vaijayanthi k.r. (viva-voce)

Molecular Genetic Analysis of Mutations That Alleviate rpsL31 Antisuppression

in Escherichia coli

Work done by REG. NO. A841014

VAIJAYANTHI KANDADAI RAGHAVAN

Under the guidance of Prof. Hussain Munavar

Department of Molecular BiologySchool of Biological Sciences

Centre for Excellence in Genomic SciencesMadurai Kamaraj University

(University with Potential for Excellence)Madurai - 625021

India

19th APRIL 2010

Previous findings• rpsL encodes the ribosomal protein S12.

Traub, P., and M. Nomura, Science (1968)

• A lesion in rpsL( rpsL31) confers streptomycin resistance, also restricts suppression ( causes antisuppression)mediated by termination suppressors like supE44 (amber suppressor)

Sullivan, M.A., and R.M. Bock, J.Bacteriol. (1985)

• Streptomycin an aminoglycoside, increases misreading.Julian Davies, Walter Gilbert, and Luigi Gorini, PNAS (1964)

Role of supE44 and effect of rpsL31

• supE an Amber suppressor –suppresses amber mutations in Streptomycin sensitive strain

• supE (glnV) encodes a glutamine tRNA• rpsL31 restricts this suppression

100 dilution- AB1157 Str R supE44

3 days incubation

Cell titre ≈108

AB1157- hisG4∆(gpt-proA) leuB6 thrB1 argE3 rpsL31 glnV44

As previously shown (Singaravelan, 2009), the rpsL restriction

(Antisuppression) effect was reconfirmed in AB1157.

10-5 dilution- AB1157 Str S supE444 days incubation

100 dilution- AB1157 Str S suppressor free5 days incubation

Streptomycin sensitive & suppressor free background

Background of the study• Previous studies from the laboratory have classified the

auxotrophic mutations of AB1157 into leaky and tight mutations. • Novel mutation identified, post plating mutagenesis (ppm) , it

increases intrinsic leakiness of markers, was mapped at 74 minutes on the E.coli genome.

(Jayaraman, 1995).

• Recent studies from the lab have shown that ppm which contributes to leakiness and supE44 , the amber suppressor, both are restricted rpsL31 mutation

(Singaravelan, 2009. Ph.D. thesis).

Present workOBJECTIVES

• To isolate mutants that can overcome the restriction posed by rpsL31 on supE44(glnV44) mediated Amber suppression

• Genetic and Molecular characterization of the mutations which counteract rpsL31 restriction.

The Necessity of the study

• To understand pathways and mutations that mediate nonsense suppression and misreading even when misreading/suppression is restricted.

• Can such a pathway affect resistance to streptomycin/other aminoglycosides?

• This understanding of termination suppression has now been extrapolated to curing diseases like Hemophilia

Paula D. James, and David Lillicrap, 2005. Aminoglycoside suppression of nonsense mutations in severe Hemophilia. Blood Journal .106: 3043-3048

Radmila Manev and Hari Manev , Aminoglycoside antibiotics and autism: a speculative hypothesis . BMC Psychiatry

Strain Relevant genotype Source/ Reference

AB1157 hisG4 ∆(gpt-proA) leuB6 thrB1 argE3 rpsL31 glnV44Berlyn MKB,CGSC, USA

MHM2001 hisG4∆(gpt-proA) leuB6 thrB1 argE3 rpsL+ glnV44 zhe511::Tn10zhe511::Tn10,rpsL+

introduced into AB1157, This work

MHM2002 hisG4∆(gpt-proA) leuB6 thrB1 argE3 rpsL+ glnV+ asnB::Tn10asnB::Tn10, glnV+

introduced into MHM2001 ,This work

MHM2003 hisG4∆(gpt-proA) leuB6 thrB1 argE3 rpsL31 glnV44 carB::Tn10carB::Tn10, thrB1 introduced into

AB1157, This workMHM2004 Same as AB1157 but argE+ srs

This work

MHM2005 Same as AB1157 but argE+argG- srsThis work

JW375 zhe511::Tn10 glnV44Lab collection

CAG12093 carB::Tn10Berlyn MKB, CGSC,

USACAG12077 asnB::Tn10 glnV44

Berlyn MKB, CGSC, USA

CAG12071 zhd::Tn10 rph1Berlyn MKB, CGSC,

USACAG12O72 zha-203::Tn10 rph1

Berlyn MKB, CGSC, USA

KL708 F', leuB6(Am), fhuA2, lacY1, glnV44(AS), gal-6, λ-, hisG1(Fs), recA1,

argG6, rpsL104, malT1(λR),xyl-7, mtlA2, metB1, Fʹ141

Lab collection

Isolation of Threonine Independent derivatives

Methodology

Occurrence of mutants

Summary of mutants obtained from independent overnight cultures

Out of 150 mutants obtained after repeated plating only 88 were able to form single colonies when restreaked

Threonine independent mutants

100 dilution- AB1157 Str R supE448 days incubation

AB1157 Str R supE44- streaked for single colonies3 days incubation

Why prolonged incubation?? ---- supE44 is a weak amber suppressor

Genetic and Molecular characterization of the mutations which counteract rpsL31 restriction.

