A Study of Phage λ Rex Regulation and Physiology. Lynn Thomason, Amos Oppenheim and Donald Court NCI-Frederick

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1 Bacteriophage λ Bacteriophage T4 A Study of Phage λ Rex Regulation and Physiology Lynn Thomason, Amos Oppenheim and Donald Court NCI-Frederick

2 Talk organization: Our genetic model system Observations about regulation Rex-dependent physiological effects Mutant hunt, library screen for multi-copy suppressors

3 rexa and rexb - expressed in λ lysogen from same transcript as ci repressor p LIT T imm rexb rexa ci P RM RexA 31 kd cytoplasmic protein RexB 16 kd 4 membrane spanning domains - inner membrane separate promoter (p LIT?)

4 What is known about Rex functions? rex + λ lysogen prevents growth of T4rII mutant phage (rii exclusion) by an unknown mechanism - Benzer 1955 Mg 2+ alleviates defects - allows rii growth - Garen 1961, Sekiguchi 1966 λ lysogen out-competes non-lysogen in glucose-limited chemostat: f(rex) -Lin, Bitner & Edlin 1976 exclusion of heteroimmune λ requires DNA replication of superinfecting phage -Toothman & Herskowitz 1980 both genes required for exclusion phenotype -Matz Schmandt & Gussin 1982

5 Goal of these studies is to determine the role(s) of the Rex system in phage λ life cycle Since rii mutants may not be very important in the environment of λ lysogens, I suspect that the natural function of this lysogenic conversion is not yet known. Bill Dove 1971 The Bacteriophage Lambda

6 a shotgun approach Let s hope we can hit the right target(s)!

7 Rex +/- strains used in growth experiments and phenotypic arrays P L luc-n ol T imm rexb rexa p LIT ci857 prm P R or cro-lacz P RM operon insertion at lac operon, linked to Kan no other λ genes present displays normal immunity and T4rII exclusion@ 37 C RexA protein levels higher than in wt lysogen - by Western blot

8 Observations on construct orientation Lambdoid prophages have a preferred orientation in the bacterial chromosome Correlates with direction of replication -Allen Campbell 2002 prm construct was made in two orientations - behave differently - one with the normal prophage orientation grows normally inverted construct has Rex-dependent growth 37 C Can we learn about Rex from the inverted construct?

9 How does construct behave at att region? gal Kan luc-n P L rexb prm rexa ci857 P R cro-lacz Tet bio ol p LIT or gal Tet lacz-cro P R prm ci857 rexa rexb P L N-luc Kan bio or p LIT or Inserted P RM construct at region of att site in both orientations Normal prophage orientation construct doesn t have growth problems but one with opposite orientation does

10 Why does this orientation have growth problems? Hypothesis: orientation of prophage with respect to direction of E. coli DNA replication affects expression o R P RM ci857 Replication fork t imm rexb rexa Normal prophage orientation o L Replication fork o R P RM t imm ci857 rexb rexa Inverted prophage orientation o L

11 Rex + Regulation: rex<>cat orf replacements: rexa<>cat, rexb<>cat, rexab<>cat luc-n P L rexb rexa ci857 prm P R cro-lacz ol rex<>cat T imm luc-n P L ol T imm p LIT cat ci857 or prm P R or cro-lacz Precise open reading frame replacements of rex genes. rexa<>cat leaves p LIT intact.

12 Rex<>cat open reading frame replacements: rexa<>cat, rexb<>cat, rexab<>cat Size of loop: ci857 o R rexa + rexb + : 2.3 kb rexa rexa<>cat rexb + : 2.2 kb rexb t imm rexa + rexb<>cat: 2.5 kb o L rexab<>cat: 1.7 kb

13 Growth of rex<>cat insertion strains on Cm plates Strain [Cm] g/ml C 37 C 42 C loop size (kb) rexa<>cat med large vv small rexb<>cat small med med 2.5 rexab<>cat small med v small 1.7 rexa<>cat small large rexb<>cat - small rexab<>cat - small Expression from p RM is strongest at 37 C 2. Presence of rexb gene may contribute to strong expression 3. See evidence of separate rexb promoter at 42 C

14 Does Rex Affect E. coli Metabolism? Rex over-expression showed significant increase in color with some Carbon sources: D-glucose, D-fructose, D-mannitol and N-acetyl-glucosamine Phenotypic Microarrays (Biolog)

15 Inverted orientation displays Rex-dependent growth problems in some liquid media Tryptone 37 C time (hours) MG1655 rex overexpresser rex- control

16 Microscopy of Stalled Cells Rex + Cells are motile and overexpress flagellin Rex -

17 Magnesium improves growth of Rex overexpressing strain tryptone tryptone 50mM Mg time (hours) Rescue by Mg ++ suggests we are looking at another manifestation of exclusion

18 Acetate Accumulates in supernatant of Rex over-expressing strain 500 In tryptone and LB (without Mg 2+), the supernatant becomes acidic and contains acetate. Acetate accumulates when carbon flux exceeds capacity of glycolysis. -H. Holms acetate, mg/liter supernatant assayed values adjusted for OD Gluconogenesis? 0 MG1655 rex - control rex +

