Molecular Biology (And the Use of E

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Genetics of the E. coli lac system (Chapter 1.3)

•The speed with which cells adapt lactose during diauxie suggested that this type of adaptation was not due to cells mutating and taking over the culture. -Why not? Ask class this. Does it make sense for mutation to be a likely explanation? What kind of mutants would these be? -How can we tell they are not mutants? Cells taken from the 2nd part of the curve undergo diauxie when grown in glucose and then in glucose+lactose. Therefore they are not mutants that have taken over the culture

• There were cases where lac- fecal bacteria could be isolated. (done by Neisser and Massini 1907) When plated on special plates lac- cells were white. When left for several days, small red, Lac+, colonies showed up on top of the white colonies (papillae). The strain that did this was called Bacterium coli mutabile SLIDES Have students read about MacConkey's agar

•Later Lwoff isolated Lac- E. coli from his own feces and showed that they mutated readily to Lac+ (~300 Lac+ when 5x10^8 were plated-this is a selection for Lac+). Most famous of this was strain ML3. Mutation rate to Lac+ was therefore ~1 in 10^6.

•In 1947 Joshua Lederberg described E. coli mutants that could not grow on lactose. Those that could not make b-galactosidase were called z-. He also isolated mutants which did not need to adapt and always made large amounts of b-gal (constitutive mutants, lacI, I for inducible). •A method for enriching for constitutive lacI mutants was developed at the Pasteur Institute which involved cycling cultures through diauxic growth conditions. (terms: Enrichment and screen) 0ec3508e0892d126893f19fa05189a66.docxPage 2 of 19 0ec3508e0892d126893f19fa05189a66.docxPage 3 of 19 Bacterial Conjugation to map genes and complement mutations

• Bacterial mating proved to be essential to understanding the genetics, physiology and biochemistry of many bacterial gene systems. Discovered by Lederberg and Tatum in 1946

Bacterial conjugation allowed: mapping of genes the ability to make diploids to test if one gene type could fix another SLIDES

Have students read about conjugation: Bacterial conjugation_An Introduction to Genetic Analysis _NCBI Bookshelf.pdf

Discuss ways of introducing DNA into bacteria in order to complement or map genes:

Hfr (mating) F' (mating) Transduction by phage Transformation: natural Introduction of plasmids 0ec3508e0892d126893f19fa05189a66.docxPage 4 of 19 Genetics In Process 9-1: Lederberg and Tatum discover genetic recombination in bacteria In eukaryotes, the sexual cycle brings together genomes from two parents into one zygotic cell; then, in this cell or in descendent cells, meiosis takes place, which allows the recombination of parental alleles. This process has been known since the time of Mendel’s paper in the 1860s. However, it was not until 1952 that a short paper by Joshua Lederberg and Edward Tatum appearing in Nature reported the discovery of “sex” in bacteria. The experiments that they used did not detect sexual union directly under the microscope, but indirectly, using a genetic method. They started with two strains of E. coli that had different nutritional deficiencies caused by mutations in genes that normally synthesize biotin, cysteine, leucine, phenylalanine, thiamine, and threonine. They wrote: . . . single nutritional requirements were established as single mutational steps under the influence of X-ray or ultra-violet. By successive treatments, strains with several requirements have been obtained. The strains that they used were both triple mutants, as shown below, where the genes are listed alphabetically:

Strains Y10 and Y24 were grown together in culture medium containing all six supplements. After a period of coincubation, cells were plated and recombinant genotypes were detected. Of the recombinants, the easiest to obtain and work with were wild types, which could be selected by plating cells on minimal medium. Wild types arose at a frequency of about one in a million cells. Wild types must have been of the following genotype:

As a control experiment, the parental strains were shown to never revert to wild type when grown individually; hence the wild types were the product of coculturing in some way. Lederberg and Tatum concluded that there were only two likely explanations for these results: 1. The two cell types fused in some type of bacterial sexual process and engaged in recombination of alleles “assortment in new combinations.” 2. Something was passing through the medium from one cell type to another, transferring hereditary material. However, they found that sterile filtrates of one culture could not “transform” the other into the wild-type genotype, so they concluded that “these experiments imply the occurrence of a sexual process in the bacterium 0ec3508e0892d126893f19fa05189a66.docxPage 5 of 19 Escherichia coli.” From: Bacterial Conjugation © 1999, W. H. Freeman and Company. The discovery of conjugation and demonstration that it is unidirectional

