Structural organization of a Bacillus subtilis operon encoding menaquinone biosynthetic enzymes

Gene, 167 (19953 105-109

1995 Elsevier Science B.V. All rights reserved.0378-1119/95/$09.50

105

GENE 09366

Structural organization of a Bacillus subtilis operon encoding menaquinone biosynthetic enzymes (Electron transport; vitamin Kz; D N A sequence)

Belinda Rowland a'b, Kex,in Hill a'*, Paul Mille#'*, Jeffrey Driscoll b and Harry Taber a'b aDeparnnent of Microbiology. lmmtotolog.rand MolecularGenetics. Alhany Medical College. 47 New Scotland Avenue. Albany. N Y 1220& USA: and bU'adsworth Center t. Department~f Biomedical Sciences. School ~f PublicHealth 2. EntpireStare Plaza. AIhan); N Y 12201-2002. USA

Receivedby M. Belfort: l0 March 1995:Revised Accepted: I Augusi 1995;Receivedat publishers: 18 September 1995

SUMMARY Menaquinone (MK) is a non-protein component of the Bacillus subtilis (Bs) electron transport chain synthesized from chorismate through a series of MK-specific reactions. The genes encoding biosynthesis of the naphthoquinone ring of M K are clustered at 273 ° on the Bs chromosome. A 3.9-kb region capable of rescuing men mutants blocked in the early stages of MK biosynthesis was sequet:ced and found to contain three major open reading frames (ORFs). The first O R F (menF) has a predicted size of 51.8 k D a and 34% amino-acid identity with the isochorismate synthases of Escherichia colt (EntC) and Aeromonas hydrophila (AmoA), O R F 2 (menD) a predicted size of 60.2 k D a and 21% identity with M e n D of E. c~l:.. O R F 3 has a predicted size of 21.4 k D a and 29% identity to triacylglycerol lipase of Psychrobacter immobilis. No sequence corresponding to menC was identified. Plasmid integrational studies of the men gene cluster had suggested the presence of promoters secondary to the previously identified pl men promoter. Sequence analysis revealed a putative promoter region upstream from ORF3.

INTRODUCTION Menaquinone (M K ) is a lipophilic, non-protein redox component of the electron transport chain of Bacillus subtilis (Bs) mediating electron transfer between dehydrogenases and cytochromes. Additionally, MK is necessary for sporulation and proper regulation of cytochrome formation in Bs (Farrand and Taber, 19733 and its formation Correspondence to: Dr. H. Taber. Wadsworth Cem,:r, Empire State

Plaza. Box 22002. Albany, NY 12201-2002,USA. Tel. 11-5183473-2760; Fax 11-5181473-1326. * Present addresses:(K.H.) Department of Radiology. Bethesda Naval Hospital, Bethesda, MD 20814, USA. Tel. (1-3013 295-5030: (P.M.I Parke-Davis, Pharmaceutical Research Division, 2800 Plymouth Rd., P.O. Box 1047,Ann Arbor, MI 48108-1047,USA. Tel. ( 1-3133996-2932. t The Wadsworth Center is a unit of the New York State Department of Health. 2 The School of Public Health is a joint venture of the New York State Department of Health and the University at Albany, State University of New York. SSDI 0378-1119195)00662-1

is highly regulated ( Farrand and Taber. 1974; Miller et al., 1988b; Qin and Taber, unpublished results). The biosynthetic steps leading to M K have been studied extensively in Escherichia colt (Ec) (Bentley and Meganathan, 1987) and appear to be similar in Bs (Meganathan et al., 1981; Taber et al., 1981; Taber, 1993; Palaniappan et al., 1994). The synthetic steps are initiated from :,'horismate, an intermediate in the c o m m o n Abbreviations: aa, amino acidls): AmoA. isochorismate synthase of Aeromonas hydrophila: B., Bacillus:bp, base pair(s};Bs, B. subtilis; CoA, coenzy~.e A; DHB, 2.3-dihydroxybenzoate;DHNA. 1,4-dihydroxy-2naphthoic acid; Ec. Escherichia colt: EntC. isochorismate synthase of Ec:kb, kilobase(sl or 1000bp; melt. :enes comprising MK operon: MenD. SHCHC synthase; menD, ~ene encoding MenD; MenF. isochorismate synthase; ntenF, gcne encoding MenF: MK, menaquinone; nt, nueleotide(s):ORF. open reading frame; OSB. o-sneeinyl benzoate; 2-OG, 2-oxoglutaratc; p. promoter; P., Psychrobacter: PABA. p-aminobenzoic acid: RBS. ribosome-binding sitels): SHCHC, 2-succinyl-6-hydroxy-2,4-eyclobexadiene-l-carboxylate; TPP, thiamine pyrophosphate; X, any aa.

