Biot'himie ( 1991 ) 73, 1493-1500

© Socirt6 franqaise de biochimie et biologie molrculaire / Elsevier, Paris

! 493

Interaction b e t w e e n 16S r i b o s o m a l R N A and ribosomal protein S12: differential effects o f p a r o m o m y c i n and streptomycin M O' Connorl, EA De Stasio2, AE Dahlberg ~ tSection of Biochemistry, Brown Universio,, Providence, R! 02912; +-Department of Genetics, Universi~' of Wisconsin. Madison, W! 53706, USA

(Received 28 August 1991; accepted 8 September 1991 )

Summary - - Strains containing a series of restrictive and non-restrictive mutations in ribosomal protein S i 2 have been transformed with plasmids carrying the 17"nB operon with mutations at positions 1409 and 1491 in 16S rRNA. The effects of the double-mutant constructs have been measured by growth rate, paromomycin and streptomycin sensitivity, resistance and dependence. The results demonstrate a functional interaction between the ! 409-1491 region of rRNA and ribosomal protein S 12. streptomycin / paromomycin / antibiotic resistance and dependence / mutagenesis / 16S rRNA / ribosomal protein S!2

Introduction

Antibiotics have been used classically as probes o f ribosome function [1]. O f particular interest has been the aminoglycoside class o f antibiotics which perturb the codon-anticodon interaction and promote misreading o f the genetic code. Within this wide group o f IlUO~V~JIII~, lllllll./It~,../IO, O l . l % ¢ l J L ~ , J I I l ~ ¢ l - ' I l l has ~ . , 1 1 ,3uojql4~,,ll.~,u to the most intensive study. Mutations in ribosomal protein S12 which confer resistance to streptomycin were first reported in 1949 [2]. However, the mechanism by which streptomycin inhibits protein synthesis is still not completely understood [3]. Streptomycin fails to bind isolated S12 in vitro and yet streptomycin does bind to ribosomal core particles lacking S12 [4]. Furthermore, streptomycin resistant ribosomes are still affected by the addition o f streptomycin both in vivo and in vitro [5]. Chemical protection experiments have shown that streptomycin protects a group o f residues in the 915 region o f 16S rRNA and mutations in this region of rRNA have been obtained which render the ribosomes resistant to streptomycin [6, 7]. These findings have given credence to the original proposal by Gorini that streptomycin binds directly to ribosomal RNA and that the observed effects o f S 12 are due to its influence on the conformation of rRNA [8]. Some mutations in ribosomal protein S12 which confer resistance to streptomycin also lead to decreased levels o f error incorporation. Conversely,

mutants o f ribosomal protein $4, which support higher levels of misincorporation, are hypersensitive to streptomycin. The decoding centre of the ribosome has been mapped to the 1400-1500 region o f 16S rRNA (reviewed in [9]). The wobble base in the anticodon loop of valyl-tRNA has been cross-linked to C1400 [10] and tRNA bound to isolated ribosomes attack [11]. In addition, error-inducing antibiotics of the kanamycin, gentamycin and neomycin (including paromomycin) groups bind to this region and protect bases 1408 and 1494 from chemical attack [6]. Resistance to paromomycin has been found to be associated with disruption o f base pairing between residues C1409-G1491 in the 3' minor domain of 16S rRNA [ 12, 13]. This is true for a wide diversity of organisms but the effects of such mutations on misreading have not been investigated. Previous work from this laboratory has described the construction and antibiotic resistance properties of a series o f mutations at positions 1409-1491 in 16S rRNA in E coli [14, 15]. All of these mutations caused some reduction in cell growth. We found that mutations which created unpaired purines were lethal while mutations which disrupted base pairing conferred resistance to paromomycin and several related antibiotics. In this study, we have taken three of the previously described mutants, 1409G- 1491 C, 1409C-I 491U and 1409C-1491C and have analysed their interactions with various mutant forms o f ribosomal protein S12.