19 Rex-dependent Plateau is also media-dependent Tryptone LB (stops at higher OD) Minimal Glucose No plateau acetogenic C-sources Glycerol Fructose not acetogenic Minor Effects Mannitol N-acetyl glucosamine less acetate than glucose No growth on acetate

20 glucose RexAB? pcka PEP poxb λci Cro serine pyruvate Acetyl ~ CoA pta acs Acetyl ~ P acka/b Acetyl-AMP acetate acs oaa TCA cycle λ is not a passive passenger! Amino acids

21 Speculation Pyruvate ----> acetate ----> acetyl phosphate Acetyl phosphate - global cellular signaling molecule Acetyl phosphate ----> OmpR-PO4 OmpR-PO4 flagella production cell division -B. Prüß 1998 But we see over-production of flagellin and inhibition of cell division - Predict low acetyl phosphate level in this Rex + construct

22 Genetic Strategies: E. coli library - multi-copy suppression Mini-Tn10 transposon mutagenesis time (hours) rex overexpresser Same general procedure for both: Introduce plasmid library or transposon: plate at 42C Inoculate tryptone 32C, shift to 37 after ~ 1 hr Harvest cells ~ 2 hrs into plateau Subculture, repeat, cycle several times

23 Library - multicopy suppression of growth defect After 3 rounds culture no longer plateaus Isolate plasmid DNA Re-transform into fresh strain - confirm rescue of growth problem Clone contains one gene: ihfb - subunit of C λ immunity T4rII exclusion Rex + Rex + [pihfb] yes no yes no

24 ihfb de-represses p L of defective prophage at att site (cro-bio) ci857 rexa rexb P L N::lacZ fusion ihfb clone causes increased expression of N::lacZ at low temperatures (32, 34, 37 C) monitored by red on Mac-lac plates Representation of 34 plate Without ihfb clone With ihfb clone

25 ihfb derepression Previously identified by Amos I found it first! The experimental system olpl rex ci pm orpr cro cii LacZ NC417 p L o L rex ci857 ind - p RM o R p R cro27 cii-lacz Introduce plasmid library of E. coli and select for red colonies at 32 C on MacConkey Lactose plates.

26 Derepression by ihfb does not require active IHF chromosomal genes. S tra in Mutat ion Pla s mid Vector pihfb NC4 17 whit e red NC4 17 ² ihfa whit e red NC4 17 ² ihfb whit e red NC4 17 ² ihfa ² ihfb whit e red Plasmids carrying ihfa (hima) or hupa (HU) do not lead to derepression.

27 ihfb mediated de-repression of p R two IHF binding sites near ol previously identified by Amos and co-workers - is IHFß binding at this site involved in relieving repression of pr? But Amos analyzed a construct deleted for ol ci pm orpr cro cii LacZ -ihfb clone causes expression from cii::lacz at 32 C for this construct also BUT IHF site is absent thus IHFß cannot be acting by binding to IHF ol or by inhibiting loop formation -Amos proposes protein-protein interactions between CI and IHF

28 Transposon mutagenesis Same general strategy as library screen one mutant from several independent pools: rho transcription termination factor teta (1) tetr tetr (3) teta Rho leader peptide rho Tn10 insertions in rho previously identified - thought to alter supercoiling and not to work via polarity suppression - Storts & Markovitz 1991

29 Phenotype of transposon rho mutants vs rho15<>amp for Rex-lac construct λ Immunity T4 rii exclusion Rex + rho::tn10 rho15<>amp yes no no yes reduced no Is the Rho effect due to transcription or supercoiling? Does transcription of P RM affect stability of looped complex and thus repression?

30 Effect of rho<>amp mutation on defective prophage at att site (cro-bio) ci857 rexa rexb P L N::lacZ fusion Representation of 37 C plate Without rho<>amp With rho<>amp

31 Why does only one orientation of construct cause growth problems? Something different about regulation in this orientation Hypothesis: RexA/RexB ratio higher- some polarity in operon High RexA/RexB triggers T4rII exclusion -L. Snyder 2x rexa strain excludes both T4rII and T4 + super Rex

32 With library and transposon mutagenesis in liquid culture, only got things that down-regulate rex expression. Interesting, but doesn t tell us about host functions targeted by Rex in the cell. New Screen - on plates - with 2xrexA strain EHA plates: tryptone + glucose + citrate strain makes tiny translucent colonies Repeated library suppressor screen on EHA plates -have candidates Tn10 mutagenesis - in process

33 Conclusions 1. Insertion of p RM operon at lac region used to probe effects of Rex on host physiology and λ regulation 2. In one orientation, lac construct confers Rex-dependent growth problems on E. coli in some liquid media - correlated with acetate production 3. Construct can be used to isolate multi-copy suppressors and host mutants that rescue the cell from Rex effects 4. The first isolates of each class (ihfb plasmid and rho mutation) down-regulate p RM and thus Rex expression: they also affect a defective prophage at the att site

34 Acknowledgements Sankar Adhya Court lab members: Nina Costantino Hanna Wierchoslawska λpapa ya