A single gene coding for resistance to both fusidic acid and chloramphenicol

J. Mol. Biol. (1982) 154, 417-425

A Single Gene Coding for Resistance to both Fusidic Acid and Chloramphenicol TONI A. V(~LKERt, SHIGERU hDA AND THOMASA. BICKLE$

Department of Microbiology Biozentrum, University of Basle Klingelbergstr. 70 CH-4056 Basle, Switzerland (Received 10 September 1981) Resistance to chloramphenicol and to fusidic acid are closely linked markers on many resistance plasmids carried by Gram negative bacteria. Here, we use a combination of classical mutagenesis and mutagenesis of isolated plasmid DNA in vitro to demonstrate that the same gene product, chloramphenicol acetyl transferase, mediates resistance to both antibiotics.

1. I n t r o d u c t i o n Plasmid-borne resistance to chloramphenicol is predominantly due to detoxification of the antibiotic by a highly specific enzyme, chloramphenicol acetyl transferase. Using acetyl-coenzyme A, this enzyme inactivates chloramphenicol b y acctylation of hydroxyl groups (Fig. l ; and see Shaw, 1967; Suzuki & Okamoto, 1967). The steroid antibiotic fusidic acid (Fig. 1; is, like chloramphenicol, an inhibitor of the translation process (Davies & Smith, 1978). I t blocks translocation by interaction with the translocation factor elongation factor G (Nierhaus & Wittman, 1980). The mechanism of plasmid-mediated resistance against fusidic acid is not understood (Davies & Smith, 1978). In the Enterobacteriaceae, the cat gene carried by plasmids of the incompatibility group F I I is often on a transposon, the so-called r-determinant, that also carries genes for resistance to several other antibiotics. With one published exception ( D a t t a et al., 1974), resistance to fusidic acid has been found to be closely linked to resistance to chloramphenicol (Datta et al., 1974; Dempsey & Willetts, 1976 ; Lane & Chandler, 1977 ; Miki et al., 1978 ; Timmis et al., 1978 ; Meyer &Iida, 1979 ; I i d a & Arber, 1980). We show here that, despite the high specificity of the protein CAT and the completely different chemical structures of chloramphenicol and fusidic acid, CAT also mediates resistance to fusidic acid, although the way in which it does this is unknown. t Present address: Merzhauserstr. 10, D-78 Freiburg i. Br. :~Author to whom all correspondence should be addressed. 417 0022-2836/82/030417~09 $02.00/0 © 1982 Academic Press Inc. (London) Ltd.

T. A. VOLKER, S. I I D A AND T. A. B I C K L E

418

IH~ COOH .

~OA~

o__o

NH--C-I I CH--~H o

I

02N

CHC(2

CHz

OH l

... H0 !

CH~

H

(a)

(b)

F~G. 1. The structures of (a) fusidie acid and (b) chloramphenicol.

2. Materials a n d M e t h o d s (a) Bacterial strains Escherichia coli WA921 met leu lac supE (Wood, 1966) was the host used in most experiments; DB10 was used for testing fusidic acid resistance (Datta et al., 1974); and POB1050 lacZam for isolating amber mutants. (b) Isolation of catam mutants E. coli POBlO5OlaeZam carrying plasmids with defective cat alleles (prepared as described in the text) were lysogenized with a ¢80 phage carrying the tyrosine inserting amber suppressor supF, and the lysogens were recognized by their red colony co]our on MacConkey lactose plates. Lysogens in which resistance to chloramphenicol had been restored were presumed to carry amber mutations in the cat gene. (c) Other methods Microbiological procedures and DNA sequencing were done using standard methods (Miller, 1972; Davis et al., 1980; Maxam & Gilbert, 1980).

