of Staphylococcus aureus - NCBI

4 downloads 0 Views 2MB Size Report
MAX LECHNER,1 JEAN-MICHEL BRAVO,3 KARL PORALLA,2 AND FRIEDRICH GOTZl* ...... Chamovitz, D., N. Misawa, G. Sandmann, and K. Hirschberg. 1992.
JOURNAL OF BACrERIOLOGY, Dec. 1994, p. 7719-7726

Vol. 176, No. 24

0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Genetic and Biochemical Analyses of the Biosynthesis of the Yellow Carotenoid 4,4'-Diaponeurosporene of Staphylococcus aureus BERND WIELAND,' CORINNA FEIL,2 EVA GLORIA-MAERCKER,1 GUNTHER THUMM,1 MAX LECHNER,1 JEAN-MICHEL BRAVO,3 KARL PORALLA,2 AND FRIEDRICH GOTZl* Mikrobielle Genetik' and Mikrobiologie, Botanisches Institut,2 Universitat Tubingen, D-72076 Tubingen, Federal Republic of Germany, and Ecole Nationale Superieure de Chimie de Mulhouse, F-68093 Mulhouse Cedex, France3 Received 25 July 1994/Accepted 6 October 1994

The major pigment produced by Staphylococcus aureus Newman is the deep-yellow carotenoid 4,4'diaponeurosporene; after prolonged cultivation, this pigment is in part converted to the orange end product staphyloxanthin. From this strain a 3.5-kb DNA fragment was identified which after being cloned into Escherichia coli and Staphylococcus carnosus conferred the ability to produce 4,4'-diaponeurosporene. DNA sequencing of this fragment revealed two open reading frames (ORFs) which are very likely cotranscribed. ORF1 encodes a 254-amino-acid hydrophobic protein, CrtM (Mf, 30,121). The deduced sequence of CrtM exhibits in three domains similarities to the sequences of Saccharomyces cerevisiae and human squalene synthases and phytoene synthases of various bacteria. ORF2 encodes a 448-amino-acid hydrophobic protein, CrtN, with an Mr of 50,853 whose deduced sequence is similar to those of phytoene desaturases of other bacteria. At the N terminus of CrtN a classical FAD-, NAD(P)-binding domain is found. Spectrophotometry and gas chromatography-mass spectrometry analyses of the carotenoid production of E. coli and S. carnosus clones containing either ORF1 or both ORFs together suggest that ORF1 and ORF2 represent the dehydrosqualene synthase gene (crtM) and the dehydrosqualene desaturase gene (crtN), respectively. The results furthermore suggest that the biosynthesis of 4,4'-diaponeurosporene starts with the condensation of two molecules of farnesyl diphosphate by dehydrosqualene synthase (CrtM); it is shown that the reaction product of this enzyme is dehydrosqualene and not squalene. Dehydrosqualene (4,4'-diapophytoene) is successively dehydrogenated by a desaturase (CrtN) to form the yellow main intermediate 4,4'-diaponeurosporene. The yellow-to-orange colony color of Staphylococcus aureus is one of the classical criteria for identification of this species. As early as 1882, Ogston connected the yellow-orange appearance of pus with the color of the infecting microorganisms (17). Later it was shown that the pigment should not be the only basis for classification, since it is not a very stable character. Pigmentation is usually apparent after 18 to 24 h of growth at 370C but is more pronounced when cultures are held at room temperature for 24 to 48 h longer. In particular, those S. aureus strains isolated from bovines or which are multiply antibiotic resistant are yellow pigmented (32). Nonpigmented (white) derivatives of S. aureus are often found in subcultures of stored organisms (23), and nonpigmented clones can arise in pigmented colonies, giving a sectored appearance (29). Nonpigmented variants are more susceptible to desiccation and to linolenic acid than the corresponding wild-type strains (14). Treatment with nitrosoguanidine can result in irreversible loss of the capacity to synthesize pigment (2). Although pigment production is a rather unstable character, the possibility that the respective genes are encoded on typical plasmids has been ruled out (14). Marshall and Wilmoth (19) isolated the pigments of S. aureus S41 and determined their chemical structures, identifying 17 compounds which are all triterpenoid carotenoids

possessing a C30 structure instead of the C40 carotenoid structure found in most other organisms. The main pigment is staphyloxanthin, an ct-D-glucopyranosyl-1-0-(4,4'-diaponeurosporene-4-oate) 6-0-(12-methyltetradecanoate), in which glucose is esterified with both triterpenoid carotenoid carboxylic acid and a C,5 fatty acid. The postulated biosynthetic pathway for staphyloxanthin starts with a head-head condensation of two molecules of farnesyl diphosphate to form dehydrosqualene (4,4'-diapophytoene) or squalene. 4,4'-Diaponeurosporene is formed by three or four successive dehydrogenation steps and is the first yellow carotenoid intermediate. The oxidation of the terminal methyl group of 4,4'-diaponeurosporene to form the carboxylic acid 4,4'-diaponeurosporene-4-oic acid proceeds via the aldehyde 4,4'-diaponeurosporene-4-al. The acyl compound staphyloxanthin, the final orange pigment in S. aureus, is formed via glucosyl-4,4'-diaponeurosporenoate (19). This pathway has been verified by the analysis of various S. aureus mutants in which intermediary products either were absent or accumulated (20). In this paper we report the characterization of two genes involved in the biosynthesis of the yellow carotenoid 4,4'diaponeurosporene and propose a pathway for its biosynthesis by analysis of intermediary products in the various clones.

* Corresponding author. Mailing address: Mikrobielle Genetik, Universitat Tubingen, Auf der Morgenstelle 28, D-72076 Tubingen, Federal Republic of Germany.