In 1944, Joshua Lederberg was interested in demonstrating mating in E. coli since this had proven useful in Neurospora genetics. His first experiments using single auxotrophs didn’t work, so he established a collaboration with Edward Tatum who had isolated double mutants (double auxotrophs) of E. coli K12. When cells of multiple auxotrophs were mixed and plated on minimal medium, colonies of prototrophs grew. Since the expected frequency of reversion of each of the parents was far too low to give rise to these prototrophs, they must have been formed by a mixing of the genetic traits of the parent strains.

Lederberg and Tatum (1946) Colonies arise upon plating of auxotrophs or a mix of auxotrophs on defined glucose media with supplements

biotin methionine proline threonine no additions Bio–Met– + + – – – Pro+Thr+ Bio+Met+Pro– – – + + – Thr– Mixed culture + + + + +

biotin, biotin and biotin and phenylalanin proline or no phenylalanin phenylalanin cysteine e and threonine additions e or cysteine e cysteine alone alone Bio–Phe–Cys– + + + – – – Pro+Thr+ Bio+Phe+Cys+Pr – – – – + – o–Thr– Mixed culture + + + + + + 0ec3508e0892d126893f19fa05189a66.docxPage 6 of 19 •Mapping by recombination:

•So, how does this relate to Lederberg's work on lac?

•Lederberg had collected ~300 lac- mutants from over .5 million screened. By mating them with each other, he was able show that they fell into 7 different groups. When the member of one group was mated with a member of another group, the cross would give Lac+ exconjugants, but when mated a member of its own group only Lac- cells would result.

• How big is lac relative to the E. coli chromosome. How likely is it to separate lacZ mutations from lacY mutations? W3110 chromosome is 4.6 x 10^6 bp lacZ is 3000 bp, lacY is 1200 bp. Therefore the middle of each are ~2100 bp apart. 2300/4.6x10^6 is ~1/2000. Therefore, about 1 in 2000 recombinants will separate average lacZ and lacY mutations. How many recombinants per mating? Hard to say, maybe 1 in 1,000 to 10,000. 0ec3508e0892d126893f19fa05189a66.docxPage 7 of 19

Classes that Lederberg writes about in 1951 chapter: Have class read Lederberg 1951 Lac1: mutants produce no LacZ when grown on lactose, but produce quite a bit when grown on alkyl galactosides (a LacI mutant) Here is a case where LacZ can clearly be made, but lactose no longer acts as an inducer, but other molecules can still induce--how could this happen? have class think about this.

Lac3: mutants unable to grow on lactose, maltose or glucose. Found ts mutants: explain ts mutations--Lac3t is wt at 25o, and Lac- at 40o. Interestingly it is Glu-Mal-Lac+ at 36o

Cst: this class of mutants produces LacZ constitutively. Cells grown on peptone, maltose, even glucose all make at least some LacZ 0ec3508e0892d126893f19fa05189a66.docxPage 8 of 19

Inducers of E. coli lac system and permease (Chapter 1.4)

•By 1950 the lac system was one of a number of model systems used to study adaptation. None of these (lac, mal, arg, trp) had a convienient biochemical assay for measuring the activity or induction of the systems. (Lactose degradation by LacZ was measured by changes in optical properties of a lactose solution, or by measuring CO2 generation as lactose was metabolized).

• In 1950 ONPG (ortho-nitrophenylgalactoside) was synthesized. This is hydrolyzed by LacZ which resultes in galactose+orthonitrophenol which is colored yellow. This allowed cheap, easy and sensitive measurement of LacZ activity in cultures (and is still used today).

•In 1951 Monod analysed all of the galactose deriviatives he could get ahold of.