3. Results (a) M u t a n t s

temperature sensitive f o r resistance to chloramphenicol are also temperature sensitive f o r resistance to f u s i d i c acid

T h e first h i n t t h a t t h e s a m e gene m i g h t code for r e s i s t a n c e to b o t h a n t i b i o t i c s c a m e f r o m o u r i n v e s t i g a t i o n of t h e t e m p e r a t u r e - s e n s i t i v e C A T m u t a n t s of N R ] d e s c r i b e d b y Mise a n d his c o l l a b o r a t o r s (Mise & S u z u k i , 1968; Mise & Y a m a d a , 1969). T a b l e 1 shows t h a t t h e s e m u t a n t s a r e also t e m p e r a t u r e s e n s i t i v e for r e s i s t a n c e to fusidic acid. M o r e o v e r , w h e n r e v e r t a n t s were s e l e c t e d for r e s i s t a n c e to e i t h e r one o f t h e a n t i b i o t i c s a t t h e n o n - p e r m i s s i v e t e m p e r a t u r e , t h e y i n v a r i a b l y p r o v e d also to be r e s i s t a n t t o t h e o t h e r a n t i b i o t i c a t t h e s a m e t e m p e r a t u r e ( T a b l e 1). T h e s e r e s u l t s c l e a r l y i n d i c a t e d t h a t t h e s a m e region o f t h e D N A codes for r e s i s t a n c e to b o t h a n t i b i o t i c s , a n d a l l o w e d o n l y t w o p o s s i b i l i t i e s for a gene for r e s i s t a n c e to fusidic a c i d : it c o u l d be t h e s a m e as t h e gene for CAT, or it m i g h t o v e r l a p w i t h t h e gene for CAT.

F U S I D I C ACID A N D C H L O R A M P H E N I C O L

RESISTANCE

419

TABLE 1

Phenotypes of different Cmts mutants and their revertants

DB10(NR1) DB10(NR1-Cmlts) DB10(NR1-Cmlts)

DB10(NR1-Cm3ts DB10(NR1-Cm3ts

DB10(NR1-Cml5ts) DB10(NR1-Cm 15ts)

Cm r 1 ( ~ Cm r 2 (a) Fa r 1 (b) Fa r 2 (b) Cm r 1 (a) Cm r 2 (a) Fa ~ 1 (b) Fa ~ 2 (b) Cm ~ 1 (a) Cm ~ 2 (a) Fa r 1 (b) Fa r 2 (b)

CE r

Fa r

8"8 × 10-1 1"4 x 10 -4 4.0×10 -1 9'4× 10 -1 9"8 × 10 -1 1"4 x 10 -2 2"1 x 10 -3 2'1 × 10 -1 9"6 × 10 -2 7"6 × 10-1 5"1 × l0 -1 7-0x 10 -s 4"0 x 10 -1 l ' 8 x 10 -~ 1"7 x 10 -2 8'1 x 10 -3

8-8 × 10 -4 3"3 × 10 -6 7-2x 10 -a 2"4x 10 -3 7"3 × 10 -3 8-0 × 10 -1 2"2 × 10 -6 1"8 × 10 -1 5"2 × 10 -1 6-8 x 10 -2 2"4 × l0 -3 8-1 × 1 0 - 6 1"5 x 10 -2 4"0x 10 -3 4"6 × 10 -1 1-6 x 10 -2