MATERIALS AND METHODS Bacterial strains, plasmids, and media. S. aureus Newman ATCC 25904 was the DNA donor strain for the pigment 7719

7720

J. BACTERIOL.

WIELAND ET AL.

biosynthetic genes. The genes were cloned in Escherichia coli JM83 by using plasmid pUC19 (31) and in Staphylococcus camosus TM300 (12, 28) by using both plasmids pCA44 (18, 25) and pCL19. pCL19 had been constructed by inserting the 320-bp PvuII fragment of pUC18, containing a multiple cloning site, into the single PvuII site of pCLP100 (5). S. camosus and E. coli were cultivated in B broth (pH 7.3) containing (per liter) 10 g of casein hydrolysate 140 (Gibco), 5 g of yeast extract (Difco), 5 g of NaCl, 1 g of K2HPO4, and 1 g of glucose. For pigment purification the strains were cultivated aerobically in a 10-liter fermentor at 370C for 24 h. S. aureus Newman was cultivated in a modified medium (19) containing (per liter) 22 g of casein hydrolysate (Gibco), 5 g of yeast extract (Difco), 5 g of NaCl, 6 ml of glycerol monoacetate, 2.1 g of NH2PO4, and 0.44 g of K2HOP4 (pH 7.2). Plasmid preparation, DNA transformation, and sequencing. Staphylococcal DNA was prepared by the cleared-lysate method essentially as described previously (22). Cells were lysed by the addition of lysostaphin (4.5 U/ml), and the DNA was isolated either by CsCl centrifugation or by using a Qiagen column 100 (Diagen GmbH, Hilden, Federal Republic of Germany [FRG]) according to the manufacturer's instructions. Transformation and cloning procedures for S. camosus have been described previously (12, 13). E. coli supercoiled plasmid DNA was prepared by modified alkaline lysis (7). E. coli cells competent for transformation were made by the method of Hanahan (15). Enzymes for molecular cloning were obtained from Boehringer (Mannheim, FRG), Bethesda Research Laboratories (Eggenstein, FRG), and Pharmacia (Freiburg, FRG); assay conditions were as recommended by the suppliers. All other recombinant DNA techniques were performed as described previously (6, 26). Both strands of the cloned insert of pUG1 were sequenced by double-stranded DNA sequencing (9), using the dideoxy procedure (27), the Pharmacia AutoRead sequencing kit, and the A.L.F. DNA Sequencer. Several sequences were determined by using specific primers synthesized with the DNA synthesizer from Pharmacia. DNA and protein sequences were analyzed by using the computer programs of Microgenie (Beckman) and PC/GENE (Intelli Genetics, Inc.). Extraction of pigments. Staphyloxanthin and intermediate carotenoids were isolated from S. camosus clones essentially as described previously (19). Cells were pelleted by centrifugation for 10 min and washed with 0.9% NaCl. The cell pellets were either used immediately or stored at -20°C; under this condition the carotenoids were stable for several months. Five grams (wet weight) of washed cells was resuspended in 80 ml of methanol, heated for 3 min in a water bath at 55°C, cooled, and centrifuged for 10 min at 20,000 X g. This extraction was repeated until all visible pigments were extracted. The methanol layers with crude pigments were pooled. For the extraction of carotenoids from S. aureus Newman and the E. coli clones, methanol was replaced by acetone (80 ml/5 g of cells [wet weight]) (21), since the carotenoids of these organisms were better extracted with acetone. The crude pigment extracts obtained were concentrated to small volumes in vacuo and extracted with ethyl acetate-1.7 M aqueous NaCl (1:1, vol/vol). The colored ethyl acetate extract was dried with anhydrous Na2SO4, and the solvent was then removed in vacuo. The residue was dissolved in ethyl acetate and subjected to silica gel column chromatography. The colored fractions were eluted with ethyl acetate by increasing the polarity (0 to 100% ethanol). The various fractions were evaporated to dryness and analyzed. All procedures were carried out at room temperature and in the dark to protect the pigments from degradation. For analysis of the product of the proposed dehydrosqual-

ene synthase (CrtM), 30 g of wet cells of E. coli(pUG9) was extracted with 200 ml of methylene chloride-methanol (1:2, vol/vol) for 1.5 h. The resulting cell pellet was extracted with 200 ml of methylene chloride-methanol-water (1:2:0.8; vol/vol! vol) for 1.5 h. To the combined extracts 200 ml of methylene chloride-water (1:1, vol/vol) was added, and the mixture was shaken. The lipids were recovered in the organic phase and concentrated to dryness under nitrogen. Before analysis, the lipid mixture was fractionated on a silica gel column, eluted with methylene chloride, evaporated to dryness, and stored at

-200C. TLC. Analytical and preparative thin-layer chromatography (TLC) procedures were employed. Carotenoids were separated on Silica Gel G/UV254 plates (Macherey-Nagel, Duren, FRG) with various solvent systems: (i) light petroleum-acetone (99:1, vol/vol), (ii) light petroleum-acetone (13:7, vol/vol), (iii) ethyl acetate-ethanol (95:5, vol/vol), and (iv) ethyl acetateethanol (99:1, vol/vol). Spots or bands from the TLC plates were recovered and analyzed by their absorption spectra in light petroleum. Enzyme assay. The qualitative assay for the farnesyl diphosphate (FPP) condensing enzyme was performed essentially as described previously (1). Cells of E. coi(pUG9) were washed and resuspended in 0.1 M Tris-HCl-50 mM EDTA (pH 8.0). After disruption of cells by X-press and centrifugation (8,000 X g), the supernatant (crude extract) was used for the assay. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.4), 10 mM MgCl2, 5 mM NADPH2, 25 puM FPP, 0.5 ,uCi of [3H]FPP, and 20 to 25 mg of protein per ml. After incubation for 2 h at 37°C, the reaction was stopped and the mixture was extracted with 400 ,ul of n-hexane-isopropanol (3:2). Separation of the extract was performed on Silica Gel 60 plates (Merck, Darmstadt, FRG). The solvent system was 8 cm with chloroform and, after drying, 16 cm with n-hexane. Radioactivity on the TLC plates was measured with a TLC radioactivity scanner (model LB 2821; Berthold Wildbad, FRG). Products were further analyzed and identified by highpressure liquid chromatography (HPLC) with diode array detection and by gas-liquid chromatography and mass spectrometry (GLCIMS). Absorption spectra. Pigments were quantitated in known volumes of light petroleum in quartz cuvettes with the Beckman DU 7500 photometer. The values for the specific extinction coefficients (E1 cm) were adapted from the molar extinction values of C30 carotenes reported previously (10). Values used for C30 carotenes are as follows: 4,4'-diapophytoene (dehydrosqualene), 1,009 at 286 nm; 4,4'-diapophytofluene, 2,105 at 347 nm; 4,4'-diapo-z-carotene, 3,415 at 400 nm; 4,4'-diapo-7,8,11,12-tetrahydrolycopene, 3,367 at 395 nm; 4,4'diaponeurosporene, 3,905 at 435 nm; 4,4'-diapolycopene, 4,450 at 466 nm; and staphyloxanthin, 1,920 at 462 nm. The extinction coefficient of 4,4'-diaponeurosporene was also used as a nominal value for 4,4'-diaponeurosporenoic acid (3,905 at 455 nm). HPLC. Carotenoids were separated by HPLC on an Li Chromsorb RP-2 (Merck) column with acetonitrile-H20 (60:40 or 90:10, vol/vol) or acetonitrile as the eluent. The various pigments were detected with a spectrum focus diode array detector (Beckman, Munich, FRG), and spectra of the elution peaks were directly recorded. GLC/MS. The GLC/MS analyses were carried out on a Finnigan MAT INCOS 50 spectrometer (70-e V electron impact). A DB-5 capillary column (30 m by 0.25 mm; film thickness, 0.1 tim) was used. Samples were injected onto the column at 50°C. The temperature program was 50 to 2200C at 20°C/min and 220 to 300°C at 6°C/min.