106 aromatic aa pathway, and proceed through a series of MK-speeific reactions following the isomerization of chorismate to isoch~.-:ismate, as shown in Fig. 1 (Bentley and Meganathan, 1987). Analysis of Bs men mutants affecting naphthoquinone ring formation sF0wed that they could be divided into two groups (I and Ii) based on differential growth responses to ~-succinyl benzoate (OSB) and lA-dihydroxy-2-naphthoic acid (DHNA) (Taber et al., 1981). These mutants were found to be clustered at 273 ° on the Bs chromosome and were subsequently cloned by plasmid rescue (Miller et al., 1988a). Plasmid integration analysis indicated that the genes are organized into at least two transcriptional units. The sequence and structure of the group-II region, called the menBE operon (Fig. 2), and identification of the menB and menE promoters has been reported (Driscoll and Taber, 1992). This operon appears to encode enzymes for the conversion of OSB to DHNA. The group-I men genes, immediately upstream from menBE (Fig. 2), are transcribed from the pl men promoter (Millet' et al., 1988b); this promoter has been shown to be responsive to extracellular pH during post exponential growth (Hill et al., 1990). Nuclease SI mapping showed that the 5' end of the transcript initiated at pl men lies 1.5 kb upstream from the first group-I men mutation that could be rescued by sube!ones (Miller ct al., 1988a). Analysis of group-I mutants showed that this region encodes at least some of the enzymes responsible for synthesis of OSB from isochorismate (Miller et al., 1988a).

EXPERIMENTALAND DISCUSSION (a) Sequence of the group-! m e n genes

The nt sequence of the 3.9-kb region upstream from menBE encompassing the group-I mutations (Taber et al.,

1981; Miller et al., 1988a) was independently determined from both strands using men DNA cloned into plasmid commona r o pathway /

¢horismate

aromaticaa

PABA

1

/ in isocborismate

vectors pBS or pSGMU2. Plasmids pAI46, pAl 79, pAl68 and pAI93 served as templates (Fig. 2). The restriction .sites at the plasmid boundaries were confirmed by sequencing the relevant portions of plasmids pAil4, pAll5 and pal39. The nt sequence of the first 238 bp of this region has been published previously and was shown to contain e, promoter named pl men (Miller et al., 1988b). Sequence analysis identified three major ORFs within the 3.9-kb region. The nt sequence reported here has been deposited with GenBank under accession Nos. M21320 (Miller et al., 1988b) and M74183. (I) ORFI

ORF1 begins at nt 143 and extends through nt 1548, potentially encoding a 471-aa protein with a predicted size of 51.8 kDa. A sequence resembling a Bs RBS lies 7 bp upstream from the AUG start codon. Previous studies of pt men promoter regulation demonstrated that the ORFI gene product is efficiently expressed in vivo (Hill et al., 19901. ORF1 shows a greater than 34% aa identity with both EntC of Ec (Ozenberger et al., 19891 and AmoA of Aerontonas hydrophila I Barghouthi et al., 1991 ) within the C-terminal half of the protein (data not shown). The entC and amoA genes encode isochorismate synthase (see Fig. 1). An ORF whose product has similarity to isochori~mate synthase is present in the same location within the Ec men gene cluster (Daruwala et al., 1994) and to maintain consistency Bs men ORFI is designated menF. Bs MenF also shows significant sequence similarity to the other chorismate-utilizing enzymes TrpE (anthranilate synthase) and PabB (p-aminobenzoate synthase) in several species of eubacteria (data not shown). These sequence similarities suggest that menF belongs to the proposed family of genes that encode chorismate-utilizing enzymes. Isochorismate is a common precursor for MK and dihydroxybenzoate (DHB)-based iron-binding compoulds or siderophores (Fig. 1); Bs uses DHB as a siderophore. In E. coli, the entC gene is located within the