Are the mutants true revertants or pseudorevertants ?

Recipient is phenotypically Thr+

In pseudo revertants, 100% of transductants should be Thr+

In true revertants, only fraction of transductants should become Thr+

Cross no. Donor Recipient Selected

marker

Unselected

phenotype

Percent

co-

transduction

1 P1/MHM2003 Mutant #1 Tetr Thr- 0% (0/98)







car::Tn10 was used as the selection marker and the TetR

transductants are grouped into Thr+/- in order to know the nature of the reversion.


marker

Unselected

phenotype

Percent

co-

transduction











marker

Threonine

independent

colonies

Percent co-

transduction










INFERENCE

The transductional crosses clearly indicate that all

the mutants used for transduction analysis are

pseudorevertants

Phenotypic Characterization Having come this far, Next step was to verify if the mutants are

CLASSES PHENOTYPE

Temperature Sensitive Can’t grow at 42oC

Cold Sensitive Can’t grow at 30oC

Resistance to other aminoglycosides Resistance to Tobramycin

Temperature sensitive in medium devoid of salt

Can’t grow at 42oC in LB without Sodium Chloride.

Cold sensitive in a medium devoid of salt Can’t grow at 42oC in LB without Sodium Chloride.

Streptomycin dependent Needs Streptomycin for Growth

Impetus for this screening was gained from the studies on transcription defective fit mutants

Cold sensitive mutantsRelative viability of cold sensitive mutants at 300C and 370C

Mutant Cfu/ml at

370C (A)

Cfu/ml at

300C (B)

Relative

viability

(B/A)

2 7.2X 108 3.2X104 0.44X10-4

31 4.11X108 2.9X104 0.705X10-4

38 8.20X108 5.0X104 0.609X10-4

Relative viability of temperature sensitive mutants at 370C and 420C

Temperature sensitive mutants

Mutant Cfu/ml at 370C

(A)

Cfu/ml at 420C

(B)

Relative

viability (B/A)

8 4.2X 108 0.32X104 0.076X10-4

10 1.41X108 0.19X104 0.134X10-4

67 3.28X108 1.06X104 0.278X10-4

95 7.61X108 2.03X104 0.266X10-4

Temperature sensitivity and cold sensitivity exhibited in LB medium without salt.

Growth at

370C

Growth at

420C

Growth at 300C

Medium dependant Temperature Sensitivity

370C 420C

LB Medium

Minimal Medium

Resistance to other Aminoglycosides

• Tobramycin resistance exhibited by one of the Thr+ derivatives (Mutant 36)

Where can the mutation be?

• The following could be the mutations that have occurred in the isolated mutants exhibiting various phenotypes.

– Lesions in ribosomal gene cluster

– Emergence of other suppressor t-RNAs

Transductional crosses to Map mutations of Mutants 3, 26 and 1

Cross no. Donor Recipient Selected marker

Unselected phenotype

(Loss of threonine

independence)

Percent co-transduction

1 JW375 (zhe511::Tn10)

Mutant#1 zhe511::Tn10 (TetR)

Thr- 0% (0/87)

2 JW375 (zhe511::Tn10)

Mutant #3 zhe511::Tn10

(TetR)

Thr- 95% (114/130)

3 JW375 (zhe511::Tn10)

Mutant #26

zhe511::Tn10

(TetR)

Thr- 65% (46/70)

4 Mutant #3 AB1157thr- zhe511::Tn10 (TetR)

Thr+ 77% (96/124)*

*Cross no. 4 indicates acquisition of threonine independence.

Possible map locations of lesions in the mutant 3 relevant to (A)right side of the insertion zhe:: Tn10

(B)left side of the insertion zhe:: Tn10

Transductional crosses to map mutations of temperature sensitive mutants


marker

Unselected phenotype (Loss of threonine independence/temperature sensitivity)

Percent co-

transduction

1 JW375(zhe511::Tn10) Mutant#8zhe511::Tn10

(TetR)Thr-/Ts+ 88% (96/108)

2 JW375 (zhe511::Tn10) Mutant #95 zhe511::Tn10 (TetR)

Thr-/Ts+

83% (90/108)

3 JW375(zhe511::Tn10) Mutant #10zhe511::Tn10

(TetR)

Thr-/Ts+

100% (21/21)

Transductional crosses involved in mapping mutations of cold sensitive mutants.

Cross

no.