S. AUREUS 4,4'-DIAPONEUROSPORENE BIOSYNTHESIS

VOL. 176, 1994

7721

1 kb

WB)

pOC1

C

.: : ..:

w::: All-::::::

................ ..

Sy St

I 1'

..: : :.::b h............::: -

-

E Sy SSH

H/HCN

S

(B)

orange

.:-e -

pOC21 (pUGi)

E

crtN

crtM

I

on

ORF2

-1

O-

yellow

pUGIO

yellow

pUG5

yellow

pUG6

white

pUG8

white

pUG9

white

FIG. 1. Restriction map of pOC1 and subfragments cloned in E. coli or-S. camosus. pOC21 was constructed by inserting the 3.5-kb SphI fragment into pCL19 for transformation into S. camosus TM300; pUG1 has the same insert in pUC19 for transformation into E. coli. The orientations of ORFi (crtM) and ORF2 (crtN) on the insert in pOC21 and pUG1 are indicated by arrows. The colony colors of the various clones are indicated on the right. Only pOC1 conferred staphyloxanthin production in S. camosus. Yellow pigmentation was observed only in those clones containing intact crtM and crtN. B, BamHI; C, ClaI; H, HaeIII; Sy, StyI; St, StuI, E, EcoRI; S, SphI; N, NruI.

Nucleotide sequence accession number. The novel DNA sequence published here has been deposited in the EMBL sequence data bank and is listed under accession number X73889. RESULTS

Cloning of the carotenoid biosynthetic genes from S. aureus Newman in S. carnosus and E. coli. S. aureus Newman chromosomal DNA was partially digested with MboI. DNA fragments of 5 to 25 kb were ligated to BamHI-digested pCA44. The ligated DNA was transformed into the unpigmented strain S. camosus TM300; several yellow- and orange-pigmented S. camosus colonies were detected. Restriction analysis of one isolated plasmid, pOC1 (Fig. 1), revealed that the orangepigmented S. camosus clone carried a 12-kb DNA insert in that plasmid. Subcloning of fragments of the 12-kb insert in E. coli and S. camosus led to yellow and unpigmented clones for both organisms (Fig. 1). The smallest fragment which caused yellow pigmentation was the 3.5-kb SphI fragment, which was inserted in SphI-digested pUC19 [for E. coli(pUG1)] and pCL19 [for S. camosus(pOC21)] vectors. The formation of the yellow pigment in E. coli JM83 indicates that in this organism the corresponding genes are expressed and the precursor substrates for the biosynthesis must be present. Subclones with pUG6, pUG8, and pUG9 in which the 3.5-kb fragment is partially deleted or absent remained unpigmented. pUG9 was created by insertion of the 1.65-kb HaeIII fragment, which contains the entire crtM gene and part of the 5' region of the

crtN gene, at the SmaI site of pUC19; in pUG9 crtM is in the same orientation as lacZ. Sequencing of the 3.5-kb SphI fragment in pUG1. The 3.5-kb SphI fragment was sequenced by using primer walking and exonuclease III-digested pUG1 derivatives (Fig. 2). The SphI fragment contains two complete open reading frames (ORFs)

(ORFi and ORF2). ORF1 starts at position 229 with ATG and terminates at position 991 with TGA. The start codon is preceded 6 bp upstream by a perfect Shine-Dalgarno sequence, AGGAGG. Upstream of this ribosomal binding site is a promoter-like sequence. The 762-nucleotide ORFi encodes a 254-aminoacid protein (CrtM) with an Mr of 30,121 which is rather hydrophobic (39% hydrophobic amino acids). The deduced amino acid sequence of CrtM was compared with those of other enzymes involved in carotenoid biosynthesis. There are three boxes with pronounced similarities to the Synechococcus phytoene synthase (8) and the squalene synthases of two Erwinia species, Saccharomyces cerevisiae, and humans. One domain represents the postulated prenyl (farnesyl) diphosphate-binding motif (16) (Fig. 3A, CrtM, domain III). ORF2 starts at nucleotide 1101 with ATG and ends at position 2445 with TAG. The start codon is preceded 7 bp upstream by a nearly perfect Shine-Dalgarno sequence, AG GTGGT. A rho-independent transcription terminator sequence after the stop codon or another longer ORF was not detected within the next 400 bp (not shown). The 1,344 nucleotide ORF2 encodes a 448-amino-acid protein (CrtN) with an Mr of 50,853. This protein also is rather hydrophobic (38% hydrophobic amino acids); however, obvious membrane-

