Mol Gen Genet (1992) 236:76-85 © Springer-Verlag 1992

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans Reinhard Sedlmeier 1 and Josef Altenbuchner 2

1 Institut ftir Genetik und Mikrobiologie, UniversitfitMiinchen, Maria Ward Strasse 1A, W-8000 Mfinchen 19, FRG 2 Institut fiir industrielle Genetik, UniversitS.tStuttgart, Azenbergstrasse 18, W-7000 Stuttgart 1, FRG Received February 6, 1992 / Accepted July 1, 1992

Summary. A broad-spectrum mercury resistance locus

(mer) from a spontaneous chloramphenicol-sensitive (CmS), arginine auxotrophic (Arg-) mutant of Streptomyces lividans 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. Iividans 1326. This element was amplified to about 20 copies per chromosome in the Cm s Arg- mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins. Key words: Streptomyces lividans - Mercury resistance -

Amplifiable element - Mercuric reductase - Organomercurial lyase

Introduction

Resistance to toxic heavy metals is widespread among prokaryotes. The resistance genes are frequently found on plasmids and transposons but also on bacterial chromosomes. This is particularly well documented for mercury resistance genes, which have been cloned from various gram-negative and gram-positive sources in recent years. Characterization of the cloned mercury resistance markers has revealed a common resistance mechanism Correspondence to: J. Altenbuchner

(for reviews see. Silver et al. 1989; Misra 1992). Mercuric ions are trapped outside the cell by mercury binding proteins (MerP) and taken up into the cell by specific transport systems (MerT). Inside the cell Hg 2+ ions are reduced to the less toxic metallic mercury (Hg °) by a mercuric reductase (MerA) in an NADPH-dependent reaction. The high volatility of elemental mercury finally leads to evaporation and detoxification of the polluted area. In some systems the resistance is limited to mercuric ions and very few organomercurial compounds. Others exhibit a broad resistance spectrum including compounds like phenylmercuric acetate (PMA). This is due to synthesis of an organomercurial lyase (MerB), which cleaves C-Hg bonds. In cases of narrow-spectrum resistance this gene is missing (Walsh et al. 1988). The five to seven resistance genes are organized in an operon structure and tightly regulated by an activator-repressor protein (MerR; O'Halloran et al. 1989). Six different mercury resistance markers have been completely sequenced so far. They provide an attractive model for study of the evolution of complex resistance mechanisms, horizontal and vertical gene transfer and adaptation in different bacteria. They have also proved to be valuable tools for transposon mutagenesis and use as selection markers in cloning experiments (Gambill and Summers 1985). In the present work we describe the cloning and DNA sequence analysis of a mercury resistance marker from Streptomyces lividans 1326, which was identified by Nakahara et al. (1985). Streptomycetes are quite different in morphology and genomic GC content to most other bacteria and therefore it is particularly interesting to elucidate the relationship between mercury resistance determinants in this group and those in other known systems. Materials and methods

Bacterial strains, plasmids and culture conditions. The strains and plasmids used are listed in Tables 1 and 2.

77 Table 1. Bacterial strains

Strain

Marker

Plasmid

Reference

S. lividans 1326 S. lividans 1326.32 S. lividans TK19 S. lividans TK20 S. lividans TK21 S. lividans TK64 E. coli JM109

Wild type Cm~ArgWild type Wild type Wild type pro, str-6 reeA1, endA1, supE44, relA1, hsdR17, thi 9yrA96, A (lac-proAB)

SLP2, SLP3 nt SLP3 SLP2

Hopwood et al. 1983 This work Hopwood et al. 1983 Hopwood et al. 1983 Hopwood et al. 1983 Hopwood et al. 1983 Yanisch-Perron et al. 1985

F" [traD36, proAB +, lacB IaeZAM15]

nt, not tested Table 2. Plasmids and mercury resistance

levels

Plasmid

Genes, Mer phenotypeb

Inhibition z o n e " Hgz+ PMA

Reference

pIJ702 pJOE796 pJOE796-7 pJOE796-11 pJOE796-17 pJOE796--32 pJOE796-33 pJOES13 pIC19H pJOE851-1 pJOE851-2

tsr, reel tsr, Merr tsr, Met (r) tsr, Met r tsr, Merr tsr, Mer~ tsr, Merhs tsr, Merr bla, lacZc~ bla, (Merr) bla, (Mer~)

10 4 6 4 4 10 14 4 9 9 9

Katz et al, 1983 This work This work This work This work This work This work This work Marsh et al. 1984 This work This work

24 10 11 nt 10 nt 25 11 nt nt nt

" Resistance to HgCI2 (Hgz+) and phenylmercuric acetate (PMA) of S. lividans TK64 carrying the plasmids listed and of E. coli JM109 bearing plasmid pIC19H, pJOE851-1 or pJOE851-2. Inhibition zone sizes (in mm) were determined as described in Materials and methods; the values are the average of at least three different determinations, nt, not tested b Met phenotype was classified according to the sizes of inhibition zones; Merr, mercury resistant; Mer"), partially resistant; Mers, sensitive; Met hs, hypersensitive. (Met r) indicates the complete mercury resistance marker from S. lividans cloned in pIC19H but not expressed in E. coil Escherichia coli K-12 strains were grown at 37°C in 2 × YT liquid medium and on 2 × YT agar plates, supplemented with ampicillin (100 rag/l), isopropylthio-[3galactoside (IPTG) (0.25 raM) and 5-bromo-4-chloro-3indoyl-13-D-galacto-pyranoside (X-gal; 20 rag/l) to select plasmids and visualize l a c Z - a - c o m p l e m e n t a t i o n (Samb r o o k et al. 1989). Streptomyces lividans strains were grown at 30 ° C in Y E M E liquid medium supplemented with 27% sucrose, 10 m M MgC12 and 0.5% glycine. F o r protoplast regeneration, matings and to obtain spore suspensions, R 2 Y E agar plates were used. Plasmids were selected with thiostrepton (12 rag/l), production of melanin was detected by adding tyrosine (400 rag/l) and CuSO 4 (15 rag/l) ( H o p w o o d et al. 1985). Mercury-resistant transformants were selected with 5 IaM mercurochrome in M40 minimal agar (Polsinelli and Beretta 1966) containing 0.2% glycerol instead of glucose and 0.05% Casamino acids. Streptomycin (10 rag/l) was added to select mercury- and streptomycin-resistant recombinants from mating experiments, whereas Casamino acids and mercurochrome were omitted when selecting for prototrophic, streptomycin-resistant recombinants. Inhibition zones with HgC12 were determined on filter discs on G M E plates containing 10 g yeast extract, 10 g malt extract, 5 g glucose, 18 g agar per 1 1 H20.

