Bacterial- and phage genetics

Bacteria Prokaryotes are unicellular organisms haploid, circular dsdna genome 70 S ribosome plasmamembrane, cytoplasm no nuclei, ER, Golgi, mitochondria asexual reproduction Present in most habitats, growing in oceans, fresh water, hydrothermal vents, hot springs, arctic regions, deep in the Earth's crust, in organic matter, live bodies of plants and animals larger biomass than plants or animals Bacteria associated with living organisms: symbionts: commensals, mutualists and parasites, probiotic, pathogenic or opportunist size: 0.2-5 μm range

Changing genomes E. coli strains exhibit 30-40 % diffference of genome all are E. coli Human and mice genomes differ less than 30-40 % In bacteria new metabolic pathways appear or genes are lost within a few generations In the genome of two, genetically very different bacteria up to 60-70 % homologous genes can be present! The species as taxonomic unit is useless in case of bacteria

Changing genomes OTU = operational taxonomic unit, based on the sequence of 16S rrna gene Vertical and horizontal gene transfer: DNA from the environment genes providing evolutionary advantage spread among microorganisms useless genes are rapidly lost The fact that natural transformation has been detected among bacteria from all taxonomic groups suggests that transformability evolved early in phylogeny 1542 nucleotides

Tree of Life based on sequence of 16S rrna genes Even extinct species fit into this system Prokaryote Archea are clearly separated from Bacteria (Woese) Organisms close to the theoretical common ancestor tolerate high temperatures well Carl Woese

Prokaroyte cells at 0, 37 and 130 C 130 C

Markers of bacteria Visible markers wild and mutant growth is different: colony size, shape wild and mutant exhibit enzyme differences, different metabolites: color or staining Selectable markers permissive and restrictive media, conditions wild or mutant allele means selective advantage (in certain conditions), cells can be selected according to genetic make up

Bakteriális kolónia formák (Eshel Ben Jacob, Tel Aviv University)

Discovery of genetic transformation Prokaryotes frequently take up genetic material from the environment to change their genome adaptation to environmental changes Discovery of gene transfer: Frederick Griffith, 1928, Streptococcus pneumoniae DNA is the genetic material: Oswald Avery (MacLeod and McCarty) 1944 Stereptococcus Enormous medical importance: spread of antibiotic resistance and pathogenicity

The bacterial metagenome The number of bacterial OTUs in a human body is estimated to be around 40 000 An average bacterium has approx. 4-5 thousand genes (0.5-10 000 ) They suppose to have close to 200 million genes, 10 000 times more than we have In reality bacterial genes probably code for less than one million genes: they have many homologous genes (genes leading to positive selection spread fast in microbial populations, even among genetically unrelated cells) Divergent species in identical environment tend to develop similar metabolism: convergent evolution

Transfer of genes, mobile genes Some genes are transfered between different cells by random events (DNA of dead cells is taken up and integrated by living cells) Other genes move around (transposons), or multiplicate (retrotransposons) to increase their chances to get into other cells Genes evolved that code for proteins forming structures (sex pili) needed for transfer of genetic material between cells Groups of genes evolved that can ensure their own survival if the environment becomes lethal for the host cells (lysogenic phages) Even more selfish genes use the host only as a means to replicate themselves (bacterial viruses = bacteriophages)

Gene exchange among prokaryotes Transformation: uptake of DNA from (dead) cells Conjugation: transfer of DNA (among living cells) Transducion: gene transfer (by infectious agents)

Recombination among auxotrophic bacteria Auxotroph cells: require compounds what wild type cells (prototrophs) do not need The experiment of Lederberg and Tatum: mixing auxotroph strains Neither strain gave rise to prototrophic cells only after mixing the strains. Exchange and recombination of genetic material.

Discovery of conjugation U-tube experiment - Bernard Davis: soluble factors are unimportant The filter allows exchange of subcellular material, but prevents mixing of the cells. No recombination detected (no prototroph colonies). William Hayes, 1953: lost capacity to recombine ( fertility ) Formation of recombinants is unidirectional and requires physical contact of cells of the strains. The transfer is one way only! "Sexual-like difference between the cells A = donor ("male"), B = recipient ("female"). Fertility frequently lost from male cells independent of other properties.

Discovery of conjugation An extrachromosomal piece of DNA, a (F= fertility) plasmid is responsible for the conjugation capability Characteristic features: donor strain has pili (pilus) One copy of F-plasmid is transferred to recipient F factor replicates independently (not linked to chromosome) Many types of plasmids: fertility, F, resistance, R, virulence (pathogenity), killer, (K or Col), degradative

Discovery of high frequency conjugation Luca Cavalli-Sforza: unexpectedly high frequency of transformation (1000 x), very low or no transfer of fertility Donor strain always exhibited high transformation frequency: Hfr strain (high frequency recombination). A number of different Hfr strains were isolated. The recipient rarely converted to Hfr

Genetic mapping During conjugation the individual genes follow each other from the donor to the recipient Interrupting the conjugation only certain genes are transferred. It took 90 min to transfer the whole E. coli genome

Genetic mapping It took 90 min to transfer the whole E. coli genome Using different Hfr strains different genes enter first. Experiments with different Hfr strains validate the results: sequence of genes.