• A few of these compounds ended up being very useful. (SLIDE OF STRUCTURES get this)

• Some of these would act as inducers of the Lac system, but couldn't be degraded by LacZ. Some could be degraded by LacZ, but couldn't induce.

•This type of result showed that the inducing machinery and LacZ were different from each other. This was one of the first lines of evidence contradicting Yudkin’s "mass action induction hypothesis" Monod pointed out that compounds other than lactose could induce the production of betagalactosidase. Some of them, such as isopropylthiogalactoside (IPTG), were not even metabolizable substrates of the enzyme. The existence of such gratuitous inducers argued against Yudkin’s hypothesis, and suggested that the inducer might not interact directly with the enzyme after all.

• The second line of evidence, repeated many times in different ways, was the demonstration that bacterial adaptation to lactose, the enzyme induction of betagalactosidase by the inducer lactose, involves synthesis of new enzyme proteins rather than assembly of precursors as Yudkin had surmised. This was shown by growing E. coli for many generations in a 35S medium without an inducer present, then transferring the cells to a nonradioactive medium and adding an inducer. The induced galactosidase did not contain any 35S. This result proves that the 0ec3508e0892d126893f19fa05189a66.docxPage 9 of 19 induced b-galactosidase is newly-synthesized (de novo) and could not have been derived from preexisting subunits, which would have contained 35S cysteine and methionine. From pdf • The pre-LacZ folding idea was finally put to rest with the following experiment: -->Take Cells -->label with radioactive SO4 --> wash out the label (cells now have labeled protein, including pre-LacZ if it exists) --> quickly induce Lac system --> precipitate LacZ with LacZ-antibodies --> Is it radioacive? If "YES" then in existed when cells were labeled, before induction. If "NO" then it was made after cells were induced. -->"NO" was the answer!, LacZ was made after induction. Therefore LacZ and "inducing machinery" are two different things. This is from Cohen_BacterRev_1957 Original probably in Hogness, Cohen, Monod BBA 16:99 1955 0ec3508e0892d126893f19fa05189a66.docxPage 10 of 19 • In 1955 Cohen and Rickenberg showed that Lac substrates are transported and concentrated ~100 fold inside E. coli cells. • They used 35S-TMG for this. TMG is not metabolized and therefore its concentration inside cells is stable and can be easily measured. •This is done by the LacY protein (aka Lac permease). •Note: this concentration of inducer by LacY is VERY important for things we will be discussing later in the term: Protein expression All or nothing gene expression Stochastic (random) gene expression

•A lacY mutation is why the Lwoff strain ML3 was Lac-

•Rickenberg and Monod submitted a paper describing the LacY protein/gene to BBA and it was rejected because the editor didn't like the name "permease" for the LacY protein. READ CITATON CLASSIC BY RICKENBERG

•Finally, it was shown that lactose itself could not induce the Lac system in a LacZ mutant! This suggested that LacZ acts on lactose to turn it into an inducer. •This was shown to be true when LacZ and lactose was added to the culture medium of a lacZ mutant--then lactose could induce. •It turns out that LacZ converts lactose to allolactose and this can act as an inducer. Lactose--> Galactose + Glucose --> Allolactose

Strange! (SLIDE SHOWING THIS) (Read paper by Muller and Rickenberg--JMB 10: 303 Grads read to middle of page 306)?

0ec3508e0892d126893f19fa05189a66.docxPage 11 of 19 •Go over Table 1. Shows lacY mutants don't induce lacZ • Go over Table 2. Shows that lactose doesn't induce lacY in a lacZ-, but allolactose does. 0ec3508e0892d126893f19fa05189a66.docxPage 12 of 19 Negative control through repressor (Book section 1.5)

•By 1957 it was clear that the inducing machinery was separate from LacZ. But how the two relalated and what the inducing machinery actually did were not yet clear.

• Following a lecture, the physicist Leo Szilard approached Monod and suggested that the inducing machinery acted through "negative" control--that is it kept LacZ off unless an inducer, like lactose, was present.