dihydroxybenzoicacid-based ironchelaUngcompounds

3

D

SHCHC

4

n- OSB

5

u, OSB-CoA

demethylMK __S ~

6

P DHNA

MK

Fig. 1. Biosynthesis of MK. Abbreviations are: 2-OG, 2-oxoglutarate: TPP, thiamine pyrophosphate: SHCHC. 2-succinyl-6hydroxy-2,4-cyclobexadien¢-1-carboxylate;OSB,o-succinylbenzoate;OSB-CoA,o-succinylbenzoate-CocnzymeA;DHNA, IA-dihydr3xy-2-naphthoic acid; MK, menaqoinone;PABA,p-aminobenzoicacid. Enzymedesignationsare: I, isochorismat¢synthas¢:2, 2-oxoglutaratedecarboxylase"3. SHCHC synthase;4, OSB synthase;5. OSB-CoAsynthase;6, DHNA synthase;7, polyisoprenyltransferase;8, methyltransferase.

107 menD 311 315 317 318

menD321 322

menF

I

ORF3

menD

me~,8

menE

I

pAl46

I

pAl39

I

pAl79

pAil5

pAll4

pal93

pAl68 1 kb

Fig. 2. Genetic and physical map of/he men locus. Bs sequencesof the pSGMU2 or pBS ÷- based plasmids used as sequencingtemplates are shown. Locations of group-I men mutants are indicated above the restriction map. Arrows. locations of known promoters (Driscoll and Taber, 1992; Miller et al., 1988bl;question mark, the putative ORF3 promoter. The locations of 1he men genes are indicated. B. BamHl; C, C/at; E, EcoRl; H, Hindlll; N, NlalV; P, Pstl; So, Sacl. Methods: Ec strains JMI01 and JMI07 were used as a cloning hosts. The plasmids pBS +t- (Stratagene) and pSGMU2 were used as vectors for men fragments required as templates for DNA sequencing. The construction of plasmids pAll4, pAll5 and pAI46 was described previously (Miller et al., 1988a). Plasmids pal25, pal23, and pAll7 (Miller et al., 1988a)were used for subcloning. Plasmld pAl39 contains the Pstl-EcoRl fragment of pal25 cloned into pBS~. Plasmid pal79 contains the Hindlll-Sacl fragment of pAl23 cloned into pBS-. Plasmid pal68 contains the HindIll fragment of pAll7 cloned into pSGMU2. Plasmid pal93 contains the Hindlll-Sacl fragment of pall4 cloned into pBS-. ent gene cluster (Ozenberger et al. 1989) and is expressed

under conditions of iron starvation. In Bs, formation of D H B is known to be regulated by iron (Walsh et al., 1971 ); however, the pl men promoter, which controls the expression of menF, is not iron-regulated; additionally, menF can be deleted without affecting the synthesis of M K or formation of D H B (B.M.R., unpublished results). The cloning of Bs genes capable of complementing several Ec ent mutants has been reported (Grossman et al., 1993). Analysis of an emC-comp!ementing clone shows the presence of an entC-like gene located within a greup of genes involved in D H B biosynthesis; as we have shown elsewhere, Bs has two isochorismate synthase genes (B.M.R., unpublished results). (2) ORF2

The possible start codon of the next O R F (ORF2) overlaps the stop codon of MenF. O R F 2 potentially

encodes a 548-aa protein with a predicted size of 60.2 kDa. Although there is no A U G start : o d o n in this reading frame there is a U U G codon at nt 1555, which can serve as a start codon in Bs. The product of O R F 2 displays significant sequence similarity with M e n d of Ec (Palaniappan et al., 1992) and has been designated MenD. Overall, Bs M e n D has 21% identity with M e n D of Ec; the C-terminal half of M e n D has 30% aa identity with Ec M e n D (data not shown). The Ec ntenD gene encodes a bifunctional enzyme (Palaniappan et al., 1992) that possesses both 2-succinyl6-hydroxy-2,4-c, ,;lohexadiene-l-carboxylate (SHCHC) synthase and TPP-dependent 2-oxoglutarate decarboxylase activities (see Fig. 1), the latter being quite separate from the 2-oxoglutarate dehydrogenase complex involved in the citric acid cycle (Bentley and Meganathan. 1987). Bs M e n D does not have significant sequence similarity to 2-oxoglutarate dehydrogenase

108 (OdhA) of Bs (Resnekov et al., 1992) except m the region of a highly conserved sequence motif shared by numerous unrelated T P P - b i n d i n g enzymes (Hav~kins et al., 1989) (data not shown). Recently, six Bs men mutants located within O R F 2 (Fig. 2) were shown to lack S H C H C synthase activity ( Palaniappan et al., 1994), which confirmed that O R F 2 is menD. The presence of the T P P - b i n d i n g motif ( H a w k i n s et al., 1989) within M e n D and the sequence similarity to 2-oxoglutarate decarboxylase (Resnekov et al., 1992) in this region suggests that Bs MenD, like the Ec homolog, is a bifunctional enzyme.

