 306-359) focuses on how the genetic information can be changed: either by mutation or by the transfer of genes from one organism to another. The successful transfer of genetic information includes two elements -- the introduction of genes from a donor cell into a recipient and recombination of those introduced genes into the recipient's genome. Microbial genetics are important because the genes are the are the basis for cell function and microorganisms are excellent tools for studying gene function.

CHAPTER NOTES

Mutations are inheritable changes in the base sequence of nucleic acid -- the genetic material. An organism with these changes is called a mutant. Genetic recombination is the process where genes from two genomes are combined together. A mutant will be different from its parent, its genotype or genetic makeup has been altered. The phenotype or visible properties of the mutant may or may not be altered. The genotype of a strain is indicated by use of three small italics letters followed by a capital letter and indicates the gene involved in a process (hisC indicates the gene for HisC protein). The phenotype of the strain is indicated by three letter code that ends in a +/-. For example Thr+indicates a strain can make its own threonine while Thr- indicates that it cannot. An auxotroph is formed when a required nutritional material (amino acid for example) that the parent strain, prototroph, could make is no longer formed.

Mutations can occur spontaneously, because of mistakes during replication or due to natural radiation at a frequency of about one in 1,000,000, or may be experimentally induced using mutagens. Mutations can be chemically induced by base analogs, compounds that are structurally similar to the purines and pyrimidines in DNA. The cell incorporates them into DNA, but during subsequent replication, the analogs have a higher probability of base pairing incorrectly, thereby inserting the wrong base into the new DNA strand. Other chemical mutagens react directly with DNA to alter the bases. UV radiation is absorbed by the purines and pyrimidines in DNA, and one of its effects is to form pyrimidine dimers in one strand, which prevents these thymine bases from pairing correctly during replication. Ionizing radiation generates free radicals in cells, and these can react with the DNA backbone to cause breaks. Biological agents, such as transposons and the bacteriophage Mu, cause mutations by inserting DNA sequences into genes, and thereby disrupting the coding information.

A mutation may be a change in a single base pair (point mutation) or involve large deletions or insertions of base pairs. The insertion of a single additional base into a gene can have dramatic effects upon the amino acid sequence of the protein produced from that gene, due to the reading-frame shift this causes in the translation of the mRNA produced from the gene. It is possible to move large sections of DNA to a second location and the process is termed translocation. If the mutated gene is part of an operon (see Section 5.10) the mutation may exert polar effects upon other genes in the operon. The effects of a specific mutation may be reversed by a second (suppressor) mutation in either the same gene, or in another gene. Note that cells do have DNA repair systems to correct damage to DNA. The SOS system is one of these, but it is error-prone and the repaired DNA may still contain mutations.

One use of mutant bacterial strains has been to determine the potential mutagenicity of chemicals -- either manufactured or natural. The Ames test utilizes back mutation in a strain of bacteria that are auxotrophic for a nutrient. When auxotrophic cells (His-) are spread on a medium that lacks histidine no growth will occur. If, however, the cells are treated with a chemical that causes a reversion mutation it can then grow.

Generalor homologous recombination requires extensive homology and is mediated by an enzyme, RecA protein. The sequence of events are (1) nicking of a DNA molecule, (2) opening of the DNA double helix, (3) pairing between homologous single strands of two DNA molecules (requires presence of RecA), and (4) breakage and rejoining of DNA strands so that portions of the DNA molecule are exchanged. An important point is that this process leads to new genotypes only if the two molecules that are recombining differ genetically in regions outside those where breakage and rejoining occurred. In order to detect recombination or exchange of DNA, the offspring must be phenotypically different from the parent.

In bacteria, the gene transfer that precedes recombination can occur by three mechanisms: transformation, transduction, or conjugation. Transformation involves the uptake by a recipient of free or naked DNA released from a donor. However, cells may only be physiologically competent to take up DNA. Competence is related to changes in the cell surface that allow strong binding of DNA. In some organisms, such as E. coli, the transformation process can be enhanced by special pre-treatment of cells. The cell can undergo electroporation where small holes or pores are open in the cell. A single strand of the transforming DNA is integrated into the chromosome, using general recombination mechanisms. A cell with a new genotype is generated when this strand is replicated and the resulting molecule forms the genome of a new cell, at cell division. Eukaryotic cells can also be treated to take up free DNA, although the specific treatments are different from those used in bacteria.

In transduction, the transferred DNA is carried in the capsid of a bacteriophage. The donor's DNA replaces part or all of the viral genome in the phage head. Thus, the particle is probably defective in viral replication because essential viral genes are missing. In the case of temperate phages such as lambda, bacterial DNA becomes associated with the virus genome when the prophage is excised incorrectly from the bacterial chromosome. When this occurs, the same set of bacterial genes is always incorporated into lambda phage. This phenomenon is specialized transduction, because it is only effective in transducing a few special bacterial genes. In contrast, generalized transduction can transfer any bacterial gene to the recipient. This process may occur with phages that degrade their host DNA into pieces the size of viral genomes. If these pieces are erroneously packaged into phage particles, they can be delivered to another bacterium in the next phage infection cycle. Phages P22 of Salmonella typhimurium and P1 and mu of E. coli carry out generalized transduction.

