Bacterial Genetics Lecture Notes PDF

BIOM 611G, Medical Microbiology PCOM Georgia BACTERIAL GENETICS Valerie E. Cadet, PhD Assistant Dean of Health Equity Integration Professor of Microbiology and Immunology...

BIOM 611G, Medical Microbiology PCOM Georgia BACTERIAL GENETICS Valerie E. Cadet, PhD Assistant Dean of Health Equity Integration Professor of Microbiology and Immunology BMS1 & BMS2 Department of Biomedical Sciences November 26, 2024 LEARNING OBJECTIVES ▪ Required Reading: ▪ Medical Microbiology by Patrick R. Murray, 8th Edition, Ch. 13 ▪ Appropriate sections of the BCMB text: The Cell, as needed 1. Describe the differences between eukaryotic and prokaryotic chromosomes 2. Describe in detail the process of bacterial DNA replication 3. Determine which processes are inhibited/interfered with by the quinolone, rifamycin, and tetracyclin families 4. Describe, to the level discussed in class, the three methods of genetic recombination in bacteria, as well as how these principles may be used in a laboratory setting 2 BACTERIAL CHROMOSOMES While still composed of DNA, bacterial chromosomes have some features which make them unique from eukaryotes (see table 12-1): ▪ Lack nucleus + other organelles ▪ Small ▪ 0.6 Mbp to 10 Mbp (eukaryotes 2.9 Mbp < 4,000+ Mbp) ▪ Single (haploid), ds, circular, supercoiled ▪ 70s ribosome ▪ Frequently also contain extrachromosomal elements ▪ Plasmids (very small, circular, self-replicating pieces of DNA) ▪ These confer non-essential factors which can be spread horizontally to other bacteria, such as antibiotic resistance ▪ Bacteriophages (bacterial viruses) 3 PROKARYOTIC GENOMES ARE ORDERED ▪ Was once (erroneously) believed that prokaryotic cells were just ‘disorganized bags of nucleic acid’ ▪ Genetic material occupies a highly structured area called the nucleoid ▪ While prokaryotic DNA is not packaged with nucleosomes, it IS arranged in a non-random way, organized by nucleoid-associated proteins. 4 GENE EXCHANGE IN PROKARYOTIC CELLS (A REVIEW) ▪ The exchange of DNA between cells allows exchange of genes and characteristics between cells, thus producing new strains of bacteria ▪ Transferred DNA can integrate into recipient chromosome or be stably maintained as plasmid or a bacteriophage ▪ Passed on to daughter bacteria as an autonomously replicating unit ▪ Transposons (jumping genes) are mobile genetic elements ( Figure 13-11 ) that can transfer DNA within a cell, from one position to another in the genome, or between different molecules of DNA (e.g., plasmid to plasmid or plasmid to chromosome) ▪ Sometimes insert into genes and inactivate those genes 5  BACTERIAL REPLICATION ▪ Replication of chromosome triggers cell division in a cascade of events ▪ The bacterial chromosome contains 2 regions which control the beginning and termination of replication ▪ OriC is the start site ▪ TerC is the termination site ▪ The replication factory is a specialized area within the nucleoid which contains the replication machinery ▪ Helicase ▪ Primase ▪ Polymerase (DdDp) ▪ Primer sequence FIGURE 12-10. Electron photomicrographs of gram-positive (Bacillus subtilis) cell division (left) and gram-negative (Escherichia coli) cell ▪ Ligase division(right). Progression in cell division from top to ▪ Topoisomerase bottom. CM, Cytoplasmic membrane; CW, cell wall; N, nucleoid; OM, outer membrane; S, septum. Bar = 0.2 µm. 6 A MODEL FOR BACTERIAL CHROMOSOME REPLICATION ▪ Note: both strands of DNA are being replicated ▪ Simultaneously ▪ Semiconservatively ▪ Bidirectionally ▪ The DNA becomes separated at the Ori C, and each separate strand will then undergo bi-directional replication ▪ one strand will be the leading strand, copied continuously ▪ one will be the lagging strand with RNA primers (Okazaki fragments) ▪ Replication is complete when the two replication forks meet 180 degrees from the origin 7 NOTE: KNOW THE FUNCTIONS OF ALL OF THESE ENZYMES AND WHY THEY’RE NECESSARY! 