Genome Plasticity Horizontal Gene Transfer Microbiology Final Study Guide I Transformation the uptake of naked DNA from dead cells from environment to competent cells A Transformasome assembled in cell envelope binds and takes up DNA thru pores i Confers natural transformation ability ii Composed of a binding protein that picks up extracellular DNA from environment and proteins that form a transmembrane pore iii Has a nuclease that degrades one strand of dsDNA and pulls the other thru pore into cell B Homologous DNA can recombine into genome intact plasmid replicates on its own C Gram cells secrete competence factor CF which induces competence i Natural transformation involves growth phase dependent assembly of transformasome complex across cell membrane ii Stress can induce competence crowding starving etc iii Quorum sensing can alert cells that Gram cell is competent D Gram cells undergo natural transformation w o CF i Can be competent all the time ii Can become competent when starved stressed iii Less likely to be competent than Gram needs more stimulation E Can treat cells w electroporation or CaCl2 to increase competence II Conjugation bacterial sex A F fertility Factor mediates conjugation B Requires sex pilus the pilin gene is on F i Cells contact each other and membranes fuse triggering F factor synthesis of enzymes ii pilus bridge contracts pulling cells together iii complex forms and ssDNA is transferred thru it iv F is the cell that carries the plasmid v F is the cell that is the recipient C DNA transfer starts at oriT site on F transferred DNA forms new F plasmid in new cell D F factor F plasmid also has other host genes that are included in new plasmid E HFR high frequency of recombination F integrates into donor chromosome Can transfer full or part of chromosome i ii Basically entire chromosome becomes the F factor iii Slow process 100 minutes in E coli due to large size conjugation usually breaks III before the entire thing and F which goes last can even be transferred Transduction mediated by bacteriophage A Phage injects DNA into host phage and its structural components are replicated inside B Some bacterial genes can get packaged by mistake and transferred thru progeny viruses C Cell lyses and releases all of the phages that infect other cells D OR viral genome is incorporated into host genome including other bacterial genetic info Microbial Genome Plasticity Recombination Mutation and Repair I Restriction Modification Systems exchanges A Safe sex approach to gene exchange conserves cell s resources but allows for genetic B Involves a pair of enzymes restriction endonuclease and corresponding methylase C Enzymatic cleavage restriction of foreign DNA and protective methylation modification of self DNA i Tags their own DNA ii Restriction endonuclease recognize specific short DNA sequences recognition sites and cleave DNA at near those sequences a Protects bacterial cells b c it will cut up foreign DNA to avoid recombination iii EcoR1 system cuts a palindromic sequence iv Modification enzymes attach methyl groups to the same sequences in itself a Makes this invisible to the restriction enzyme even when one strand is methylated b Protects bacterial genome from being cut by endonucleases c Marks this DNA as self foreign DNA will not be marked so it will be non self D hsdM methylase modifies self hsdR endonuclease cuts foreign DNA E How can a bacteriophage w restriction site ever successfully infect a host i Depends on what gets to phage DNA first endonuclease or methylase ii Foreign DNA can get methylated quickly enough to not get cut II Recombination A Incoming plasmid can replicate on its own has its own origin of replication B If not a replicon often is degraded my nuclease or incorporated into genome by recomb C Generalized homologous recombination the two DNAs have a long homologous region i can replace chromosomal DNA if sequences are very similar ii variable lengths can recombine iii used to repair damaged DNA mutations restarting replication forks iv can sample genes from other species to enhance their competitive fitness v cointegrate molecule single recomb event joins 2 DNA molecules to make 1 large circle a a linear donor and circular genome require 2 crossovers D Process i RecA recombinase coats incoming ssDNA and scans for homology a forms a triplex and helps exchange strands where homology is found b RecA ssDNA filaments are formed ii RecBCD unwinds and scans entering DNA and unwinds till chi site is found nicks that site a Chi signals endonucleolytic cleavage by RecBCD b 1000 chi sites in E coli genome c Chi where homologous recombination is unusually LIKELY iii RecA filament forms and starts strand invasion a 3 end must be free iv RuvAB carries out branch migration extends base pairing and crossing over v Holliday junction displaced recipient strand is cleaved and attached to donor strand a RuvC protein cleaves across junction and ends are joined b heteroduplex region one strand from donor and opposite is from genome E Site specific needs short sequence of homology inserts phage genome into host via short attachment site i ii doesn t use RecA only moves a few genes III Mutations any permanent stable change in DNA sequence A Must be change in base sequence cell does not repair change before next replication B Point mutations single nucleotide change often less damaging to cell i Transition pur pur or pyr pyr ii Transversion pur pyr iii Missense mutation one change in base 1 AA change iv Nonsense one base change causes stop codon C Frameshift mutations far more damaging to cell Insertion changes chain of Aas past insertion site i ii Deletion changes chain of Aas past deletion site iii Inversion flips sequence D Silent mutations do not change AA sequence due to codon redundancy E Loss of function mutation reduces or eliminates a function of the protein F Gain of function mutation makes protein more active or gives it new function G Knockout mutation eliminates a function H Mutations can be spontaneous or induced i spontaneous mutations are very rare 10 6 10 8 per generation ii Spontaneous mutations can be due to changes in bonding properties water reactions metabolic activities methylation iii mutagens chemical agents that damage DNA a deamination cytosine uracil due to chemical reaction with water b depurination depyrimidation lose the base on the sugar abasic site water rxns c Growth in O2 Reactive oxygen species that modify nucleotide residues