Some temperate phages cause phenotypic changes in the bacteria they infect even without transducing bacterial genes. In the lysogenic state, viral genes are expressed which confer new properties on the cell. Examples of this phage conversion are toxin production by pathogenic bacteria such as Corynebacterium diphtheriae and surface polysaccharide structure in Salmonella anatum.

Conjugation , the third means of gene transfer is mediated by special genetic elements called plasmids. Plasmids are defined as small, circular DNA molecules that reproduce autonomously. While plasmids are DNA, they control their own replication separately from that of the chromosome. The presence of plasmids in cells can be detected by techniques that separate them from chromosomal DNA. This involves buoyant density differences due to the tight supercoiling of these rather small DNA circles; the density difference can be enhanced by adding compounds that intercalate between DNA base pairs, such as ethidium bromide. The tightly wound plasmid DNA cannot bind as much ethidium bromide as the chromosomal fragments. Adding ethidium bromide, or other treatments that affect DNA, to whole cells at the appropriate concentration may cure cells of their plasmids. If plasmid replication is more sensitive to these agents than chromosome replication, plasmids may not segregate to all progeny cells during cell division (see Figure 7.18).

Some (but not all) have genes that can direct their transmission from one cell to another by conjugation. Finally, plasmids may have genes that confer novel phenotypes on cells, such as resistance to antibiotics, production of toxins, or the capacity to metabolize unusual substrates such as pesticides or industrial solvents. Antibiotic resistance is conferred by R plasmids. These plasmids have diminished the effectiveness of antibiotics in combating infectious diseases because (i) they may confer resistance to as many as five different antibiotics at once upon the cell, and (ii) by conjugation, they can be rapidly disseminated through the bacterial population. Multiple antibiotic resistance is a consequence of their construction -- they contain several transposons, each of which confers resistance to a unique antibiotic. The genes in the transposons generally specify an enzyme that inactivates the drug before it enters the cell and reaches its target. This differs from chromosomal mutations that result in antibiotic resistance. These generally are modifications of the antibiotic's target of action.

Plasmids are autonomously replicating molecules. What elements are necessary to control DNA replication? There must be an origin of replication, where the frequency of replication can be regulated. The number of plasmid copies is tightly regulated at a few copies with some plasmids, whereas in others, initiation of replication is relatively uncontrolled, and twenty to thirty plasmid copies may be present in a cell. In general, the enzymes used for DNA replication are those coded by the chromosome -- it is the regulatory genes that are plasmid encoded.

Conjugative plasmids initiate gene transfer by altering the cell surface to allow contact between the plasmid-containing donor cell and a plasmid-less recipient. A plasmid gene codes for the production of a sex pilus that initiates pair formation. Subsequently, a conjugation bridge is formed through which DNA is transferred. The transfer of plasmid DNA is accompanied by its replication. That is, the donor cell does not lose its plasmid but transfers a copy to the recipient. In actual fact, replication is shared between donor and recipient. A single DNA strand is transferred as a consequence of rolling circle replication in the donor; this strand is used as a template by the recipient to generate a double stranded DNA molecule. Therefore, the consequence of conjugation is that both the donor and the recipient cells contain the plasmid. The recipient is now competent to serve as a plasmid donor in other conjugations.

Some conjugative plasmids, such as the F factor in E. coli, can also direct transfer of chromosomal genes by conjugation. E. coli strains which have this property are Hfr strains. The F factor can integrate into the chromosome to form one DNA molecule. This occurs at regions of homology between F and the chromosome. These regions are insertion sequences located on both molecules. F factor can now transfer chromosomal genes during a conjugation, because in effect, the chromosome has become part of the F factor. It is the F factor that has the genetic information to drive gene transfer. Specifically, there is a nucleotide sequence on F that specifies the origin of transfer. The host chromosome was inserted just downstream from this region. DNA is transferred just as described above for plasmid transfer. It is important to note that chromosomal genes are transferred before any of the plasmid genes. Thus, if the cytoplasmic bridge is broken before the entire chromosome is transferred, the recipient remains F-.

Just as improper excision of lambda prophage leads to high frequency transfer of some chromosomal genes by specialized transduction, the improper excision of F factor results in the F factor containing a few chromosomal genes. Conjugation involving such F' factors transfers these particular chromosomal genes at high frequency.

