MCBL121_24W_lecture 3_genomes and genetic information (1).pdf

BIOL/MCBL 121 INTRODUCTORY MICROBIOLOGY Lecture 3 Genomes and Acquisition of Genetic Information Copyright Ansel Hsiao 2021 Objectives • Compare and contrast eukaryotic and bacterial genomes • Distinguish between genes and operons • Distinguish monophyletic vs polyphyletic ancestries for bacteri...

BIOL/MCBL 121 INTRODUCTORY MICROBIOLOGY Lecture 3 Genomes and Acquisition of Genetic Information Copyright Ansel Hsiao 2021 Objectives • Compare and contrast eukaryotic and bacterial genomes • Distinguish between genes and operons • Distinguish monophyletic vs polyphyletic ancestries for bacteria • Compare and contrast mechanisms of horizontal gene transfer and gene acquisition, vertical and horizontal transmission • Describe experiments leading to the discovery of transformation • Describe mechanisms of defense against foreign DNA in bacterial cells Copyright Ansel Hsiao 2021 Bacterial Genomes • The entire genetic complement of DNA in a cell = genome • First bacterial genome sequenced was Haemophilus influenzae in 1995 • There is tremendous diversity in prokaryotic genomes Copyright Ansel Hsiao 2021 Microbial genome is highly variable Copyright Ansel Hsiao 2021 Welch, et al. 2002, PNAS, 99:17020 Genome Organization • Bacterial and archaeal chromosomes range in size from 490 to 9,400 kilobase pair (kb) • For comparison, eukaryotic chromosomes range from 2,300 kb (2.3 megabase pair Microsporidia) to over 100 billion bp (flowering plants) • The human genome is over 3 billion kb Genome size (Mb) Number of genes Gene density Bacteria 0.12-9.4 120-8000 1 : 1-1.5 kb Budding yeast 12 6275 1 : 2 kb Human 2900 21000 1 : 100 kb Copyright Ansel Hsiao 2021 Genome Organization • Bacterial genomes have relatively little noncoding DNA (untranscribed) • Typically > 90% in eukaryotes, < 15% in prokaryotic genomes • No introns • Fewer repeated sequences • More compact genomes Copyright Ansel Hsiao 2021 Genes • A typical bacterial gene is ~ 1 kb in length • A structural gene produces (expresses) a functional RNA molecule, which usually encodes a protein for translation • Some untranslated RNAs have specific functions as well • Genotype – sequence/organization of genetic material • Gene product leads to a phenotype – observable characteristics exhibited by the organism • A DNA control sequence regulates the expression (transcription) of a structural gene – the promoter • Does not encode an RNA Copyright Ansel Hsiao 2021 Functional Units of Genes • A gene can operate independently of others or be expressed in tandem with other genes in a unit called an operon • Operons are usually polycistronic (many genes transcribed together on a single transcript) Copyright Ansel Hsiao 2021 Genes and Proteins • Genes in bacteria are lower case and italics (luxC) • The proteins that are produced from the gene are capitalized regular case (LuxC) • Operons that include many genes can be written together as they appear: luxCDABE (contains luxC, then luxD, then luxA, etc.) or described as one operon (lux operon) Copyright Ansel Hsiao 2021 Nucleiod • No membrane separates DNA from cytoplasm • Single loop of double-stranded DNA • Single molecule of DNA • E. coli: ~4x106 bp (4000 kb) • Attached to cell membrane: DNA origin • Replicates once for each cell division - starts from the DNA origin Copyright Ansel Hsiao 2021 Nucleiod Organization • The nucleoid forms about 50 loops of chromosome called domains • Within each domain, the DNA is supercoiled and partly compacted by DNA-binding proteins Copyright Ansel Hsiao 2021 Nucleiod Organization • Plenty of exceptions to this idealized model • Some species have multiple large circular chromosomes • Genome copy number varies • Episomes (plasmids) – circular extrachromosomal DNA • Some species have multiple linear chromosomes (e.g. Borellia –Lyme Disease pathogen) Copyright Ansel Hsiao 2021 Plasmids • Extrachromosomal DNA, usually circular • Size varies: several kilobase (Kb) to megabase (Mb) • Copy numbers varies: 1 to several hundred per cell • Can contain genes that benefit the host cell Copyright Ansel Hsiao 2021 The Mosaic Nature of Genomes • A surprise arising from bioinformatic studies is the mosaic nature of all microbial genomes • This is the result of heavy horizontal gene transfer, DNA recombination, mutation, and DNA repair strategies Copyright Ansel Hsiao 2021 The Mosaic Nature of Genomes • Microbial genomes have undergone extensive gene loss and gain • Example: Escherichia coli’s genome has many genomic islands, inversions, deletions, and paralogs and orthologs • Genomic islands – regions of the genome with signs of horizontal transfer • Homologs – genes with shared