7722

J. BACTERIOL.

WIELAND ET AL. 1

TACTTGAGCTATACTACACAATTTATTTATCTGCATCGAAGGGTCGGCCAATTTTCTAATTTATTAATGGTATGTCATCCATTGTTATTTATGTTTTTTA

101

CTAAAATTTTCATCCAATCTTGGAAACAAACGCATCGTTATGGTGTAGTTGAATGGAAAGGTCGTCAATATTCTATATCTAAAGAACAATAAATCAAGGT

Cr:tM >

S/D

201 AATGGCATTTCAATATAGGAGGACTAGTATGACAATGATGGATATGAATTTTAAATATTGTCATAAAATCATGAAGAAACATTCAAAAAGCTTTTCTTAC M T M M D M N F K Y C H K I M K K H S K S F S Y

24

301 GCTTTTGACTTGTTACCAGAAGATCAAAGAAAAGCGGTTTGGGCAATTTATGCTGTGTGTCGTAAAATTGATGACAGTATAGATGTTTATGGCGATATTC A F D L L P E D Q R K A V W A I Y A V C R K I D D S I D V Y G D I

57

401

AATTTTTAATCCAAATAAAAGAAGATATACAATCTATTGAAAAATACCCATATGAACATCATCACTTTCAAAGTGATCGTAGAATCATGATGGCGCTTCA Q

501

F

Q

I

I

E

K

D

Q

I

S

I

E

Y

K

Y

P

E

H

H

H

Q

F

D

S

R

R

M

I

M

Q

L

A

91

GCATGTTGCACAACATAAAAATATCGCCTTTCAATCTTTTTATAATCTCATTGATACTGTATATAAAGTCAACAT1ACAATGTTTGAAACGGACGCTG H

601

L

A

V

Q

H

K

N

I

A

F

Q

S

Y

F

N

L

I

D

T

Y

V

K

N

V

I

L

Q

L

C

K

R

T

L

124

GAATTATTCGGATATTGTTATGGTGTTGCTGGTCGTAGGTGAAGTATTGACGCCGATTTTAGTGATCATGAAACACATCAGACATACGATGTCGCAAGAA E

L

F

G

Y

C

Y

G

V

A

G

R

R

S

S

I

D A

D

F

S

D

H

E

T

H

Q

T

Y

D

V

A

R

157

701 GACTTGGTGAATCGTTGCAATTGATTAATATATTAAGAGATGTCGGTGAAGATTTTGACAATGAACGGATATATTTTAGTAAGCAACGATTAAAGCAATA R L G E S L Q L I N I L R D V G E D F D N E R I Y F S K Q R L K Q Y

191

801 TGAAGTTGATATTGCTGAAGTGTACCAAAATGGTGTTAATAATCATTATATTGACTTATGGGAATATTATGCAGCTATCGCAGAAAAAGATTCAAGAT E V D I A E V Y Q N G V N N H Y I D L W E Y Y A A I A E K D F Q D

224

901 GTTATGGATCAAATCAAAGTATTTAGTATTGAAGCATCACCAATCATAGAATTAGCAGCACGTATATATATTGAAATACTTGGACGAAGTTGAGACAGGC V M D Q I K V F S I E A S P I I E L A A R I Y I E I L G R S *

254

1001

II

III

S/D TAACTATACATTACATGAACGTCTTTTGTGATAAGAGAAAAAGGCAAAGTTGTTTCATGAAAATAAATAGTAAATATCATAGAATATAGGTGGTTGAATA CrtN >

1101 ATGAAGATTGCAGTAATTGGTGCAGGTGTCACAGGATTAGCAGCGGCAGCCCGTATTGCTTCTCAAGGTCATGAAGTGACGATATTTGAAAAAAATAATA M K I A V I G A G V T G L A A A A R I A S Q C H E V T I F E K N N

33

1201 ATGTAGGCGGGCGTATGAATCAATTAAAGAAAGACGGCTTTACATTTGATATGGGTCCCACAATTGTCATGATGCCAGATGTTTATAAAGATGTTTTTAC N V G G R M N Q L K K D G F T F D M G P T I V M M P D V Y K D V F T

67

1301 AGCGTGTGGTAAAAATTATGAAGATTATATTGAATTGAGACAATTACGTTATATTTACGATGTGTATTTTGACCACGATGATCGTATAACGGTGCCTACA A C G K N Y E D Y I E L R Q L R Y I Y D V Y F D H D D R I T V P T

100

1401 GATTTAGCTGAATTACAGCAAATGCTAGAAAGTATAGAACCTGGTTCAACGCATGGTTTTATGTCCTTTTTAACGGATGTTTATAAAAAATATGAAATTG D L A E L Q Q M L E S I E P G S T H G F M S F L T D V Y K K Y E I

133

1501 CACGTCGCTATTTCTTAGAAAGAACGTATCGCAAACCGAGTGACTTTTATAATATGACGTCACTTGTGCAAGGTGCTAAGTTAAAAACGTTAAATCATGC A R R Y F L E R T Y R K P S D F Y N M T S L V Q G A K L K T L N H A

167

1601 AGATCAGCTAATTGAACATTATATTGATAACGAAAAGATACAAAAGCTTTTAGCGTTTCAAACGTTATACATAGGAATTGATCCAAAACGAGGCCCGTCA D Q L I E H Y I D N E K I Q K L L A F Q T L Y I G I D P K R G P S

200

1701 CTATATTCAATTATTCCTATGATTGAAATGATGTTTGGTGTGCATTTTATTAAAGGCGGTATGTATGGCATGGCTCAAGGGCTAGCGCAATTAAATAAAG L Y S I I P M I E M M F G V H F I K G C M Y C M A Q G L A Q L N K

233

1801 ACTTAGGCGTTAATATTGAACTAAATGCTGAAATTGAGCAAATTATTATTGATCCTAAATTCAAACGGGCCGATGCGATAAAAGTGAATGGTGACATAAG D L G V N I E L N A E I E Q I I I D P K F K R A D A I K V N G D I F

267

1901 AAAATTTGATAAAATTTTATGTACGGCTGATTTCCCTAGTGTTCCGGAATCATTAATGCCAGATTTTGCACCTATTAAAAAGTATCCACCACATAAAATT K F D K I L C T A D F P S V A E S L M P D F A P I K K Y P P H K I