Irrespective of their ability to drive conjugation, plasmids confer interesting properties on cells. We have said that conjugative plasmids encode the production of pili. Note that these pili may make the cell susceptible to phage infection, as some RNA phages use these pili as receptors.

A cell can contain several different plasmids. However, some plasmids are incompatible with each other. That is, they cannot be maintained together in a single cell. This may be a consequence of similarity between the regulatory elements that control plasmid replication.

Conjugation with Hfr strains was used to map the E. coli chromosome. Genes are transferred in a linear, sequential process. Conjugants were deliberately broken apart at different time intervals, and the recipients were analyzed for the genes they received. The shorter the time interval of the interrupted mating in which two genes were successfully co-transferred, the closer they must physically be located on the chromosome.

Transposons and insertion sequences are genetic elements capable of moving within cells. Transposons differ from insertion sequences in that they contain additional genes, such as ones for antibiotic resistance. The frequency with which these elements move is rather low, but 10 to 100 fold greater than the frequency of spontaneous mutation. The ends of these elements contain repeated sequences. In addition, they code for a transposase enzyme that can insert the elements at any point into a DNA molecule.

When these transposable elements insert into a DNA target sequence, that target sequence is duplicated. In addition, elements that undergo replicative transposition also are duplicated. That is, a copy remains at the original site, and the other copy is inserted at a new site. The transposase makes single strand cuts in the inverted repeat sequences at the ends of the transposable element, and at the target site. The element is joined to the target via the single strand ends, and the gaps are filled in by DNA replication. Finally, the cointegrate formed by recombination is resolved to generate a copy of the transposable element at the new site. In other transposons (such as Tn5), transposition is conservative, and the transposon is excised from its original location, and is reinserted at a new site. If the site of transposon insertion is within an existing bacterial gene, it is likely to be inactivated, and a mutation has occurred.

Another type of genetic rearrangement is the inversion of a DNA segment in the genome. This has regulatory significance if the segment contains a promoter. Remember that the orientation of the promoter determines the direction in which mRNA synthesis will occur. Therefore, inverting a promoter sequence leads to expression of a different gene. This mechanism is responsible for changes in the type of flagellar protein produced by Salmonella.

The genetic transfer mechanisms discussed in this chapter have been used to map the order of genes on bacterial chromosomes. Interrupted mating was useful in coarsely determining gene order, because large DNA segments can be transferred in conjugation. To accurately determine the order of closely linked genes, transduction has been most useful, because small DNA pieces are transferred.

The three genetic exchange mechanisms described above can and have been used to map the location of genes in the bacterial chromosome. In E. coli, the location of some 1900 genes has been identified. This type of mapping has revealed that the genes that control a given pathway are often clustered or closely linked on the bacterial chromosome. This grouping of genes has lead to the operon concept that theorizes that the closely linked genes are under a common control mechanism.

In eukaryotic organisms, new combinations of genes are assembled on a regular basis, as a consequence of sexual reproduction. Eukaryotic microorganisms undergo an alternation of generations, in which the number of chromosomes per cell varies by a factor of two. At some point, the haploid cells, which contain one copy of each chromosome, function as gametes and fuse to form a diploid zygote. In this zygote, new gene combinations can arise from the mixture of the gametes' genomes.

Gametes are generated by meiosis. A diploid cell divides into 2 cells without replicating its chromosomes -- each chromosome pair the cell possesses is divided between the two daughter cells. Thus, they are now haploid. These cells replicate their chromosomes and divide again, so that four gametes are produced from the diploid cell.

More is known about the genetics of Saccharomyces cerevisiae (yeast) than of other eukaryotes. For most of their life cycle, yeasts are haploid. These cells can serve as gametes, if cells of the two different mating types fuse. Mating types differ in the hormones they excrete, and the receptors on their surface that interact with the complementary hormone. The resulting diploid zygote undergoes meiosis to form four haploid ascospores, each of which can germinate to form a vegetative cell. Yeast genetics is attractive because the four ascospores can be isolated and germinated separately. Thus, the consequences of sexual reproduction can be unequivocally determined.

The mating type of yeast cells is regulated by an insertion of DNA segments, called the cassette mechanism. The DNA sequence transcribed from the MAT promoter can be replaced with a copy of the gene of one of the two mating types located elsewhere on the yeast genome. These genes are inactive unless copied to the "reading head", the MAT promoter.

Eukaryotes contain DNA not only in the nucleus but also in two organelles -- mitochondria and chloroplasts. Although most of the genetic information to produce these structures is in the nucleus, there are a few functions retained in the organelles' DNA. For progeny cells to obtain these organelles, the existing structures must divide. Thus, the genetic information in these structures is inherited separately from that in the nucleus. This information does not code for many proteins, but does specify the ribosomal and transfer RNA used for protein synthesis within the organelles. A unique feature of protein synthesis in mitochondria is that some mRNA codons are translated differently than in all other systems.