P o c k formation and matings. To demonstrate pock formation, spores of S. lividans strains were plated on R 2 Y E agar plates to obtain single colonies. Sporulated colonies were replica plated on R2YE plates previously seeded with 107 spores of S. lividans TK64 and incubated at 30 ° C. When aerial mycelium and spores had formed the plates were examined for pocks around the transferred colonies. Mating experiments were carried out by plating 107 spores of both parental strains on R 2 Y E plates. After sporulation, dilutions of the spore suspensions were plated on M40 agar plates to select recombinants. Determination o f mercury resistance by inhibition zone assays. A b o u t 107 spores of S. lividans strains or 107 E. coli cells of an overnight culture in 2 z YT medium were plated on G M E plates. A filter disc (5 m m diameter) bearing 50 nmoles HgC12 or 10 nmoles phenylmercuric acetate was laid on the agar. The diameters of inhibition zones in m m (less 5 m m filter diameter) were determined after 12 h at 37 ° C (E. coli) or 48 h at 30 ° C (S. lividans) incubation. D N A manipulations. Restriction enzymes, T4 ligase, alkaline phosphatase and Klenow polymerase were pur-

78 chased from Boehringer and used as recommended by the manufacturer. Unidirectional deletions in p JOE851-1 and p JOE851-2 were generated by cleavage of plasmid D N A with a tenfold excess of SacI and EcoRI and treatment of the D N A with exonuclease III (New England Biolabs) and mung bean nuclease (Pharmacia) as described by Henikoff (1984). Then the D N A was sizefractionated on 0.7% agarose gels (in 90 mM TRISborate, 2 mM EDTA pH 8.0) and fragments recovered by the freeze-squeeze method (Tautz and Renz 1983). After ligation overnight, the D N A was transformed into E. coli JM109 as described by McKenney et al. (1981). Deletions with Bal31 exonuclease in p JOE796 were generated in the following way: 4 lag plasmid DNA was cleaved with ScaI to completion, the D N A extracted with phenol, precipitated with sodium acetate/isopropanol, resuspended in 40 gl Bal31 buffer and incubated with 2.5 units Bal31 for 7 rain at 37 ° C. The reaction was stopped with 4 gl EGTA (0.2 M, pH 8.0), the D N A was precipitated and resuspended in 20 gl 10 mM TRIS-HC1 0.1 mM EDTA pH 8.0 (TEl0.01), treated with 5 units Klenow polymerase in the presence of 0.1 mM dNTPs and again precipitated. After resuspension in TEl0.01 about 1 ~tg of this D N A was ligated to 1 gg phosphorylated HindIII linker (Boehringer) overnight at roomtemperature with 2 units T4 ligase; the D N A was precipitated, treated with 20 units HindIII endonuclease for 2 h and fractionated on an agarose gel; the linearized plasmid D N A was recovered by the freeze-squeeze method, ligated and transformed into S. lividans TK64. A genomic library of S. lividans 1326.32 was constructed from D N A fragments obtained by digesting 10~tg chromosomal D N A in 100gl with 0.5 units Sau3AI for l h at 37° C. The DNA was separated on a low melting point agarose gel, fragments in the range of 5-10 kb were recovered by melting the appropriate region of the gel at 65 ° C, extracting twice with phenol in the presence of 0.5 M NaC1 and precipitating DNA with ethanol. About 2 lag of this D N A was ligated to 1 lag of pIJ702 (cleaved with Bg/II and treated with alkaline phosphatase) in a volume of 50 lal and transformed into protoplasts of S. lividans TK64. Protoplasting and transformation was done as described in Hopwood et al. (1985). Transformants were selected by overlaying the R2YE plates, after 12 h incubation, with 3 ml R2YE soft agar containing 1.25 mg thiostrepton (kindly provided by Squibb and Sons). DNA Preparation. For small-scale plasmid preparations from S. lividans and E. eoli the method of Kieser (1984) was used. For D N A sequencing, plasmid D N A was prepared according to a protocol of Holmes and Quigley (1981) with the following modifications. After boiling and centrifugation of the cells, the supernatant was treated with 10 gg RNase for 20 min at 37°C and for 10 min at 55 ° C, twice extracted with phenol and once with chloroform/isoamylalcohol (24:1), precipitated with isopropanol, resuspended in 200 gl TEl0.01 and again precipitated and washed with 70 % ethanol. Chromosomal D N A from Streptomyces strains was prepared from 15 ml liquid cultures. The cells were col-

lected by centrifugation and resuspended in 8 ml lysis buffer (10% sucrose, 25 mM TRIS-HC1, 25 mM EDTA, pH 8.0), 40 mg lysozyme in 1 ml lysis buffer was added and after 45-60 mint at 37 ° C the cells were lysed with 1 ml 10% N-laurylsarcosine at 65°C for 10rain. The DNA was further purified by dye buoyant density centrifugation (Sambrook et al. 1989). DNA sequencing. Double-stranded plasmid D N A was sequenced with the chain termination method according to a protocol of Zagursky et al. (1986) using AMV reverse transcriptase (Pharmacia) for the sequencing reactions. Slight modifications concerned the labeling of D N A with [a -35S]dATP, lowering the ddNTP concentrations to 0.63gM ddATP, 1.6laM ddCTP, 0.8gM ddGTP and 3.2 gM ddTTP in the reaction mixes and raising the incubation temperature for the sequencing reactions to 50 ° C for 20 min followed by another 25 min at 50 ° C after addition of the chase solution. The D N A was separated on 6 % polyacrylamide gradient gels in a Macrophore electrophoresis unit and processed for autoradiography as recommended by the manufacturer (Pharmacia). The D N A sequences were analysed on a MicroVAX 3200 using the software program U W G C G (Devereux et al. 1984). For the codon preference program a codon usage table was set up based on eight Streptomyces gene sequences: tsr, vph, aph (Bibb et al. 1985), ORF438, mel (Bernan et al. 1985), endoH (Robbins et al. 1984), sph and apt (Tohyama et al. 1987). For multiple sequence alignment the program CLUSTAL in the software package H U S A R at the German Research Center in Heidelberg was used. Results

Mercury resistance in Streptomyces lividans strains An inducible mercury resistance was identified by Nakahara et al. (1985) in the S. Iividans strain 1326. This strain carries two plasmids called SLP2 and SLP3 revealed by its ability to form two different pocks on strains lacking one or the other plasmid (Hopwood et al. 1983). To determine whether the mercury resistance is encoded by one of the two plasmids, we tested S. lividans 1326 and some derivatives cured for one or both pock-forming activities (Table 1), for resistance to HgC12 in an inhibition zone assay. Only strains carrying SLP3, i.e.S, lividans 1326 and TK19, exhibited resistance (inhibition zone: 4 ram), the strains S. lividans TK20, TK21 and TK64, which lack SLP3, were sensitive (inhibition zone: 10 ram). To confirm this obvious correlation between SLP3 and mercury resistance, mating experiments were performed between TK19 (Mer 9 and TK64 (Mer e, pro, str). Mercury- and streptomycin-resistant, as well as mercuryresistant, proline prototrophic recombinants were selected on agar plates. Neither type of recombinant was obtained (recombination frequency < 10 -v per parental spore), whereas in a control mating between 1326 and TK64 both types of recombinants arose at a frequency of around 10 -4 per parental spore.