i 494

M O'Connor et al

We f o u n d that strains c o n t a i n i n g the 1 4 0 9 C - 1 4 9 1 U m u t a n t d i s p l a y p a r o m o m y c i n r e s i s t a n c e o n l y in the presence of a stteptomycin resistant rpsL allele while the p a r o m o m y c i n r e s i s t a n c e c o n f e r r e d by the 1 4 0 9 C 1491C m u t a t i o n is i n d e p e n d e n t o f the p r e s e n c e o f a m u t a t i o n in S12. T h e c o m b i n a t i o n o f a h i g h l y restrictive r p s L allele a n d the 1491U a n d 1491C r R N A mutants leads to the g e o e r a t i o n o f s t r e p t o m y c i n p s e u d o dependent and streptomycin dependent phenotypes r e s p e c t i v e l y . By contrast, n o n - r e s t r i c t i v e o r w e a k l y restrictive s t r e p t o m y c i n resistant strains c a r r y i n g the 1491U o r 1491C m u t a t i o n s d i s p l a y a partial r e v e r s a l o f the a b i l i t y o f s t r e p t o m y c i n to inhibit cell g r o w t h . Materials and methods Media and bacterioh~gical methods LB medium 1161, was used for routine cultivation of all strains. Ampicillin was added to media at a final concentration of

200 mg/l, paromomycin was used at a concentration of 5 mg/i and tetracycline at 12.5 mgll. Minimal (E) medium [17] was used for the selection of prototrophic recombinants. Matings, preparation of transducing phage lysates and transductions were performed as described previously [ 161. Sensitivity to UV light was used to screen for the REC- phenotype. Strains and plasmids All strains, plasmids and phages used in this study are described in table I. Derivatives of DH 1 carrying known rpsL alleles were constructed in several steps. DH ! was first made recA + by mating with BW5660, an Hfr strain which transfers srl::Tnl0 and recA + markers at high frequency. REC ÷ tetracycline resistant recombinants were selected on minimal agar plates containing tetracycline. The srl::Tnl0 marker was subsequently eliminated by transduction to SRL ÷ using PI prepared on a wild type strain of E coil An aroE- derivative of DHI recA ÷ was then constructed by transducing DH1 recA+ to tetracycline resistance with P1 prepared on CH30 and subsequently screening for co-inheritance of the ARO- phenotype. DH1 recA + aroE-, TnlO was then transduced in turn to ARO + with PI prepared on each streptomycin resistant strain,

Table I. Bacterial strains, plasmids and phage used in this study. Strain

Genotype

Source~Reference

HBI01

F - hsdS20 supE44 a r a l 4 galK2 lacYl proA2 rpsL20 x y l l 5 leu mti recA13 mcrB mrr

[20]

DH 1

F- recA 1 endA 1 gyrA96 thi hsdR 17 supE44 relA 1

[21 ]

DH 1 rpsL221

As DH 1, rpsL221 recA srl::Tnl0

This study

DHi rpsL224

As DH1, rpsL224 recA s r l ' : T n l 0

This study

DH l rpsL226

As DH 1, rpsL226 recA srl::Tnl0

This study

DH 1 rpsL282

As DH 1, rpsL282 recA s r l ' : T n l 0

This study

JC10240

Hfr KL16 thr leu recA srl::Tnl0

[40]

BW5660

Hfr del(gpt-lac)5 supE44 thi I srlC300:'Tnl0 rel A I spoT

[41 ]

C~ 130

aroE TnlO (30% linkage)

D Hughes, Uppsala

UK235

rpsL282

[22]

UK325

rpsL22 !

[22]

UK327

rpsL224

[22]

UK328

rpsL226

[22]

Plasmids and phage pKK3535

[42]

pKK09G91C

[14, 15]

pKKI491U pKKI491C Phage P 1

[14, 15] [14, 151 Laboratory stock

Antibiotic resistant rRNA and ribosomal protein mutants screening for co-inheritance of streptomycin resistance and loss of the linked tetracycline resistant transposon. Each DH1 recA+ rpsl- strain was then made recA- by mating with JC10240, selecting for inheritance of srl::Tnl0 and screening for coinheritance of the recA- allele. Analysis of rRNA Total RNA was extracted from cells as described by Tapprich et al [18]. Primer extension analysis of the 1491 mutants, to determine the proportions of mutant and wild type rRNA was performed as described by Sigmund et al [191 using an oligonucleotide complementary to bases 1524-1494 of 16S rRNA.

