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The ribulose monophosphate pathway operon encoding formaldehyde fixation in a thermotolerant methylotroph, Bacillus brevis S1
FEMS Microbiology Letters 214 (2002) 189^193
www.fems-microbiology.org
The ribulose monophosphate pathway operon encoding formaldehyde ¢xation in a thermotolerant methylotroph, Bacillus brevis S1 Hiroya Yurimoto a , Reiko Hirai a , Hisashi Yasueda b , Ryoji Mitsui a , Yasuyoshi Sakai a , Nobuo Kato a; a
Division of Applied Life Sciences, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan b Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc., Kawasaki 210-0801, Japan Received 30 April 2002; received in revised form 29 June 2002 ; accepted 12 July 2002 First published online 9 August 2002
Abstract The hps and phi genes encoding 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase, the key enzymes of the ribulose monophosphate (RuMP) pathway for formaldehyde fixation, were cloned from the chromosomal DNA of a thermotolerant methylotroph, Bacillus brevis S1. Enzyme induction and Northern blot analyses revealed that both the hps and phi genes are induced by methanol or ethanol, and that their expression is controlled polycistronically at the transcription stage. Sequence analysis also suggested that the hps and phi genes constitute an RuMP operon. The gene organization of the RuMP operon and its surrounding region are unique among bacteria possessing the RuMP pathway genes. 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : 3-Hexulose-6-phosphate synthase; 6-Phospho-3-hexuloisomerase; Ribulose monophosphate pathway ; Formaldehyde-¢xing operon; Methylotroph; Bacillus brevis S1
1. Introduction Methylotrophic bacteria that ¢x formaldehyde through the ribulose monophosphate (RuMP) pathway consist of three groups; Gram-negative obligate methylotrophs, Gram-positive facultative methylotrophs, and thermotolerant Bacillus spp. The Bacillus strains belonging to the third group are distinct from the others in several respects, their growth ability at higher temperature, the methanol oxidation system catalyzed by NADþ -dependent methanol dehydrogenase [1], etc. Also, Bacillus strains are attractive for application to the production of useful compounds [2]. Genetic studies on the RuMP pathway have been per-
* Corresponding author. Tel. : +81 (75) 753 6385; Fax : +81 (75) 753 6385. E-mail address : [email protected] (N. Kato).
formed with two methylotrophic bacteria belonging to the former two groups, Methylomonas aminofaciens 77a [3] and Mycobacterium gastri MB19 [4], respectively. A homology search of protein databases revealed that the enzymes of the RuMP pathway, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), exhibit high similarity to a variety of unidenti¢ed proteins of non-methylotrophic prokaryotes, including Bacteria and Archaea [4]. In a non-methylotrophic B. subtilis, both HPS and PHI are induced by formaldehyde and indeed function in the detoxi¢cation of harmful formaldehyde [5]. Comparison of the RuMP pathway at the molecular level between methylotrophic and non-methylotrophic organisms, e.g., comparison between B. brevis S1 and Bacillus subtilis, will provide valuable information on the regulation of C1 -metabolism in prokaryotes. In this study, we cloned the gene cluster encoding HPS and PHI from a methylotroph, B. brevis S1, and revealed that the hps and phi genes are expressed as a polycistronic operon. The results also provide some basic information for molecular breeding of methylotrophic Bacillus strains for their application.
0378-1097 / 02 / $22.00 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 8 2 - 0
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2. Materials and methods
2.3. DNA manipulation
2.1. Microorganisms, culture conditions, and vectors
Upstream and downstream primers were designed on the basis of the N-terminus and internal amino acid sequences of HPS, respectively, to PCR-amplify the DNA fragment coding for the HPS gene (hps) derived from B. brevis S1 chromosomal DNA. The sequences of the primers used were as follows : N-terminal, 5P-GARGTICARGARTAYGTIGAYATHGTIGA-3P; and internal, 5P-TTYTTICCIACIGCYTGIARRTCRTA-3P. The PCR product was approximately 400 bp in length and used as a probe. Through Southern hybridization [10] and inverse PCR [11], one positive clone containing a 3.0-kb SalI fragment on pGEM-T Easy (Promega Co.) was isolated, the resultant plasmid being named pGivS1. Inverse PCR was performed using primers hps-ivB1 (5P-TAACCGGAGTACCGATTTCC-3P) and hps-ivS1 (5P-CACGCTGGATACGATCTCCA-3P). The 3.0-kb SalI fragment was subcloned from pGivS1 into pBluescript II SKþ (Stratagene) and sequenced. Nucleotide sequencing was performed with a Thermo Sequence Fluorescent-Labeled Cycle Sequence Kit (Amersham Pharmacia) and a DNA sequencer model DSQ2000L (Shimadzu Co., Ltd.). The sequence data were analyzed with the BLAST and FASTA programs. The nucleotide sequence data reported in this paper have been deposited in the GenBank nucleotide sequence database under accession number AX343668.