8 ANTIBIOTICS AND BACTERIAL REPLICATION ▪ While the enzymes and overall process of replication is similar between prokaryotes and eukaryotes, there are subtle variations in our proteins ▪ We take advantage of this in our antibiotics ▪ Quinolones (ex: ciprofloxacin) work by inhibiting bacterial DNA gyrase (ie: topoisomerase) ▪ Rifamycins (ex: rifampin) bind to and inhibit prokaryotic RNA polymerase, blocking genes from being transcribed ▪ Tetracyclins (ex: Tetracyn ©) bind to and interfere with prokaryotic ribosomes, blocking genes from being translated into proteins 9 GENETIC RECOMBINATION IN BACTERIA ▪ Because bacteria are asexual, they are not capable of creating genetic diversity through meiosis/chromosome crossing over ▪ There are three mechanisms by which they are able to get genetic recombination in bacteria: 1. Transformation 2. Conjugation 3. Transduction 10  1. TRANSFORMATION ▪ Uptake of naked DNA into a bacterial cell ▪ New DNA must have homologous region with which to recombine into host chromosome ▪ Note: this is not reciprocal, so it simply replaces the DNA that was on the existing chromosome 1. DNA must be competent DNA must be large enough and double- stranded to be incorporated into chromosome 2. Cell must be competent Has Competence Factor cell-surface protein used to transport DNA into the cell 11 GRIFFITH’S EXPERIMENT ▪ 1927 Frederick Griffith was working with Streptococcus pneumoniae ▪ Strains that secrete capsules look smooth and can cause fatal infections in mice ▪ Strains that do not secrete capsules look rough and infections are not fatal in mice 12 1. Smooth strains (S) with capsule 🡪 death of mouse ▪ Capsule prevents immune system from killing bacteria ▪ Living bacteria found in blood 2. Rough strains (R) w/o capsule 🡪 survival of mouse ▪ No living bacteria found in blood 3. Heat-killed S 🡪 survival of mouse 4. Live R + heat-killed S 🡪 death of mouse ▪ Blood contains living S bacteria => Transformation 13 TRANSFORMATION: A CLOSER LOOK 14 2. CONJUGATION Gene transfer from donor to recipient requiring cell-to cell contact mediated by sex-pilus Amount of genetic information transferred can be significantly larger than in transformation or transduction Occurs when recipient has acquired Fertility Factor (F+) ▪ Typical conjugation mechanism ▪ 1: Initial contact mediated by conjugal F pilus (donor cell to recipient cell) ▪ 2: Conjugal DNA processing begins with donor plasmid nicked in site- and strand-specific way at oriT (origin of transfer) ▪ 3: Single strand of plasmid DNA transferred in 5' to 3' direction to recipient cell, followed by re-ligation of nicked DNA ▪ Replacement DNA strand synthesis occurs in both the donor (leading strand) and recipient (lagging strand) cell, concurrently with DNA transfer ▪ 4: After mating pair separate, both the donor and recipient contain the conjugal plasmid ▪ The transfer stops as soon as the cells lose contact with each other 15  CONJUGATION: HFR Donor F plasmids integrate into recipient chromosome of host cell via transposable elements (either insertion sequences-IS- or transposons-Tn). ▪ If the F plasmid inaccurately excises from the chromosome after formation of an Hfr, it can take a portion of the chromosome with it, which then becomes part of the plasmid itself. ▪ This form of the F plasmid is called F' (F prime) ▪ High Frequency conjugation mechanism ▪ 1: Transfer of DNA from donor to recipient can occur by transfer mediated by ▪ Hfr (where the chromosomal DNA transferred is dependent on site of F factor insertion into the chromosome for origin of replication) ▪ or by an F' (where only a restricted amount of chromosome carried on plasmid is transferred) ▪ 2: Conjugal DNA processing begins with donor plasmid nicked in site- and strand-specific way at oriT (origin of transfer) ▪ 3: Single strand of Hfr chromosome is