( 3 ) ORF3

Downstream from menD is a sequence resembling that of promoters capable of being recognized by the ~-A form of Bs RNA polymerase. The sequence 3-'tZ-TCAAGA-(Nrz)-TATGCT-3Z4o has the putative - 10 and - 35 elements with 4/6 and 3/6 matches with consensus, respectively, and the ideal 17-bp spacing. A potential RBS exists immediately upstream from the third O R F (ORF3), which initiates at nt 3271 and stops at nt 3858. O R F 3 potentially encodes a 195-aa protein witB a predicted size of 21.4 kDa. There is an O R F in the Ec men genes between m e n d and menB which shows 28% identity with O R F 3 (M.E.S. Hudspeth and R. Meganathan. personal communication) (data not shown). Sequence conservation and location of this gene suggest that it is involved in M K biosynthesis. O R F 3 has sequence simiiarity to triacylglycerol lipase of Psychrobacter immobilis (Arpigny et al., 1993) consisting of 290/0 identity across 120 aa res? "lues (data not shown). The O R F3 product has the consensus peptide GIy-X~-Ser-X_,-GIy (containing the active Ser), which is present within all known lipases, and the consensus peptide His-Gly, which is present in all nonfungal lipases (Arpigny ct al., 1993 ) (data not shown). This strongly suggests that O R F 3 encodes a lipase, perhaps required to prepare membrane lipids for proper insertion of M K into the bacterial inner membrane.

(b) Organization of the m e n operon These, results taken with those published previously (Driscoll and Taber, 1992~ have defined the organization of the Bs men gone cluster as shown in Fig. 2. Both nt seque,lce analysis and enzymatic analysis of men mutants blocked in the early stages of M K biosynthesis (Palaniappan et al., 1994) have failed to identify menC. The menC gone product, OSB synthase, converts S H C H C to OSB (Fig. 1 ). In Ec, the menC gene lies immediately downstream from menB (Sharma et al., 1993). The possibility that meuC lies downstream from menE in Bs is being pursued.

(c) Conclusions In this paper we report the complete nt sequence of a 3.9-kb region that overlaps the published sequence of the ntenBE operon at an N l a l V site (Driseo!l and Taber, 1992). Sequence analysis revealed the presence of three major O R F s that potentially ence:le an isochorismate synthase, a S H C H C synthase and a lipase. The putative promoter elements identified upstream to O R F 3 may be responsible for the readthrough transcription at menB promoter (Driscoll and Taber, 1992) or these transcripts may initiate from the pl men promoter.

ACKNOWLEDGEMENTS We thank M. Belfort, L. Flaherty and J. Errington for strains and piasmids, A. Arrow for oligonucleotides, X. Qin and C. Ricard for D N A sequence acquisition, and M. Hudspeth and R. Meganathan for comraunication of unpublished results and critical discus~ic,ns This investigation was supported in part by a grant from The National Institutes of Health (GM44547).

REFERENCES Arpigny. J.L.. Feller. G. and Gerday. C.: Cloning. sequence and structural features of a lipase from the antarctic facultative psychrophile Psychrohacter immubilis BI0. Biochim. Biophys. Acta 1171 119931 331-333. Barghouthi. S., Payne. S.M.. Arceneaux.J.E.L and Byers.B.R.: Cloning. mutagenesis,and nucleotide sequenceof a siderophore biosynthetic gone lamoAI from Aeromonas hydrophila. J. Bactcriol. 173 11991~ 5121-5128. Bentley, R. and Meganathan, R.: Biosynthesis of the isoprenoid quinones ubiquinone and menaquinone. In: Neidhardt. F.C.. Ingraham. J.L., Low, K.B., Magasanik. B.. Schaeehter. M. and Umbarger, H.E. (Eds.), Escherichia coil and Sahmmella o'phimurium: Cellular and Molecular Biology.American Societyof Microbiology.Washington, DC, 1987. pp. 512 520. Daruwala. R., Kwon. O.. Hudspeth, M.E.S. and Meganathan. R.: Menaquinone (vitamin K21 biosynthesis:evidence for a nov, gone ImenFI encoding an alternate isochorismatesynthase. Abstract. 94th ASM General Meeting. 1994. Driscoll, J.R. and Tabor. H.W.: Sequence organization and regulation of the Bacillus suhtilis menBE operon. J. Bacteriol. 174 (1992l 5063 5071. Farrand. S.K. and Tabor. H.W.: Physiologicaleffects of menaquinone deficiencyin Baciilu.~subtilis. J. Bacteriol. 1151197311035 le,M4. Farrand. S.K. and Taber. H.W.: Changes in menaquinone conc.zn~ration during growth and early sporulation in Bacillu.ssubtilis. J. Bacteriol. 11711974) 324 326. Grossman, T.H., Tuckman, M.. EIIcstad. S. and Osburne, M.S.: Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: relationship between B. subtilis .~n° and Escherichia coil entD genes. J. Bacleriol, 175 {199316203-6211. Hawkins. C.F., Borges, A. and Perham. R.N.: A common structural motif in thiamin pyrophosphate-bindiug enzymes. FEBS Lett. 255 11989l 77-82.

