Biology 105/Summer Bacterial Genetics 8/12/ Bacterial Genomes p Gene Transfer Mechanisms in Bacteria p.

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1 READING: 14.2 Bacterial Genomes p Gene Transfer Mechanisms in Bacteria p. 486 Suggested Problems: 1, 7, 13, 14, 15, 20, 22 BACTERIAL GENETICS AND GENOMICS We still consider the E. coli genome as a fairly typical Bacterial genome, and given the extensive information available about this organism and it's lifestyle, the E. coli genome is a useful point of departure for subsequent discussion of bacterial genome diversity. Physical Form of the Genome vs. the Genetic Map: The circularity of the E. coli genetic map reflects the physical form of the DNA genome. All of the genes in the genome of E. coli are coded in a single double-stranded DNA molecule that has no ends. (Contrast this with the linear genetic maps typical of eukaryotic chromosomes.) Although we sometimes refer to the DNA as being "circular", this distorts physical reality. If the genome were laid out in a perfect circle, the circumference would be about 1 mm, at least several hundred times the dimensions of an E. coli cell. Clearly the DNA is not in a true circle while inside a cell, but highly folded and compacted. So, while the sophisticated E. coli geneticist refers to the genetic map as circular, they describe the genomic DNA as being "covalently continuous". Vital Statistics Derived from Complete Genome Sequencing of E. coli The classic genetic map is arbitrarily divided into 100 "minutes", corresponding to 4,639 kbp of DNA containing 4,288 ORF's. The average size of a gene is close to 1 kbp. Therefore, approximately 90% of the DNA in the genome is coding sequence. For a searchable online database of all E. coli genes go to: < 1 of 6

2 Functional Distribution of E. coli genes FUNCTIONAL CATEGORY # Proteins % DNA Replication, Recombination, Repair Regulatory Proteins Enzymes for Cell Structural Components Translation Physiological Responses to Environment Energy Metabolism Other Enzymes of Intermediary Metabolism Biosynthetic Enzymes Transport Proteins Other known Unknown 1, TOTAL 4, Bidirectional replication from fixed origin (oric at 84') and terminus opposite. Gene Orientation In the E. coli genome, highly expressed genes tend to be transcribed in the same direction in which they are replicated. Transcription-Translation Coupling The lack of a nuclear compartment in prokaryotes leads to one of the most important molecular differences between prokaryotes and eukaryotes, namely, in prokaryotes transcription and translation of protein coding genes is coupled (simultaneous). In other words, ribosomes are translating the 5' end of a mrna before the 3' end of the mrna has been transcribed. Coupling of transcription to translation reduces opportunity for RNA processing (intron splicing). Introns are exceedingly rare in the bacterial genomes sequenced so far. There are no introns in the E. coli genome. 2 of 6

3 Distribution of genome sizes in bacteria Mitochondria (Human) Carsonella ruddii 16 kb; 37 genes 160 kb; 182 genes an endosymbiont present in cells of all species of sap-feeding insects known as psyllids Mycobacterium genitalium 580 kb; 525 genes smallest known genome for "free-living" organism Escherichia coli K12 Bradyrhizobium japonicum 4, 640 kb; 4,467 genes 9,106 kb; 8,373 genes Average gene size in prokaryotes = 1 kb 3 of 6

4 MECHANISMS OF GENETIC EXCHANGE IN BACERIA Note that none of these mechanisms should be referred to as "bacterial sexuality" as you frequently see in the older literature. Sex entails an equal genetic contribution by two organisms to "offspring". Bacterial mechanisms all involve a unidirectional transfer of genetic information, usually of only a small portion of the genome, from a "donor" to a "recipient". In the case of transformation and transduction, the donor cells die in the process. These mechanisms operate most efficiently when the donor and recipient cells are fairly closely related. (i.e. when the donor and recipient cells are the same "species"). The effectiveness of gene transfer mechanisms may be diminished by restriction/modification systems. Genetic TRANSFORMATION in Bacteria Definition: Uptake and incorporation of exogenous ("free") DNA from solution results in genotypic (heritable) change in genotype of a recipient cell. The free DNA results from lysis, rather than "excretion" of DNA by donor cells. Review the historical account of the work by Frederick Griffiths and by Oswald Avery and his collaborators. Transformation involves two phases: DNA uptake (competence) followed by the stable integration of the exogenous DNA sequences by homologous genetic recombination. Natural Transformation Artificial Transformation Natural Transformation is a genetically programmed physiological mechanism permitting the efficient uptake and incorporation of DNA. It is NOT simply a nutrient uptake system. Competence requires the coordinated expression of specific genes. Competence is induced by the laboratory manipulation of cultured cells. Widely applied to transforming E. coli with artificial recombinant plasmids as part of cloning project. You are not responsible for knowing the functions of each specific protein (ComG, ComF, etc.) in Fig. 8.20/8.12 Step 2. 4 of 6

5 Biology 105/Summer Bacterial Genetics 8/12/13 The text gives a cursory description of how genetic transformation can be applied to determining the relative positions and distances between bacterial genes (gene mapping). We will not cover this. Conjugation Characteristics of the classic F Plasmid in E. coli: Present in the original E. coli K12 isolate. low copy number (1-2 copies per cell). 100 kb; 30 kb approx. required for conjugal transfer Transposable elements in F: Tn 1000; IS3 [2 copies]; IS2 facilitate recombination with cellular genome to produce Hfr strains Fusion of F with E. coli chromosome is primarily driven by homologous recombination between shared transposable elements (IS and composite transposons). 5 of 6

6 Fusion of F with Bacterial Chromosome and Mobilization of Bacterial Genes (Hfr Strains) Transduction Generalized Transduction: Note the similarities to transformation; i.e. any region of the donor genome may be transferred. Specialized transduction: Can be carried out by "temperate" bacterial viruses only. Requires "defective excision" of viral genome from bacterial chromosome. Transfer of bacterial genes is restricted to those adjacent to viral genome insertion site. 6 of 6