79 Bell Bell

Bell

JI

I



tsr

A

MluI Mlul ScaI Mlul I

BamHI BclI BamH[

Bgl[I

I)

BgllI BamHI

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mer

reel'

0

p JOE813

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I

mel"

[]

Mort

D

p JOE796-11

Mer r

----'--]

pJOE796-17 p JOE796-7

I

pJOE796-32

H

Fig. 1. Restriction map of pJOE796 and its derivatives. The cloned fragment with the rner genes is indicated by the hatched bar, the thiostrepton resistance marker tsr and the inactivated melanin genes (rnel' and reel" Katz et al. 1983) and their orientations are symbolised by arrows. The open bars below show the sizes and locations of several deletions introduced into pJOE796 (the deletion endpoints of pJOE796-32 were not exactly defined, they lie somewhere within the brackets) and the corresponding phenotypes and plasmid designations as in table 2. Phenotypes: Mer r, mercury resistance; Met (rf, partial resistance; Mers, sensitive; Merh~, hypersensitive

Mlul

Mer r I

p JOE796-33

Mer (r)

t--4 [

The strain T K 1 9 was further tested for its ability to form pocks on a lawn of the plasmid-free strain T K 6 4 as described in Materials and methods. The same was done with 1326 and T K 6 4 controls. Only colonies of 1326 clearly produced pocks. These results indicate that the correlation between SLP3 and mercury resistance is coincidental. Another correlation was fortuitously found. S. lividans strains are k n o w n to segregate spontaneously, in two steps, C m ~ A r g - m u t a n t s at a very high frequency. The double mutants usually have a 5.7 kb element amplified to a concentration of several hundred copies per c h r o m o s o m e (Altenbuchner and Cullum 1985). In one of these mutants (S. lividans 1326.32) we found additional amplification of a very long stretch of D N A to a b o u t 20 copies per chromosome. A 2 kb BylII fragment cloned f r o m this amplified D N A into an E. coli vector hybridized to c h r o m o s o m a l D N A of 1326 and TK19, but not to D N A derived f r o m TK20, TK21 and T K 6 4 in Southern hybridisation experiments (data not presented). As shown below, the mercury resistance genes are localized on this long amplifiable element.

Clonin9 o f the mercury resistance 9enes

The mer genes were cloned in order to characterize them in m o r e detail and to prove that they are encoded by an amplifiable element present in S. lividans 1326 and TK19. D N A of S. lividans 1326.32 containing this amplified element was inserted into the vector pIJ702 and transformed into the mercury-sensitive strain TK64. Transformants were selected with thiostrepton. A b o u t 3000 transformants were obtained, one third of them carrying insertions in the vector, as estimated from the inactivation of the melanin m a r k e r of pIJ702. Several of these clones showed increased mercury resistance after replica plating on to mercurochrome-containing M40 plates, but only one of them had the same high resistance level as S. liviclans 1326, when tested with HgC12 in the inhibition zone assay (4 m m each). The plasmid isolated f r o m this clone was n a m e d p JOE796. R e t r a n s f o r m a t i o n of pJOE796 into T K 6 4 reproducibly gave the same high level of resis-

]

0

MerhS

1

2

3

4

5

6

1

2

3

4

5

6

Fig. 2. Agarose gel with genomic DNA from Streptomyces lividans 1326 (lanes 1, 3, 5) and the Cm~Arg- mutant S. lividans 1326.32 (lanes 2, 4, 6) and the corresponding autoradiogram of a Southern hybridisation analysis with 32P-labelled pJOE796 DNA (right). The genomic DNA was digested with BglII lanes (1 and 2), BamHI lanes (3 and 4) and SacI lanes (5 and 6). Lane 0 contains a molecular weight marker ()~ DNA digested with HindIII) tance to HgC12 (data not shown). Furthermore, T K 6 4 with pJOE796 exhibited the same broad-spectrum resistance as S. lividans 1326 (inhibition zones caused by P M A were 10 m m each, compared to 24 m m for T K 6 4 with pIJ702, Table 2). The restriction m a p of p JOE796 shown in Fig. 1 revealed the presence of a 6 kb insertion in pIJ702. Southern hybridization of 32P-labelled pJOE796 D N A to S. lividans 1326 wildtype and the C m ~ A r g - derivative gave a much stronger signal with D N A of the mutant, which proves that the cloned mercury resistance genes are indeed located on the amplified element (Fig. 2). The restriction pattern of the cloned fragment coincided with that of the hybridizing c h r o m o s o m al D N A in the wild-type and m u t a n t strains.

Deletion analysis o f the cloned mercury resistance 9enes

In order to localize the mer genes more precisely several deletions were introduced into pJOE796, the plasmids

8O 1

GGGCCGAGCAAGCCGCCCAGGAGCAGCAGGAGCGCGAAGCCACGGTTGGCGCCG ATCGGGGCGTGCGGGCTTCTCCGCTCGGCCGGGGAG

91

GCCG TGTCGCGGGCTGCTGTCGTGGTCA TGTGGGGTTCCGTTCCGGCAGTTG CCCGCGC.CGGGGGC T TGGTCTG TTCCCGC-CCGCAGCGG

181

GT~TGGGCGT(?~GGCGCGGTCAGCAGCAGTGGCCGTCGCTGTCGCGGTCGGTTGAGGCCGGTGTGGGGACGGGCAGGGGCGCGCA~AGTC

271

D A A A A S P A T G T P T 8 H R A P Q R T R H R I A A A A V GTCCGCTGCGGCGGCGGAGGGGGCTGTCCCGGTGGGAGTGGAGTGGCGGGCCGGCTGCCGTGTGCGGTGGCGGATGGCGGCGGCCGCCAC

361

A L V V V V A T V A I A T T H A Q I W A T A T A S F T A L G AGCGAGGACGACCA CCACGGCC,G TGACGGCGA TGGCGG TGGTA T G G ~ C T G G A TCCAGGCGG T G G ~ G TGGCGGAGAAGGTGG~AGGCC

451

G G P A A G C C G C ~ G G

541

T I R T G H R A L A T I K R T L A A G A A A T T V A V L L L GGTGAT~TGCCGTGCCGGGCCAGGGCGGTGATCTTGCGGGTGAGGGCGGCGCCCG~TGGTGACCGCGACGAGCAGCAG