Donor Recipient Selected

marker

Unselected

phenotype

Percent co-

transduction

1 CAG12075(zhd::Tn10) Mutant#2 TetR Loss of Cold

sensitivity 45%(37/82)





4 CAG12071(zhc::Tn10) Mutant#2 TetR Loss of Cold





sensitivity 86%(87/101)7 CAG12153(zhb::Tn10) Mutant#2 TetR Loss of Cold

sensitivity13%(13/97)

8 CAG12153(zhb::Tn10) Mutant#31 TetR Loss of Cold sensitivity

13.9%(13/93)

9 CAG12153(zhb::Tn10) Mutant#38 TetR Loss of Cold sensitivity

0.05%(4/72)

Possible map locations of lesions in the Temperature Sensitive mutants 8, 95 relevant to

left side of the insertion zhe:: Tn10 (A) right side of the insertion zhe:: Tn10 (B)

Possible map locations of lesions in the cold sensitive mutants ( 2,31 and 38 )

relevant to the insertions – zhb::Tn10, zhc::Tn10, zhd::Tn10

Cold sensitive mutation designated as (suppressor of rpsL restriction- srs)

Summary of map locations as calculated from co-transduction frequencies

Mutant

number

Relevant

phenotype

Possible map

location

#8 Ts 74.3±0.092

#95 Ts 74.3±0.138

#2 Cs 74.09

#31 Cs74.18

#38 Cs74.07

Cold sensitive phenotype and threonine independence Is it because of a single lesion?

• Transductants that lost cold sensitivity also lost Threonine Independence.

• Demonstrating the fact that the same lesion is responsible for cold sensititvity and Threonine independence as well

Is the Cold Sensitive mutation (srs) Dominant or Recessive ?

• F’ 141( 68 minute to 77 minute of E. coli chromosome) from

the Strain KL708 bearing the Wild type alleles of the mapped

region 74.1- 74.8 minute.

• Strains constructed to allow complementation by argG+ of the

Fʹ in order to select ex-conjugants.

– The mutants, argE- were made argE+ by transduction using a wild type

lysate made on MG1655

– argE+ transductants were made argG- by transduction using a lysate

made on CSH57 harboring a Tn10 close to argG-

Inference drawn from the conjugational crosses – the lesion conferring cold sensitivity and threonine independence is

dominant over wildtype.

Conjugational crosses involved in characterizing srs (suppressor of rpsL restriction)

Cross no. Donor Recipient Selected marker Phenotype

exhibited by

merodiploids

1 KL708 MHM2005(2) argG- / StrepR Thr+/Cs+

2 KL708 MHM2005(31) argG-/StrepR Thr+/Cs+

3 KL708 MHM2005(38) argG-/StrepR Thr+/Cs+

Discussion and Outcome of the present study

• The cold sensitivity conferring mutation could possibly be in the essential genes coding for ribosomal proteins rplQ- rpsM operon, rpsN-rpsH operon

• Some of the proteins coded by these operons are involved in the m-RNA decoding, they mediate interaction of the codon-anticodon pairs ( Potapaov, 1988.) & ( Ramakrishnan, 2000) .

• S10 (rpsJ), S11 (rpsK), S14 (rpsN), S5 (rpsE), S4 (rpsD), L15 (rplO).

Genes coding for protein present in the Decoding centre

It to be noted that S12 ( rpsL) is closest to the decoding centre

Model proposed Genes coding for

ribosomal proteins of the decoding centre, in their

allelic forms could code for proteins which can allow misreading even

when misreading/non-

sense suppression is restricted through

interactions with the protein S12.

Taken from Ogle et al., 2000.

Future prospects of the study

• Complementation studies to locate srs by cloning

• Characterization of the mutations of the other phenotypic classes of mutants.

• Biochemical analysis to reveal effects of mutation on polysome formation, decoding interactions.

ACKNOWLEDGEMENTS

• Prof. M.Hussain Munavar• Sr.Prof. P. Gunasekaran & Prof. Sripathi Kandula• Mr. B. Singaravelan• CEGS for the instumentation facility and financial

assistance.• CGSC for the E. coli strains.• All the members at Laboratory of Molecular Biology

KEY REFERENCES

1.Anetta N., E. Grzesiuk, 2000. Reversion of argE3 ochre strain Escherichia coli as a tool for studying the stationary phase mutations. Acta Biochemica Polonica. 47:459-4692.Enrico Gallucci, Guido Pacchetti and Sandro Zangrossi, 1970. Genetic studies on temperature sensitive nonsense suppression. Molecular and General Genetics. 106: 362--3703.Gudmundur Eggerrtsson and Dieter soll, 1988. Transfer Ribonucleic acid-Mediated Supression of termination codons in Escherichia coli. Microbiological rev.52:354-3734.Gorini L.,1970. Ribosomal discrimination of tRNAs. Nature New Biol.234:261-2645.Haritha V., J.Philip, 2009. Accuracy modulating mutations of ribosomal protein S4-S5 interface do not necessarily destabilize protein interactions.RNA.15:1100-11096.Kirthi N., Teresa Kelly, 2006. A novel single Amino acid change in Small subunit ribosomal protein S5 has profound effects on translational fidelity. RNA.12:2080-20917. Jayaraman R, 1995. Leakiness of genetic markers and susceptibility to post plating mutagenesis in Escherichia coli. J.Genet.74:85-978.Rosa Nagel, Ana Chan, 2006. Mistranslation and genetic variability the effect of Streptomycin, Mutation research.601:162-1709.Singaravelan B, Ph.D., thesis 2009. Molecular genetic studies on Suppression and related aspects in Escherichia coli. MKU. 10.Strigni P., Gorini L. 1970. Ribosomal mutations affecting efficiency of amber suppression. J. Mol. Biol. 47: 517-530

vaijayanthi k.r. (viva-voce)

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