Biochimie (1990) 72, 835-843 © Soci6t6 franqaise de biochimie et biologie moldculaire / Elsevier, Paris

835

Original article

Isolation and characterization of a new temperature-sensitive polynucleotide phosphorylase mutation in Escherichia coli K-12 SD Yancey, SR Kushner* Department of Genetics, University ~'Georgia. Athens. GA, 30602, USA (Received 19 November 1990; accepted 26 November 1990)

Summary - - Polynucleotide phosphorylase (PNPaseJ has been studied in detail since its discovery in 1955 [11. In an attempt to determine what role, if any, it has in mRNA decay in Escherichia coil, we have isolated and characterized a temperature-sensitive mutation, pnp-200, in the pnp gene. ;n vitro phosphorolysis, polymerization and exchange acti,~ities of the partially purified Pnp-200 enzyme are all reduced to 30-40% of wild-type activity at 50°C compared to 32°C. The pnp-200 mutation alone does not affect cell growth or mRNA stability. A triple mutant strain containing pnp-200 in combination with other temperature-sensitive mutations in genes known to affect mRNA metabolism (rnb-500 and ares-l) is conditionally lethal and shows increased mRNA stability after shift to the non-permissive temperature. PNPase / mRNA decay / phosphorolysis

Introduction Polynucleotide phosphorylase (PNPase) catalyzes both the synthesis (5' to 3') and phosphorolysis (3' to 5') of oligoribonucleotides as well as an exchange reaction between nucleoside diphosphates and inorganic phosphate [2]. PNPase has been isolated from a variety of both prokaryotic [31 and eukaryotic [41 sources. It is unique in that it can polymerize long random polymers in a processive fashion from ribonucleoside diphosphates without any template requirement [5]. The cloning [61 and sequencing 171 of the pnp gene from E coil has facilitated analysis of its various activities. Our interest in PNPase relates to its role in general m R N A turnover in E coli. It has been shown that the presence of either PNPase or RNase I1 (an exonuclease that degrades m R N A in the 3' to 5' direction) is required for cell viability in E coil [81. Partially degraded m R N A s are stabilized in strains containing the null pnp-7 [9] mutation and the temperature-sensitive rno-500 (RNase II) allele after a shift to nonpermissive temperature [8]. When a temperaturesensitive mutation in the ams (altered m R N A stability) [10] gone was introduced into a pnp-7 rnb-500 genetic background, the chemical stability of bulk

*Correspondence and reprints

m R N A as well as specific m R N A s was significantly increased [11]. In addition, we found that any multiple mutant strain that contained the pnp-7 allele had a generation time in rich medium at 30°C of approximately 63-67 min, while those that were pnp ÷ had generation times of 35-43 min [11]. We also found that multiple mutant strains containing "--t,~ct, nt,-7 ,,,-"A'^,~,~ were unstable and yielded temperature-resistant revertants after extended periods of growth at 44°C. Since pnp-7 was the only null mutation used, it seemed likely that the absence of PNPase was responsible for the slower growth at the permissive temperature. Other investigators have also found that the pnp-7 mutation has an effect on growth at 45°C, although not at 43°C [ 121. Although Reiner had isolated a temperature-sensitive PNPase mutation (pnp-27) using whole-cell nitrosoguanidine mutagenesis [9], he found that the enzyme was only heat-inactivated in vitro, while retaining at least 80% of its activity in cells grown at 45°C [131. Only when cultures were starved for glucose at 44°C and grown to pH 6, was PNPase activity in a pnp-27 mutant temperature-sensitive in vivo. In addition, Krishna et al found that growth and RNA metabolism in the pnp-27 mutant was identical to a wild-type control at 45°C [ 141.

836

SD Yancey, SR Kushner

W e therefore sought to isolate a temperature-sensitive pnp mutation that w o u l d allow n o r m a l g r o w t h at 30°C, but would be P N P a s e deficient at 44°C. The w o r k presented here describes the identification and characterization o f a temperature-sensitive P N P a s e mutation (pnp-200) g e n e r a t e d by h y d r o x y l a m i n e m u t a g e n e s i s o f the c l o n e d wild-type pnp+ gene. W h e n the pnp-200 e n c o d e d P N P a s e was partially purified, in vitro polymerization, phosphorolysis and exchang.e activities were all r e d u c e d to 30--40% after 30 m m incubation at 50°C. In addition, we f o u n d that pnp200 rnb-500 and ares-! pnp-200 rnb-500 multiple mutants ~ e w n o r m a l l y at 30°C. H o w e v e r , following shift to 44°C, ~ o w t h stopped after two g e n e r a t i o n s in a pnp-200 rnb-500 ams-I triple mutant, while the half-life o f bulk m R N A w a s essentially identical to that o f a pnp-7 rnb-500 ams-I control strain. Interestingly, the pattern o f m R N A b r e a k d o w n for a specific m e s s a g e (trxA) w a s different w h e n c o m p a r e d with the triple m u t a n t strain containing pnp-7. The significance o f this d i f f e r e n c e will be discussed.