631

V A A S G A A Y A A F V A L L G A F S A T A Q A Q A I V A L GACGGCGGCCGATCCGGCGGCGTAGGCGGCGAAGACCGCGAGGAGCCCGGCGAAGCTGGCGGTGGCCTGGGCCTGGGCGATGACGGCGAG

721

L V G F T C S L S A A A Y G V G F A A H R 8 A T P P CAGCACGCCGAAGGTGCACGACAGGGACGCGGCGGCGTAGCCGACGCCGAAGGCCGCCA T G C C ~ C G ~ T C ~ C C C ~ A ~

811

R T S T T L H L R L S P T R G T L H V A G L L L L L I G r A GCGGGTGCTGGTGGTCAGGTGCAGCCGCAGGCTGGGTGTGCGGCCGGTGAGCATGACGGCGCCGAGGAGCAGCAGGAGGATGCC~T~

901

L G L W P A A Q I L A R A G A S V I L G A A A L T G A F G L CAGGCCCAGCCAGGGGGCGGCTTGGA TCAGGGCGCGGGCTCCGGCGCTGACGATGAGTCCGGCGGCTGCGACCGTGCCGGCGAAGCCGAG

991

T L A A G S R L A R T L R V P L P S GGTGAGGGCGGCGCCC~AGCGCA~TGAGGCGTACCC~A~AGGCGTCG

1081

P L L A F G C P N V P A L H G A A F A L A L L G N H e- ORF4 • A G A GGGCAGCAGGGCGAAGCCGCAGGGGTTGACCGGGGCGAGCATCCCC~CGAAGGCGAGGGCGAGCAGGCCG~TCATCACGCCCCTGC~



1171

C

C

H

G

D

S

D

8

D

T

S

A

P

T

P

V

P

L

P

A

C

C

D

T T A G G T A A P Y W Y W A L Y A G T L V L V A A TGG TGGCGCCGCCGGTGGCAGCGGGGTACCAG T ACCAGGCCAGGTAGGCGCCGGTGAGGACGAGGACGGCGGC

A

D

G

A

S

R

S D G L A A T I W A CTGTCGCCCAGGGCGGCGG TGATCCAGGC

K K L A D Q I Q D A S P D T A R Y T V K G O P A V V I L T S TTC~TCAGGGCGTCCTGGA~CTGGTCGGCGGAGGGGTCC~TGGCCCGGTAGGTGACCT~GCC~TGC~ACGACGATCAGTGTGGAC RBS--

1261

L A A V Q Y 8 Q S L A A G K D I T A P L A P A K I Y D L F Q AGCGCC~CGACCTGGTAGCGCTGGGAGAGGGCGGCGCCCTTGTCGATGGTGGCGGGCAGAGC~CCTTGATG~AGTCGAGGAACTGC

1351

H I T Q K S E N P D H D V A L F N A K K G A K D F A Q A A K ATGATCGTCTGCTTCGACTCGTTCGGGTCC ATGTCCACCGCG AGGAAG T TGGCCT TCT TCC CC~CTTGTCGAACGCCTGGGCC~CTTG

1441

D L S K A G G A C E G C G V S F F F L A S P K D G P V A L T TCCAGGCTCTTGGCCCCGCCX3C~GCACTCGCCGCAGCCCACCGAGAAGAAGA~CAGGGCGGAGGGCTTGTCCC~ACCGCCAGCGTG

1531

T O D L L A V T" D A K A A A A N G S G A R T A P T N A K T D GTGTCGTCGAGGAGGGCGACGGTGTCCGCCTTGGCAGCGGCGGCGTTGCCGCTGCCAGCCCTCGTGGC~GGGGTGTTCGCCTTGGTGTCG

1621

T G C A S L T L A A T A A A A L A L A T R R R L R T R R A T G TACCGCAGGCGGACAGGGTGAGCGCAGCGGTGGCAGCCGCAGCCAGAGCCAGC~CGG T A ~ A G G C G ~ T ~ C G G T G

1711

P S P S T H ~-- ORF3 GGGGACGGTGAGGTCATGGGTGGTGCC, GGCCT T CCTCGAAGTCCGGGACTGGACGGGCGGTCCCC~TCC,C C A G G G A G C G A A ~ A ~ RBS

1801

" q K R L L D R D I.I q O GCGGCGGTGACCGGCAGAACGGC~TGAGCCGGGCGCCGC~CGGGTCACTGCTTGCGG AGCAGGTCGCC~TCGTGCTGGTCG

1891

T R P A P L C C GTG~CCGGGAGGCAGCAGGCA

1981

A P A L L W P S V L V G G L G A L A G S A L L V P G A C C I GCCC~CCAGCAGCCAGGCL;CTGACCAGCACCCCGCCGAGTCCGGCC AGGGCGCCGCTGGCCAGCAGCACCC~-,CCCGGCGCAGCAGATG

A

D G N G T R R R R L W W T L A G A L L V A TCGCCGTTGCCGG T G C C C ~ ~ C A G C C A C C A G G T G A G A G ~ C C ~ C C A G C A G G A C C C ~

(-- ORF2 I

2071

2161

P

L

L

A

V

G

V

V

A

L

T

G

L

L

G

G

IR R

D

G

P

Q

T

P

P

P

T

H " R ATCGGCAGCAGGGCGACGCCGACGACC~CGAGGG TGCCGAGCAGGCCGCCGCGGCGGTCACCCE3GCTGGGTGGGCC~A~TCAT~T Q E G E G P I R T C C D L A A A N D A A L C R A L H V L D A GC~CCCTCTCCGGGGATGCGGGTG~AGCAGT~CAGGGCC~TTGTCGGCGG~CAGGCACC~CAGCATGACCAGATCCC~A RBS

2251

V R CGC~

2341

S V R P Q S I G A H E V C E A S r R E G 8 L I F Q L L K L R ACACCCC~TGGGAGATACCGGCGTGCTCGACGCACTCCGCGCTGGTGCGCTCGCCACGCAGGATGAAC~A~A~TTCA~

2431

T P D A L A R F F R A T T D P H T C P V E A T A L S G A L A TGGGGTCGGCCAGCGCGCGGAAGAACC~CCGTGGTGT C C ~ T C ~ T G C A C G G C A C C T C G G C C G T G G C C A G G G A T C C G G C ~ A G

2521

2611

P

D G V S Y R L K K G D R R A S V Y G C D V L C S L H V TCGCCGACGGAGTAGCGCAGTTT CTTGCCGTCCCGC~GGC~CTCA CATAGCCGCAG TCCACGAG-ACAGGACAGGTGGACCG

(--- ORFI P S K M - 352 - 10 GGGATTTCATGGG~AGGGC~TT~CCACCAGCAGCTATT~GTCTGCCCGCATAGTAAGGTGGCGGTCTGCAATACGC~AAGCCGCATAGGA RBS - 10 35