Temperature sensitivity of strains harbouring NR1-Cmts and temperature-resistant revertants. The NR1 derivatives carrying the thermosensitive cat alleles Cmts-1, Cmts-3 and Cmts-15 have been described (Mise & Suzuki, 1968; Mise & Yamada, 1969). Temperature sensitivity of DB10 carrying these derivatives for resistance to either chloramphenicol or fusidic acid was determined by measuring the relative number of colony formers at 43°C versus those at 32°C on LA plates containing 50 tLg chloramphenicol/ml or 20 t~g fusidic acid/ml. Also shown are the resistance properties of 2 independent revertants of each allele selected for (a) chloramphenicol resistance and 2 selected for (b) fusidic acid resistance at the non-permissive temperature. I t should he noted that wild-type fnsidic acid resistance (first line of Table) is relatively temperature sensitive, and that some of the revertants are more resistant than the wild type. These observations prompted us to determine the DNA sequence of a small t r a n s p o s o n , Tncam204 ( n o w c a l l e d Tn981) d e r i v e d f r o m t h e r - d e t e r m i n a n t o f N R 1 ( I i d a & A r b e r , 1 9 7 7 ; A r b e r et al., 1978). T h i s t r a n s p o s o n c o n f e r s r e s i s t a n c e t o c h l o r a m p h e n i c o l a n d f u s i d i c a c i d , a n d w a s f o u n d t o c o n s i s t o f 921 b a s e - p a i r s o f D N A f l a n k e d b y d i r e c t r e p e a t s o f I S / ( M a r c o l i et al., 1980). C o m p u t e r a n a l y s i s showed that only one protein larger than 50 amino acid residues could be coded by this DNA sequence, and that this protein was CAT, whose amino acid sequence h a d r e c e n t l y b e e n d e t e r m i n e d ( S h a w et al., 1979). T h e D N A s e q u e n c e a l l o w e d t h r e e p o s s i b i l i t i e s f o r r e s i s t a n c e t o f u s i d i c a c i d : (1) i t m i g h t b e d u e t o o n e o f t h e s m a l l peptides (smaller than 50 amino acid residues) that the sequence could theoretically p r o d u c e ; (2) i t m i g h t b e m e d i a t e d b y C A T i t s e l f ; o r (3) i t m i g h t b e d u e t o a truncated protein consisting of the C-terminal two-thirds of CAT produced by initiation of protein synthesis at an internal methionine codon (Met77) of the CAT g e n e , w h i c h is p r e c e d e d b y a r e a s o n a b l e h o m o l o g y t o t h e 3' e n d o f 16 S r i b o s o m a l R N A ( S h i n e & D a l g a r n o , 1974). (b) The isolation of c a t amber mutants by i n v i t r o mutagenesis To distinguish between these possibilities, we have selected mutations that are amber for chloramphenicol resistance and screened their behaviour towards fusidic

420

T . A . V()LKER, S. I I D A AND T. A. B I C K L E

acid. S h o u l d c h l o r a m p h e n i c o l a m b e r m u t a t i o n s also be a m b e r for fusidic a c i d r e s i s t a n c e , t h i s w o u l d p r o v e t h a t t h e genes for r e s i s t a n c e to b o t h a n t i b i o t i c s were in t h e s a m e r e a d i n g fl'ame. Moreover, if such a m u t a t i o n were N - t e r m i n a l to t h e o n l y possible i n t e r n a l t r a n s l a t i o n a l s t a r t , t h i s w o u l d d e m o n s t r a t e t h e i d e n t i t y of t h e t w o genes. A s a m u t a g e n i c agent, we u s e d h y d r o i y l a m i n e in vitro, w h i c h p r o d u c e s p r e d o m i n a n t l y C t o T t r a n s i t i o n s ( B u d o w s k y , 1976), a n d t h e r e f o r e c a n m u t a t e t h e t r y p t o p h a n c o d o n (TGG) and one of t h e g l u t a m i n e c o d o n s (CAG) to a m b e r (TAG). A c c o r d i n g t o t h e D N A sequence, t h e r e a r e t e n such c o d o n s d i s t r i b u t e d f a i r l y e v e n l y t h r o u g h o u t t h e gene. T h e m u t a g e n e s i s was c a r r i e d o u t w i t h t h e p l a s m i d pB1~325 (Bolivar, 1978; P r e n t k i et al., 1981), which c o n t a i n s a gene for C A T ( A l t o n & V a p n e k , 1979; M a r c o l i et al., 1980), cloned i n t o t h e v e c t o r p B R 3 2 2 a n d confers r e s i s t a n c e to tetracycline, chloramphenicol and ampicillin. Treatment of the DNA with h y d r o x y l a m i n e lowered t h e t r a n s f o r m a t i o n a b i l i t y of t h e p l a s m i d w i t h single h i t k i n e t i c s (Fig. 2). Because s e g r e g a t i o n o f m u t a n t s is a slow p r o c e s s w i t h m u l t i c o p y p l a s m i d s (Ishii et al., 1978), D N A was i s o l a t e d f r o m t r a n s f o r m e d c u l t u r e s a f t e r o v e r n i g h t g r o w t h in t h e p r e s e n c e ' o f a m p i c i l l i n a n d u s e d t o t r a n s f o r m Escherichia coli K 1 2 POBIO5OlacZam. Cm s m u t a n t s were d e t e c t e d b y r e p l i c a p l a t i n g , a n d t h e i r frequency increased with time of treatment with hydroxylamine from none out of 1500 (no t r e a t m e n t ) o v e r 0"5% (10 min) t o 1"5°/o (30 rain). O f t h e Cm s clones i s o l a t e d in this w a y , 30 were l y s o g e n i z c d w i t h a ¢80 p h a g e c a r r y i n g t h e t y r o s i n e i n s e r t i n g a m b e r s u p p r e s s o r supF, a n d t h e lysogens were r e c o g n i z e d b y t h e i r r e d