Results Paromomycin resistance in two strains containing rRNA mutations In E coli 16S rRNA one 'end' o f the decoding region contains the base pair C1409-G1491. Strains carrying a cloned copy o f the rrnB operon in which these bases are reversed (pKK09G-91C), have a slightly increased doubling time and a four-fold increase in the level o f resistance to neamine as measured by the minimal inhibitory concentration. The paromomycin resistant rRNA mutants studied here, p K K 1 4 9 1 U and p K K 1491C, have substantially increased doubling times and are resistant to paromomycin (> 5 mg/l) as well as neamine and se~,ral other related aminoglycoside antibiotics [15]. In all previous analyses o f these mutants the E coli strain HB101 was used as a host [14,15]. Since this strain contains a mutant form o f ribosomal protein S12 [20] which confers resistance to streptomycin and restricts misreading considerably, we have re-examined the properties of these mutants i n a ,t~tzild t , l n , = r i l . ' w ~ c n m ~ l h a e l . - c t r n H n d ~brhon +hese rRNA mutants are expressed in the streptomycin sensitive strain DH1 [21] the pattern o f growth rates observed is similar to that seen with H B I 0 1 (data not shown). However, pKK1491U, which confers resistance to paromomycin in HB101, does not do so in DH 1. In addition, resistance to other aminoglycosides conferred by p K K 1 4 9 1 U is substantially reduced in DH1 compared with H B I 0 1 (table II). By contrast, pKK1491C, which causes very slow growth, does confer resistance to paromomycin in both HB 101 and DH 1 and resistances to other aminoglycosides remain roughly the same in both strains (table II). These observations suggest that genetic differences between these two strains determine the aminoglycoside sensitivity or resistance of ribosomes carrying plasmid encoded rRNA. A major difference between strains HB101 and DH1 is the presence of a streptomycin resistant rpsL allele in HB101. We have, therefore, constructed a series o f streptomycin resistant derivatives of the DH 1 strain and examined the paromomycin resistance phenotypes associated with the rRNA mutants in this set o f strains.

1495

Table II. Changes in resistances to aminoglycosides conferred by pKKI491U and pKK1491C in DH! relative to HBi01. Resistances to these aminoglycosides were measured by determining the minimal inhibitory concentration (MIC) for each antibiotic, using the tube dilution method as previously described [14, 15]. Antibiotic Paromomycin Neomycin Neamine Kanamycin Gentamycin Apramycin Hygromycin

pKK I 491 U $8-fold ,1.16-fold No change ,[,2-fold .l.4-fold ,l.8-fold ,l.2-fold

pKK I 491C No change ,l.2-fold $2-fold $2-fold No change ,l,4-fold No change

Effects of protein S12 mutations on phenotypes of rRNA mutants A series o f streptomycin resistant derivatives of DH 1 were constructed as described in Materials and methods. The ,~ derivatives carry one of four rpsL alleles which differ from one another both in primary sequence and in the degree o f restriction which they impose on misreading in vivo and in vitro [22]. Each o f the DH1 derivatives was transformed in turn with each o f the mutant rn~B plasmids and transformants were screened for r e istance to paromomycin. The data presented in table III indicate that cells containing the wild type pla::mid, pKK3535, or pKK09G91C are sensitive to tr?.omomycin regardless of the presence of a mutatio+: in ribosomal protein S l2. However, p K K I 4 9 1 U , which does not confer resistance to paromomycin in a wild type ribosomal background, does confer resistance to the antibiotic in all o f the streptomycin resistant DH1 derivatives. Resistance to paromomycin is observed in both streptomycin resistant and streptomycin sensitive strains containing the very slow growing mutant, pKK1491C. In the strain carrying a severely restrictive rpsL allele, (rpsL282), we were unable to test the antibiotic resistance (see below). Ribosomal protein S12 mutants do not alter the proportions ~.f plasmid and host coded rRNA The data presented above indicate that the paromomycin resistance of ribosomes with a 1491 G--+U change in 16S rRNA is conditional upon the presence o f a streptomycin resistant form o f ribosomal protein S 12. The effect o f the mutant S 12 is not, however, due to a change in the proportion o f mutant and wild type