~TC~TCCGGCGTCC~GCGCTGGA~C~CA~TGAGGGAGGTAGTGCTGT~CTCCAGGCACACACCGGTT~CGACCTGG~I~BS G~TCAT~GGCTC ORF5 (merA)--)

H

L

~

A

H

_T G

Y

D.D. L

_A I

I

G_ S_

2701

GGGCGCCGGCGCATTCGCCGCGGCCATCGCGGCCCGCAACAAGGGCAGGAGTG•GGTGATGG•CGAGCGCGGCACCACCGGCGGCACCTG G A G A F A A A I A A IR N K G R S V V H V E R C T T G G T C

2791

CGTGAACGTCGGCTGCGTGCCGTCCAAGGCGCTGCTGGCCGCCGCCGAGGCCCGTCACGGCGCCCAGGCGGCGAGCCGGT•CCCCGGCAT V N V G C V P S K A L L A A A E A R H G A Q A A S R F P G I

Fig. 3. Nucleotide sequence of the m e r genes and predicted polypeptides. Only the upper D N A strand is shown. The predicted amino acid sequences are given in the standard one-letter code, stop codons are indicated by asterisks. The amino acids are written above the first base of the corresponding codon when genes are transcribed from right to left and below for genes transcribed in the opposite direction. Putative ribosomal binding sites (RBS) and promoter sequences ( - 1 0 , - 3 5 ) are underlined, palindromic D N A sequences are marked by arrows. The sequence will appear in the GenBank Nucleotide Sequence Database under accession number X65467

81 2881

ACA~CCAC~GAGC~CGCGCT~ACTTCCCCGCGCTGATCAGC~CAA~ACACGCT~TC'C`C`GCA~TGCGGGC~AGAAGTA~A~A Q A T E P A L D F P A L I S G K D T L V G Q L R A E K Y T D

2971

~CT~CCG~CGAGTAC~CT~AGATCGTG~AC~CAC~GCCACC~TCG~CGAC~CCCCA~GCT~A~TCG~CTGAACGA~C~ L A A E Y G W Q I V H G T A T F A D G P H L E V A L N D G G

3061

CACCGCCACCGTCGA~CCGCCCACTACCTGATCG~CACC~CTC~CCC~CACCGCGCCGCACATCGAC~CCT~ACCA~T~ACTA T A T V E A A H Y L I A T G S A P T A P H I D G L D Q V D Y

31SI

CCTGACCTCCACCAC~CCAT~AACTCCAGCAGCTCCCCGAGCACCTGCTGATCCTC~C~C~CTACGTC~TCT~A~A~CCCA L T S T T A M E L Q Q L P E H L L I L G G G Y V G L E Q

A

3241

G~TCTTC~CGCCTC~CAGCCGGG~CACCCT~CCGTCCG~T~C~GCC~CCTCCCGGGAAGAGCC~AGAT~TCC~T~GA L F A R L G S R V T L A V R S R L A S R E E P E I S A G I E

3331

GAACATCTTCCG~GA~AGGGCATCACCGTCCACACCCGCACC~AGCTCCGTGC~TC~CCGCGAC~CGAG~AT~CTC~ACCCT N I F R E E G I T V H T R T Q L R A V R R D G E G I L A T L

3421

~ACC~CCC~AC~CGATCAGCA~TGCGCGCCAGCCACCTGCTCAT~CCACC~A~GAC~TCCGTCACCAAC~CCTC~CT~A T G P D G D Q Q V R A S H L L I A T G R R S V T N G L G L E

351t

GCGGGTCGGGGT~AAGACC~CGA~C~GA~TC~T~T~ACGAGTACCT~ACC~ACAA~CCCCGCATCTGGGC~CC~GA R V G V K T G E R G E V V V D E Y L R T D N P R I W A A G D

5601

CGTCACCTGCCACCC~ACTTCGTGTACGTCG~CG~CGCGCAC~CACCCT~TCGCCGACAACGCCCT~AC~CGCCGA~GCACCCT V T C H P D F V Y V A A A H G T L V A D N A L D G A E R T L

3691

~A~TACACCG~CCTGCCGAA~TCACCTTCACCAGCCCCGCCATC~CTC~T~ACTGACCGAGGCCCAGCTGA~CGAGGCC~TAT D Y T A L P K V T F T S P A I A S V G L T E A Q L T E A G I

3781

CGC~CACCAGACCCG~ACCCTGTCCCT~AGAACGTGCCCC~GCT~TCAACCGTGACACCCGC~CCT~TCAAGCTCATCGCCGA A H Q T R T L S L E N V P R A L V N R D T R G L V K L I A E

3871

ACGCGGCACCGGGAAGCTGCT~CCGCCCACGTCCTCGCCGAGGGAG~GGGCGACGT~ATCACCGCCGC~ACCTAC~GATCACCGCC~ R G T G K L L A A H V L A E G A G D V I T A A T Y A I T A G

3961

~CT~A~CGT~GA~AGCT~GC~CG~ACCTGGCA~CCCTACCTGAC~AT~CG~A~GTTGAAG~TCGCCGCCCAGAC~TT~A~CT~CGA L T V D Q L A R T W H P Y L T M A E A L K L A A Q T F T S D

4051

CG~CGCCAAGC~CTCCTGCTG~GC~GGCTGACCCACCCCACCCACCACCGCAAA~AC~CGCGCCCAT~ACTCCCA~CCCAGCA~TC

Q

R~ V

A

K

L

S

C

C

A

G

"

O~6(~rB)~

M

D

S

Q

A

Q

Q

L

4141

GCCA~CC~CTGACCACCGCGTTCAAC~C~C~AGCCGCCAGCTCTCGCCC~T~CTGT~CGCCCGCTACTC~AACTC~TCGC~CAG

4231

~GACCCGTGACCGTCGAGCAGATCGCCCA~CCACCGACCGCACC~CGACCA~TCCGCGA~CTCGCCGCC~C~CGACACC G R P V T V E Q I A Q A T D R T P D Q V R E A L A A N P D T

4321

GAATACGACGAGA~AT~ACC~CAGC~CCTCACCCAGAACCCCACCCCGCACCA~TT~A~T~ACGGGCAGCAGCTCTAC E Y D E R G R I T G S G L T Q N P T P H H F E V D G Q Q L Y

4411

ACCT~TGCGCGCT~ACACCCTGATCTTCCCCGCCATC~TC~CCGCCCCGCCCACGTGACGTCCC~T~CA~CCACC~CACCCCC T W C A L D T L I F P A I L G R P A H V T S P C H A T G T P

4501

GTCCGCCTCACCGTCGAAC~ACCA~TCACCAGCGT~A~C~C~ACCGCCGTTGTCTCGATCGTCA~CCCCGA~GCC~C~C~T~ V R L T V E P D Q V T S V E P A T A V V S I V T P D A P A S