300

2001 GCAGACTTAGATTACTCTTGTTCAGCATTTTTAATGTATATCGGTATAGATATTGATGTGACAGATCAAGTGAGACTTCATAATGTTATTTTTTCAGATG A D L D Y S C S A F L M Y I G I D I D V T D Q V R L H N V I F S D

333

2101 ACTTTAGAGGCAATATTGAAGAAATATTTGAGGGACGTTTATCATATGATCCTTCTATTTATGTGTATGTACCAGCGGTCGCTGATAAATCACTTGCGCC D F R G N I E E I F E G R L S Y D P S I Y V Y V P A V A D K S L A P

367

2201 AGAAGGCAAAACTGGTATTTATGTGCTAATGCCGACGCCGGAACTTAAAACAGGTAGCGGAATCGATTGGTCAGATGAAGCTTTGACGCAACAAATAAAG E G K T G I Y V L M P T P E L K T G S G I D W S D E A L T Q Q I K

400

2301 GAAATTATTTATCGTAAATTAGCAACGATTGAAGTATTTGAAGATATAAAATCGCATATTGTTTCAGAAACAATCTTTACGCCAAATGATTTTGAGCAAA E I I Y R K L A T I E V F E D I K S H I V S E T I F T P N D F E Q

433

2401 CGTATCATGCGAAATTTGGTTCGGCATTCGGTTTAATGCAACTTTAGCGCAAAGTAATTATTATCGTCACAAAATGTATCGCGAGATTATAAAGATTTAT T

Y

H

A

K

F

G

S

A

F

G

L

M

Q

L

*

448

2501 ATTTTGCAGGTGCAAGTACGCATCCAGGTGCAGCGCTTCCTATTGTCTTAACGAGTGCGAAAATAACTGTAGATGAAATGATTAAAGATATTGAGCGGGC 2601 GTATAAGGGAGTAGTCTAAGAGAAAGATGTGAGAAAAGTATAAGGGGAACGTAAAAGGCATAGTGAAATAAGTATGTGCTTAAGAAGTTTTATTTAACAA

FIG. 2. Nucleotide and deduced amino acid sequences of ORF1 (CrtM) and ORF2 (CrtN). Potential Shine-Dalgarno sequences (S/D) are underlined; stop codons are indicated by asterisks. The amino acid sequence is written below the nucleotide sequence. Roman numerals mark the approximate positions of conserved amino acid sequences (see Fig. 3).

S. AUREUS 4,4'-DIAPONEUROSPORENE BIOSYNTHESIS

VOL. 176, 1994

A

7723

3.0

Eh-CrtB Sc-Syn Hs-Syn

34 46 20 33 65 66

Sa-CrtM

125

Ss-Pys

133 116 129 173

Sa-CrtM Ss-Pys Eu-CrtB

RKAVWAIYAVCRKIDDSID RQAIWAIYVWCRRTDELVD RRSVLALYAWCRHCDDVID RRSVLMLYTWCRHCDDVID RNCVTLFYLILRALDTIED RNAVCIFYLVLRALDTLED

52 64 38 51 83 84

I

0 Cl)

II

Eu-CrtB Eh-CrtB Sc-Syn Hs-Syn

Sa-CrtM Ss-Pys Eu-CrtB

III

Eh-CrtB Sc-Syn Hs-Syn

166

159 180 151 164 214 207

E-LFGYCYGVAG EDLYTYCYRVAG DDTLRYCYHVAG EDTLRYCYHVAG HDYDVYCHYVAG QEWDKYCHYVAG

.Ja

135 144 127 140 184 177

LGESLQLINILRDVGEDFDNERIYFS LGIANQLTNILRDVGEDARRGRIYLP LGLAFQLTNIARDIVDDAHAGRCYLP

LGLAFQLTNIARDIIDDAAIDRCYLP MGLFLQKTNIIRDYNEDLVDGRSFWP MGLFLQKTNIIRDYLEDQQGGREFWP

0.0 184 205 176 189 239 232

Wavelength (nm)

0.10

B

ID

297

275 Sa-CrtN Eh-CrtI Rc-CrtI

3 4 11

IAVIGAGVTGLAAAARIASQGHEVTIFEKNNNVGGRMN TVVIGAGFGGLALAIRLQAAGIPTVLLEQRDKPGGRAY

AVVIGAGLGGLAAAMRLGAKGYKVTVVDRLDRPGGRGS

40 41 48

o0.05 FIG. 3. (A) The CrtM (proposed dehydrosqualene synthase) sequence (Sa-CrtM) was aligned with the sequences of the Synechococcus phytoene synthase (Ss-Pys) (8) and the squalene synthases of E. herbicola (Eh-CrtB) (3), E. uredovora (Eu-CrtB) (21), S. cerevisiae (Sc-Syn) (16), and humans (Hs-Syn) (24). There are three moderately conserved domains, domains I to III; domain III represents the postulated prenyl (farnesyl) diphosphate-binding motif (16). The entire CrtM sequence shows only a low degree of similarity (7.2%) to the other enzymes. (B) The CrtN (proposed dehydrosqualene desaturase) sequence was aligned with the sequence of the carotenoid desaturases of E. herbicola (3) and R. capsulatus (Rc-CrtI) (4). Only the NH2-terminal region of CrtN is shown; it reveals a conserved FAD-, NAD(P)-binding motif with the characteristic 1-sheet--helix,B-sheet structure. The entire CrtN sequence exhibits 15.4% identical and 34.1% similar amino acids. Identical and homologous amino acids are indicated by asterisks and points, respectively.

spanning domains characteristic of integral membrane proteins are absent. The deduced amino acid sequence was compared with those of other carotenoid biosynthetic enzymes. CrtN shows a pronounced similarity (30 to 35%) to the phytoene desaturases (CrtI) of Erwinia herbicola and Rhodobacter capsulatus. The NH2 terminus of CrtN (amino acids 2 to 29) is distinguished by a conserved amino acid sequence with homology to the FAD-, NAD(P)-binding domains of a series of dehydrogenases and oxidases (Fig. 3B). The phytoene desaturases catalyze the dehydrogenation reactions from phytoene to neurosporene (lycopene). Identification of carotenoids in S. carnosus(pOC21), E.