The thermotolerant methanol-utilizing B. brevis S1 (NCIMB12524) isolated by Al-Awadhi et al. [6] was used as the source of genomic DNA for the cloning procedure. The proposed name for this strain is Bacillus methanolicus [7]. B. brevis S1 was cultivated at 45‡C for 16 h in a 2-l shake £ask containing 500 ml of medium of the following composition (per liter): methanol (1.6 g), K2 HPO4 (4.65 g), NaH2 PO4 WH2 O (1.5 g), (NH4 )2 SO4 (1.5 g), MgSO4 W7H2 O (0.2 g), yeast extract (0.5 g), peptone (0.5 g), casamino acids (0.5 g), vitamin solution, and metal solution, pH 7.0 [8]. 5 ml of a culture on trypton soya broth CM129 (Oxid Ltd.) was inoculated into the above medium at 37‡C. The growth conditions for Escherichia coli, transformation, gene cloning, and gene expression were described previously [4]. 2.2. Protein manipulation HPS and PHI activities were assayed at 30‡C as described previously [2]. The amount of protein was determined using a Bio-Rad protein assay kit with bovine serum albumin as the standard. Sodium dodecyl sulfate^ polyacrylamide gel electrophoresis (SDS^PAGE) was performed on a 15% polyacrylamide gel. The apparent molecular mass of a native enzyme was determined by gel ¢ltration on a Superdex 200 column (Pharmacia Biotech) in a fast protein liquid chromatography system. PHI was puri¢ed through the following steps at 4‡C, all bu¡ers used containing 1 mM dithiothreitol, 5 mM MgCl2 , and 0.15 mM phenylmethylsulfonyl £uoride. After harvesting, cells grown under the conditions given above were washed with 50 mM potassium phosphate bu¡er (pH 7.5), disrupted by sonication at 180 W for 30 min, and then centrifuged at 110 000Ug for 60 min. The soluble fraction was dialyzed against 20 mM Tris^Cl (pH 7.5), and then chromatographed on a Q-Sepharose column (1.6U10 cm) pre-equilibrated with 20 mM Tris^Cl (pH 7.5). Elution was performed with a linear gradient of increasing KCl concentration (0^0.5 M). The active fraction, to which ammonium sulfate was added to 1.7 M, was applied on a Butyl-Toyopearl column (2.2U20 cm). The enzyme was eluted with a linear gradient of decreasing ammonium sulfate concentration (1.7^0 M). After concentration of the active fractions by ultra¢ltration with a Centriprep (Millipore Co.), the enzyme was dialyzed against 100 mM Tris^Cl (pH 7.5) containing 0.1 M KCl, and then chromatographed on a Superdex-200 column (1.6U60 cm) with the dialyzed bu¡er. The active fractions were collected and used as the puri¢ed enzyme. The N-terminus of the puri¢ed enzyme and an internal amino acid sequence were determined as previously described [9].
2.4. Expression of the hps and phi genes in E. coli The coding region of each of the hps and phi genes (see below) was ampli¢ed using chromosomal DNA of B. brevis S1 as the template. The upstream and downstream primers for the ampli¢cation of these genes were as follows: for hps, 5-GGAATTCCTAAGGAGGTTTTTATATGCAACTTCAATTAGCTCTA-3P (the Shine^Dalgarno sequence is italicized and the EcoRI site is underlined) at the N-terminal and 5P-GGAATTCCTCATAACCCTTGTTTAACTAAT3P- (the EcoRI site is underlined) at the C-terminal; and for phi, 5P-GGAATTCCTAAGGAGGTTTTTATATGATGCAGACAACTGAATTC-3P (the Shine^Dalgarno sequence is italicized and the EcoRI site is underlined) at the N-terminal and 5P-GGAATTCCCTACTCGAGATTGGCATGTCT-3P (the EcoRI site is underlined) at the C-terminal. Each PCR product was digested with EcoRI, ligated into the EcoRI site of pKK223-3, and then introduced into E. coli JM109 cells. The resultant plasmids were designated as pKHS1 and pKPS1 for hps and phi, respectively. Each transformant of E. coli JM109 was grown on 2UYT medium containing ampicillin (50 Wg ml31 ) and 0.5 mM IPTG at 37‡C for 14 h.
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Table 1 Puri¢cation of HPS from B. brevis S1 Puri¢cation
Total protein (mg)
Total activity (Ua )
Speci¢c activity (U mg31 )
Puri¢cation (fold)
Yield (%)
Cell-free extract Q-Sepharose Butyl-Toyopearl Superdex 200
550 73.7 20.2 4.70
10 100 3 550 1 630 425
17.0 49.4 87.0 91.1
1 2.90 5.12 5.36
100 35.1 16.1 4.20
a
U: Wmol min31 .
2.5. RNA analysis B. brevis S1 was grown to the mid-exponential phase in the medium mentioned above containing one of the following carbon sources: methanol (50 mM), ethanol (100 mM), or glucose (100 mM) with formaldehyde (0.1 mM) or formate (0.1 mM). Total RNA preparation and Northern blot analysis were performed as described previously [4]. Hybridization was carried out with an AlkPhos DIRECT (Amersham Pharmacia Biotech, Tokyo, Japan), using the entire coding region of hps or phi as a probe. rRNA was used as a standard for loaded total RNA.
