Microbial genetics: recombination and plasmids

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Ch. 14 in Prescott et al, Microbiology, 4th Ed.

Note: These notes are provided as a guide to topics the instructor hopes to cover during lecture. Actual coverage will always differ somewhat from what is printed here. These notes are not a substitute for the actual lecture! Copyright !!!. Thomas ". Terry

Gene transfer and Recombination

General problem

#acteria don$t have se%. &ormal mechanism of cell division is clonal 'one cell produces many identical offspring(. )hat possibilities e%ist for transfer of *&A from one cell to another+ ,ow common are they in nature+ -riginally thought that bacteria lac.ed any significant gene transfer. #ut discovery of /0plasmid transfer was shoc.ing revelation of rapid gene transfer, not 1ust among individuals of same species but among 2uite different genera of bacteria. ,ave discovered 3 mechanisms for *&A transfer in bacteria4 transformation, transduction, con1ugation. &ot clear how important these are in nature. 5n laboratory they are very important. 6enetic 7ngineering relies critically on ability to move *&A from one cell to another. Any modern biologist should be familiar with basic mechanisms, possibility of e%tending these with laboratory 8tric.s8.

Recombination

*istinguish two stages of gene transfer4 1. getting *&A from *onor cell to recipient cell . getting *&A integrated into recipient 'or into a different type of stable form, typically a plasmid(. 7ven if 1 happens, no detectable result unless also happens. 9ee figure below4

,ow does recombination wor.+ /e2uires specific proteins produced in recipient cell 'rec gene products, others(. /e2uires regions of very similar *&A se2uence between donor and recipient *&A 'homology(. Possible for ds *&A to separate, one strand of donor to pair up with one strand of recipient, allow cross0over that attaches donor in place of normal recipient *&A for some distance. )hy would such a system ever evolve+ Probably as a repair mechanism for situations when cell faces double stranded damage to chromosome 'can$t be fi%ed by normal repair mechanisms, since don$t have a remaining 8good strand8 to copy from(. #ut if cell had duplicated chromosome but not yet divided, could copy good *&A from other chromosome, use to replace damaged section.

Transformation

:pta.e of *&A fragments from medium surrounding cell. /e2uires specific proteins in cell membrane, energy. -nly found naturally in certain cells; e.g. Pneumococcus, Hemophilus, etc. Not found in E. coli. )hy did this evolve+ "aybe as way to scrounge *&A from dead cells, save trouble of having to ma.e nucleotides from scratch.

Competence

Cells that can ta.e up *&A are said to be competent. /e2uires induction of several genes

Typically occurs during e%ponential growth, shuts off in stationary phase.

Mechanisms for transforming cells without natural competence systems

<ery few bacterial species have evolved highly efficient transformation systems 5n laboratory, can devise techni2ues to enhance *&A upta.e = 8artificially induced competence8 7%ample4 treat E. coli with high Ca>> concentrations, then chill. *&A upta.e now occurs 'though not as well as with naturally competent bacteria(. lectroporation4 pulsed electric fields produce short0lived membrane pores, allows *&A movement 'either in or out of cell(. :seful way to move small pieces of *&A and plasmids between cells.

Transduction
Mechanism of generalized transduction

*&A phages normally replicate in cell, produce many copies of phage *&A, many copies of phage head coat proteins, finally assemble phage heads by pac.ing phage *&A into new phage heads. Attach tail 'if present(, open cell, release progeny. ,ost *&A often degraded. #ut occasionally, piece of partially degraded bacterial *&A is correct si?e to be pac.ed inside phage coat proteins, phage erroneously pac.s up a 8mista.e8 = transducing phage.

This 8mista.e8 phage can$t cause infection; but it can be transferred to a different bacterium, get *&A into cell without ris. of being degraded in environment 7ven if this occurs, chances are slim that successful e%pression of *&A will occur 000 still needs to undergo recombination. 5f most cells are .illed by phage, not much use.

Con!ugation

Con1ugation = plasmid0directed transfer of *&A from one cell to another.

"roperties of "lasmids

Circular *&A elements, always double0stranded *&A, 9upercoiled Can occur in as few as 1 copy per cell 'single copy plasmids( to as many as several do?en 'multicopy plasmids(. <ariable si?es; small plasmids about !.1@ si?e of host chromosome, large plasmids can be as much as 1!@ the si?e of host chromosome. 9maller plasmids have few genes '3! or less(. 9i?e ranges from 1!!! bp '1 .bp( to 1!!! .bp. :bi2uitous; almost all cells isolated in nature carry plasmids, often more than one .ind. '5n E. coli alone, more than 3!! different plasmids isolated.( ,ave a replicon 'origin for *&A replication(, number of copies per cell regulated. Aarge plasmids typically only 10B copiesCcell 'stringent control(; small plasmids D1!0B! copiesCcell 'rela%ed control( "any plasmids are incompatible; if one is present, cell cannot support another plasmid of same compatibility group. &ot essential to cell under all circumstances; can be 8cured8 by agents that impair *&A replication 0000E cured cell lac.ing plasmid. Can be spontaneously lost over time unless some selection ma.es plasmid valuable to cell. 7%tend range of environments in which a cell can live 'e.g., by degrading antibiotics, or providing en?ymes for digestion of novel catabolites(.

