Reading_Genome&HGT_1 - The Bacterial Genome and...

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Unformatted text preview: The Bacterial Genome and Horizontal Gene Transfer Annotating a Genome Sequence Textbook Reading See hand out provided in class The points to be remembered from this text: Know how open-reading frames are identified in prokaryotes. Know what DNA and protein alignments are used for. Know the difference between ortholog and paralog. Detecting Horizontal Gene Transfer Horizontal gene transfer (HGT) is defined as the exchange of DNA between bacteria. Vertical gene transfer is defined as the inheritance of a gene from a progenitor. There were many arguments against HGT playing a major role in bacterial evolution, but sequencing of bacterial genomes has revealed its major role. Genes acquired by HGT may show the following properties: 1. Such genes may have a very similar sequence in two distantly related bacteria. 2. Such genes may have a different %GC content as compared to the rest of the genome. 3. Such genes may have a different codon usage pattern than genes on the rest of the genome. As a case in point, for how genome sequencing is demonstrating the major role of HGT in shaping bacteria, consider the genome comparison of E. coli MG1655 and E. coli 0157:H7. MG1655 is a domesticated strain of E. coli that was derived from a strain that is thought to be part of the normal biota of our colon. 0157:H7 is a virulent strain of E. coli that causes a malabsorptive diarrhea or even a bloody diarrhea that typically affects children. Remember from an earlier lecture that scientists have determined the relatedness of bacteria based on the 1 sequence of their rRNA. By this measure E. coli MG1655 and E. coli 0157:H7 are different strains of the same species. Many other genes besides the rRNA gene bear out this relationship. But compare the genomes of these two bacteria in the figure below. Legend to the Figure. Outer circle shows the distribution of regions that are shared or different between 0157:H7 and MG1655: shared co-linear backbone (blue); position of 0157:H7-specific sequences (red); MG1655-specific sequences (green); 0157:H7- and MG1655-specific sequences at the same locations in the backbone (tan); hypervariable (purple). Second circle shows the G+C content calculated for each gene longer than 100 amino acids, plotted around the mean value for the whole genome, color-coded like outer circle. Third circle shows the GC skew for third-codon position, calculated for each gene longer than 100 amino acids: positive values, lime; negative values, dark green. Fourth circle gives the scale in base pairs. Approximately 30% of the 0157:H7 genome is not present in the MG1655 genome. Each strainspecific sequence might be ancestral and lost from the reciprocal genome; however, atypical base composition suggests that most strain-specific sequences are the result of relatively recent HGT from a donor species with a different intrinsic base composition. Similar results are obtained for analysis of the third-codon position composition. (See the course website for a link to the article on the 0157:H7 genome sequence if you would like more information.) Definition of species in the face of HGT How to define a bacterial species is a matter of hot debate among scientists. What they agree on most is that our current methods are not adequate. Should species be defined based on a coreconserved group of genes? Should ecology of the bacterium be considered in definition of species? Are their limits to horizontal gene transfer and would this help define a species? Gene Transfer Mechanisms Between Bacterial Cells Transformation Textbook Reading Pages 439-441, “Transformation” The points to be remembered from this text: Know the difference between natural and artificial transformation. Know the basic mechanism of DNA uptake into the cell and introduction into the cellular genome. Know that some bacteria will bind and take up any DNA available and others recognize a unique sequence in their own DNA and only take this up. Understand why uptake of a single strand of DNA is advantageous for recombination of DNA into the chromosome (see section on homologous recombination if this is not clear, pg. 429-432). Phage Transduction Phage are either temperate (lysogenic) or virulent (lytic). Virulent phage are phage that, on entry into a new host cell, immediately start the production of new phage particles (i.e. a lytic 2 cycle). Typically, the lytic cycle will result in death of the host cell. However, some phage, like the filamentous phage (see Cholera section below), bud from the host cell without killing the host. When a temperate phage, infects a cell it can either immediately undergo a lytic cycle or remain dormant. In this dormant state, the phage genome may integrate into the host genome or the viral genome may replicate as a plasmid. Temperate phage have an important role in evolving functions for bacteria. For example, many temperate phage carry the genes for toxins that cause human disease. Thus, when a host bacterium is lysogenized by a temperate phage carrying the gene for a toxin, this bacterium can become more pathogenic. Some temperate and virulent phage are capable of packaging host genomic DNA and transducing that to another cell. Textbook Reading Pages 441-442, “Transduction” The points to be remembered from this text: Know the mechanism of generalized transduction. Know how host genomic DNA gets into phage particles and that packaging of host genomic DNA is a relatively rare event. Know what happens to host genomic DNA when transduced to a new cell. You will not be responsible for knowing the mechanism of specialized transduction. Conjugation Conjugation is defined as the direct cell-to-cell transfer of DNA, involving intimate cell contact. Self-Transmissible Plasmids Textbook Reading Pages 434-439 The points to be remembered from this text: Know the general mechanism of conjugative plasmid transfer. Know that self-transmissible plasmids encode the genes to making the mating complex and transfer the DNA from the host cell to the recipient cell. Know that a single strand of DNA is transferred to the recipient cell while the plasmid is being replicated by a rolling-circle mechanism of replication (recall