•Monod realized that if this was true, then the w.t. inducing machinery (I+) should be dominant over the constitutive inducing machinery (I-). That is, I+ should fix I-. Note this would not be true if the I- mutation caused E. coli to constantly make an internal inducer (like lactose), which is what Monod thought the I- mutation did.

PaJaMo

• This idea was tested in the famous PaJaMo (Pardee, Jacod, Monod) experiment of 1959 (JMB 1:165). First published in French in 1958.

Read: (Grads)

• Hfr mating was used to transfer genes and make temporary diploids (heteromerozygotes)

•To show the systems worked Z+Y+I+SmS Hfr was mated to Z-Y+I+SmR cells in the presence of IPTG and Strep. The Hfr couldn't make b-gal because the Strep stopped protein synthesis (SmS) and the recipient couldn't make b- gal because it was Z- (but protein synthesis wasn't inhibited in the recipient by strep because it was SmR). Therefore only diploids which acquired Z+ from the Hfr could make b-gal. 0ec3508e0892d126893f19fa05189a66.docxPage 13 of 19 • These experiments showed that the Z+ gene was transfered into the SmR recipient started at about 15 min (see Z+SmR "recombinants" curve), and that b-gal enzyme formation began shortly after the Z+ was transfered (enzyme showed up at ~20 to 30 min). THEREFORE THE TRANSFER SYSTEM WORKS!)

•Next they tested whether 8 Z- strains were recessive (ie could Z+ fix it when transferred into a Z- strain): Show vin complementation slide to explain 0ec3508e0892d126893f19fa05189a66.docxPage 14 of 19

Note that no enzyme was made when the different Z- alleles were put into the same cell-->this was because the muations were in the same gene. So... Z+ was dominant over all 8 Z- alleles, and the 8 alleles were in the same gene.

• Next the following experiments were done to test the whether I+ was dominant (can I+ cause a constitutive I- strain to become shut off again)

Experiment 1

Hfr Z+I+ x Z-I-

•Neither strain makes b-gal before mating (DISCUSS WHY-no inducer) •Z+ and I+ are transferred together into the Z- I-(constitutive) strain. •If I+ is dominant it should keep the Z+ turned off following transfer (Szilard's POV). If I- (constit) is dominant, then Z+ should be turned on when it is transferred (Monod's POV).

•Was b-gal made? Yes, but it slowly shut off. This was hard to interpret, I+ didn't cause Z+ to stay shut off, but it did seem to shut it off slowly. Hmm.

Experiment 2 (the opposite of Expt 1)

Hfr Z-I- x Z+I+-

•Neither strain makes b-gal •Z- and I-(constitutive) are transferred together into the Z+ I+ strain. •If I- is dominant it should allow Z+ to be turned on (and make b-gal) when it enters the Z+I+ recipient.

•Was b-gal made? NO, none at all!. So I-(constitutive) was not dominant.

• Together the two experiments show that I+ is dominant. The reason that repression by I+ was slow in Experiment 1 was that it took a while for LacI 0ec3508e0892d126893f19fa05189a66.docxPage 15 of 19 protein to be made after it was transferred into the recipient. PaJaMo didn't know this was why shutoff was slow, but they did realize that something had to happen, after transfer into the recipient cytoplasm in order for the I+ to work. That "something" wasn't needed in experiment 2 because I+ was already there, in the cytoplasm, ready to go.

Note that PaJaMo thought that LacI probably made a galactose-type molecule which inhibited b-gal synthesis, and which was antagonize by real inducers like lactose (allolactose).

Bottom line: In the case of adapation to lactose, E. coli keeps the lac genes off using LacI. When inducer is present LacI doesn't work and the lac system is turned on. lacI- strains don't make the LacI and can't keep the lac genes off. LacI--> known now as "Lac repressor"

So far we have the following Lac genes:

Explain why LacY mutants are inducible by IPTG. 0ec3508e0892d126893f19fa05189a66.docxPage 16 of 19 Further evidence for negative control (Lac Book 1.6) SLIDES about Cis/Trans tests Operator mutants: The model predicts that mutations in operator DNA should exist and give a Lac constitutive phenotype and these should be cis- dominant, that is, not fixed by a good operator provided in trans.