44 kDa on gel ¢ltration. The N-terminal amino acid sequence, MQLQLALDLVNIEEAKQVVAEVQEYVDIVE, was determined on Edman’s degradation of HPS. The amino acid sequence of an internal peptide fragment was MGVDYIXVHAGYDLQAVGKN (X represents any amino acid). Judging from the results, HPS is a homodimeric enzyme. The enzyme was moderately thermostable, 20% of its activity being retained after incubation for 10 min at 60‡C. The maximum activity was found at 55‡C and pH 7.5. These enzymatic properties are comparable to those of the enzyme from Bacillus C1, except for the subunit structure, the latter being reported to be a monomeric enzyme with a molecular mass of 32 kDa [12]. 3.2. Nucleotide sequence and structural analysis of the SalI fragment
3. Results 3.1. Puri¢cation and general properties of HPS from B. brevis S1 Through the puri¢cation procedures, HPS was puri¢ed 5.36-fold from the soluble fraction to apparent homogeneity (Table 1). The relative molecular mass of the puri¢ed HPS was estimated to be 25 kDa on SDS^PAGE and
Determination of the entire nucleotide sequence of the 3.0-kb SalI insert in pGivS1 revealed two complete (orf2 and orf3) and one partial ORF (orf1) (Fig. 1). orf2 consists of 636 bp, and the deduced amino acid sequence includes 211 amino acid residues with a theoretical molecular mass of 22 652 Da. This value is close to the molecular mass
Fig. 1. Gene diagram of RuMP operons in (A) B. brevis S1, (B) B. subtilis [5], (C) M. gastri MB19 [4], and (D) M. aminofaciens 77a [3]. Products of genes: orf1, glucose-6-phosphate dehydrogenase; hps (orf2), HPS; phi (orf3), PHI ; yckH, a putative DNA-binding protein; hxlA, HPS ; hxlB, PHI; rmpR, a putative regulatory protein ; rmpB, PHI; rmpA, HPS; and rmpI, transposase (IS10-R). A: The arrows in the diagram downstream of the phi gene indicate a putative transcriptional terminator as an inverted repeat with subsequent T residues (dashed line). The numbering of nucleotides starts from hps.
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determined by SDS^PAGE of the puri¢ed HPS from B. brevis S1, i.e. 25 kDa. The N-terminal and internal amino acid sequences of the puri¢ed HPS were found in the deduced amino acid sequence. From this and the results of gene expression studies (see below), orf2, named hps, was shown to encode HPS. The deduced primary structure is similar to those of the HPSs of B. subtilis (66% identity and 83% similarity), M. gastri MB19 (40% identity and 61% similarity), and M. aminofaciens 77a (37% identity and 59% similarity). orf3 consists of 555 bp corresponding to 184 amino acid residues with a predicted molecular mass of 19 942 Da. The amino acid sequence is similar to the deduced amino acid sequences of the PHIs of B. subtilis (64% identity and 82% similarity), M. gastri MB19 (39% identity and 63% similarity), and M. aminofaciens 77a (33% identity and 63% similarity). The orf3 gene was named phi according to these high similarity scores as to other PHIs and the expression of PHI activity in E. coli. The phi gene was located downstream of the hps gene with a space of 5-bp. A partial ORF (orf1), which is in the reversed orientation to hps and phi, exhibits signi¢cant similarity with the glucose 6-phosphate dehydrogenase gene from Vibrio cholerae. This enzyme is also known to be involved in the RuMP pathway [4]. As shown in Fig. 1, one set of typical promoter sequences (335 and 310 sequences) exists upstream of hps and a putative transcriptional terminator with subsequent T residues (a rho-independent transcriptional terminator) is present downstream of the termination triplet of the phi gene. Shine^Dalgarno sequences exist upstream of the hps and phi genes, respectively, one for the phi gene being within the coding region of the hps gene. These gene organization analysis results raised the possibility that hps and phi are transcribed as a polycistronic operon. 3.3. Gene expression of hps and phi in E. coli A cell-free extract of E. coli JM109 [pKHS1] harboring the hps gene, prepared from cells induced by IPTG, showed a signi¢cant level of HPS activity (2.25 Wmol min31 mg of protein31 ). The recombinant HPS was puri¢ed 16.6-fold from E. coli [pKHS1] by almost the same procedure as described above. The puri¢ed preparation gave a single band on SDS^PAGE. The N-terminal sequence, MQLQLALDLVNIEEAKQVVA^, was identical to that of the deduced amino acid sequence of the corresponding gene, hps, and that of the puri¢ed HPS from B. brevis S1. The speci¢c activity of the puri¢ed enzyme and the temperature characteristics of the activity were the almost same as those of the parent strain. The transformant of E. coli JM109 [pKPS1] harboring the phi gene was grown under the same conditions as those for hps expression. The cell-free extract exhibited a high level of PHI activity (0.5 Wmol min31 mg of protein31 ).
