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14 Microbial Genetics: Recombination and Plasmids

 

CHAPTER OVERVIEW

This chapter begins with a general discussion of bacterial recombination, plasmids, and transposable elements, and then examines the acquisition of genetic information by conjugation, transformation, and transduction. The way these recombination procedures are used to map the bacterial genome is explained. Finally, viral recombination and genome mapping are discussed.

 

CHAPTER OBJECTIVES

After reading this chapter you should be able to:

o discuss the nature of procaryotic recombination

o discuss the three ways (conjugation, transformation, and transduction) that bacteria acquire new genetic material

o discuss how plasmids and transposable elements can move genetic material between bacterial chromosomes and within a chromosome to cause changes in the genome and the phenotype of the organism

o discuss the use of these gene transfer procedures to map the bacterial genome

o discuss the sequencing of microbial genomes

o discuss the recombination that occurs when two viruses simultaneously infect the same host

 

CHAPTER OUTLINE

I. Bacterial Recombination: General Principles

A. Recombination is the process by which a new chromosome (with a genotype different from either parent) is formed when genetic material from two parent organisms combine

1. General recombination usually involves a reciprocal exchange in which a pair of homologous sequences break and rejoin in a crossover

2. Nonreciprocal recombination involves the incorporation of a single strand into the chromosome to form a stretch of heteroduplex DNA

3. Site-specific recombination is the nonhomologous insertion of DNA into a chromosome

a. Often occurs during viral genome integration into the host

b. The enzymes responsible are specific for the virus and its host

4. Replicative recombination accompanies replication and is used by genetic elements that move about the genome

B. Terminology

1. Horizontal gene transfer-transfer of genes from one mature, independent organism to another

2. Vertical gene transfer-transmission of genes from parents to offspring

3. Exogenote-donor DNA that enters the bacterium by one of several mechanisms

4. Endogenote-the genome of the recipient

5. Merozygote-a recipient cell that is temporarily diploid for a portion of the genome during the replacement process

C. Types of horizontal exogenote transfer

1. Conjugation is direct transfer from another bacterium

2. Transformation is transfer of a naked DNA molecule

3. Transduction is transfer from a bacteriophage

D. Intracellular fates of exogenote

1. Integration into the host chromosome

2. Independent functioning and replication of the exogenote without integration (a partial diploid clone develops)

3. Survival without replication (only the one cell is a partial diploid)

4. Degradation by host nucleases (host restriction)

II. Bacterial Plasmids-small, circular DNA molecules that are not part of the bacterium's chromosome

A. Plasmids have their own replication origins; they replicate autonomously and are stably inherited

B. Curing is the elimination of a plasmid; it can occur either spontaneously or as a result of treatments that inhibit plasmid replication but do not affect host cell reproduction

C. Episomes are plasmids that can either exist independent of the host chromosome or be integrated into it

D. Conjugative plasmids have genes for pili and can transfer copies of themselves to other bacteria during conjugation

E. Types of plasmids

1. Fertility plasmids (e.g., the F factor) are episomes that can direct the formation of sex pili and can transfer copies of themselves during conjugation

2. Resistance factors (R plasmids) have genes for resistance to various antibiotics; some are conjugative; however, they are not episomal (they do not integrate into the host chromosome)

3. Col plasmids carry genes for the synthesis of colicins that destroy Escherichia coli; other similar plasmids carry genes for bacteriocins that are directed against other bacterial species; some are conjugative and may also carry resistance genes

4. Virulence plasmids make the bacterium more pathogenic by conferring resistance to host defense mechanisms or by carrying a code for the production of a toxin

5. Metabolic plasmids carry genes for enzymes that utilize certain substances as nutrients (aromatic compounds, pesticides, etc.)

III. Transposable Elements-transposons

A. Segments of DNA that can move about chromosomes within a single organism or between different organisms

B. Differ from bacteriophages in that they lack an infectious viral life cycle

C. Differ from plasmids in that they are unable to reproduce independently

D. Types of transposable elements

1. Insertion sequences (IS elements) contain genes only for those enzymes required for transposition; they are bound on both ends by inverted terminal repeat sequences

2. Composite transposons carry other genes in addition to those needed for transposition (e.g., for antibiotic resistance, toxin production, etc.)

E. Movement is typically by replicative transposition, during which a replicated copy of the transposon inserts at the target site on the DNA, while the original copy remains at the parental site

F. Effects of transposable elements

1. Insertional mutagenesis, including deletion of genetic material at or near the target site

2. Arrest of translation or transcription due to stop codons or termination sequences located on the inserted material

3. Activation of genes near the point of insertion due to promoters located on the inserted material

IV. Bacterial Conjugation-the transfer of genetic information via direct cell-cell contact; this process is mediated by fertility plasmids (F plasmids)

A. F+ ´ F- mating

1. Nonreciprocal exchange between donor (F+) and recipient (F-)

a. Recipients usually become F+ (plasmid is transferred)

b. Donors remain F+ (plasmid is retained)

c. The plasmid DNA replicates by the rolling-circle mechanism, and the displaced strand is transferred and then copied to produce double-stranded DNA; the other strand and its complement are retained by the donor