4591

ATC~GCA~CGCCTT~TGCAACCA~TCCACTTCTT~GC~AE~CCCGACGCGGGCAAGGGCT~CT~A~AGCA~CCCGTC~CAC~TC

4681

CTG~C~T~CCGATGCCTACCAACTC~CCGACCCCTCACCGAC-GC~T~CTCACCGGCGACACCC~CCCCC~CT~TG~TG~TCC~ L P V A D A Y Q L G R P L T E A L L T G D T P P G C C "

4771

TCCCC~ACGGG~AAC~CTGTGTGACACAG~TTAAGTATTCTGTGTCACACATGGGCTGAGCCGCTCCGAGCGACCT~A~AGATC~

A

T

R

L

T

T

A

F

N

G

G

G

A

A

S

S

R

P

W

L

W

R

P

L

L

Q

L

L

A

Q

I R T A F C N Q V H F F A T P D A G K G W L E E H P V A T V

4861

~TC~T~CGACGA~C~CTC~CAACGTTCTGTGTCACC~C~CCC.GA~TGCCCGCAC~CGTCCGTGATCCCTCTCCCGCGCACCTG

4951

GATC~CGA~ACGTACG~GAAACGGGCCC~AGATCACCGCGTACTTCCCGCGC~CC~CCGTGCCCCCAC~CG~A~ACC~CTAG

8041

CACGAC~ACA~CC~CTCACCCGCCCGCAGCT~TTCC

were transformed into TK64 and cells were analysed for mercury resistance. The deletion of a 2.1 kb Bg/II fragment (pJOE796-33) not only led to the complete loss of resistance to HgC12 and PMA but resulted in transformants that were even more sensitive to HgC12 than was TK64 carrying only the vector pIJ702 (mercury hypersensitive, Fig. 1 and Table 2). A small deletion involving both ends of the cloned fragment was obtained by inserting the 5.5 kb MluI fragment from pJOE796 between the two MluI sites in the tyrosinase gene of pIJ702 (p JOE813). In this case, no change in the resistance level was observed. An attempt to delete the 2 kb BamHI internal fragment in the cloned 6 kb region failed. Of 12 transformants analysed, all showed a larger deletion than expected, covering almost the whole insertion in

Fig. 3 (continued)

pJOE796 and including vector DNA. A typical example is pJOE796-32. Finally a series of derivatives was obtained by cutting D N A of pJOE796 at the single ScaI site and progressively deleting D N A from both ends with Bal31 nuclease. Before ligation, a double-stranded octanucleotide with a HindIII recognition site was added to facilitate the mapping of the deletion endpoints. Three examples are shown in Fig. 1. Deletions of 250 bp (pJOE796-11) and 850bp (pJOE796-17) had no influence on mercury resistance, whereas the deletion of 2.1 kb in pJOE796-7 significantly decreased the resistance level (Table 2). The deletion analysis thus localized the mercury resistance genes to a 5 kb region extending from the right deletion endpoint of pJOE796-17 to the right hand Mlul site of pJOE796 (Fig. 1).

82 DNA sequence analysis of the mercury resistance determinant For DNA sequencing, the 5 kb HindIII/MluI fragment from p JOE796-17 encoding the mer genes was inserted in both orientations into the Sinai site of the plasmid pIC19H (Marsh et al. 1984) after the 5' protruding ends of the fragment had been filled in with Klenow polymerase. The two resulting plasmids, pJOE851.1 and p JOE851.2, conferred no mercury resistance to the transformed E. coli strain JM 109 (Table 2). A set of overlapping deletions was generated in these two plasmids by unidirectional digestion with exonuclease III (Henikoff 1984), starting from the polylinker region to the left of the integration site and extending into the insert fragment in steps of about 250 bp. The deletion derivatives allowed the sequencing of both strands of the DNA cloned in pIC19H with the chain termination method modified for double-strand sequencing according to Zagursky et al. (1986). The 5081 bp DNA sequence is shown in Fig. 3. Because of its high GC content, Streptomycetes use mainly codons with G or C at the third position (Bibb et al. 1984). A codon usage table generated from eight Streptomyces genes was used to scan all six possible frames of the determined DNA sequence using the codon preference program (Gribskov et al. 1984). Six ORFs matching the codon usage of a typical Streptomyces gene were indentified (data not shown). One of them (ORF5) begins with GUG as translation initiation codon. GUG as a start codon has also been reported for several other Streptomyces genes, such as two protease-encoding genes from S. 9riseus (Henderson et al. 1987). ORF2 begins in a region with many rare codons followed by a second region lacking them. Because there is a another ATG codon at the junction, preceded by a potential ribosomal binding site (Bibb 1986), only the second half of this ORF was considered to represent a gene. Potential ribosomal binding sequences were also identified in front of all other ORFs (Fig. 3). Interestingly, ORF1 and ORF2, as well as ORF3 and ORF4, have overlapping start-stop codons. The ORFs are clustered in two divergently oriented sets of four and two genes. Another ORF begins the end of the group of four genes that are transcribed leftwards (not shown), and might represent a further gene involved in mercury resistance but of undefined function. The two ORFs transcribed from left to right were identified as rnerA, for coding mercuric reductase and merB encoding organomercurial lyase by amino acid sequence comparisons (see Discussion). They are followed by a palindromic sequence; such palindromes are considered to act as transcriptional terminators in Streptomyces (Bibb et al. 1985; Tohyama et al. 1987). A further palindromic sequence found in the region between the two divergently transcribed sets of ORFs may be involved in regulation. In the same region, two putative promoter sequences were identified by sequence comparison with known streptomycete promoters. The promoter for O R F 1 0 R F 4 matches the consensus sequence of E~7°-like promoters, the divergently orientated promoter for ORF5-ORF6 has only sequence sim-

ilarities in the - 1 0 but not in the - 3 5 hexameric sequence. This is not unusual, as streptomycete promoter regions contain a wide diversity of sequences (Strohl 1992).