109 Hill. K.F, Mueller. J.P. and Taber, H.W.: The Bacillus subtilis menCD promoter is responsive to extracellular pH. Arch. Microbiol. 153 ( 19901 355-359. Meganathan. R., Bentley, R. and Taber. H.: Identification of Bacillus subtilis men mutants which lack o-succinylbenzoyl-coenzyme A synthetase and dihydroxynaphthoate synthase. J. Bacteriol. 145 119811 328-332. Miller, P., Rabinowitz, A. and Taber, H.: Molecular cloning and preliminary genetic analysis of the men gone clusler of Bacillus subtilis. J. Bacteriol. 170 (1988a) 2735-2741. Miller, P., M ueller, J.. Hill, K. and Taber, H.: Transcriptional regulation of a promoter in the men gene cluster of Bacillus subtilis. J. Bacteriol. 170 ( 1988bl 2742 2748. Ozenberger, B.A.. Brickraan. T.J. and Mclntosh, M.A.: Nuclo,~tide sequence of Escherichia coil isochorismate synthetase gene c u t ( and evolutionary relationship of isochorismate synthelase and other chorismate-utilizing enzymes. J. Bacteriol. 171 ( 19891 775-783. Palaniappan, C.. Sharma. V., Hudspeth, M.E.S. and Meganathan, R.: Menaquinone (vitamin K_,) biosynthesis: evidence that the Escherichia coil menD gene encodes both 2-succinyl-6-hydroxy-2,4cyclohexadiene-l-carboxylic aci~ synthase and ~t-ketoglutarate decarboxylase activities. J. Bacteriol. 174 (1992) 8111-81 i8. Palaniappan. C., Taber. H. and Meganathan. R.: Biosynthesis of

o-succinylbenzolc acid in Bacillus subtilis: identification of menD mutants and evidence against the involvement of the ~z-ketoglutarate dehydrogenase complex. L Bacteriol. 176 (1994) 2648-2653. Resnekov, O., Melin, L., Carlsson, P., Maanerlov, M., yon Gabain, A. and Hcderstedt. L.: Organization arid regulation of the Bacillus subtilis odhAB opcron, which encodes two of the subenzymes of the 2-oxoglutarate dehydrogenase complex. Mol. ,~en. Geoet. 234 119921 285-296. Sharma, V.. Meganathan, R. and Hudspeth, M.E.S.: Menaquinone (vitamin K,) biosynthesis: cloning, nucleotide sequence, and expression of the menC gene from Escherichia coil J. Bacteriol. 175 (1993) 4917-4921. Tabor, H.W., Dellers, E.A, and Rivoire Lombardo, L.: Menaquinone biosynthesis in Bacillus subtilis: isolation of men mutants and evidence for clustering of men genes. J. Bacteriol. 145 ( 1981 ) 321-327. Taber. H.W.: Respiratory Chains. In: Sonenshein, A.L., Hoch, J.A. and Losick. R. (Eds.), Bacillus subtilis and Other Gram-Positive Becteria: Biochemistry. Physiology, and Molecular Genetics. American Society for Microbiology, Washington. DC. 1993. pp. 177-187. Walsh, B.L.. Peters, W.J. and W~rren, R.A.J.: The regulation of phenolic acid synthesis in Bacilh¢s subtilis. Can. J. Microbiol. 17 (1971) 53-59.