the viral genome replication lecture). This requires the DNA to be nicked at an oriT sequence (the origin of transfer). Know that the end result of conjugation is a copy of the plasmid in the host cell and the recipient cell. Know that if a conjugative plasmid integrates into the chromosome, this can result in transfer of chromosomal DNA to the recipient cell. Mobilizable Plasmids Mobilizable plasmids lack the genes to make the mating bridge between the host and recipient cells. These plasmids do encode an oriT sequence and the gene for the protein to nick the DNA at oriT. Thus, in a host cell with a self-transmissible plasmid, the mobilizable 3 plasmid can be transferred to a recipient cell using the mating bridge formed by the selftransmissible plasmid. Conjugative Transposons (pg. 447) Conjugative transposons, like other transposons (see below), are elements of DNA that do not replicate independently. They replicate as part of a larger DNA element (i.e. the chromosome or the plasmid) into which the transposon has integrated. The conjugative transposon, unlike other transposons, encode genes to make a mating bridge, an oriT sequence and the protein to nick the oriT. During the process of transfer of these transposons, the transposon excises from the DNA element in which it resides and circularizes. Now the transposon looks like a plasmid. It is nicked and transferred with rolling circle replication just like the conjugative plasmids. Once a double stranded copy of the transposon is present in the recipient cell. This transposon will “hop” into the recipient chromosome or plasmid (if present). Gene Transfer Mechanisms Between Segments of DNA This section considers DNA elements that move between segments of DNA rather than between cells. These mobile DNA elements are important agents of genome evolution. Insertion sequence and transposons mediate chromosomal rearrangements, duplications, and deletions. This can occur through homologous recombination between identical insertion elements or transposons on the same chromosome. Insertion Sequences and Transposons Textbook Reading Pages 433-444 The points to be remembered from this text: Be able to describe the differences and similarities between an IS element (IS) and a transposon (Tn). Know that the key features of an IS or a Tn is a gene encoding a transposase and inverted repeats at the ends of these elements. Be able to describe the difference between a Tn and a composite Tn. Be able to explain how an IS can mediate integration of plasmids into a chromosome. Know the function carried out by transposase. Know what the net effects of non-replicative transposition versus replicative transposition. Integrons Textbook Reading Pages 444-447, “Integrons” The points to be remembered from this text: Know that integrons are transposons with a mechanism to easily acquire new genes and express those genes. Know the function of integrase for integrons. Know the function of the attI site on the integron (see Figure 10.34). 4 Know that gene cassettes that integrate into the integron have an att sequence that is recognized by the integrase. Where gene cassettes come from is unknown. Evolution of a Bacterial Pathogen: Vibrio cholerae Vibrio cholerae is the causative agent of cholera. Two major virulence factors for this organism are Cholera Toxin (CTX) and the Toxin-Co-Regulated Pilus (TCP pilus). The genes for CTX were known to be in a cluster of genes that were flanked by an inverted repeat (the CTX element). Within the cluster were genes that appeared to encode a site-specific recombination system. Was this cluster of genes a transposon or a phage genome? The genes for the TCP pilus were in a cluster of genes that have been termed a pathogenicity island, reflecting acquisition of these genes by apparent HGT. 1. 2. 3. 3. 4. To address whether the CTX element was a self-transmissible phage, scientist inserted a kanamycin-resistance gene in the genes for CTX. This would allow selection of recipient cells (initially lacking the CTX element) that had received the CTX element from a donor cell. The following experiments were carried out to demonstrate the CTX element is a phage (think about what you can conclude from each experiment and what would be appropriate control groups for the experiments – we will go over the answer to this in class): Mix donor (CTX+) cells with recipient (CTX-) cells. Result: Recipient cells become CTX+. Harvest cell-free culture supernatant from the growth of CTX+ donor cells. Incubate CTX- recipient cells with the supernatant. Result: Recipient cells become CTX+. Treat cell-free supernatant from the growth of CTX+ donor cells with DNase and RNase. Incubate CTX- recipient cells with the treated supernatant. Result: Recipient cells become CTX+. Analyze the cell-free supernatants of CTX+ and CTX- cells by electron microscopy. Long, filamentous structures are observed in the CTX+-cell supernatant that were not observed in the CTX--cell supernatant. Purify the filamentous structures, extract DNA and sequence. Result: DNA was present in the preparations of the filamentous structure, and the sequence of this DNA matched the DNA sequence of the chromosomal CTX element. Scientists noticed that strains lacking the TCP pilus could not be transduced by the CTX phage, indicating that the TCP pilus was a receptor for the phage. They also knew that the El Tor Biotype of V. cholerae, the cause the current 7th pandemic of cholera, poorly expressed TCP pili in the laboratory. Correspondingly, the ability of the El Tor strain to be lysogenized by the CTX phage was low, compared to strains that expressed high levels of the TCP pili. To test the hypothesis that the TCP pili are expressed at high levels in a host animal and that in the host animal the El Tor strain could be lysogenized at high levels by the CTX phage, the El Tor strain lacking the CTX phage was co-inoculated with a CTX phage donor strain into the intestines of mice. After 24 hours, the contents of the intestine were collected and the percent of the El Tor cells that were lysogenized by the CTX phage was determined. The scientist found a 100-fold increase in the frequency of lysogenization of the El Tor strain after growth in the host animal. 5 ...
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