~o

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0

to 3

o_ E L

0

i

I0 20 Hydroxylomine treotrnenf (rnin)

o

30

FIG. 2. Inactivation of plasmid DNA by hydroxylamine. Mutagenesis of the plasmid pBR325 with hydroxylamine followed the method ofVSlker & Showe (1980) with some modifications. Two volumes of DNA in a Tris-EDTA buffer were mixed with 1 vol. 3 ~-hydroxylamine, 5 mM-EDTA (pH 6.0) and incubated at 75°C in a glass tube. At the indicated times, portions were removed, mixed with 3 vol. cold tryptone medium supplemented with 5 mM-EDTA and dialyzed first against the same medium and then against a Tris-EDTA buffer. This DNA was used for transformation using the calcium shock procedure of Cohen et al. (1973). After shaking at 37°C in tryptone medium for 1 h, the cells were titrated on plates containing ampicillin at 25/~g/ml. The different symbols indicate the inactivation curves from 2 independent experiments.

FUSIDIC ACID AND CHLOBAMPHENICOL RESISTANCE

421

colony colour on MacConkey lactose plates due to suppression of the lacZ a m b e r mutation. Five of the Cm s clones became Cm R when lysogenized, and thus contained tyrosine-suppressible a m b e r mutations in the cat gene.

(c) Resistance to fusidic acid is in the same reading frame as resistance to chloramphenicol Plasmid D N A from these five clones was transferred to the fusidic acid-sensitive E. coli K12 strain, D B ] 0 (Datta et al., 1974). All five strains were as sensitive to chloramphenico] and fusidic acid as the non-transformed host. After lysogenization with ¢80supF, resistance to both antibiotics was restored in four cases, while the fifth, catamH32, became CmRFa s. A possible explanation of this CmRFa s p h e n o t y p e is t h a t when this m u t a t i o n is .suppressed by tyrosine, missense incorporation leads to a partially active protein, giving sufficient protection against chloramphenicol but not against fusidic acid. The following d a t a prove this interpretation. A m o n g Cm R r e v e r t a n t s of DB 10 harbouring catamH32, we isolated a strain t h a t suppressed a set of phage T4 a m b e r m u t a n t s with exactly the same efficiency p a t t e r n as supE of E. coli C600 (supE inserts glutamine). Plasmid D N A of this r e v e r t a n t was isolated and used to transform both E. coli DB10 and E. coli C600 (supE). In the first case, the t r a n s f o r m a n t s were sensitive to chloramphenicol, in the second case, they were resistant. We can therefore conclude t h a t the reversion to the Cm R phenotype was an intergenic suppression due to a chromosomal m u t a t i o n to a glutamine-inserting a m b e r suppressor~ m o s t likely supE. This chromosomal m u t a n t was also fully resistant to fusidic acid, making it likely t h a t the original m u t a t i o n was in a glutamine codon. As shown below, the m a p p i n g d a t a of catamH32 confirmed these results. The fact t h a t any a m b e r m u t a n t in one function is also a m b e r for another function proves t h a t both functions are read in the same frame. Thus, the genes for chloramphenicol and fusidic acid resistance are in the same reading frame. Should one of our a m b e r m u t a n t s m a p to the N-terminal side of the possible internal translational start, this would show t h a t t h e y are the same gene.