Discussion Six ORFs were identified in the DNA sequence encoding mercury resistance in S. lividans. They were translated into the corresponding amino acid sequences and compared to known Mer proteins of other species using the "BESTFIT" program (Devereux et al. 1984). Similar size (215 versus 216 amino acids) and 54 % identity in amino acid sequence with the organolyase from the S. aureus plasmid pi258 (Laddaga et al. 1987) clearly identifies ORF6 as merB, responsible for the broad resistance spectrum in S. lividans 1326 and strains carrying pJOE796. A high degree of similarity (53.5% identity at the amino acid level) was also found to the organolyase of Bacillus RC602 (Wang et al. 1989) and somewhat less (42%) to MerB from pDU1358 (Griffin et al. 1987). A further similarity was found between ORF5 and the mercuric reductase encoded by merA in other systems (Fig. 4). The protein encoded by ORF5 shares 49% identity over a total of 473 amino acids to the mercuric reductase from pi258 and 46% to that from TnS01 (Brown et al. 1983). The main difference between MerA from S. lividans and all others sequenced so far is the lack of an about 80 amino acid N-terminal sequence, which is present in all other enzymes and even occurs twice in the MerA from Bacillus RC607 (Wang et al. 1989). The function of this domain is not clear. Fox and Walsh (1983) showed that the N-terminal domain of the Tn501encoded mercuric reductase can be cleaved off proteolytically, without loss of in vitro activity. Bogdanova and Mindlin (1989) have described a group of mercuric reductases that are resistant to this protease treatment, and therefore postulated the existance of a class of enzymes lacking this domain. To verify the N-terminal sequence of the S. lividans MerA protein it was sequenced by D. Tribie (Hoechst AG) using purified MerA protein kindly provided by M. Moore and E. Pai (unpublished). For purification the merA gene was overexpressed in S. lividans using a DNA amplification vector system developed in our laboratory. Using this product we also confirmed the function of MerA in enzyme assays (oxidation of NADPH in the presence of Hg2+; Eichenseer et al. 1991). The first 18 amino acids determined from the N-terminus of MerA by Edman degradation were identical to these deduced from the DNA sequence and are underlined in Fig. 3. The MerA protein of S. lividans very probably represents the first sequenced member of the class of mercuric reductases proposed by Bogdanova and Mindelin (1989) on the basis of biochemical evidence. The other four ORFs found on the 5 kb fragment are transcribed in the opposite orientation to merA and merB. Deletion ofmerA and merB from p JOE796 results in hypersensitivity to mercury caused by the remaining mer genes. In other mer operons this phenotype is asso-

83 Tn501

pi258 Bacillus Tn501 p1258 Bacillus

Tn501

pi258 Bacillus S. lividans

Tn501 pi258 Bacillus S. lividans

Tn501 pi258 Bacillus S. lividans

Tn501 p1258 Bacillus S. lividans

TnSOl p1258 Bacillus S. lividans

TnSOl pi258 Bacillus S. lividans

Tn501 p1258 Bacillus S. lividans

Tn501 pi258 Bacillus S. lividans

Tn501 p1258 Bacillus S. lividans

MTH LKITGMTCDSCAAHVKEALEKVPGVQSALVSYPKGTAQLAIVPGTSPDALTAA MTQNSYKIPIQGMTCTGCEEHVTEALEQA GAKDVSADFRRGEAIFELSDD QIEKAKQN M KKYRVNVQGMTCSGCEQHVAVALENM GAKAIEVDFRRGEAVFELPDDVKVEDAKNA

56 58

VAGLGYKATLADAPLADNRVGLLDKVRGWMAAAEKHSGNEPPVQVA ISAAGYQPGEEESQPSENSVDFNRDG IADANYHPGEAEEFQSEQKTNLLKKYRLNVEGMTCTGCEEHIAVALENAGAKGIEVDFRR

102 83 117

VIGS DYDLLIIGS GEALFELPYDVDIDIAKTAITDAQYQPGEAEEIQVQSFIiRTDVSLNDEGNYDYDYIIIGS MLQAHTGYDLAIIGS

106 92 177 15

GGAAMAAALKAVEQGAQVTLIERGTIGGTCVNVGCVPSKIMIRAAHIAHLRRESPFDGGI GGAAFSAAIKANENGAKVAMVERGTVGGTCVNIGCVPSKTMLRAGEINGLAQNNP FTGL GGAAFSSAIEAVALNAKVAMIERGTVGGTCVNVGCVPSKTLLRAGEINHLAKNNP FVGL GAGAFAAAIAARNKGRSVVMVERGTTGGTCVNVGCVPSKALLAAAEARHGAQAASRFPGI

57

166

152 237

75 226

AATVPTIDRSKLLAQQQARVDELRHAKYEGILGGNPAITVVHGEARFKDDQSLTVRLNEG QTSTGAADLAQLTEQKDGLVSQMRQEKYIDLIEEY GFDLIRGEASFIDDKTIQVNGQN HTSASNVDLAPLVKQKNDLVTEMRNEKYVNLIDDY GFELIKGESKFVNENTVEVNGNQ QATEPALDFPALISGKDTLVGQLRAEKYTDLAAEY GWQIVHGTATFADGPMLEVALNDG

210 295 134

GERVVMFDRCLVATGASPAVPPIPGLKESPYWTSTEALASDTIPERLAVIGSSVVALELA ITSKSFLIATGASPAVPEIPGMNEVDYLTSTSALELKEVPQRLAVIGSGYIAAELG ITAKRFLIATGASSTAPNIPGLDEVDYLTSTSLLELKKVPNRLTVIGSGYIGMELG GTATVEAAHYLIATGSAPTAPHIDGLDQVDYLTSTTAMELQQLPEHLLILGGGYVGLEQA

266 351 194

286

QAFARLGSKVTVLARNT LFFREDPAIGEAVTAAFRAEGIEVLEHTQASQVAHMDGEFVL QMFHNLGTEVTLMQRSERLFKTYDPEISEAIDESLTEQGLNLITGVTYQKVEQ NGKSTS QLFHNLGSEVTLIQRSERLLKEYDPEISEAITKALTEQGINLVTGATYERVEQ DGDIKK QLFARLGSRVTLAVRS RLASREEPEISAGIENIFREEGITVHTRTQLRAVRR DGEGIL

345 325 410

TTTHGE LRADKLLVATGRTPNTRSLALDAAGVTVNAQGAIVIDQGMRTSNPNIYA IYIEVNGQEQVIEADQVLVATGRKPNTETLNLESAGVKTGKKGEVLTNEYLQTSNNRIYA VHVEINGKKRIIEAEQLLIATGRKPIQTSLNLHAAGVEVGSRGEIVIDDYLKTTNSRIyS ATLTGPDGDQQVRASHLLIATGRRSVTNGLGLERVGVKTGERGEVVVDEYLRTDNPRIWA

400 385 470 312

AGDCTDQPQFVYVAAAAGTRAAIN MTGGDAALDLTAMPAVVFTDPQVATVGYSEAEAHH AGDVTLGPQFVYVAAYEGGIVANNALGLAKRKIDLRFVPGVTFTNPSIATVGLTEQQAKE AGDVTPGPQFVYVAAYEGGLAARNAIGGLNQKVNLEVVPGVTFTSPSIATVGLTEQQAKE AGDVTCHPDFVYVAAAHGTLVADNALDGAERTLDYTALPKVTFTSPAIASVGLTEAQLTE