same direction as the pnp promoter (fig IA). SK5015 (rnb-296, RNase II-) was the recipient for the conjugal cross, since insertion of the pnp-200 allele into the chromosome should generate conditionally lethal exconjugants. Exconjugants were selected on LB plates containing chloramphenicol (to select for recombinants) and kanamycin (to counterselect against the Hfr donor strain) at 30°C. P I transducing phage lysates were made according to Willetts and Mount [241, with two passes before titering. Growth curves and viability tests of different strains were all performed in LB. Cells were inoculated to a Klett reading of 5 at 30°C, incubated with shaking at 30°C to a Klett of 40 (No 42 green filter, mid-log) then shifted to 44°C. Cells were kept in log phase growth by diluting with pre-warmed LB. Cell viability was determined by removing an aliquot of cells, diluting with M56/2 buffer and plating on LB agar at 30°C overnight.

DNA manipulations

Growth media were LB [ 15], LB solidified with 2% agar or YT broth containing 0.2% glucose [9, 13]. Ampicillin (100 lag/ml), chloramphenicol (20 lag/ml), tetracycline (20 lag/ml) and kanamycin (59 l.tg/mi) were added when required. The minimal medim for selection of arginine prototrophs was M56/2 [16] supplemented with thiamine, glucose, required amino acids and thymine or uracil when necessary. Restriction endonucleases, T4 DNA ligase~ DNA po!y_m__era_~ ! K!enow fragment and Na2ADP were from Boehringer Mannheim (Indianapolis, IN). Ampicillin, chloramphenicol, kanamycin, l ifampicin, naladixic acid, tetracycline and hydroxylamine were from Sigma (St Louis, MO). Carrier-free inorganic 132Plphosphate, 15-3H]CDP, [5,6-3Hluridine, and [tx-32PIdATP were from New England Nuclear (Wilmington, DE). Poly[8-3H]adenylic acid was from Amersham (Arlington Heights, IL). Plasmid pCMI [171, cartier polyA and Na2CDP were from Pharmacia LKB Biotechnologies (Piscataway, NJ). GTG agarose was from FMC (Rockland, ME). Acrylagel and bis-acrylagel were from National Diagnostics (Manville, NJ). Ultra-pure ammonium sulfate was from United States Biochemical Corp (Cleveland, OH). All other chemicals were reagent grade.

The plasmid pK~AK7 contains a 5.3-kb Hindlll-EcoRl fragment isolated from a chromosomal DNA clone carrying the argGpnp region inserted in pBR322 (K Armstrong, MS Thesis, Univ of GA, ! 981). Five lag of plasmid DNA was treated with hydroxylamine at 37°C for 24 h [18], dialyzed against TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) buffer overnight, extracted with phenoi/chloroform/isoamyl alcohol (25/24/1) and precipitated with ethanol. Bacterial transformations followed the procedure of Kushner [19]. 0.2 to 1.0 lag of DNA was used to transform strain SK5017 (rnb-296 pnp-7/pDK33 Cm r rnb÷). After expression in LB for 2 h at 30°C, ampicillin was added to 100 lag/mi and growth continued at 30°C for 6-8 h to allow displacement of the resident plasmid (pDK33, rnb ÷Cmr). Dilutions of transformed cells were plated on LI:~ containing ampicillin and incubated at 30°C ovemight. Colonies were patched onto LB plates, incubated at 30°C for 8--10 h, then replica-plated to plates with/without ampicillin at 30°C ~ 44°C and chloramphenicol at 30°C. Small and large scale preparations of plasmid DNA were t:urried out using the method of ish-Horowicz and Burke [20]. All restriction enzyme digests and other DNA treatments followed the manufacturers' directions. DNA fragments were isolated from 0.8% GTG agarose gels by electroelution into TAE (40 mM Tris-HCl, 20 mM NaOAc, 2 mM Na2EDTA, pH 8.0) buffer. Chromosomal DNA for Southern analysis was isolated from 20 ml of cells using a modification of the Marmur procedure [211. [tx-32p]dATP-labelled DNA probes for the Southern analysis were generated from gel-purified DNA fragments (fig 1) using the random primer method [22]. BioTrans nylon membrane (ICN; Costa Mesa, CA) was used, with the manufacturers recommended procedures. Blots were exposed to Kodak X-OMAT AR film (Rochester, NY) at -70°C for various periods.