ancestry • Orthologs – genes derived from a single gene separated by a speciation event • Paralogs – genes created by duplication within the genome Copyright Ansel Hsiao 2021 The Mosaic Nature of Genomes DNA sequence is not static • Mutations of single bases – slow, but accumulated through time • Large deletions • Large insertions of sequence – genetic islands • Transferred from other species – horizontal gene transfer • Introduce new functions • Maintained if they confer a growth advantage in specific environmental conditions – natural selection • Plasmids (episomal circular DNA molecules, separate from the chromosome) Copyright Ansel Hsiao 2021 Prokaryotic genomes are very malleable • Prokaryotic genetic information is not simply passed from generation to generation • Genomes change by mutation or acquisition of genetic material within generations Copyright Ansel Hsiao 2021 Bacteria and Gene Acquisition • Genes can be transferred between bacteria: • Vertical transmission: from parent to child • Horizontal transmission: transfer of small pieces of DNA from one cell to another • Bacteria exchange or acquire information in the form of DNA that can contain different genes Copyright Ansel Hsiao 2021 Wikimedia Commons Bacteria and Gene Acquisition • These genes can be retained and confer advantageous traits • Antibiotic-resistance genes • Spread wherever antibiotics are overused (hospitals, farms) • Pathogenicity islands (PAIs) • Encode virulence genes for pathogen to cause disease • Genes to use or produce special metabolites • Genes are then passed on via vertical transmission Copyright Ansel Hsiao 2021 Wikimedia Commons Bacteria and Gene Acquisition • Genetic information can be transferred from the environment • Some events are beneficial and initiated by bacteria themselves • Some events are harmful and imposed on bacterial cells Copyright Ansel Hsiao 2021 Gene Transfer Processes • Transformation: free DNA taken up from the environment • Can be induced • Conjugation: cell-cell contact (sex pilus) • Transduction: DNA transfer is mediated by virus that targets bacteria (bacteriophage) Copyright Ansel Hsiao 2021 Transformation of DNA • Many cells are capable of natural transformation • The cells need to be competent • Others require artificial manipulations • Why do species undergo natural transformation? • Use indiscriminate DNA as food • Use specific DNA to repair damaged genomes • Acquire new genes through horizontal gene transfer Copyright Ansel Hsiao 2021 Discovery of Transformation • First demonstrated with Streptococcus pneumoniae by Griffith (1928) • Mortality experiment with mice using two kinds of the same bacterium, distinguished by colony morphology: • smooth (with capsule, caused disease) • rough (no capsule, did not cause disease) Copyright Ansel Hsiao 2021 Discovery of Transformation • Rough bacteria mixed with heat-killed smooth bacteria caused disease and became smooth bacteria • Identified a “transforming principle” that made rough bacteria virulent (able to cause disease) Copyright Ansel Hsiao 2021 Discovery of Transformation • Avery and McLeod (1941) • Tested polysaccharide, protein, RNA, and DNA, and only DNA from smooth strain conferred the ability to kill on rough strain • The smooth strain DNA made the rough strain able to kill -> changed the phenotype Copyright Ansel Hsiao 2021 Gene Transfer: Natural Transformation • Uptake of DNA directly from the environment • Cells that can take up DNA are competent • Protein complexes at the cell surface take up foreign DNA • Stress can induce competence – acquisition of additional traits as a method of adaptation Copyright Ansel Hsiao 2021 DNA Uptake Copyright Ansel Hsiao 2021 Inducing Competence in Gram-Negative Bacteria • CaCl2 and low temperature (4oC) enhances competence by making the cell membrane more permeable to DNA • DNA can be driven into competent bacteria cells by heat-shock (42oC for E. coli) or electroporation (pulse of high-voltage electricity makes cell membrane permeable) • Physiological state of cells greatly affects competence • Competence peaks during early log phase growth Copyright Ansel Hsiao 2021 Conjugation • Conjugation is the transfer of DNA from one bacterium to another, following cell-to-cell contact. • Often called “bacterial sex.” • It is typically initiated by a special pilus protruding from the donor cell Copyright Ansel Hsiao 2021 Conjugation • Pilus proteins can be encoded by genes on a fertility plasmid or F factor Donor (F+) • F factor is self-transmissible • Makes its own transfer machinery • In recipient cell (F-), transferred DNA receives a copy of F factor and becomes F+ and able to transfer DNA Copyright Ansel Hsiao 2021 Recipient (F-) DNA Transfers between Bacteria and Eukarya • Some bacteria can actually transfer genes across biological domains. • Agrobacterium tumefaciens, which causes crown gall disease • Contains a tumor-inducing plasmid (Ti) that can be transferred via conjugation to plant cells Copyright Ansel Hsiao 2021 Gene Transfer by Phage Transduction • Bacteriophages (phages) • “Phage”from “eat” in Greek • Viruses that attack bacteria but do not harm eukaryotes • Injects viral DNA into host (bacterial) cell • Replicated viral DNA is packaged into new viral particles to be released to infect other bacterial cells Copyright Ansel Hsiao 2021 Gene Transfer: Transduction • Transduction is the process in which bacteriophages carry host DNA from one cell that has been infected to another cell • This occurs accidentally as an offshoot of the phage life cycle • Sometimes package bacterial DNA by mistake • Virus carrying host DNA can act as transducing particles Copyright Ansel Hsiao 2021 Gene Transfer by Phage Transduction • Generalized transduction: can transfer any gene from a donor to a recipient cell • Specialized transduction: can transfer only a few closely linked (adjacent) genes between cells Copyright Ansel Hsiao 2021 Gene Transfer by Phage Transduction • Generalized transduction: can transfer any gene from a donor to a recipient cell • Host genome is hydrolyzed and some fragments get moved into new phage particle Copyright Ansel Hsiao 2021 Gene Transfer by Phage Transduction • Generalized transduction: can transfer any gene from a donor to a recipient cell • Host genome is hydrolyzed and some fragments get moved into new phage particle • Specialized transduction: can transfer only a few closely linked genes between cells • Phage integrates into the bacterial genome • When the phage genome is excised, some parts of the host genome move with it into a phage particle Copyright Ansel Hsiao 2021 Fate of the DNA Entering the Cell • Plasmids can coexist and replicate in the cell as extrachromosomal DNA • DNA can incorporate into the chromosomal DNA by recombination Copyright Ansel Hsiao 2021 Recombination • Combination of two DNA molecules • Replaces variable-sized section of the endogenous DNA • Could be used to repair damaged DNA • Requires specific recombination proteins (Rec) • Homologous (matching) DNA sequences must be present Copyright Ansel Hsiao 2021 Recombination • RecBCD protein machine unwinds donor DNA • Many copies of RecA bind to revealed single strand • RecA finds homology matches to recipient DNA • RecA bound strand invades recipient and donor strand base-pairs to homologus stretch of recipient (crossover) • DNA rearranges, junction is cleaved and any breaks repaired Copyright Ansel Hsiao 2021 Generalized vs site-specific recombination • Generalized: requires long stretch of sequence homology (>50bp) • Site-specific: requires little sequence homology but a short 10-20bp sequence recognized by recombination enzyme (recombinase) Copyright Ansel Hsiao 2021 Fate of the DNA Entering the Cell • Plasmids will coexist in the cell as extrachromosomal DNA • DNA can incorporate into the chromosomal DNA by recombination • Foreign DNA can be degraded in the recipient cell Copyright Ansel Hsiao 2021 DNA Restriction and Modification • How can bacteria deal with foreign DNA that might encode proteins harmful for the cell? • Bacteria have developed a kind of “safe sex” approach to gene exchange. This protection system, called restriction and modification, involves: • Enzymatic cleavage (restriction) of alien DNA, by restriction endonucleases • Protective methylation (modification) of host DNA Copyright Ansel Hsiao 2021 Defense against transferred DNA • Restriction / modification system • Bacteria cut foreign DNA to pieces using restriction endonucleases • Cut at specific DNA sequences (restriction sites) • “Endo” – cuts within a DNA sequence • Bacteria add methyl groups to its own DNA using matching methylation enzymes • Protects restriction sites • Foreign DNA without native DNA methylation pattern is destroyed Copyright Ansel Hsiao 2021 EcoRI restriction/ modification site Copyright Ansel Hsiao 2021 Defense against transferred DNA • CRISPR • “Clustered Regularly Interspaced Short Palindromic Repeats” • Bacterial immune system against viral DNA • On infection, bacteria cuts up invading viral DNA and inserts pieces (“spacers”) into their own genome • “memory” against infection Bio-rad Copyright Ansel Hsiao 2021 Defense against transferred DNA • Spacers are transcribed and the Cas9 enzyme uses these to monitor DNA sequences complementary to these transcribed spacers • Matching sequences are then degraded to prevent infection Bio-rad Copyright Ansel Hsiao 2021