(d) The same gene product mediates resistance to both antibiotics The m a p p i n g of these m u t a t i o n s was aided b y the fact t h a t three of the ten positions within the cat gene m u t a t a b l e to a m b e r b y h y d r o x y l a m i n e lie within restriction enzyme cleavage sites. These are in the codons for Gln38, Trp150 and Glnl90, which affect P v u I I , B s t N I and H i n f I sites, respectively (Alton & Vapnek, 1979: Marcoli et al., 1980). For three of the a m b e r m u t a n t DNAs, no alteration could be observed in the electrophoresis p a t t e r n s of digests generated by these three enzymes. The m u t a n t catamH32 (Fig. 3(b)) has lost the H i n f I site between fragments 1 and 5 of pBR325 (Fig. 3(a)). We conclude t h a t the codon for Glnl90 has been

422

T. A. V O L K E R , S. I I D A A N D T. A. B I C K L E

(a)

(b)

(c)

-..~ 1.1.5

(d)

(e)

(f)

i I

1 ~="-

la~--

lb,.~ 5 ,,"--

FIG. 3. Restriction cleavage analysis of pBR325 and 2 of its cat amber derivatives. Electrophoresis was carried out in a 0'7% (w/v) agarose/2°/o (w/v) acrylamide gel. Arrowheads indicate the positions of bands affected by the amber mutations. The numbers next to the bands refer to the numbering of the H i n f I .fragments of pBR325 (see Fig. 4). (a). (c) and (e) Digests of pBR325 DNA with HinfI, P v u I I and both enzymes together, respectively. (b) A H i n f l digest of pBR325 catam H32. (d) A P v u I I digest of pBR325 catamH22. (f) A double digest of pBR325 catamtI22 with P v u I I and HinfI.

FUSIDIC ACID AND CHLORAMPHENICOL RESISTANCE Va[GtnLeu ..GTT~AGCTG.--CAASTCGAC.

--.~

)__ s'0

X

423

AtalteG[n T 6CGATTCAG,CGCTAAGTC-

,~0

1~0

'

Amino acid numbering of 'theCa¢ gene /

Pvull

Fro. 4. A map of pBR325 showing the positions of the mutations characterized in this paper. The

HinfI fragments (Prentki et al., 1981) are shown in the circle representing the plasmid pBR325. The 2 PvuII sites of the plasmid are also indicated. The expanded region of the map represents the 657 basepair structural gene for CAT, the start codon to the left, and with the putative internal start indicated with an arrow. The 2 inserts show the detailed DNA and amino acid sequences around Gln38 and Glnl90 (the hyphens have been omitted for clarity): the restriction enzyme recognition sites in these regions are indicated together with the C residues that, after transition to T, create the amber stop codon.

m u t a t e d to a m b e r (see Fig. 4). This result confirms our i n t e r p r e t a t i o n of the p h e n o t y p e of the p s e u d o r e v e r t a n t of catamH32 described above. The i n s e r t i o n of g l u t a m i n e b y the s u p p r e s s o r p r o d u c e s a p r o t e i n w i t h t h e w i l d - t y p e a m i n o acid sequence. F i g u r e 3(d) shows t h a t in the m u t a n t catamH22, one of t h e two P v u I I sites of p B R 3 2 5 (Fig. 3(e)) has been lost. A P v u I I / H i n f I d o u b l e digest (Fig. 3(e) a n d (f)) shows t h a t it is the site in the cat gene t h a t has b e e n m u t a t e d . W e conclude, therefore, t h a t the a m b e r m u t a t i o n in catamH22 is in t h e codon for Gln38. This last result has b e e n confirmed b y direct D N A sequencing. These results are s u m m a r i z e d in F i g u r e 4. Since b o t h of these a m b e r m u t a t i o n s for e h l o r a m p h e n i c o l resistance are also a m b e r for resistance to fusidic acid, the gene for fusidic acid resistance m u s t be in the s a m e r e a d i n g f r a m e as t h a t for resistance to e h l o r a m p h e n i c o l . Since the m u t a t i o n in catamH22 (Gln38) precedes t h e o n l y possible i n t e r n a l t r a n s l a t i o n a l s t a r t a t Met77, the s a m e gene p r o d u c t , CAT, m u s t m e d i a t e r e s i s t a n c e to b o t h antibiotics.