coli(pUG1), and S. aureus Newman. In the yellow-pigmented E. coli (pUG1), 4,4'-diaponeurosporene was identified by its characteristic absorption spectrum as a main carotenoid (Fig. 4A). 4,4'-Diapo-7,8,11,12-tetrahydrolycopene and 4,4'-diapolycopene were present in smaller amounts (Table 1). These results are consistent with the presence of the two genes, crtM and crtN, on the 3.5-kb DNA insert of plasmid pUG1. In TLC

0

I

240

280

320

Wavelength (nm) FIG. 4. (A) Absorption spectra of carotenoid extracts of E. coli with and without pUG1; only in the presence of the plasmid are the characteristic 4,4'-diaponeurosporene peaks present. (B) Absorption spectrum of dehydrosqualene after isolation of compound V (see Fig. 5) from the TLC plate and purification by HPLC.

with [3H]farnesyl diphosphate as a substrate and E. coli(pUG1) extracts, dehydrosqualene (compound V) and 4,4'diaponeurosporene (compound IV) were produced (Fig. 5, lane 3), indicating that the proposed dehydrosqualene synthase (CrtM) is catalyzing the condensation of two farnesyl diphosphates to dehydrosqualene and that the proposed dehydrosqualene desaturase (CrtN) is catalyzing all oxidation steps between dehydrosqualene and 4,4'-diaponeurosporene. In the yellow S. camosus(pOC21) we found essentially the same carotenoid spectrum (not shown) as in E. coli(pUG1), identifying 4,4'-diaponeurosporene as the main product at 22.3 ,ug/g of cells (dry weight) (Table 1). In S. aureus Newman extracts we found two main fractions by TLC analysis: one fraction was identified as 4,4'-diaponeurosporene, and the other contained the orange end product staphyloxanthin. The concentration of staphyloxanthin was only 50% of that of 4,4'-diaponeurosporene. This can be explained by the observation that 4,4'-diaponeurosporene is formed already after 12 h of cultivation, while staphyloxathin is

7724

WIELAND ET AL.

J. BACTERIOL.

TABLE 1. Identification and quantification of carotenoids in various staphylococci and E. coli(pUG1) Color of ofarotenoi content content

crtColor

Organisma crude Organism'

extract

(p~g/g [dry ~vt]) Carotenoid

TLC Rf valueb with the following light petroleum/acetate ratio (vol/vol): 99:1

S. aureus Newman

Yellow

S. camosus(pOC21) S. camosus(pC194) E. coli(pUG1) E. coli(pUC19)

Orange Yellow Pale yellow Yellow Pale yellow

a

b

50.0 24.3 22.3

21.8

Carotenoid identification

412, 433, 464 (412, 435, 465) 460, 491 (461, 491) 412, 434, 464 (412, 435, 465)

4,4'-Diaponeurosporene

Staphyloxanthin 4,4'-Diaponeurosporene

412, 434, 464 (412, 435, 465)

4,4'-Diaponeurosporene

13:7

0.34

0.99

-c

0.28(0.38) 0.98 0.96 0.98 0.96

0.36 0.1 0.36 0.28

km. (nm)b in light petroleum

S. aureus Newman was cultivated for 72 h aerobically; all other strains were cultivated for 16 h aerobically. Values in parentheses are from the literature (10, 19). c, not separated in this solvent system.

produced later, after 24 h of incubation. The amounts of the main carotenoids were estimated spectrophotometrically by using their specific extinction coefficients. The main carotenoids, their amounts in various strains, their Rf values on TLC, and the characteristic main peaks in the absorption spectra are summarized in Table 1. Identification of dehydrosqualene in E. coli(pUG1) and E. coli(UG9). In E. coli JM83 extracts no squalene was detectable. On the other hand, we identified squalene in S. camosus TM300 extracts, which is in agreement with earlier observations (30). This indicates that a squalene synthase must be chromosomally encoded in S. camosus. The finding of squalene in S. carnosus extracts raises the question as to the enzymatic activity of CrtM, which exhibits similarities with phytoene and squalene synthases. In order to answer the question of whether the C30 carotenoid biosynthesis starts with squalene or, in analogy to the C40 carotenoid biosynthesis, with dehydrosqualene (4,4'-diapophytoene), we constructed plasmid pUG9 containing only the intact crtM gene. In enzyme assays with cell extracts of E.

coli(pUG9), we never obtained squalene, irrespective of the presence of NAD(P)H. However, we could identify a compound with an Rf value slightly lower than that of squalene (Fig. 5, lanes 2 and 3, compound V) which was found in a very hydrophobic fraction of the lipid extract of E. coli(pUG9). The samples were separated by HPLC and analyzed by diode array spectroscopy (Fig. 4B). Only one peak occurred; it had an absorption maximum at 287 nm and shoulders at 275 and 297 nm. On the basis of the characteristic absorption spectrum (20), we identified this compound as dehydrosqualene. No dehydrosqualene was detected in control experiments with E. coli(pUC19). In GLC/MS analysis the isolated dehydrosqualene showed two peaks with similar retention times and with molecular ions at mlz 408, corresponding to C30H48. The fragmentation patterns are nearly identical, suggesting that the two compounds are the cis and trans isomers of dehydrosqualene. Since no squalene is found in the E. coli(pUG9) extracts, these results show that CrtM represents a dehydrosqualene synthase. Squalene is very likely not a substrate for CrtN, the proposed dehydrosqualene desaturase. With squalene as a substrate and with cell extracts of either E. coli(pUG1) or S. camosus(pOC21), we never observed in an in vitro assay the formation of 4,4'-diaponeurosporene, irrespective of the presence of cofactors (such as flavin adenine dinucleotide, NADP, NAD, and flavin mononucleotide) or various divalent metal ions. We therefore think that the proposed dehydrosqualene desaturase is specific for dehydrosqualene. Since dehydrosqualene is not available as a substrate, the positive control reaction was not possible. DISCUSSION

-IV

12

3

5

FIG. 5. Determination of the S. aureus dehydrosqualene synthase in crude extracts by ThC. Lanes: 1, E. ccli JM83(pUC19); 2, E. col [3H'farnesyl diphosphate; 5, JM83(pUG9); 3, E. ccli JM83(pUG1)* 4, the start; [3H]squalene. Roman numerals: I, farnesyl diphosphate atII,

farnesol; III, unknown; IV, 4,4'-diaponeurosporene; V, dehydrosqual-

ene; VI,

squalene.