transferred in 5' to 3' direction to recipient cell, followed by re-ligation of nicked DNA ▪ As the bacterial chromosome is replicated, the leading portion of the F factor becomes the ‘locomotive’ that drags the DNA across the pilus into the donor cell ▪ The rest of the DNA which follows is the ‘caboose’ ▪ This transfer moves at a rate of about 1 gene per second and while fast, it would still take >4000 seconds to completely transfer the chromosome, so only a portion of it gets transferred ▪ 4: When cells become separated, the transferred portions of the donor genome will replace the recipients DNA via double crossover ▪ Often, because the remaining F factor usually does not get 16 transferred, the receiving cell remains F- 3. TRANSDUCTION ▪ Process of moving bacterial DNA from one cell to another using a bacteriophage ▪ Bacteriophage are bacterial viruses ▪ Consist of DNA inside a protein coat ▪ Protein coat binds to the bacterial surface, then injects the phage DNA ▪ The phage DNA takes over the cell’s machinery and replicates many virus particles 17 FORMS OF TRANSDUCTION ▪ In generalized transduction, any piece of the bacterial genome can be transformed ▪ Occurs when bacteriophage undergoes a normal lytic lifecycle ▪ In specialized transduction, only some specific pieces of the genome will be transformed ▪ Occurs when the bacteriophage becomes lysogenic for a time 18 GENERALIZED TRANSDUCTION 1. Phage attaches to the cell and injects its DNA. 2. Phage DNA replicates, and is transcribed into RNA, then translated into new phage proteins. 3. New phage particles are assembled. 4. Cell is lysed, releasing about 200 new phage particles. Total time = about 15 minutes. Some phages will break up the bacterial chromosome into small pieces, and then package it into some phage particles instead of their own DNA. The phage containing the bacterial DNA can then infect a fresh host bacteria 19 SPECIALIZED TRANSDUCTION ▪ In specialized transduction, the phage DNA will be incorporated into the host chromosome as part of the lysogenic phase of the lifecycle ▪ The phage genes are quiescent during this time – no protein expression occurs to make phage proteins ▪ During a period of stress, the phage DNA will loop out and have a crossover, resulting in its removal from the of the bacterial chromosome ▪ Sometimes, the viral DNA excises imperfectly, and some of the bacterial chromosome will come out with it ▪ If this occurs, the resulting phage that infect new hosts will also be transducing those bacterial genes into the host, where a double crossover can occur to insert the DNA into the new host chromosome 20  LYSOGENIC PHAGE LIFE CYCLE 1. Phage attaches to the cell and injects its DNA. 2. Phage DNA becomes incorporated into the host chromosome via homologous recombination This is considered a ‘stable transduction’, so the phage DNA will be replicated along with the bacterial chromosome, and all daughter cells will contain it 3. In periods of stress, the phage DNA can be excised from the chromosome, and the phage will then go through the lytic cycle, eventually resulting in lysis. 21  SUMMARY: MECHANISMS OF BACTERIAL GENE TRANSFER 22 Figure 13-12 GENETIC MECHANISMS OF EVOLUTION OF METHICILLIN- AND VANCOMYCIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA AND MVRSA) ▪ VRE ((Enterococcus faecalis (in blue)) contains plasmids with multiple antibiotic resistance and virulence factors ▪ During co-infection, MRSA (in pink) may have acquired the enterococcal resistance plasmid (e-plasmid) (in purple) by transformation (after lysis of the enterococcal cell and release of its DNA) or, more likely, by conjugation. ▪ A transposon in the e-plasmid containing the vancomycin resistance gene jumped out and inserted into the multiple antibiotic resistance plasmid of the MRSA ▪ The new plasmid is readily spread to other S. aureus bacteria by conjugation 23 Figure 13-13