Table 2 Speci¢c activities of HPS and PHI in extracts of cells grown on several carbon substrates Carbon source
HPS activity (Wmol min31 mg protein31 )
PHI activity
Methanol Ethanol Glucose
7.4 3.7 0.67
11 3.9 0.97
SDS^PAGE of the cell-free extract gave a major distinct protein band corresponding to a molecular mass of 21 kDa, this value being close to the theoretical molecular mass obtained for the amino acid sequence. The control strain harboring pKK223-3 exhibited no PHI activity. 3.4. Regulation of gene expression in B. brevis S1 B. brevis S1 was grown on medium containing each carbon source, methanol, ethanol, or glucose, until the mid-exponential phase. The speci¢c activities of HPS and PHI are shown in Table 2. Cells that had been grown on methanol or ethanol showed high activities of both enzymes. Negligible HPS and PHI activities were detected in the cells that had been grown on glucose. These data indicate that HPS and PHI are induced by methanol, as well as ethanol, when added to the medium. Northern blot analysis of the cells grown on the medium containing methanol, ethanol, or glucose+formaldehyde with the DNA fragment of hps or phi as the probe revealed a identical hybridizing band, the size corresponding to the entire length of the transcription product of hps and phi (1.2 kb) (data not shown). On the other hand, the hps and phi probes did not hybridize with the total RNA of cells grown on glucose or glucose+formate. These results are in fair agreement with the enzyme induction pro¢le described above, and imply that expression of hps and phi is regulated at the mRNA level, and that the gene cluster is transcribed as a polycistronic operon, which responds to methanol (or formaldehyde) and ethanol.
4. Discussion Genetic studies on the RuMP pathway have been performed with two methylotrophic bacteria, M. gastri MB19 [4] and M. aminofaciens 77a [3,9], and a non-methylotroph, B. subtilis [5] (Fig. 1). Although the primary structures of HPS and PHI show high similarity among these three strains, the gene organization and regulatory mechanism for gene expression are quite di¡erent between the obligate methylotroph and the others. In M. aminofaciens 77a, a transposase, which is present between hps and phi, is related to regulation of the adjacent gene expression in a unique manner, and both genes are expressed monocistronically [3]. In B. brevis S1, the expression of hps and
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phi is induced by methanol and regulated under the same control at a transcriptional level as a polycistronic operon, which is similar to the cases of M. gastri MB19 [4] and B. subtilis [5]. Such a regulatory system responding to the growth substrate is indispensable for facultative methylotrophs. The arrangement of the genes on the RuMP operon in B. brevis S1 was the same as that in B. subtilis. On the other hand, the phi gene exists upstream of the hps gene in M. gastri MB19 (Fig. 1). The ORFs present upstream of the RuMP operon are di¡erent among the three strains. In the cases of B. subtilis and M. gastri MB19, the gene products are putative regulatory proteins encoded by the yckH and rmpR genes, respectively [4]. In contrast, we could not detect any ORF that encodes a regulatory protein within 2 kb upstream and 1 kb downstream of the RuMP operon region. Although the primary structures of HPS and PHI of B. brevis S1 are highly homologous to those of B. subtilis, the expression levels of hps and phi in B. brevis are much higher than those in B. subtilis. Further studies on the RuMP gene cluster are necessary to elucidate the mechanism of transcriptional regulation in facultative methylotrophic bacteria. Considerable HPS and PHI activities were present in ethanol-grown cells, and the transcription of the RuMP operon responded to ethanol. In methylotrophic Bacillus C1, NADþ -dependent methanol dehydrogenase, which could catalyze the oxidation of methanol and ethanol, is induced in methanol and ethanol medium [12]. On the other hand, HPS and PHI are assumed to be speci¢c enzymes for methanol assimilation but not for ethanol assimilation. The physiological signi¢cance of induction of the RuMP operon by ethanol remains unclear.
Acknowledgements This study was partly supported by a grant to Y.S. from the Ministry of Education, Culture, Sports, Science and Technology.