! 49'J

M O'Connor et al

Table I!i. Effects of mutant S i 2 on 1409-1491 mutations. Plasmid

rpsL wt

rpsL 221 a

rpsL224

rpsL 282

pKK3535

CG

Par sens~

Par sens

Par sens

Par sens

pKK09G91C

GC

Par sens

Par sens

Par sens

Par sens

pKK 1491U

CU

Par sens

Par res

Par res

Par resc

pKK 1491C

CC

Par res

Par res

Par res

nd~

aAli rpsL alleles confer resistance to streptomycin. The level of restrictiveness increases from rpsL221 (non restrictive) to rpsL224 (mildly restrictive) to rpsL282 (highly restrictive), bParomomycin resistance (res) was measured by growth on LB plates containing 200 mg/l of ampicillin and paromomycin at a concentration of 5 mg/l. CGrowth of this strain was stimulated by the addition of streptomycin (streptomycin pseudo-dependence), and = not determined. Strains which require streptomycin to grow cannot grow in the presence of both paromomycin and streptomycin, see text.

rRNA. Primer extension analysis of total RNA from streptom)cin resistant and streptomycin sensitive strains carrying pKKI491U or pKK1491C showed that 50 to 60% of the total 16S rRNA was of the mutant form, regardless of the host genotype (data not shown). Thus, the effect of ribosomal protein S 12 on the resistance of pKK1491U to paromomycin must be due to an altered interaction between S12 and this region of 16S rRNA. Cumulative effect of mutants of S12 and rRNA on growth rate, streptomycin dependence and translational hdelity Mutants of ribosomal protein S 12 which give rise to streptomycin resistant ribosomes fall into several ela~nnn.re.~trlrtlvP Cao rpsL22! and ,-,,~i "~'~ mildly restrictive (eg rpsL224) and highly restrictive (eg rpsL282) alleles used in this study, as well as streptomycin pseudo-dependent and streptomycin dependent classes. These mutations have increasingly detrimental effects on the level of missense incorporation in vivo and in vitro [22-24]. Each of the three mutant rRNAs used in this study gives rise to a unique pattern of growth rates in all strains examined: pKK09G-91C has the least effect, pKK1491U has an intermediate effect on the growth rate and pKK 1491C invariably causes its host to grow very slowly. This relative difference in growth rate is most dramatic in the DH1 strain carrying a severely restrictive rpsL allele, rpsL282 (table III). In this background, pKK09G-91C grows slowly and is unstable, giving rise to faster growing clones which have lost the rRNA mutation. In the same host strain, pKK1491U displays a streptomycin pseudt;dependent phenotype; the transformants grow very slowly in media lacking streptomy2in but the growth rate can be increased by the addition of the antibiotic. When DH1 rpsL282 is transformed with pKK1491C,

no transformants are obtained unless streptomycin is added to the transformation plates. This suggests that the mutant rRNA interacts with the mutant S12 to increase the fidelity of these ribosomes to such an extent that the strains now require streptomycin for growth. The slow growing transformants that do arise on plates containing ampicillin and streptomycin are extremely unstable and segregate faster growing derivatives which have lost the rRNA mutation. These streptomycin dependent transformants do not grow when transferred directly from plates containing ampicillin and streptomycin to plates containing ampicillin and paromomycin, showing that paromomycin cannot substitute for streptomycin to relieve the antibiotic dependence of this strain. (Similar results were obtained with another highly restrictive S12 streptomycin dependent transformants do not grow when transferred directly from plates containing ampicillin and streptomycin to plates containing ampicillin, streptomycin and paromomycin. Thus paromomycin not only cannot substitute for streptomycin but it is also lethal when combined with streptomycin to these streptomycin dependent cells. Effect of rRNA mutations on S12-mediated streptomycin resistance DH 1 strains carrying either of the two non-restrictive rpsL alleles, rpsL221 or rpsL226, grow well on media containing at least 500 mg/1 of streptomycin. The DH1 derivative containing the mildly restrictive rpsL224 allele can grow well on media containing 250 mg/l of the antibiotic. Identical levels of streptomycin resistance are seen when these strains are transformed with pKK3535 or pKK09G-91C. However, when these strains are transformed with either pKK 1491U or pKK 1491 C, streptomycin resistance is decreased considerably (table IV). These data suggest