Genetic mapping F-plasmids can integrate into different positions within the chromosome. Depending on the site and orientation different genes are transfered in different sequences. If the plasmid integrated into the chromosome Fertility genes are transferred last. The chances for this are very low, usually conjugation is interrupted earlier. With F-plasmids that are free replicons the situation is the opposite: donor property is transmitted frequently, as origo is not separated from the fertility gene by the whole bacterial chromosome. Conjugation is the most frequent and most important genetic event concerning antibiotic resistance and pathogenicity Conjugating bacteria

Phage genetics Work with bacteriophages (phages) was very simple. Some of the most important and fundamental genetic informations resulted from studies on E. coli phages T2, T4 and lambda. After infection with one single phage particle withing a couple of hours tens of thousands of phages are released from the lysed bacterial cell. The new phages infect new cells and soon a clear plaque is formed in the bacterial lawn.

Phage genetics To find genetic markers was a problem Most phage mutations are "lethal. Phages have a haploid genome. A mutation can be inherited only if it is conditionally lethal: in permissive conditions (proper host, low temperature) it can function. ts mutations (ts = temperature sensitive) are very useful: they have wild type phenotype at low temperature, but render essential gene products inactive at higher, restrictive temperatures (37-42 C). 40 C 30 C

Lysogenic and lytic infection

Phage-mediated gene transfer: general transduction Recombination of DNA is a frequent event in bacteria, phage DNA might also be involved Recombination of phage DNA with chromosomal genes might create transducing phages. These phages carry host sequences but lost phage gene(s), so are defective. The transducing phage-infected cell will be the recipient. Any host gene can be carried by these phages. General transduction was discovered by Lederberg and Zinder (1951) working with the P22 phage of Salmonella.

Phage-mediated gene transfer: special transduction Some phages integrate at a given sequence into the chromosome (att). Inversion of a small DNA fragment involving host and integrated phage sequences generate special transducing phages In case of special transduction the transducing phage can carry only specific host genes: genes neighboring the phage integration site. The phenomenon was discovered in the lambda phage - E. coli system. Lambda phage integrates at a unique site in the coli chromosome: (attb).

Special transduction In case of incorrect removal of the phage DNA from the chromosome, gal gene, which is very close to the attb region will replace part of the phage genome. Only this gene will be carried by special transducing phages. This transducing phage is a defective phage, lambda dgal, as it lost phage genes. This phage can not integrate, unless a "helper phage" can complement its lost functions.

Use of bacterial and phage genetics New knowledge resulting from bacterial and phage genetics led to the discovery of the restriction-modification enzymes and the development of in vitro gene technologies. Today sequence of any gene of any organism can be determined. Study of genetics of different prokaryotic organisms is still an important goal: especially bacteria causing diseases, living in extreme environments, or in symbiosis with higher organisms. We want to know everything about virulence genes and genes that code for exotic enzymes.

Circular Genome Map represents circular bacterial genome (and plasmids) E. coli Outer ring (red): genes on direct strand, next (yellow): genes on complementary strand trnas (green arrows), rrnas (pink or orange stripes), GC content (brown lines), GC skew (yellow lines). Replication origin and terminus predicted from the GC skew shift points are also labeled.

The genome of Clostridium perfringens, which shows extremely biased GC skew and gene orientation. E. coli: non biased gene orientation

GC content usually does not vary much within the genome, but local area of abnormal GC content is sometimes indicative of horizontally transferred genes or insertions. For example, the lower left region with low GC content in Corynebacterium glutamicum is a known large insertion region.

Islands of pathogenicity (PAI) Pathogenic bacteria have virulence factors encoded by pathogenicity islands of genes. Pathogenicity islands are encoded by either mobile genetic elements (transposons, retrotransposons) plasmids (eg. invasion factors) bacteriophages (eg. Shiga toxin) or by the host chromosome

Research on bacteria help following human migration A multilocus haplotype tree and quantitative sources of nucleotides from three ancestral populations for 119 H. pylori isolates from Ladakh, East Asia, and Indo-Europe.

Taiwan By analyzing both genetic variations in human gut bacteria and linguistic evidence, scientists found that people migrated to the Pacific Islands approximately 5,000 years ago from Taiwan Read more: http://www.the-scientist.com/blog/display/55350/#ixzz1enodqbu3

Bacterial and phage genetics Bacterial genome is very flexible, genes are rapidly lost and aquired Analysis of ribosomal gene sequences help to identify bacteria Genes are transmitted by conjugation (frequent event), F+ and Hfr strains Bacteria take up and integrate genes from the environment (from lysed cells) Defective phages transmit genes between host cells (specific and general transduction, very rare event) Most bacteria living on/in us are beneficial. Very few bacteria cause disease: only those equipped with virulence factors Virulence factors are encoded by genes of pathogenicity islands (sets of genes) Pathogenicity islands are carried by mobile genetic elements, plasmids and bacteriophages The list of genome-sequenced microorganisms: http://www.tigr.org/tdb/mdb/mdbcomplete.html Ongoing microbial genome projects: http://www.tigr.org/tdb/mdb/mdbinprogress.html