Jacob and Adelberg (1959) found stable F' elements that carried lac genes. Using these they could show easily that lacI+ was dominant over lacI-. (PaJaMao did this with unstable genetics where the Hfr was present for a shortish time, providing lacI+ in trans) lacI mutants that could no longer bind allolactose were found by Jacob and Allan Campbell (1959). These were "super-repressor mutants (lacIs) and stayed permanently bound to the lac operator. Thus they were dominant over lacI+ and lacI-. (SLIDEs) Read this paper Wilson_JMB_1964? (Grads) 0ec3508e0892d126893f19fa05189a66.docxPage 17 of 19 Triumph of the Jacob-Monod Theory (Lac Book 1.7)

A summary of the Lac genetics leading to Jacob and Monod's model of negative regulation. Read Monod’s Nobel Lecture (G) Mo

Minor defects in the Jacob Monod Theory (Lac Book 1.8)

•Jacob and Monod thought that Lac repressor was a RNA because some was made in the presence of chloramphenicol. -This was brought into question when lambda repressor cI was shown to be a protein because some cI mutations (11/300) could be fixed with a nonsense suppressor. -The same thing was shown by Muller for lacI mutants three years later. 2/187 mutants could be fixed by nonsense suppressors, therefore Lac repressor is a protein (1966).

Summary: LacI is a repressor LacI binds to DNA LacI is a protein LacI binds to inducer

•What are non-sense suppressors?

•Nonsense mutation: a mutation that results in a stop codon in a gene. The mutation causes translation to stop prematurely

•Certain codons can mutate readily to stop codons. For example: -UAU & UAC (tyr) can mutate to UAG and UAA (stop) -UGU and UGC (cys) can mutate to UGA (stop) -there are others that are one mutation away--try to find them

•What if this happens in an essential gene? -->Death! •Can they be rescued? --> Yes, in a nonsense suppressor strain -This is a strain with an anticodon mutation in a tRNA gene that allows it to recognize a stop codon and put in an amino acid. (Go over 5' to 3' antiparallel to figure out anti-codon sequence). The amino acid inserted depends on the suppresor strain. IMPORTANT: becaue the nonsense 0ec3508e0892d126893f19fa05189a66.docxPage 18 of 19 suppressing tRNA no longer recognizes its own codon, the must be another, unaffected tRNA gene in the cell that can still recognize the altered tRNA's codon.

EcoWiki page on tRNA nonsense suppressors: http://ecoliwiki.net/colipedia/index.php/Nonsense_suppressor http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/rev- sup/nonsense-suppressors.html

-These strains are often sick because they often don’t recognize stop codons and instead put in an amino acid and keep translating. Note, however that hey cannot suppress a particular class of stop codons too efficiently or else too many ORFs with that stop codon would have long proteins due to non-stoppage. SLIDES

• Nonsense mutations and suppressor strains are not usually used as conditional lethals for bacteria because you would have to have your mutated gene and the supressor strain, and move it into a non-suppressor for analysis. That is kind of a headache to do.

•Instead, they are often used for bacteriophage genetics. You have a phage with an nonsense mutation and it can be used to infect wt. bacteria and nonsense suppressor bacteria. The phage(nonsense) will only give rise to new phage progeny when the nonsense suppressor bacteria is used as the host.

An aside-- LacZ complementation and cloning Read Lac Book 2.23 0ec3508e0892d126893f19fa05189a66.docxPage 19 of 19 • Describe blue-white screening for clones. Show pGEM T Easy map and ponder how this works given lacZ is 3000 bp. Segue to a-complementation.

• A deletion of lacZ codons 21 to 41 lacM15 is completely LacZ-. A deletion of all but the codons1-60 is also completely LacZ-. However, the two mutations can complement each other in trans. That is, the two protein products can somehow fix each other. This is called alpha-complementation and was described by Agnes Ullman and Jacob and Monod in 1967.

SLIDES ON A-COMP

XL1Blue: endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK- mK+)