459 445 530

DGIETDSRTLTLDNVPRALANFDTRGFIKLVIEEGSIIRLIGVQAVAPEAGELIQTAALAI KGYDVKTSVLPLDAVPRALVNHETTGVYKLVVNAQTQKLIGAHIVSENAGDVIYAATLAV KGYEVKTSVLPLDAVPRALVNRETTGVFKLVADAKTLKVLGAHVVAENAGDVIYAATLAV AGIAHQTRTLSLENVPRALVNRDTRGLVKLIAERGTGKLLAAHVLAEGAGDVITAATYAI

519 505 590 432

RNRMTVQELADQLFPYLTMVEGLKLAAQTFNKDVKQLSCCAG QFGLTIEDLTDSFAPYLTMAEGLKLAALTFDKDVSKLSCCAG KFGLTVGDLRETMAPYLTMAEGLKLAVLTFDKDVSKLSCCAG TAGLTVDQLARTWHPYLTMAEALKLAAQTFTSDVAKLSCCAG

ciated with a functional Hg 2÷ transport system but an inactive mercuric reductase, leading to enhanced accumulation of toxic Hg 2+ relative to strains lacking all mer genes (Nakahara et al. 1979). Therefore one or several of the four remaining ORFs should encode a mercury uptake system. According to the Kyte and Doolittle (1982) hydropathy plot (data not shown) two hydrophobic polypeptides are specified by ORF2 and ORF4, which may be responsible for mercury uptake. The polypeptide encoded by ORF2 has a deduced molecular weight similar to that of MerT in gram-negative systems, but lacks significant sequence similarity. The protein encoded by ORF4 shows 29% identity in amino acid sequence with a polypeptide predicted from an O R F of unknown func-

561 547 632 474

252

372 Fig. 4. Alignment of the mercuric reductase sequences (merA) from TnS01 (Brown et al. 1983), pi258 (Laddaga et al. 1987), Bacillus sp. RC607 (Wang et al. 1989) and S. lividans. Amino acids common to all four proteins are marked by asterisks. (Parameters for C L U S T A L pairwise similarity scores: K-tuplelength = 1, gap penalty = 3, window size = 10; multiple alignment, gap penalty = 10)

tion in the mer operon of pi258 (fortuitously also designated ORF4) (Babich et al. 1991). The similarity between these two proteins is also seen in the nearly identical patterns of hydrophilic and hydrophobic regions in the Kyte and Doolittle (1982) hydropathy blot (not shown). The two presumptive transport proteins from S. lividans might act independently of each other, as is supposed for the mer locus of T n 2 1 . Here the Hg 2+ ions are taken up mainly by MerT, but there is a second gene m e r C (Misra et al. 1985). This second gene was also found in Thiobacillus ferrooxidans, where it seems to be the only mercury uptake system (Kusano et al. 1990). Another protein involved in Hg 2÷ uptake of gramnegative bacteria is MerP, the periplasmic Hg 2÷ binding

84 Table 3. Properties and possible functions of the six predicted polypeptides encoded in the mer region of Streptomyces lividans

Coding sequence

Calculated MW

Properties

Similarity to

Percentage identity"

Possible function

ORF1 ORF2 ORF3

13483 10353 19345

ArsR b -

29 -

ORF4 ORF5 ORF6

31552 23025 49652

Hydrophilic Hydrophobic Hydrophilic signal sequence? Hydrophobic Hydrophilic Hydrophilic

ORF4 c MerB c MerAc

29 54 49

Regulator Hg 2+ transport Extracellular Hg2+ binding Hg 2+ transport Organolyase Mercuric reductase

a Percentage amino acid sequence identity according to the BESTFIT program (Devereux et al. 1984) between predicted S. lividans Mer proteins and related proteins b ArsR from Staphylococcus xylosus (Rosenstein et al. 1992) c ORF4, MerA and MerB encoded by pi258 (Laddaga et al. 1987)

protein. A similar function has been proposed for the mer operons o f pi258 and Bacillus RC607 (Wang et al. 1989; Laddaga et al. 1987). In S. lividans the function o f this extracellular binding protein m a y be carried out by the gene p r o d u c t encoded by ORF3. O R F 3 specifies a protein with a calculated molecular weight o f 19345 and a potential signal peptide for export, i.e. five arginine residues at the N-terminus followed by a short hydrophobic region and a signal peptide cleavage site (LSA+C) at position 31-34. The cleavage site is typical for lipoproteins like the 13-1actamase I I I f r o m Bacillus cereus 569/H (Hussain et al. 1987), which are processed by signal peptidase II (von Heijne 1989) and anchored to the m e m brane at the first cysteine residue. Besides the transport system a regulatory protein should be encoded by one of the four ORFs. The m o s t likely candidate is the protein encoded by O R F 1 with a calculated molecular weight of 13483 and an amino acid composition typical of cytoplasmic proteins. Despite its similar size, no h o m o l o g y was found between this and the other related and relatively uniform M e r R proteins sequenced so far. However, S. Silver (personal c o m m u n ication) has noticed a 25 % identity between the S. lividans ORF1 and the A r s R regulatory protein of an arsenical resistance m a r k e r f r o m the E. coli plasmid R773 (San Francisco et al. 1990) and an even higher degree of identity (29 %) with a related system f r o m Staphylococcus xylosus (Rosenstein et al. 1992), which we have confirmed. Preliminary results with x y l E transcriptional fusions indicate an autoregulatory repressor function for O R F 1 , in contrast to the repressor-activator function of all other M e r R proteins. The molecular weights, hydrophobicity and identity in amino acid sequence to other k n o w n proteins o f the six O R F s are summarized in Table 3. By Southern hybridization it was shown that the cloned fragment determining mercury resistance is located on a large amplifiable D N A sequence. Spontaneous amplification o f D N A sequences up to several hundred copies is quite c o m m o n in Streptornyces. The nature of these amplifiable D N A sequences and the molecular mechanism of amplification are still unknown. At the beginning of this work a correlation between mercury resistance and the plasmid SLP3 was noticed but we were unable to demonstrate the presence of this

plasmid in our strains by mating experiments or pock formation. The pocks formed by SLP3 are not easy to see (T. Kieser, personal communication) and they might have been missed under the conditions we used. It is also k n o w n that SLP3 does not mobilise c h r o m o s o m a l D N A , in contrast to most other Streptomyces plasmids (Hopw o o d et al. 1984), thus precluding the use of this property to demonstrate the presence o f the plasmid. Therefore, precise relationship between this plasmid and the mer locus remains unresolved. Acknowledgements. We thank Mrs. Youvenal and Dr. D. Tripier for the protein sequencing, Drs. M. Moore and E. Pai for the sample of purified MerA protein and Prof. D. Hopwood and Dr. T. Kieser for the S. lividans strains. We are also grateful to Prof. S. Silver, Prof. R. Schmitt and Prof. R. Mattes for helpful discussions, support and encouragement. This work was supported by the Deutsche Forschungsgemeinschaft.

References

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Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans.

A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cms), arginine auxotrophic (Arg-) mutant of Streptomyces...
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