Bacterial strains and genetic analysis

Enzyme ass~,s

The E coli strains used are listed in table I. The insertion of the pnp-200 mutation into the chromosome was carried out via conjugation [23]. To facilitate the insertion of the pnp-200 allele into the chromosome, a selectable marker near pnp was required. Accordingly, the 1.2-kb Hhal-BclI fragment containing the entire cat gene (chloramphenicol acetyl transferase)

Cells were grown in YT broth with 0.2% glucose [9, 13] containing ampicillin. Crude extracts were made from either 20 mi of overnight cultures for screening purposes or 4 1 grown to an A650 of 1.3-1.5 when PNPase was being isolated for ammonium sulfate precipitation. Cell extracts were prepared as described in [3], except that a lysozyme (400 lag/mi)/freezethaw treatment replaced the homogenization and French press step. When cell extracts were made from 20-ml samples, the debris was spun out in a Beckman JA-20 rotor at 10 000 rpm for 20 min. For large scale isolations, S I00 extracts were prepared in a Beckman Ti60 ultracentrifuge rotor spun at 35 000 rpm for 1 h. Isolation of the ammonium sulfate fraction

Materials and Methods Media and Materials

was isolated from pBR325, treated with the Klenow fragment of DNA polymerase I to generate blunt ends, and ligated into the Asp718 site of pSYK8 (pnp-200, Apt). Plasmid DNA was isolated from Cm r, Apr transformants of AB312 (Hfr POI2) and screened for the correct orientation of the cat fragment. pSYKI 1 (in SK6629) contains the cat promoter oriented in the

Polynucleotidephosphorylasemutationin E coli K-12

837

Table I. Escherichiat'oli strains. Both pKAK7 and pDK33 are derivatives of pBR322. Strain

Genotype

Source/Reference

AB312

Hfr POI2 thi-1 thr-I leu-6 lacZ4 str-8 supE44

A J Clark O9SC*

MG1693 t h y A T l 5 ~" PR27

pnp-27 rna-19 thr-I leu-6 thi-1 lacY1 rpsL32

O3SC*

SK4900

thr leu argG6 zgi-203:'Tnl O

This

laboratory

SK5015

thr leu argG6 zgi-203:'TnlO rnb-296 pyrF::Tn5

This

laboratory

SK5017

thr leu rnb-296 pyrF:'Tn5 pnp-71pDK33 Cm r rnb +

This laboratory

SK5033

thr leu rnb-296 pyrF:'Tn5 pnp-7/pKAK7 Ap r pnp +

This

SK5662

thyA715 argG6 zgi-203::TnlO ~,"

This laboratory

SK5693

thyAT15 argG6 zgi-203::TnlO rnb-500 ~"

This laboratory

SK5715

thyA715

This laboratory

SK5726

thyA715 pnp-7 rnb-500/pDK39 Cm r rnb-500

This laboratory

SK6623

thr leu rnb-296 pyrF:'Tn5 pnp-7/ pSYK7 Ap r pnp-200

This work

SK6629

AB312/pSYKll

~T.,rLL,'~rt

,,h--

1. . . . . . .

O,&'i~OOJU

¢:nd

t~U

rnb-500 ams-1 ~-

IL".."IP~¢

pytr

.. m z m j

This work

Ap r Cmrpnp-200 .--k

~

I t~o-~Tu

,,-, . , . ,,-.

")/')/1

pt~p-~u~

laboratory

g'~..~r

~.,,mmm-

~,.l Jt

,,J 1.1

SK6631

same as SK6630

Km r Cm r exconjugant P1-SK6630xSK5015 A r g G + transductant

SK6632

thyA715

rnb-500 pnp-200 Cm r ~,-

P1.SK6631xSK5693

SK6634

thyA715 rnb-500 ams-1 argG6 zgi-203"'TnlO

A r g G + transductant P1.SK4900xSK5715 Tc r transductant

SK6638

SK6631/pSYK8

SK6639

thyA715

Aprpnp-200

pnp-200 Cm r Z,-

This work P1-SK6631xSK5662

SK6640

thyA715 pnp-200 rnb-500 ams-1 Cm r

ArgG + transductant PI.SK6631xSK6634 A r g G + transductant

SK6649

SI~5015/pKJ~K7 Ap r p n p +

This work

*Coli Genetic Stock Center, Yale University

838

SD Yancey, SR Kushner

(A) 5 3 kb

SK5015

H III

Eco (-- o r f /--11

pBR322 I--/

H III

I E¢o ~

I

•

pnp-200 I

I

I II Pv Dr Pv

I H III

/ Ocl I

I

(---cat

(

I

"1

E¢o

I

pnp

I

I I II Pv DrPv

I I E¢o Asp

I

1.8 kb

I

Eco

4.7 kb

I

I H III

SK6630131

I

E¢o

H III

k-~\.,.\\\\~\-.\.~

(B)

pSYK11

I Hh= I