M2 L11: bacterial genetics and mapping Flashcards
why is it important to study bacterial genetics
90 of the 100 trillion cells in the human body are bacteria
can be commensal (bac +/host 0), beneficial, or pathogenic
tech, med, bio relevance
6 reasons bacteria are better subjects than elephants
1) genomes are simpler (less genes but more gene dense)
2) haploid –> can study effects of recessive mutations (not masked by dominant alleles)
3) short generation times
4) lots of progeny
5) easy to propagate
6) easy to manipulate/mutate
what is complete media
has all nutrients necessary for growth
what is minimal media
only has a carbon and nitrogen source
what’s replica plating
using sterile velvet to stamp colonies onto different plates
what’s a prototroph
organism that can live on minimal media
what’s an auxotroph
organism that cannot grow on minimal media - has a mutation (auxotrophy)
what are bacterial plasmids? are they part of the bacterial genome?
circular DNA molecules –> nonessential genes/genes important for certain enviros
not considered part of bacterial genome - not in bacterial chrom and readily gained/lost
what’s lateral/horizontal transfer? effect?
transfer of genetic material between distantly related species
lateral transfer of plasmids can spread antibiotic resistance
do plasmids replicate autonomously?
usually yes - replicate at dif time than bacterial chrom and present in many copies/cell
sometimes replicate at same time –> present in 1-2 copies/cell
bacteria reproduce clonally - can they do recombination?
yes - but one way, not reciprocal like eukaryotes
3 methods of bacterial recombination
1) conjugation: transfer through conjugation pilus
2) transformation: take up DNA from enviro
3) transduction: DNA transferred from virus/phage
how was conjugation discovered? what proposition did it lead to?
2 types of bacteria that can’t grow separately on minimal media (have dif auxotrophies) –> but can grow on minimal media when mixed together
proposed genetic info could be transferred between cells
how did researchers know bacterial growth after mixing them wasn’t just from reversion mutations (define rev muts)?
reversion mutations are mutations that reverse other mutations
mixing bacteria with several auxotrophies –> still growth when mixed on minimal media –> would require several reversion mutations at same time (highly unlikely)
does conjugation require physical contact between cells? How do we know?
yes - U tube experiment mixed cells in media –> filter prevented phys contact between cells but suction and pressure ensured media mixing –> auxotrophs did not complement/no growth on minimal media –> phys contact req for conjugation
who discovered bacterial conjugation is unidirectional? what determines if a cell is a donor or recipient? can any cell donate to any other cell?
william hayes
have F factor/F plasmid (F+) –> can donate
don’t have F factor –> can receive
F+ cannot donate to F+ bc of surface exclusion (proteins from F+ on surface prevent conjugation)
what is transferred in bacterial conjugation? what is the result?
F plasmid gets transferred –> donor and recipient both F+
what protein complexes are involved in conjugation
1) exporter complex: exporter and pilus proteins –> bridge for DNA
2) relaxosome complex: cuts one strand donor DNA at OriT –> partially degrades, leaves relaxase –> helps move 5’ end cut DNA to recipient
3) coupling factor: binds relaxase, aids in DNA transfer
what’s rolling circle replication in conjugation?
replication from the 3’ end where the DNA was cut and the 5’ end is going to the recipient –> replicate strand that’s going to be transferred –> push/spool T strand into recipient
what’s the recipient of conjugation called
exconjugant
if the F plasmid is not part of the genome, how do we get recombination within genomes?
F plasmid integrates into genome via insertion sequences –> chrom w/ bacterial DNA and F plasmid = Hfr (high freq recomb) chrom
what is Hfr cojugation? what’s the process?
Hfr conj: transfer of bacterial genes
process: same rolling circle recomb method as F plasmid conjugation (start at OriT)
result of Hfr recomb? how much DNA transferred? why?
part of F plasmid and part of bac chrom transferred
never make Hfr or F+ exconjugants bc whole F plasmid or whole Hfr chrom never transferred (pilus breaks before possible)
in Hfr conjugation, is the transferred DNA circular or linear?
linear bc whole circular chrom never transferred
how does Hfr conjugation allow for recombination?
recipient undergoes recomb at homologous seq between their own genome and the transferred DNA
how can Hfr strains be used for mapping relative gene order?
interrupted mating: disrupt Hfr conjugation at different times –> observe time req to transfer individual genes (time of entry mapping)
what 2 properties of Hfr conjugation make time of entry mapping possible
1) transfer always starts at OriT
2) amount of DNA transferred is linear with time
What are F’ cells? how are they produced? what can they lead to?