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References [1] Arfman, N., de Vries, K.J., Moezelaa, H.R., Attwood, M.M., Robinson, G.K. and van Geel, M. (1992) Environmental regulation of alcohol metabolism in thermotolerant methylotrophic Bacillus strains. Arch. Microbiol. 157, 272^278. [2] Lee, G.H., Hur, W., Bremmon, C.G. and Flickinger, M.C. (1996) Lysine production from methanol at 50‡C using Bacillus methanolicus: Modeling volume control, lysine concentration, and productivity using a three-phase continuous simulation. Biotechnol. Bioeng. 49, 639^653. [3] Sakai, Y., Mitsui, R., Katayama, Y., Yanase, H. and Kato, N. (1999) Organization of the genes involved in the ribulose monophosphate pathway in an obligate methylotrophic bacterium, Methylomonas aminofaciens 77a. FEMS Microbiol. Lett. 176, 125^130. [4] Mitsui, R., Sakai, Y., Yasueda, H. and Kato, N. (2000) A novel operon encoding formaldehyde ¢xation: the ribulose monophosphate pathway in the Gram-positive facultative methylotrophic bacterium Mycobacterium gastri MB19. J. Bacteriol. 182, 944^948. [5] Yasueda, H., Kawahara, Y. and Sugimoto, S. (1999) Bacillus subtilis yckG and yckF encode two key enzymes of the ribulose monophosphate pathway used by methylotrophs, and yckH is required for their expression. J. Bacteriol. 181, 7154^7160. [6] Al-Awadhi, N., Egli, T., Hamer, G. and Wehrli, E. (1989) Thermotolerant and thermophilic solvent-utilizing methylotrophic, aerobic bacteria. Syst. Appl. Microbiol. 11, 207^216. [7] Arfman, N., Dijkhuizen, L., Kirchhof, G., Ludwig, W., Schleifer, K.H., Bulygina, E.S., Chumakov, K.M., Govorukhina, N.I., Trotsenko, Y.A., White, D. and Sharp, R.J. (1992) Bacillus methanolicus sp. nov., a new species of thermotolerant, methanol-utilizing, endosporeforming bacteria. Int. J. Syst. Bacteriol. 42, 439^445. [8] Kato, N., Miyamaoto, N., Shimao, M. and Sakazawa, C. (1988) 3-Hexulose phosphate synthase from a new facultiative methylotroph, Mycobacterium gastri MB19. Agric. Biol. Chem. 52, 2659^ 2661. [9] Yanase, H., Ikeyama, K., Mitsui, R.R.S., Kita, K., Sakai, Y. and Kato, N. (1996) Cloning and sequence analysis of the gene encoding 3-hexulose-6-phosphate synthase from the methylotrophic bacterium, Methylomonas aminofaciens 77a, and its expression in Escherichia coli. FEMS Microbiol. Lett. 135, 201^205. [10] Southern, E.M. (1978) Detection of speci¢c sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503^517. [11] Ochamn, H., Gerber, A.S. and Hartl, D.L. (1988) Genetic application of an inverse polymerase chain reaction. Genetics 120, 6221^6223. [12] Arfman, N., Bystrykh, L., Govorukhina, N.I. and Dijkhuizen, L. (1990) 3-Hexulose-6-phosphate synthase from thermotolerant methylotroph Bacillus C1. Methods Enzymol. 188, 391^397.
FEMSLE 10611 11-9-02
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The ribulose monophosphate pathway operon encoding formaldehyde ¢xation in a thermotolerant methylotroph, Bacillus brevis S1 Hiroya Yurimoto a , Reiko Hirai a , Hisashi Yasueda b , Ryoji Mitsui a , Yasuyoshi Sakai a , Nobuo Kato a; a
Division of Applied Life Sciences, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan b Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc., Kawasaki 210-0801, Japan Received 30 April 2002; received in revised form 29 June 2002 ; accepted 12 July 2002 First published online 9 August 2002
Abstract The hps and phi genes encoding 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase, the key enzymes of the ribulose monophosphate (RuMP) pathway for formaldehyde fixation, were cloned from the chromosomal DNA of a thermotolerant methylotroph, Bacillus brevis S1. Enzyme induction and Northern blot analyses revealed that both the hps and phi genes are induced by methanol or ethanol, and that their expression is controlled polycistronically at the transcription stage. Sequence analysis also suggested that the hps and phi genes constitute an RuMP operon. The gene organization of the RuMP operon and its surrounding region are unique among bacteria possessing the RuMP pathway genes. 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : 3-Hexulose-6-phosphate synthase; 6-Phospho-3-hexuloisomerase; Ribulose monophosphate pathway ; Formaldehyde-¢xing operon; Methylotroph; Bacillus brevis S1
1. Introduction Methylotrophic bacteria that ¢x formaldehyde through the ribulose monophosphate (RuMP) pathway consist of three groups; Gram-negative obligate methylotrophs, Gram-positive facultative methylotrophs, and thermotolerant Bacillus spp. The Bacillus strains belonging to the third group are distinct from the others in several respects, their growth ability at higher temperature, the methanol oxidation system catalyzed by NADþ -dependent methanol dehydrogenase [1], etc. Also, Bacillus strains are attractive for application to the production of useful compounds [2]. Genetic studies on the RuMP pathway have been per-
* Corresponding author. Tel. : +81 (75) 753 6385; Fax : +81 (75) 753 6385. E-mail address : [email protected] (N. Kato).
formed with two methylotrophic bacteria belonging to the former two groups, Methylomonas aminofaciens 77a [3] and Mycobacterium gastri MB19 [4], respectively. A homology search of protein databases revealed that the enzymes of the RuMP pathway, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), exhibit high similarity to a variety of unidenti¢ed proteins of non-methylotrophic prokaryotes, including Bacteria and Archaea [4]. In a non-methylotrophic B. subtilis, both HPS and PHI are induced by formaldehyde and indeed function in the detoxi¢cation of harmful formaldehyde [5]. Comparison of the RuMP pathway at the molecular level between methylotrophic and non-methylotrophic organisms, e.g., comparison between B. brevis S1 and Bacillus subtilis, will provide valuable information on the regulation of C1 -metabolism in prokaryotes. In this study, we cloned the gene cluster encoding HPS and PHI from a methylotroph, B. brevis S1, and revealed that the hps and phi genes are expressed as a polycistronic operon. The results also provide some basic information for molecular breeding of methylotrophic Bacillus strains for their application.