The aim of this work was to genetically characterize the first steps of staphyloxanthin biosynthesis in staphylococci. We chose S. camosus TM300 as the cloning host because the colonies of this strain are white and therefore should not produce staphyloxanthin or colored intermediates. Staphyloxanthin-producing clones of S. carnosus harboring pOC1 were easily detected by their orange colonies. By subcloning fragments of the 12-kb DNA insert of pOC1 in S. carnosus or E. coli, we isolated yellow and unpigmented clones. The smallest DNA fragment which led to yellow pigmentation in both E. coli and S. camosus was the 3.5-kb SphlI fragment. The DNA sequence revealed two ORFs, ORFi and ORF2, both of which are very likely components of a gene cluster starting upstream of ORF1 and ending with ORF2. In the 400 nucleotides downstream of ORF2, no conspicuous transcription terminator sequences or additional ORFs were found. Between the

VOL. 176, 1994

S. AUREUS 4,4'-DIAPONEUROSPORENE BIOSYNTHESIS

end of ORF1 and the beginning of ORF2, no rho-independent transcription terminator sequences and no classical promoter-

like sequences can be found. It is therefore very likely that ORF2 is cotranscribed with ORF1. The deduced protein sequences of CrtM and CrtN were compared with the protein data bank. The amino acid sequence of CrtM exhibits similarity, within three domains, to the Synechococcus (8) and Erwinia (3, 4, 21) phytoene synthases and to the squalene synthases of human origin (24) and of S. cerevisiae (16). In addition, by mass and absorption spectroscopic data for products of the enzymatic reaction, we identified dehydrosqualene. These results strongly indicate that ctrM encodes a dehydrosqualene synthase. Furthermore, we have indications from GLC/MS that there is a cis-trans isomerization of dehydrosqualene, suggesting that the C30 carotinoid biosynthesis starts in a manner analogous to that of the C40 carotinoid biosynthesis (3, 21). Since dehydrosqualene is formed by the condensation of two molecules of farnesyl diphosphate, the question arises as to whether E. coli is able to synthesize this precursor substrate. Indeed, it was shown that E. coli possesses the gene encoding farnesyl diphosphate synthase (ispA) (11). The deduced amino acid sequence of CrtN was similar to those of the phytoene desaturases of E. herbicola, Erwinia uredovora, and R. capsulatus (3, 4, 21). We therefore assume that CrtN represents the dehydrosqualene desaturase of S. aureus. This assumption is in agreement with the accumulation of the deep-yellow 4,4'-diaponeurosporene in both the E. coli(pUG1) and S. camosus(pOC21) clones; in the corresponding plasmidless strains, this carotenoid was not found. The presence of a classical FAD-, NAD(P)-binding domain at the N terminus of CrtN also supports the identification of CrtN as a desaturase. Small amounts of the intermediate products 4,4'-diapophytofluene and 4,4'-diapo-7,8,11,12-tetrahydrolycopene were detected in both clones and not in the plasmidless strains, indicating that 4,4'-diaponeurosporene is formed from dehydrosqualene by three successive dehydrogenation reactions catalyzed by a single desaturase. This is in accord with a previous study (20) in which no mutants which accumulated direct precursors of 4,4'-diaponeurosporene were found. Furthermore, the desaturase should also be able to catalyze the cis-to-trans isomerization of the double bond in position 12 of dehydrosqualene. Otherwise, the cis isomer would accumulate. We could also detect small amounts of 4,4'-diapolycopene in E. coli(pUG1), indicating that the desaturase is principally able to further dehydrogenate 4,4'-diaponeurosporene (data not

shown).

In in vitro assays with squalene as a substrate and S. camosus(pOC21) or E. coli(pUG1) extracts, it was not possible to observe the formation of 4,4'-diaponeurosporene in detectable amounts. We therefore think that CrtN, the proposed dehydrosqualene desaturase, reacts specifically with dehydrosqualene. Because of the nonavailability of dehydrosqualene, the in vitro assay could not be carried out with this substrate. To further verify this result, we plan to express separately the crtN gene in S. camosus. On the basis of the results presented here, we propose the enzymatic reactions of the dehydrosqualene synthase (CrtM) and dehydrosqualene desaturase (CrtN) outlined in Fig. 6. This proposed pathway is in agreement with the results of Marshall and Wilmoth (19, 20) and also with the pathway for C40 carotenoids (3, 21). Marshall and Wilmoth (19, 20) have shown that 4,4'diaponeurosporene is an intermediary product in staphyloxanthin biosynthesis in S. aureus. Our growth studies with S. aureus Newman have shown that the deep-yellow 4,4'-diaponeuro-

7725

2 Farnesyl-PP I

Dehydrosqualene

synthase (CrtM) Dehydrosqualene (= 4,4'-Diapophytoene)

4,4'-Diapophytofluene

Dehydrosqualene desaturase (CrtN)

-2H+ 4,4'-Diapo-7,8,11,12tetrahydrolycopene

4,4'-Diaponeurosporene -2H

|

!

+

(4,4'-Diapolycopene) FIG. 6. Proposed enzymatic functions of the S. aureus dehydrosqualene synthase (CrtM) and dehydrosqualene desaturase (CrtN) in the biosynthesis of 4,4'-diaponeurosporene. sporene is the first detectable major carotenoid, found in high concentrations after 12 h of cultivation. The final orange end product, staphyloxanthin, is detectable only after prolonged cultivation for 24 to 36 h. This observation might indicate that the staphyloxanthin biosynthetic genes are organized in two gene clusters, one responsible for the synthesis of 4,4'-diaponeurosporene from farnesyl diphosphate (described here) and the other responsible for the synthesis of staphyloxanthin from 4,4'-diaponeurosporene. Perhaps a critical amount of 4,4'diaponeurosporene must accumulate before the genes for staphyloxanthin biosynthesis are expressed. ACKNOWLEDGMENTS We thank Arielle Ferrandon and Vera Augsburger for technical assistance and Karen A. Brune for critically reading the manuscript. We thank A. Hager, H. Stransky, and H.-J. Bigus from the Botanical Institute for the use of their HPLC/diode array spectroscopy and radioactivity-scanning facilities. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 323). REFERENCES 1. Agnew, W. S. 1985. Squalene synthetase. Methods Enzymol.