Antibiotic resistant rRNA and ribosomal protein mutants

1497

Table IV. Effect of 1409-1491 mutations on streptomycin resistance. Strain + plasmid

Streptomycin cmwentration (rag~l) 0

50

100

250

500

1000

rpsL221/226

pKK3535

+++

+++

+++

+++

-I-d-

pKK09G-91C

+++

+++

+++

+++

++

pKK 1491U

+++

+++

++

+

pKK 1491C

+++

+++

++

+

pKK3535

+++

++

++

+

pKK09G-91C

+++

++

++

+

pKK 1491U

++

-

-

-

pKK 1491C

++

-

-

-

m

rpsL224

+++ indicates good growth of the strain on LB plates containing ampicillin (200 mg/1) and streptomycin at the indicated concentration after overnight incubation at 30°C. ++ and + indicate successively poorer growth on the same media and - indicates an absence of growth.

that interaction between certain p a r o m o m y c i n resistant r R N A mutations and streptomycin resistant f o r m s o f S 12 can result in a partial restoration o f the sensitivity to streptomycin in these double-mutant ribosomes. Discussion In this report, we have utilized mutations in both r R N A and ribosomal protein S I 2 to show that there is a functional interaction between S 12 and the 1 4 0 9 1491 region o f 16S rRNA. This interaction is observed as a modulation of the response o f r R N A mutants at position 1491 to the antibiotics p a r o m o m y c i n and streptomycin. The 1400-1500 region of 16S r R N A has been implicated as the site o f codon-anticodon interaction by a n u m b e r o f studies utilizing mutagenesis, crosslinking and chemical protection [9, 25, 26]. t R N A bound to the A site protects bases 1408, 1492, 1493 and 1494 in this region [11]. Aminoglycoside antibiotics which affect codon-anticodon recognition by promoting misreading also protect bases 1408 and 1494 [11]. S12 plays a crucial role in the selection of cognate t R N A s at the ribosomal A site and its omission from reconstituted particles leads to an abnormally low level o f error incorporation [27]. Additional evidence for effects o f S12 on the 1 4 0 0 1500 region c o m e s f r o m the work o f Allen and Noller

[28]. In their study, ribosomes from a series o f streptomycin resistant strains were probed chemically and the results were correlated with the degree of misreading imposed by each rpsL mutation, rRNA from ribosomes containing a streptomycin dependent S l2 mutation exhibited significantly decreased reactivity to chemical probes at positions 1413 and 1487, but no effects of wild type S12 on the 1409-1491 region of r R N A have been observed [29]. Furthermore, neutron diffraction measurements place S 12 distant from the 1490 region [30]. Nonetheless, the effects of streptomycin dependent rpsL alleles on the accessibility o f this region o f r R N A to chemical probes as well as the data presented here suggest some interaction between S 12 and this region of rRNA. An even more distant interaction has been reported for a mutation at position 2661 in 23S rRNA, the phenotypic expression o f which is dependent on the the nature o f the S 12 background. Chemical footprinting o f p a r o m o m y c i n shows that the binding site for the antibiotic lies in the 14091491 region [6]. The isolation of drug dependent ( p a r o m o m y c i n , streptomycin or ethanol dependent) S l 2 mutants indicates that S12 affects the binding site for p a r o m o m y c i n . The co-operative binding o f streptomycin and p a r o m o m y c i n to 70S particles in vitro also suggests that there is considerable interplay between the binding sites for both antibiotics [32]. One possible explanation for the differing responses o f 1491U and 1491C to p a r o m o m y c i n (tables II and