0378-1097 / 02 / $22.00 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 8 2 - 0
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H. Yurimoto et al. / FEMS Microbiology Letters 214 (2002) 189^193
2. Materials and methods
2.3. DNA manipulation
2.1. Microorganisms, culture conditions, and vectors
Upstream and downstream primers were designed on the basis of the N-terminus and internal amino acid sequences of HPS, respectively, to PCR-amplify the DNA fragment coding for the HPS gene (hps) derived from B. brevis S1 chromosomal DNA. The sequences of the primers used were as follows : N-terminal, 5P-GARGTICARGARTAYGTIGAYATHGTIGA-3P; and internal, 5P-TTYTTICCIACIGCYTGIARRTCRTA-3P. The PCR product was approximately 400 bp in length and used as a probe. Through Southern hybridization [10] and inverse PCR [11], one positive clone containing a 3.0-kb SalI fragment on pGEM-T Easy (Promega Co.) was isolated, the resultant plasmid being named pGivS1. Inverse PCR was performed using primers hps-ivB1 (5P-TAACCGGAGTACCGATTTCC-3P) and hps-ivS1 (5P-CACGCTGGATACGATCTCCA-3P). The 3.0-kb SalI fragment was subcloned from pGivS1 into pBluescript II SKþ (Stratagene) and sequenced. Nucleotide sequencing was performed with a Thermo Sequence Fluorescent-Labeled Cycle Sequence Kit (Amersham Pharmacia) and a DNA sequencer model DSQ2000L (Shimadzu Co., Ltd.). The sequence data were analyzed with the BLAST and FASTA programs. The nucleotide sequence data reported in this paper have been deposited in the GenBank nucleotide sequence database under accession number AX343668.
The thermotolerant methanol-utilizing B. brevis S1 (NCIMB12524) isolated by Al-Awadhi et al. [6] was used as the source of genomic DNA for the cloning procedure. The proposed name for this strain is Bacillus methanolicus [7]. B. brevis S1 was cultivated at 45‡C for 16 h in a 2-l shake £ask containing 500 ml of medium of the following composition (per liter): methanol (1.6 g), K2 HPO4 (4.65 g), NaH2 PO4 WH2 O (1.5 g), (NH4 )2 SO4 (1.5 g), MgSO4 W7H2 O (0.2 g), yeast extract (0.5 g), peptone (0.5 g), casamino acids (0.5 g), vitamin solution, and metal solution, pH 7.0 [8]. 5 ml of a culture on trypton soya broth CM129 (Oxid Ltd.) was inoculated into the above medium at 37‡C. The growth conditions for Escherichia coli, transformation, gene cloning, and gene expression were described previously [4]. 2.2. Protein manipulation HPS and PHI activities were assayed at 30‡C as described previously [2]. The amount of protein was determined using a Bio-Rad protein assay kit with bovine serum albumin as the standard. Sodium dodecyl sulfate^ polyacrylamide gel electrophoresis (SDS^PAGE) was performed on a 15% polyacrylamide gel. The apparent molecular mass of a native enzyme was determined by gel ¢ltration on a Superdex 200 column (Pharmacia Biotech) in a fast protein liquid chromatography system. PHI was puri¢ed through the following steps at 4‡C, all bu¡ers used containing 1 mM dithiothreitol, 5 mM MgCl2 , and 0.15 mM phenylmethylsulfonyl £uoride. After harvesting, cells grown under the conditions given above were washed with 50 mM potassium phosphate bu¡er (pH 7.5), disrupted by sonication at 180 W for 30 min, and then centrifuged at 110 000Ug for 60 min. The soluble fraction was dialyzed against 20 mM Tris^Cl (pH 7.5), and then chromatographed on a Q-Sepharose column (1.6U10 cm) pre-equilibrated with 20 mM Tris^Cl (pH 7.5). Elution was performed with a linear gradient of increasing KCl concentration (0^0.5 M). The active fraction, to which ammonium sulfate was added to 1.7 M, was applied on a Butyl-Toyopearl column (2.2U20 cm). The enzyme was eluted with a linear gradient of decreasing ammonium sulfate concentration (1.7^0 M). After concentration of the active fractions by ultra¢ltration with a Centriprep (Millipore Co.), the enzyme was dialyzed against 100 mM Tris^Cl (pH 7.5) containing 0.1 M KCl, and then chromatographed on a Superdex-200 column (1.6U60 cm) with the dialyzed bu¡er. The active fractions were collected and used as the puri¢ed enzyme. The N-terminus of the puri¢ed enzyme and an internal amino acid sequence were determined as previously described [9].