110:359-373. 2. Altenbern, R. A. 1967. Genetic studies of pigmentation of Staphylococcus aureus. Can. J. Microbiol. 13:389-395. 3. Armstrong, G. A., M. Alberti, and J. E. Hearst. 1990. Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotosynthetic and photosynthetic prokaryotes. Proc. Natl. Acad. Sci. USA 87:9975-9979. 4. Armstrong, G. A., M. Alberti, F. Leach, and J. E. Hearst. 1989. Nucleotide sequence, organisation, and nature of the protein products of the carotenoid biosynthetic gene cluster of

7726

WIELAND ET AL.

Rhodobacter capsulatus. Mol. Gen. Genet. 216:254-268. 5. Augustin, J., R. Rosenstein, B. Wieland, U. Schneider, N. Schnell, G. Engelke, K. D. Entian, and F. Gotz. 1992. Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis. Eur. J. Biochem. 204:1149-1154. 6. Ausubel, F. M., R. Brent, R. F. Kingston, D. D. More, J. G. Seidmann, J. A. Smith, and K. Struhl. 1987. Current protocols in molecular biology. John Wiley and Sons, New York. 7. Birnboim, H. C. 1983. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 100:243-255. 8. Chamovitz, D., N. Misawa, G. Sandmann, and K. Hirschberg.

9. 10. 11.

12. 13. 14.

15. 16. 17.

18.

1992. Molecular cloning and expression in Escherichia coli of a cyanobacterial gene coding for phytoene synthase, a carotenoid biosynthetic enzyme. FEMS Lett. 296:305-310. Chen, E. J., and P. H. Seeburg. 1985. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4:165170. Davies, B. H. 1977. C30 carotenoids, p. 51-100. In T. W. Goodwin (ed.), International review of biochemistry. University Park Press, Baltimore. Fujisaki, S., T. Nishino, H. Katsuki, H. Hara, Y. Nishimura, and Y. Hirota. 1989. Isolation and characterization of an Escherichia coli mutant having temperature-sensitive farnesyl diphospate synthase. J. Bacteriol. 171:5654-5658. Gotz, F. 1990. Staphylococcus camosus. A new host for gene cloning and protein production. J. Appl. Bacteriol. Symp. Suppl. 69:49-53. Gotz, F., and B. Schuhmacher. 1987. Improvements of protoplast transformation in Staphylococcus camosus. FEMS Microbiol. Lett. 40:285-288. Grinsted, J., and R. C. Lacey. 1973. Ecological and genetic implications of pigmentation in Staphylococcus aureus. J. Gen. Microbiol. 75:259-267. Hanahan, D. 1987. Technique for transformation of E. coli. IRL Press, Oxford. Jennings, S. M., Y. H. Tsay, T. M. Fisch, and G. W. Robinson. 1991. Molecular cloning and characterization of the yeast gene for squalene synthetase. Proc. Natl. Acad. Sci. USA 88:6038-6042. Kloos, W. E., K.-H. Schleifer, and F. Gotz. 1991. The genus Staphylococcus, p. 1369-1420. In B. Balows, H. G. Triiper, M. Dworkin, W. Harder, and K.-H. Schleifer (ed.), The prokaryotes. Springer-Verlag, New York. Kreutz, B., and F. Gotz. 1984. Construction of Staphylococcus plasmid vector pCA43 conferring resistance to chloramphenicol, arsenate, arsenite and antimony. Gene 31:301-304.

J. BACTERIOL. 19. Marshall, J., and G. Wilmoth. 1981. Pigments of Staphylococcus aureus, a series of triterpenoid carotenoides. J. Bacteriol. 147:900913. 20. Marshall, J., and G. J. Wilmoth. 1981. Proposed pathway of triterpenoid carotenoid biosynthesis in Staphylococcus aureus: evidence from a study of mutants. J. Bacteriol. 147:914-919. 21. Misawa, N., M. Nakagawa, K. Kobayashi, S. Yamano, Y. Izawa, K. Makamura, and K. Harashima. 1990. Elucidation of the Erwinia uredovora carotenoid biosynthesis pathway by functional analysis of gene products expressed in Escherichia coli. J. Bacteriol 172: 6704-6712. 22. Novick, R. P., and D. Bouanchaud. 1971. Extrachromosomal nature of resistance in Staphylococcus aureus. Ann. N.Y. Acad. Sci. 182:279-294. 23. Pinner, M., and M. Voldrich. 1932. Derivation of Staphylococcus albus, citreus and roseus from Staphylococcus aureus. J. Infect. Dis. 50:185-202. 24. Robinson, G. W., Y. H. Tsay, B. K. Kienzle, C. A. Smith-Monroy, and R. W. Bishop. 1993. Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation. Mol. Cell. Biol. 13:2706-2717. 25. Rosenstein, R., A. Peschel, B. Wieland, and F. Gotz. 1992. Expression and regulation of the antimonite, arsenite, and arsenate resistance operon of Staphylococcus xylosus plasmid pSX267. J. Bacteriol. 174:3676-3683. 26. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 27. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 28. Schleifer, K. H., and U. Fischer. 1982. Description of a new species of the genus Staphylococcus: Staphylococcus camosus. Int. J. Syst. Bacteriol. 32:153-156. 29. Servin-Massieu, M. 1961. Spontaneous appearance of sectored colonies in Staphylococcus aureus. J. Bacteriol. 82:316-317. 30. Suzue, G., K. Tsukada, C. Nakai, and S. Tanaka. 1968. Presence of squalene in Staphylococcus. Arch. Biochem. Biophys. 123:644. 31. Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268. 32. Willis, A. T., S. I. Jacobs, and G. M. Goodburn. 1964. Pigment production, enzyme activity and antibiotic sensitivity of staphylococci. Sub-division of the pathogenic group. J. Pathol. Bacteriol. 87:157-167.