F plasmid with some bacterial DNA
can reverse integration that made Hfr chrom (cut out F plasmid) –> if part of bac chrom removed w/ F plasmid –> F’ plasmid
If transferred into another cell –> recipient will be partial diploid
what term describes a cell that can take up DNA from the enviro in transformation
competent
in transformation, how does the exogenous DNA get into the cell?
it binds to a receptor site –> one strand degraded as it enters recipient
What are strand invasion and heteroduplexes?
in transformation, the ssDNA (from enviro) base pairs w/ homologous region in bacterial chrom –> displaces one strand of bacterial chrom –> heteroduplex (partly mismatched double helix from 2 dif parent molecules)
what is the result of replication of a heteroduplex?
1 daughter has just the bacterial genome, other daughter has the exogenous DNA
how can transformation be used for gene mapping?
upper limits on size of exogenous DNA acquired –> if 2 genes co-transform, they must be close
what is a vector? what is the vector in transduction?
vector: carrier of genetic info
phages are the vectors in transduction
how does transduction work?
phage takes up piece of bacterial DNA –> injects into another bacteria –> transductant cell integrates donor DNA into own genome via homologous recomb
why can phages take up bacterial DNA?
phage apparatuses package DNA based on strand size, not sequence
how can we use transduction to map bacterial genes?
size limit of fragments that can be transferred –> genes that are cotransduced more often are closer together
who researched the fine structure of genes? what were the 2 questions being asked?
seymour benzer
1) are genes the fundamental units of mutations or can parts of genes be mutated?
2) is recomb only between genes or can it occur within genes?
describe benzer’s complementation tests and results - any exceptions?
2 phages w/ mutations in same gene (can’t lyse bacteria individually or after coinfection - no plaques)
2 phages w/ mutations in different genes (can’t lyse bacteria individually, but CAN lyse after coinfection - plaques)
—> 2 complementation groups
sometimes phages w/ mut in same gene did lyse and produce plaques
how did benzer discover intragenic recombination? how did he know if mutations were in the same nucleotide?
sometimes phages w/ mut in same gene did lyse and produce plaques –> intragenic recombination generated one allele w/ 2 mutations and one WT allele
if 2 phages never produced plaques –> mut in same nucleotide (recombination just trades mutants)
who discovered penicillin
alexander fleming
How does antibiotic misuse lead to resistance?
- not finishing course doesn’t kill all bacteria –> leaves the ones that are most resistant
- overprescribing introduces an unnecessary selective pressure –> increase freq of resistant genotypes
An E. coli colony grows on complete media but not minimal. It can grow if it’s supplemented with alanine and leucine but not each separately. It cannot grow in lactose media even when supplemented with alanine and leucine. What is the genotype? Explain
Ala- leu- lac-. It’s deficient in both alanine and leucine because it can only grow if supplemented with BOTH. It’s lac- because it cannot grow with lactose, even if it has the other supplements that allow it to grow with glucose.
What is the significance of oriT, and why is it important that the 5’ end of the T strand always be transferred?
OriT is the origin of transformation - it’s where the relaxosome cuts the donor DNA and it’s the beginning of the sequence that the recipient is going to get. The 5’ strand always has to be transferred because that leaves an open 3’ end in the donor → donor can remake the strand it’s losing (has 3’ OH to add to).
How is an Hfr chromosome generated? What sequences does this rely on?
The F plasmid integrates into the bacterial chromosome. It relies on Insertion Sequences in the F plasmid.
Explain the process of transformation. Use heteroduplex and strand invasion in your answer.
DNA from enviro binds to a receptor site on competent cell → one strand of DNA degraded as it enters the cell. It base pairs with the recipient’s DNA and goes through homologous recombination. This displaces part of the recipient’s DNA → makes a heteroduplex (that spot in the recipient’s genome now has one strand of its own DNA and one strand from the environment). The rest of the transformational DNA and the sequence that got displaced in the transformant gets degraded and the transformational sequence gets ligated into the transformant’s genome.
If a bacterium is only sensitive to a concentration of a drug that exceeds what can be tolerated by a patient, is that bacterium treatable by that drug? Why or why not?
No, you cannot safely give the patient a high enough dose to kill the bacteria.