2.4. Expression of the hps and phi genes in E. coli The coding region of each of the hps and phi genes (see below) was ampli¢ed using chromosomal DNA of B. brevis S1 as the template. The upstream and downstream primers for the ampli¢cation of these genes were as follows: for hps, 5-GGAATTCCTAAGGAGGTTTTTATATGCAACTTCAATTAGCTCTA-3P (the Shine^Dalgarno sequence is italicized and the EcoRI site is underlined) at the N-terminal and 5P-GGAATTCCTCATAACCCTTGTTTAACTAAT3P- (the EcoRI site is underlined) at the C-terminal; and for phi, 5P-GGAATTCCTAAGGAGGTTTTTATATGATGCAGACAACTGAATTC-3P (the Shine^Dalgarno sequence is italicized and the EcoRI site is underlined) at the N-terminal and 5P-GGAATTCCCTACTCGAGATTGGCATGTCT-3P (the EcoRI site is underlined) at the C-terminal. Each PCR product was digested with EcoRI, ligated into the EcoRI site of pKK223-3, and then introduced into E. coli JM109 cells. The resultant plasmids were designated as pKHS1 and pKPS1 for hps and phi, respectively. Each transformant of E. coli JM109 was grown on 2UYT medium containing ampicillin (50 Wg ml31 ) and 0.5 mM IPTG at 37‡C for 14 h.
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Table 1 Puri¢cation of HPS from B. brevis S1 Puri¢cation
Total protein (mg)
Total activity (Ua )
Speci¢c activity (U mg31 )
Puri¢cation (fold)
Yield (%)
Cell-free extract Q-Sepharose Butyl-Toyopearl Superdex 200
550 73.7 20.2 4.70
10 100 3 550 1 630 425
17.0 49.4 87.0 91.1
1 2.90 5.12 5.36
100 35.1 16.1 4.20
a
U: Wmol min31 .
2.5. RNA analysis B. brevis S1 was grown to the mid-exponential phase in the medium mentioned above containing one of the following carbon sources: methanol (50 mM), ethanol (100 mM), or glucose (100 mM) with formaldehyde (0.1 mM) or formate (0.1 mM). Total RNA preparation and Northern blot analysis were performed as described previously [4]. Hybridization was carried out with an AlkPhos DIRECT (Amersham Pharmacia Biotech, Tokyo, Japan), using the entire coding region of hps or phi as a probe. rRNA was used as a standard for loaded total RNA.
44 kDa on gel ¢ltration. The N-terminal amino acid sequence, MQLQLALDLVNIEEAKQVVAEVQEYVDIVE, was determined on Edman’s degradation of HPS. The amino acid sequence of an internal peptide fragment was MGVDYIXVHAGYDLQAVGKN (X represents any amino acid). Judging from the results, HPS is a homodimeric enzyme. The enzyme was moderately thermostable, 20% of its activity being retained after incubation for 10 min at 60‡C. The maximum activity was found at 55‡C and pH 7.5. These enzymatic properties are comparable to those of the enzyme from Bacillus C1, except for the subunit structure, the latter being reported to be a monomeric enzyme with a molecular mass of 32 kDa [12]. 3.2. Nucleotide sequence and structural analysis of the SalI fragment
3. Results 3.1. Puri¢cation and general properties of HPS from B. brevis S1 Through the puri¢cation procedures, HPS was puri¢ed 5.36-fold from the soluble fraction to apparent homogeneity (Table 1). The relative molecular mass of the puri¢ed HPS was estimated to be 25 kDa on SDS^PAGE and
Determination of the entire nucleotide sequence of the 3.0-kb SalI insert in pGivS1 revealed two complete (orf2 and orf3) and one partial ORF (orf1) (Fig. 1). orf2 consists of 636 bp, and the deduced amino acid sequence includes 211 amino acid residues with a theoretical molecular mass of 22 652 Da. This value is close to the molecular mass
Fig. 1. Gene diagram of RuMP operons in (A) B. brevis S1, (B) B. subtilis [5], (C) M. gastri MB19 [4], and (D) M. aminofaciens 77a [3]. Products of genes: orf1, glucose-6-phosphate dehydrogenase; hps (orf2), HPS; phi (orf3), PHI ; yckH, a putative DNA-binding protein; hxlA, HPS ; hxlB, PHI; rmpR, a putative regulatory protein ; rmpB, PHI; rmpA, HPS; and rmpI, transposase (IS10-R). A: The arrows in the diagram downstream of the phi gene indicate a putative transcriptional terminator as an inverted repeat with subsequent T residues (dashed line). The numbering of nucleotides starts from hps.