#amples of "lasmid genes

1. $ntibiotic resistance genes 'en?ymes that modify or degrade antibiotics( 00 plasmids with these genes are called / factors . %ea&y metal resistance 'en?ymes that deto%ify metals by redo% reactions( 3. 6rowth on unusual substrates 'en?ymes for hydrocarbon degradation, etc.( 4. Restriction'modification enzymes 'protect *&A, degrade unprotected *&A( B. (acteriocins 'proteins to%ic to other bacteria lac.ing the same plasmid( F. To%ins 'proteins to%ic to other organisms; e.g. humans( 00 called &irulence plasmids. 9ome 7%amples4 1. Staph aureus virulence factors4 coagulase, hemolysin, enteroto%in, others . pathogenic E. coli strains4 hemolysin, enteroto%in G. Proteins that mediate plasmid transfer to uninfected strains

Con!ugati&e "lasmid transfer

9ome plasmids have specific transfer genes, can copy *&A, transfer one copy to a plasmid0 cell )e# pilus4 rigid fiber, stic.s out from cell wall. Acts as recognition molecule to locate sensitive cell 'plasmid minus(. )hen > and 0 cell are attached by pilus, cells pulled together. '6rappling hoo. analogy(. Plasmid *&A replicates during transfer; one copy remains in donor cell, identical copy transferred to recipient. -nce recipient receives plasmid, it grows pili, becomes a donor. -ne plasmid could potentially infect a whole population, convert all cells to plasmid0 containing cells.

*+factors: *,- *+- and %fr strains

H is a con1ugative plasmid. &ormally, H> % H0 0000E all progeny H> <ery rarely, H plasmid becomes integrated into host *&A = ,fr. <ery stable, doesn$t 8pop8 bac. out 9till retains ability to transfer to another cell, but in doing so, drags host *&A with it Transfer is always unidirectional; always moves from plasmid > cell to plasmid 0 cell Transfer always originates at same point 'due to location of H factor in chromosome at one site( Transfer occurs with fairly high fre2uency ',fr = abbreviation(.

Mating of %fr with *+

5n 7. coli, when ,fr mates with H0, transfer of entire chromosome ta.es D1!! minutes. Aocation of different genes can be mapped by time of transfer.

To detect transfer, must design donor and recipient cells cleverly. *onor cell4 must have easily selectable mar.ers 'typically, carries wild0type genes that allow growth(. #ut must somehow prevent donor cells from growing after *&A transfer. Tric.4 ma.e donor sensitive to some antibiotic 'e.g., streptomycin sensitive(, add that antibiotic to plates to loo. for successful gene transfer. /esult4 donor cells all .illed on plates. /ecipient cell4 must carry mar.er genes to identify portions of map. Typically use au%otroph mar.ers 'e.g., his0, leu0, thr0, ser0, etc.(, plate cells onto plates lac.ing one needed metabolite 'e.g. histidine( but containing all other metabolites. /esult4 none of 8native8 recipient cells alone can grow. -verall result4 -nly cells that will be able to grow are recipient cells that received a wild type gene by transfer, can grow in absence of missing metabolite. &ote4 in practice, must plate cells at different time points on a variety of different plates, all containing antibiotic4 one set of plates lac.ing histidine, another set lac.ing leucine, still another set lac.ing threonine, etc. Aoo. at I of colonies growing on each plate, plot results vs. time. 9ee graph. 7%trapolate curve for each mar.er bac. to earliest possible time of entry.

Chapter 14
1. /ecombination is the process in which a new recombinant chromosome is formed by combining genetic material from two organisms. True Halse . The transfer of genetic material between bacteria in direct physical contact is called con1ugation transformation transduction 3. The transfer of a na.ed piece of *&A between bacteria is called con1ugation transformation transduction 4. The transport of bacterial *&A to other bacteria via bacteriophages is called con1ugation transformation transduction B. Transposable elements are plasmids viruses pieces of *&A F. 6enetic material is moved between bacterial chromosomes and within chromosomes by plasmids transposable elements plasmids and transposable elements G. /ecombination of virus genomes occurs by transformation by transduction when two viruses with homologous chromosomes infect a host cell simultaneously

J. Plasmids are small, circular *&A molecules that can e%ist independently of host chromosomes. True Halse K. Plasmids are stably inherited. True Halse 1!. Plasmids have proved invaluable to scientists in constructing and transferring new genetic combinations and in cloning genes. True Halse 11. Transposable elements cause mutations bloc. translation carry antibiotic resistance genes all of the above
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