Chapter 10 (continued) Bacterial Genetics

Transformation vs. Transduction vs. Conjugation

Genetic Transformation
Genetic Transformation: process by which free DNA is incorporated into a recipient cells and brings about genetic change. A number of prok. are naturally transformable, including gram-pos. and gram-neg. Bacteria and some Archaea. Only a small number of genes from one cell can be transferred to another by a single transformation event.

Competent Cells
Competent cells: able to take up a molecule of DNA via transformation. Only certain strains are competent and this ability is genetically determined. Competence is regulated with special proteins playing a role in DNA uptake and processing. ssDNA or dsDNA may taken up by cells, though it must be in ssDNA form to be incorporated into the genome by recombination. Competent cells bind up to 1000X more DNA than noncompetent cells (dsDNA binds better to cells). DNA fragments compete with each other for uptake. While the max. frequency of transformation = 20% of the population, actual values = 0.1-1.0%. Min. conc. of DNA yielding detectable transformants = 0.00001 g/ml.

Integration of Transforming DNA

DNA is either taken up single-stranded or dsDNA is taken up and one strand is degraded ssDNA. Next, the ssDNA associates with competence-specific protein that remains attached to the DNA to protect it from nuclease attack until it reaches the chromosome, where RecA takes over. DNA is then integrated into the genome of the recipient by recombination. During replication of this heteroduplex DNA, one parental and one recombinant DNA molecule are formed. On segregation at cell division, the recombinant DNA molecule is present in the transformed cell.

Transfection
Transfection: Bacteria transformed by bacteriophage DNA instead of DNA from another bacterium. Transfection by a lytic bacteriophage leads to normal virus production.

Artificially Induced Competence/Electroporation

Only a few bacteria exhibit natural transformation. Other bacteria can be made competent through artificially induced competence. High conc. of cold Ca2+ ions causes E. coli to become competent at low efficiency. Electroporation: a technique in which cells are exposed to pulsed electric fields to open small pores in their membranes. DNA present outside the cells can enter through these pores. This method works for a variety of prok. and euk. Plasmids can be transferred directly from one cell to another because DNA can exit as well as enter through these pores.

Transduction
Transduction: DNA is transferred from cell to cell via viruses. A variety of prok. can undergo transduction and a variety of phages can transduce. 2 types of transduction virus ends up defective and homologous recomb. can occur in either case: (1) generalized transduction: host DNA derived from virtually any portion of the host genome becomes a part of the DNA of the mature virus particle in place of the virus genome. (2) specialized transduction: occurs only in some temperate viruses; DNA from a specific region of the host chromosome is integrated directly into the virus genome usually replacing some of the virus genes.

Generalized Transduction

Specialized Transduction

Phage Conversion
Phage conversion: phenotypic alterations made in a lysogenized cell, can be acquisition of immunity to further infection by the same type of phage or can be some other change. ex. toxin production in Corynebacterium diphtheriae.

Plasmids
Plasmids: genetic elements that replicate independently of the host chromosome. Thousands of different types of plasmids are known, almost all of which are dsDNA, most of which are supercoiled and circular, are vary in size from 1-1000 kbp. Different plasmids are present in cells in a particular number of plasmid molecules per cell = copy number, which can vary from 1-100. Most gram-neg. plasmids replicate similar to the chrom., although some replicate unidirectionally. Most gram-pos. plasmids replicate by the rolling circle mechanism similar to a phage.

Plasmids (cont.)
Cells can contain different types of plasmids. A cell in which two plasmids cannot be maintained together are said to be incompatible. Curing: elimination of a plasmid from a cell. Curing can occur spontaneously or with the help of chemicals or electroporation. Plasmids lack extracellular form. The main mechanism of cell-to-cell plasmid transfer = conjugation. Plasmids that govern their own transfer by cell-to-cell contact = conjugative.

Types of Plasmids
While all plasmids carry genes that ensure their own replication, some carry genes for conjugation, as well as other unique properties conferred upon the cell. Resistance (R) plasmids: confer resistance to antibiotics and other inhibitors of growth. These plasmids often transfer resistance to other cells via cell-to-cell contact, resulting in antibiotic resistant populations. R plasmids with genes for resistance to most antibiotics are known. The following virulence factors of pathogenic bacteria can be encoded on plasmids: (1) ability of microorganisms to attach and colonize specific sites in the host (2) formation of substances (ex. toxins, etc.) that cause damage to the host. What is this similar to that we just discussed?

Bacteriocins
Bacteriocins: agents produced by bacteria that inhibit or kill closely related species or different strains of the same species. They are different from antibiotics, which have a wider spectrum of activity. Bacteriocins are often plasmid-encoded. Bacteriocins are named according to the organism that produces them. They can interfere with another cells proton motive force and, thus, have practical uses such as food preservatives.

Conjugation and Chromosome Mobilization

What is bacterial conjugation also known as? Conjugation is a plasmid-encoded mechanism, but can mobilize host chromosome as well. The F plasmid of E. coli first confirmed the occurrence of conjugation. Conjugation involves a donor cell containing a conjugative plasmid and a recipient cell, which does not. What are these cells also known as? Sex pilus: may be specified by the plasmid, allowing for specific pairing between donor and recipient. The pilus formed by the F plasmid is called the F pilus.