! 4q8

M O'Connor et al

lid is that the 1491U mutant requires an additional confonnational perturbation of this region, afforded by an S!2 mutation, in order to render it resistant to the effects of paromomycin, whereas in the 1491C mutant the conformation of this region is already sufficiently disrupted to make it insensitive to the antibiotic. Previous work on the 1409-1491 series of mutants indicated that the higher order structure is perturbed in the region surrounding the site of the mutation [ 14]. When ribosomes containing the 1491U or 1491C mutant rRNAs were probed with DMS, si~maificant differences in the reactivities of bases in this region were observed. In particular, the reactivity of 1408 was decreased and the normally unreactive bases, 1409, 1483 and 1418 became reactive in both mutants. In pKKI491C, position 1491C became reactive and 1494G displayed decreased reactivity. Thus, residues outside the mutant base itself respond differently to chemical probes, suggesting that this region has an altered conformation in these mutants. Streptomycin has been cross-linked to the 892-917 and 1394--1415 regions of 168 rRNA using nitrogen mustard as a cross-linking agent [33]. In addition, streptomycin protects residues in the 912 region both in 308 and 708 particles and residues 1413, 1487 and 1494 in 308 subunits [6]. Together the cross-linking and protection data suggest that the 1490 region of 168 rRNA may constitute part of the streptomycin binding site. This offers a reasonable explanation for the altered response to streptomycin by streptomycin resistant strains containing the 1491U and 1491C mutants (table IV). All strains examined here which are resistant to both streptomycin and paromomycin will grow on m e _ d i n enntalnlng a l t h a r ~ntlki~lq~ .alan. h,,* I,,~il nntgrow on media containing a mixture of both antibiotics (table Ill). This finding is reminiscent of the behaviour of drug dependent rpsL alleles isolated in Gorini's laboratory io the 1960s [34]. In that study, mutants of E coli were selected which required either streptomycin or paromomycin to grow. In a proportion of these cases, either drug would suffice to allow growth, but a combination of paromomycin and streptomycin was invariably found to be lethal, presumably due to the co-operative promotion of high levels of misreading by the two antibiotics [35]. The lethal effect of a combination of the two antibiotics is also observed in the streptomycin dependent strain resulting from the combination of a restrictive S12 mutant (rpsL282) and the paromomycin resistant rRNA mutation, pKKI491C (table III). All of Gorini's drug dependent mutants were due to changes in ribosomal protein S12 [36, 37]. Here we provide an example of co-operativity between a mutant ribosomal RNA and a mutant of S12 which results in the generation of a streptomycin dependcat phenotype.

The interaction between the highly restrictive rpsL282 S12 protein and the 1491U /1491C mutant rRNAs (streptomycin pseudo-dependence and streptomycin dependence respectively) argues a role for this region of 168 rRNA in the maintenance of translational fidelity. In keeping with this proposal, in vitro translation experiments with poly U-programmed ribosomes from DH1 rpsL wild type strains carrying each of the three mutants studied here, showed that 1491U ribosomes supported higher levels of misreading than the corresponding wild type ribosomes whereas the levels of error incorporation were decreased in the 1409G-91C mutant (our unpublished data). Furthermore, Weiss-Brummer and Huttenhofer [38] have observed a drastic decrease in the level of spontaneous frameshifting in yeast mitochondria in the presence of a C--->G change in yeast 158 rRNA at the position corresponding to 1409. Finally, Allen and Noller [39], have isolated a ram-like C--->U mutation at position 1469 on a plasmid carrying the rrnB operon which relieves the streptomycin dependence of several rpsL alleles and which, in the absence of any rpsL mutations, supports a high level of misincorporation in in vitro translations. These results support a model in which the interaction of the 1400-1500 region of 168 rRNA with ribosomal protein S12 is crucial in the control of translational fidelity.

Conclusions 1) An interaction between 168 ribosomal RNA and ribosomal protein S12 has been demonstrated by utilizing mutants which give differential responses to paromomycin and streptomycin; 2) a 1491G--->U mutation in i68 rRNA confers resistance to paromomycin only in strains containing a streptomycin resistant rpsL allele; 3) a G---~C mutation at position 1491 confers resistance to paromomycin irrespective of the presence of a mutation in S 12; 4) the combination of a highly restrictive rpsL allele and the 1491U and 1491C rRNA mutations leads to the generation of streptomycin pseudo-dependent and streptomycin dependent phenotypes respectively; 5) when the same rRNA mutations are placed in non-restrictive or weakly restrictive streptomycin resistant strains, a partial restoration of streptomycin sensitivity is observed.

Acknowledgments We gratefully acknowledge the contributions from Mans Ehrenberg, Charles Kurland, Diarmaid Hughes, Barbara Bachmann and George Q Pennabble. This work was supported by a grant from the US National Institutes of Health (GM19756) to AED.

Antibiotic resistant rRNA and ribosomal protein mutants

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19

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