M2 L11: bacterial genetics and mapping

Question 1

why is it important to study bacterial genetics

Answer 1

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

Question 2

6 reasons bacteria are better subjects than elephants

Answer 2

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

Question 3

what is complete media

Answer 3

has all nutrients necessary for growth

Question 4

what is minimal media

Answer 4

only has a carbon and nitrogen source

Question 5

what’s replica plating

Answer 5

using sterile velvet to stamp colonies onto different plates

Question 6

what’s a prototroph

Answer 6

organism that can live on minimal media

Question 7

what’s an auxotroph

Answer 7

organism that cannot grow on minimal media - has a mutation (auxotrophy)

Question 8

what are bacterial plasmids? are they part of the bacterial genome?

Answer 8

circular DNA molecules –> nonessential genes/genes important for certain enviros

not considered part of bacterial genome - not in bacterial chrom and readily gained/lost

Question 9

what’s lateral/horizontal transfer? effect?

Answer 9

transfer of genetic material between distantly related species

lateral transfer of plasmids can spread antibiotic resistance

Question 10

do plasmids replicate autonomously?

Answer 10

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

Question 11

bacteria reproduce clonally - can they do recombination?

Answer 11

yes - but one way, not reciprocal like eukaryotes

Question 12

3 methods of bacterial recombination

Answer 12

1) conjugation: transfer through conjugation pilus

2) transformation: take up DNA from enviro

3) transduction: DNA transferred from virus/phage

Question 13

how was conjugation discovered? what proposition did it lead to?

Answer 13

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

Question 14

how did researchers know bacterial growth after mixing them wasn’t just from reversion mutations (define rev muts)?

Answer 14

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)

Question 15

does conjugation require physical contact between cells? How do we know?

Answer 15

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

Question 16

who discovered bacterial conjugation is unidirectional? what determines if a cell is a donor or recipient? can any cell donate to any other cell?

Answer 16

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)

Question 17

what is transferred in bacterial conjugation? what is the result?

Answer 17

F plasmid gets transferred –> donor and recipient both F+

Question 18

what protein complexes are involved in conjugation

Answer 18

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

Question 19

what’s rolling circle replication in conjugation?

Answer 19

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

Question 20

what’s the recipient of conjugation called

Answer 20

exconjugant

Question 21

if the F plasmid is not part of the genome, how do we get recombination within genomes?

Answer 21

F plasmid integrates into genome via insertion sequences –> chrom w/ bacterial DNA and F plasmid = Hfr (high freq recomb) chrom

Question 22

what is Hfr cojugation? what’s the process?

Answer 22

Hfr conj: transfer of bacterial genes

process: same rolling circle recomb method as F plasmid conjugation (start at OriT)

Question 23

result of Hfr recomb? how much DNA transferred? why?

Answer 23

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)

Question 24

in Hfr conjugation, is the transferred DNA circular or linear?

Answer 24

linear bc whole circular chrom never transferred

Question 25

how does Hfr conjugation allow for recombination?

Answer 25

recipient undergoes recomb at homologous seq between their own genome and the transferred DNA

Question 26

how can Hfr strains be used for mapping relative gene order?

Answer 26

interrupted mating: disrupt Hfr conjugation at different times –> observe time req to transfer individual genes (time of entry mapping)

Question 27

what 2 properties of Hfr conjugation make time of entry mapping possible

Answer 27

1) transfer always starts at OriT

2) amount of DNA transferred is linear with time

Question 28

What are F’ cells? how are they produced? what can they lead to?

Answer 28

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

Question 29

what term describes a cell that can take up DNA from the enviro in transformation

Answer 29

competent

Question 30

in transformation, how does the exogenous DNA get into the cell?

Answer 30

it binds to a receptor site –> one strand degraded as it enters recipient

Question 31

What are strand invasion and heteroduplexes?

Answer 31

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)

Question 32

what is the result of replication of a heteroduplex?

Answer 32

1 daughter has just the bacterial genome, other daughter has the exogenous DNA

Question 33

how can transformation be used for gene mapping?

Answer 33

upper limits on size of exogenous DNA acquired –> if 2 genes co-transform, they must be close

Question 34

what is a vector? what is the vector in transduction?

Answer 34

vector: carrier of genetic info

phages are the vectors in transduction

Question 35

how does transduction work?

Answer 35

phage takes up piece of bacterial DNA –> injects into another bacteria –> transductant cell integrates donor DNA into own genome via homologous recomb

Question 36

why can phages take up bacterial DNA?

Answer 36

phage apparatuses package DNA based on strand size, not sequence

Question 37

how can we use transduction to map bacterial genes?

Answer 37

size limit of fragments that can be transferred –> genes that are cotransduced more often are closer together

Question 38

who researched the fine structure of genes? what were the 2 questions being asked?

Answer 38

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?

Question 39

describe benzer’s complementation tests and results - any exceptions?

Answer 39

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

Question 40

how did benzer discover intragenic recombination? how did he know if mutations were in the same nucleotide?

Answer 40

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)

Question 41

who discovered penicillin

Answer 41

alexander fleming

Question 42

How does antibiotic misuse lead to resistance?

Answer 42

• 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

Question 43

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

Answer 43

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.

Question 44

What is the significance of oriT, and why is it important that the 5’ end of the T strand always be transferred?

Answer 44

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).

Question 45

How is an Hfr chromosome generated? What sequences does this rely on?

Answer 45

The F plasmid integrates into the bacterial chromosome. It relies on Insertion Sequences in the F plasmid.

Question 46

Explain the process of transformation. Use heteroduplex and strand invasion in your answer.

Answer 46

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.

Question 47

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?

Answer 47

No, you cannot safely give the patient a high enough dose to kill the bacteria.