DNA Transfer During Conjugation

DNA synthesis is necessary for DNA transfer to occur. Rolling circle replication: model best explains DNA transfer during conjugation. This process is triggered by cell-to-cell contact. At the end of the process, both donor and recipient possess plasmids and the recipient can become a donor, spreading the plasmids between populations like infectious agents.

Transfer of Plasmid DNA by Conjugation

Hfr (High Frequency of Recombination) Strains

F plasmid: conjugative, can integrate into host chromosome (= episome), and can also mobilize chromosome transfer. Cells with an unintegrated F plasmid = F+, while those having a chromosome-integrated F plasmid = Hfr, and cells without and F plasmid = F-. Conjugation with Hfr donor transfer of host chromosome. After transfer, an Hfr strain remains Hfr since it retains a copy of the F plasmid in the chromosome. Note: ori = origin, ex. of replication or of transfer

F Plasmid
Cells having an F plasmid are able to synthesize and F pilus, mobilize DNA for transfer to another cell, and alter surface receptors so that the cell can no longer serve as a recipient. The F plasmid can integrate into the host chromosome at sites called insertion sequences (IS) . Once integrated, the F plasmid no longer controls its own replication. Usually, because of breakage of the DNA strand during transfer, only part of the donor chromosome is transferred. Although Hfr strains transmit chromosomal genes at high frequency, they usually do not convert F- strains to F+ or Hfr because the entire F plasmid is rarely transferred. However, F+ strains can convert F- strains to F+ because the entire F plasmid is transferred.

Interrupted Mating
Recombinants from conjugation can be selected for. In an Hfr strain, the transfer of chromosomal genes will always occur in the same order from a fixed site on a given Hfr strain. Interrupted mating: interrupt mating pairs by agitation after a certain time that conjugation has occurred. Genes present closer to the origin enter the F- cell first. This technique leads to genetic mapping since you can determine the order in which the genes occur by the order in which they are transferred. Genes at certain points can be referred to as positioned at so many minutes. Genetic recombination is dependent on the occurrence of homologous recombination and is not a result of genetic transfer alone. F = cells in which the F plasmid has been excised from the chromosome and takes some chromosomal DNA with it.

Interrupted Mating Experiment

Other Conjugation Systems

Conjugative transposons can be transferred from the chromosome of a donor to a recipient and can mobilize other genetic elements. Conjugative transposons are common to grampos. cells.

Complementation
Complementation is used to determine whether or not two mutations are in the same gene by restoring function of a gene by complementing the defective (mutant) gene with a normal (nonmutant) copy of that gene. Homologous recombination can restore gene function (unless both of the mutations include changes in exactly the same base pairs) but cannot reveal whether or not the mutations were in the same gene. The two mutations are said to complement each other. Bacterial gene transfer must be done in order to conduct this test. Complementation does not involve recombination. Cistron = gene two mutations in the same cistron cannot complement each other. In diploid organisms: Cis = 2 mutations from the same parent, Trans = 2 mutations from different parent.

Complementation (cont.)

Transposons and Insertion Sequences

Some genes are capable of moving under certain conditions. The process by which a gene moves from one place to another in the genome = transposition. Transposition is relatively rare. Not all genes are capable of transposition. Transposition of genes is linked to the presence of special genetic elements called transposable elements. There are 3 types of transposable elements in Bacteria: (1) insertion sequences (2) transposons (3) some special viruses (ex. Mu) Transposable elements have 2 features in common: (1) transposase enzyme necessary for transposition (2) inverted terminal repeats and the ends of their DNA.

Mechanisms of Transposition
Two mechanisms of transposition: (1) Conservative: the transposable element is excised from one location in the chromosome and becomes reinserted at a second location. The copy number of a conservative transposon remains at one. Direct repeats are formed in the target site at the ends of the transposon. (2) Replicative (ex. bacteriophage Mu): transposons are duplicated and a new copy is inserted at another location. A composition structure called a cointegrate is formed. Transposition is essentially a recombination event, but one that does not occur between homologous sequences or use the general recombination system of the cell. It is called site-specific recombination and involves transposase instead of RecA.

Mechanisms of Transposition (cont.)

Mutagenesis with Transposable Elements

If the insertion site for a transposable element is within a gene, insertion of the transposon will result in loss of linear continuity of the gene, leading to mutation = transposon mutagenesis = means of creating mutants throughout the chromosome.

Integrons
Integrons = genetic elements that can capture and express genes from other sources. Integrons code for integrase, which catalyzes a type of sitespecific recombination. Integrase can integrate gene cassettes and a promoter that can then express the newly integrated gene cassette. The genes in the gene cassette that are captured are not captured randomly, but have specific DNA sequences recognized by the integrase and genes that are not expressed until they become part of an integron.