424

T . A . V()LKER, S. IIDA AND T. A. BICKLE 4. D i s c u s s i o n

The isolation of cat amber mutants described above clearly demonstrates the power of classical mutagenesis when combined with the techniques of molecular cloning of DNA. Mutations leading to an inactive gene product could be isolated in this gene of about 800 base-pairs using hydroxylamine-induced mutagenesis at a frequency higher than 1%, so t h a t selective techniques were not needed. More extensive mutagenesis would be inadvisable. Amber mutations in the cat gene arose at a frequency of one in 500, and thus the probability that any of our isolates is a double amber is about l0 -6. The G" C base-pairs mutatable to amber represent about 3% of all the G" C base-pairs in the gene. Assuming t h a t all G" C base-pairs are equally mutatable by hydroxylamine, about one in 15 of the clones has a single C to T transition somewhere within the gene. I t is surprising at first sight that the same gene product should confer resistance against such structurally dissimilar antibiotics as chloramphenicol and fusidic acid. The mechanism of resistance to chloramphenicol has been well-characterized, and consists of an acetylation of the antibiotic by CAT, which renders it biologically inactive (Shaw, 1967; Suzuki & Okamoto, 1967). Although cat gene amplification can confer higher resistance to fusidic acid (Meyer & Iida, 1979) the mechanism of resistance to fusidic acid in gram negative bacteria has not been established. However, McKell & Rownd have observed t h a t fusidic acid inhibits the acetylation of chloramphenicol in vitro, and suggest t h a t resistance to fusidic acid might simply be due to binding of the antibiotic to CAT (R. Rownd & J. McKell, personal communication). I f this were so, one might expect t h a t resistance to fusidic acid analogues would also be mediated by CAT. However, we have found t h a t the antibiotics helvolinic acid (6-hydroxy-3,7-dioxo-ll-desoxyfusidic acid) and cephalosporin.P1 (6-acetoxy-7-hydroxy-ll-desoxyfusidic acid) will inhibit the growth of E. coli DB10 even in the presence of a functional CAT carried by pBR325. This observation makes it unlikely t h a t fusidic acid resistance is entirely due to a relatively non-specific binding of steroid antibiotics by CAT. I t is not at all clear why members of the Enterobacteriaceae should carry a resistance gene against fusidic acid, since they are impermeable to the drug and thus intrinsically resistant. However, the genes for resistance to fusidic acid and other antibiotics must have evolved long before the clinical use of antibiotics, most likely in an environment in which the antibiotics were naturally present, that is, in the soil, and probably in organisms very different from the medically important bacteria in which we now find them. One may speculate t h a t in the carbon-poor environment in which they evolved, m a n y of these genes may have had a catabolic function, enabling the organisms to use antibiotics as a carbon source. At least two antibiotic resistances have been shown to be catabolite repressible in E. coli (Harwood & Smith, 1971; de Crombrugghe et al., 1973), and m a n y of the mechanisms of antibiotic inactivation (acetylations, phosphorylations, adenylations, etc. (Davies & Smith, 1978)) resemble the activation steps of classical catabolic pathways. The genes would then have been mobilized by the acquisition of flanking IS elements and transposed to plasmids, giving them the potential of spreading to other bacterial species (Datta & Hedges, 1972; Calos & Miller, 1980;

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