2. Chromosomal genes are not transferred

B. Hfr conjugation

1. F plasmid integration into the host chromosome results in an Hfr strain of bacteria

2. The mechanics of conjugation of Hfr strains are similar to those of F+ strains

3. The initial break for rolling-circle replication is at the integrated plasmid's origin

a. Part of the plasmid is transferred first

b. Chromosomal genes are transferred next

c. The rest of the plasmid is transferred last

4. Complete transfer of the chromosome takes approximately 100 minutes, but the conjugation bridge does not usually last that long; therefore, the entire F factor is not transferred, and the recipient remains F-

C. F¢ conjugation (sexduction)

1. An integrated F plasmid leaving the chromosome incorrectly may take with it some chromosomal genes from one side of the integration site; this is called an F¢ plasmid

2. The F¢ cell retains all of the plasmid genes, although some of them are in the host chromosome and some are on the plasmid; in conjugation, it behaves as an F+ cell, mating only with F- cells

3. The chromosomal genes included in the plasmid are transferred with the rest of the plasmid, but other chromosomal genes are not

4. The recipient becomes an F¢ cell, and a partially diploid merozygote

V. DNA Transformation

A. A naked DNA molecule from the environment is taken up by the cell and incorporated into its chromosome in some heritable form

B. A competent cell is one that is capable of acting as a recipient

C. This process naturally occurs in a limited number of species, but can be induced in other species in the laboratory

D. The mechanics of the process differ from species to species

E. Species that are not normally competent (such as E. coli) can be made competent by calcium chloride treatment, which makes the cells more permeable to DNA

VI. Transduction-transfer of bacterial genes by viruses

A. Generalized transduction-any part of the bacterial genome can be transferred during lytic infection

1. The phage degrades host chromosome into randomly sized fragments

2. During assembly, fragments of host DNA of the appropriate size can be mistakenly packaged into the phage head

3. When the next host is infected, the bacterial genes are injected and a merozygote is formed

a. Preservation of the transferred genes requires their integration into the host chromosome

b. Much of the transferred DNA does not integrate into the host chromosome, but is often able to survive and be expressed; the host is called an abortive transductant

4. Because fragmentation is random, any bacterial gene can be transferred; therefore, this is called generalized transduction

B. Specialized (restricted) transduction-only temperate phages that have established lysogeny (their DNA has been integrated into the host chromosome and replicates along with it) are capable of specialized transduction

1. A prophage (an integrated, nondestructive viral genome) is sometimes excised incorrectly and contains portions of the bacterial DNA that was adjacent to the phage's integration site on the chromosome

2. The excised phage genome is defective because it lacks some of its own genes and carries some of the bacterial genes

3. When the next host is infected, the bacterial genes lead to the formation of a merozygote, and the phage cannot reproduce

4. Because only those genes adjacent to the integration site can be carried, and because integration sites are at specific locations, the number of transferable genes is limited; therefore, this is called specialized transduction

VII. Mapping and Sequencing the Genome

A. Hfr mapping involves the use of an interrupted mating experiment

1. Chromosome transfer occurs at a constant rate

2. The frequency of a particular recombinant indicates the position of that gene relative to the plasmid integration site

a. High-frequency recombinants indicate that the gene is close to the integration site

b. Low-frequency recombinants indicate that the gene is farther from the integration site

3. The instability of the conjugation bridge makes it nearly impossible to map genes that are very distant

4. The use of several Hfr strains with different integration sites can generate overlapping maps, which can then be pieced together to form the entire genome map

B. Transformation mapping-the frequency with which two genes simultaneously transform recipient cells indicates the distance between the genes; overlapping maps can then be pieced together

C. Generalized transduction maps-as with transformation mapping, the frequency of cotransduction indicates the distance of two genes from each other

D. Specialized transduction maps provide distances from integration sites, which themselves must be mapped by conjugation mapping techniques

E. Sequencing a genome by the whole-genome shotgun approach is a multi-step process

1. Small fragments are generated and sequenced

2. Sequenced fragments are clustered into longer contiguous groupings (contigs) by analysis of sequence overlaps

3. Gaps between contigs are sequenced until the entire genome sequence has been determined

F. Annotation involves identifying open reading frames (ORFs), determining potential amino acid sequences and comparison to known proteins

G. Comparison among sequenced organisms allows estimates of functional similarity and determination of the minimum set of genes necessary to sustain life

VIII. Recombination and Genome Mapping in Viruses

A. Recombination maps are generated from crossover frequency data obtained when cells are infected with two or more phage particles simultaneously

B. Denaturation maps-the effect of mild denaturing conditions is greater on AT-rich regions than on GC-rich regions; bubbles are thus formed, and can be seen in electron micrographs; maps are generated by comparing mutants for differences in bubbling

C. Heteroduplex mapping-wild type and mutant chromosomes are denatured and allowed to reanneal together; homologous regions pair normally, but mutant regions form bubbles that can be seen in electron micrographs

D. Restriction and endonuclease mapping-locates deletions and other mutations by examining the electrophoretic mobility (size) of the fragments generated

E. Sequence mapping-small phage genomes can be directly sequenced to map mutations

 

 

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