Restriction Enzymes
Protect prok. from foreign DNA, ex. viruses. Restriction enzymes recognize certain sequences of DNA and cut the DNA. Palindrome: sequence of bases that reads the same when read from either right or left. Palindromes are often the target of REs. Introduce double stranded breaks. In a random DNA molecule, one would expect any 4-bp sequence to occur ~ once every 256 bps based on the probability of 1/4 X 1/4 x 14 x 1/4. A 6 bp sequence would appear every 4096 bps in random DNA and a 8 bp sequence would appear once every 1000 bps, so NotI cuts the E. coli genome (4600 bps) 21x, therefor the recognition sequence for NotI occurs more often than predicted. There are over 2000 REs known with over 200 specificities.

Protection from Restriction

Cells protect their own DNA from their REs by methods such as methylation of their own sequences that would be targeted by their REs.

RE Analysis of DNA
RE analysis is done by gel electrophoresis - whats the procedure for this? Can be used to generate a physical map of DNA. Nucleic acids can be purified from gels and used to transform cells or for nucleic acid hybridization as nucleic acid probes to find similar sequences from different genetic elements = Southern blot (RNA hybridization = Northern blot, protein hybridization = Western blot).

Sequencing and Synthesizing DNA

2 procedures: (1) Maxim and Gilbert method (2) Sanger dideoxy method Both methods generate DNA fragments that end at each of the four bases (G, A, T, C) and that are radioactive. The fragments are subjected to gel electrophoresis in which 4 sample lanes are featured, one for each base. Maxim-Gilbert method: used chemicals that break the DNA preferentially at each of the four nucleotides. Sanger dideoxy method: sequence is determined by making a copy of the ssDNA using DNA pol., which used dNTPs as substrates, adding them to a primer. The dNTPs feature a dideoxy sugar analog that prevent lengthening of the chain and acts as a specific chain-termination reagent. Fragments of variable length are obtained. Either the dNTPs or primer are radioactive. This method can be used to sequence RNA as well. Sequencing by the Sanger method is now automated and fluorescent labels have replaced radioactive ones.

Molecular Cloning
= Gene cloning Purpose: isolate large quantities of specific genes or chromosomal fragments in pure form. Basic strategy: move the desired gene or region from a large, complex genome to a small, simple one. Tools used: restriction enzymes, DNA ligase, synthetic DNA (see #2 below). Steps: 1. Isolation and fragmentation of the source DNA. 2. Joining the DNA fragments to a cloning vector (ex. plasmid or virus) with DNA ligase. Blunt or sticky ends may be created on the ends of the source and/or vector DNA - what does this mean and how do you deal with each? 3. Introduction and maintenance in a host organism. What types of organisms are used as host organisms (what are their characteristics)?

Molecular Cloning (cont.)

What makes plasmids good cloning vectors? What is plasmid pBR322 a good cloning vector? How does insertional inactivation work?

Polymerase Chain Reaction (PCR)

PCR requires that the nucleotide sequence of a portion of the desired gene be known because short oligonucleotide (- what does this mean?) primers complementary to sequences in the gene or genes of interest must be available for PCR to work. Steps: 1. Two oligonucleotide primers flanking the target DNA are made (how?) and added to excess to heat-denatured target DNA. 2. As the mixture cools, the target strands anneal mostly to a primer, which are in excess, and not to each other. 3. DNA pol. then extends the primers using target strands as template. 4. After an appropriate incubation period, the mixture is heated again to separate the strands. The mixture is then cooled to allow the primers to hybridize with complementary regions of newly synthesized DNA, and the whole process is repeated.

Polymerase Chain Reaction (PCR)

PCR (cont.)
Taq pol. is stable at 95C and is unaffected by the denaturation step. However, it has no proofreading activity. Pfu pol. is stable at 100C and has proofreading activity, and is therefore more accurate. PCR is often conducted in automated thermocycling machines. It can be used to amplify very small quantities of DNA present in a sample. It is not necessary for the organism to be grown in the lab, so it is important for environmental studies. It can also be used for DNA fingerprinting, a powerful forensic tool permitting ID of individuals (crime scene/suspects) or relationships between individuals (paternity testing).

In Vitro and Site-Directed Mutagenesis

In Vitro = in glass, i.e. in the lab external to the organism, as opposed to in vivo = in the living organism. In other words, you can remove genes from and organism, manipulate them, engineer in mutations and put them back into an organism. Site-directed mutagenesis: Mutations can be introduced at precisely determined sites on genes. Mutagenesis studies are often done on at the gene level to make amino acid changes to study protein structure. Cassette mutagenesis: segments of DNA can be manipulated in which synthetic fragments of DNA have replaced the wild-type sequence. These cassettes can be used for insertion inactivation, causing gene disruption. How is this done?

The Bacterial Chromosome

The entire genome of E. coli K-12 has been sequenced and has been found to be 4,639,221 bp with 4288 open reading frames, corresponding to 88% of the genome (what are these and what is the rest of the DNA used for?). The map distances on this genome are given in minutes of transfer, in which 0 time = origin of transfer and 100 min. = time the whole chromosome takes to be transferred from an Hfr strain to an F- strain.

The Bacterial Chromosome