The Fertility Factor, or F Pili Contains pili genes, tra genes, replication genes, but no genes essential for cell survival or growth. Chromosome F factor 100,000 bp Closely related R factor contains multiply antibiotic resistance genes, responsible for the rapid spread of antibiotic resistance in bacteria
F + F - Pili -(protein tubes) bring F + and F - cells together so that there can be an exchange of genetic information
Overview of Conjugation F + x F - F +, F + products
Genes are transferred in one direction William Hayes, 1953 A Strain A (met - thr + leu + thi + ) B streptomycin (antibiotic) kills cells slowly wash out strep. Strain B (met + thr - leu - thi - ) MIX Plate on minimal media (colonies grew)
F+ cells cant receive DNA. Strain B (met + thr - leu - thi - ) A B streptomycin (kills slowly) wash out strep. Strain A (met - thr + leu + thi + ) MIX Plate on minimal media (no colonies)
Within a population of F+ cells, approximately 1/1000-1/100,000 has an integrated F factor. There are many sites of integration scattered around the E. coli genome.
Origin of transfer goes back from the arrow. F+ integrated into the genome
F + x F - F + factor transferred at high frequency (1/3 of all F - become F + ) In 10 7 F + cells, 10 2-10 4 cells with F + integrated. This represents many different integration events F + x F - yields a low frequency transfer of all genomic markers (~1/10 7 ).
Integrated F factor: If separated from the rest of the F+ cells, this is known as an Hfr strain. Special class of F + strains The F factor has been integrated into the bacterial chromosome at a particular site. High frequency transfer of chromosomal markers Named Hfr = high frequency of transfer. Useful for mapping genes in the bacterial chromosome
Transfer of the integrated F factor results in transfer of genomic markers Must be two recombinations to maintain circle. Linear piece gets degraded. Recipient cell very rarely converted to F+. Hfr a+ x F - a- Hfr a+ + F - a+
Bacterial Mating Hfr str s a+ x F - str r a - str r - selectable marker in F - strain (need to kill Hfr strain) a+ - selectable marker in Hfr strain exconjugants: recombine a+ marker into F - parent, so F - str r a+
Hfr x F - X X
1. Grow mixture of cells on plates that contain Streptomycin 2. Plates also contain media which selects for the a+ marker.
Hfr x F - summary In Hfr cells, all cells have the F integrated at the same site. These cells are all descended from a single integration event Hfr x F - yields a high frequency transfer of genomic markers located near the origin of transfer (1/10 4 ) and a very low frequency of transfer of the F factor (<1/10 7 ).
How can Hfr strains be used to determine the order of genes in bacteria? In any one particular Hfr strain, markers are transferred into an F- cell in a particular order. Can one measure which markers are transferred first, second third etc.
Ellie Wollman and Francois Jacob, 1957 Hfr str s thr + azi r ton r lac + gal + x F - str r thr - azi s ton s lac - gal - Blend at various times to disrupt conjugation
10 min 20 min 30 min
Replica Plate Screen Selection 15 min. StrR thr+ thr+ is used because its already known to be transferred first, close to origin of transfer. Plate with streptomycin but without threonine StrR thr+lac+ StrR thr+gal+ StrR thr+azir StrR thr+tonr Lactose plate Galactose plate Azide plate plate + T1
Plateau is because they eventually fall apart. Time became the distance measurement
The order of gene transfer of Hfr chromosome during conjugation Gene transfer starts with the origin of the F factor and ends with the fertility genes. Genes close to the origin are transferred before distal genes.
Creating Genetic Maps in bacteria through conjugation A. Interrupted mating (distance in minutes) B. Natural gradient of transfer (order, not distance) C. Three point crosses + reciprocal crosses = (order, not distance)
Natural gradient of Transfer Same concept as interrupted matings. The gradient of transfer allows the spontaneous disruption of mating pairs. Select for the first marker transferred. Screen for additional markers through replica plating. Markers close to the first marker have higher transfer frequency than markers distal to the first marker.
Determine the order of Arg and Leu Master plate Hfr str s met + leu + arg + x F - str r met - leu - arg - + Streptomycin +Leucine +Arginine (- Methionine) (160 colonies) Replica A + Leucine - Arginine (128 colonies) Replica B + Arginine - Leucine (64 colonies) met + =100%, arg + = 80%, leu + = 40%
Natural gradient of transfer met + Hfr str s met + leu + arg + x F - str r met - leu - arg - leu + arg + arg + arg + met + met + met + Select str r met + leu + leu - arg + arg - met + met - Str r