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determined by SDS^PAGE of the puri¢ed HPS from B. brevis S1, i.e. 25 kDa. The N-terminal and internal amino acid sequences of the puri¢ed HPS were found in the deduced amino acid sequence. From this and the results of gene expression studies (see below), orf2, named hps, was shown to encode HPS. The deduced primary structure is similar to those of the HPSs of B. subtilis (66% identity and 83% similarity), M. gastri MB19 (40% identity and 61% similarity), and M. aminofaciens 77a (37% identity and 59% similarity). orf3 consists of 555 bp corresponding to 184 amino acid residues with a predicted molecular mass of 19 942 Da. The amino acid sequence is similar to the deduced amino acid sequences of the PHIs of B. subtilis (64% identity and 82% similarity), M. gastri MB19 (39% identity and 63% similarity), and M. aminofaciens 77a (33% identity and 63% similarity). The orf3 gene was named phi according to these high similarity scores as to other PHIs and the expression of PHI activity in E. coli. The phi gene was located downstream of the hps gene with a space of 5-bp. A partial ORF (orf1), which is in the reversed orientation to hps and phi, exhibits signi¢cant similarity with the glucose 6-phosphate dehydrogenase gene from Vibrio cholerae. This enzyme is also known to be involved in the RuMP pathway [4]. As shown in Fig. 1, one set of typical promoter sequences (335 and 310 sequences) exists upstream of hps and a putative transcriptional terminator with subsequent T residues (a rho-independent transcriptional terminator) is present downstream of the termination triplet of the phi gene. Shine^Dalgarno sequences exist upstream of the hps and phi genes, respectively, one for the phi gene being within the coding region of the hps gene. These gene organization analysis results raised the possibility that hps and phi are transcribed as a polycistronic operon. 3.3. Gene expression of hps and phi in E. coli A cell-free extract of E. coli JM109 [pKHS1] harboring the hps gene, prepared from cells induced by IPTG, showed a signi¢cant level of HPS activity (2.25 Wmol min31 mg of protein31 ). The recombinant HPS was puri¢ed 16.6-fold from E. coli [pKHS1] by almost the same procedure as described above. The puri¢ed preparation gave a single band on SDS^PAGE. The N-terminal sequence, MQLQLALDLVNIEEAKQVVA^, was identical to that of the deduced amino acid sequence of the corresponding gene, hps, and that of the puri¢ed HPS from B. brevis S1. The speci¢c activity of the puri¢ed enzyme and the temperature characteristics of the activity were the almost same as those of the parent strain. The transformant of E. coli JM109 [pKPS1] harboring the phi gene was grown under the same conditions as those for hps expression. The cell-free extract exhibited a high level of PHI activity (0.5 Wmol min31 mg of protein31 ).
Table 2 Speci¢c activities of HPS and PHI in extracts of cells grown on several carbon substrates Carbon source
HPS activity (Wmol min31 mg protein31 )
PHI activity
Methanol Ethanol Glucose
7.4 3.7 0.67
11 3.9 0.97
SDS^PAGE of the cell-free extract gave a major distinct protein band corresponding to a molecular mass of 21 kDa, this value being close to the theoretical molecular mass obtained for the amino acid sequence. The control strain harboring pKK223-3 exhibited no PHI activity. 3.4. Regulation of gene expression in B. brevis S1 B. brevis S1 was grown on medium containing each carbon source, methanol, ethanol, or glucose, until the mid-exponential phase. The speci¢c activities of HPS and PHI are shown in Table 2. Cells that had been grown on methanol or ethanol showed high activities of both enzymes. Negligible HPS and PHI activities were detected in the cells that had been grown on glucose. These data indicate that HPS and PHI are induced by methanol, as well as ethanol, when added to the medium. Northern blot analysis of the cells grown on the medium containing methanol, ethanol, or glucose+formaldehyde with the DNA fragment of hps or phi as the probe revealed a identical hybridizing band, the size corresponding to the entire length of the transcription product of hps and phi (1.2 kb) (data not shown). On the other hand, the hps and phi probes did not hybridize with the total RNA of cells grown on glucose or glucose+formate. These results are in fair agreement with the enzyme induction pro¢le described above, and imply that expression of hps and phi is regulated at the mRNA level, and that the gene cluster is transcribed as a polycistronic operon, which responds to methanol (or formaldehyde) and ethanol.
4. Discussion Genetic studies on the RuMP pathway have been performed with two methylotrophic bacteria, M. gastri MB19 [4] and M. aminofaciens 77a [3,9], and a non-methylotroph, B. subtilis [5] (Fig. 1). Although the primary structures of HPS and PHI show high similarity among these three strains, the gene organization and regulatory mechanism for gene expression are quite di¡erent between the obligate methylotroph and the others. In M. aminofaciens 77a, a transposase, which is present between hps and phi, is related to regulation of the adjacent gene expression in a unique manner, and both genes are expressed monocistronically [3]. In B. brevis S1, the expression of hps and
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phi is induced by methanol and regulated under the same control at a transcriptional level as a polycistronic operon, which is similar to the cases of M. gastri MB19 [4] and B. subtilis [5]. Such a regulatory system responding to the growth substrate is indispensable for facultative methylotrophs. The arrangement of the genes on the RuMP operon in B. brevis S1 was the same as that in B. subtilis. On the other hand, the phi gene exists upstream of the hps gene in M. gastri MB19 (Fig. 1). The ORFs present upstream of the RuMP operon are di¡erent among the three strains. In the cases of B. subtilis and M. gastri MB19, the gene products are putative regulatory proteins encoded by the yckH and rmpR genes, respectively [4]. In contrast, we could not detect any ORF that encodes a regulatory protein within 2 kb upstream and 1 kb downstream of the RuMP operon region. Although the primary structures of HPS and PHI of B. brevis S1 are highly homologous to those of B. subtilis, the expression levels of hps and phi in B. brevis are much higher than those in B. subtilis. Further studies on the RuMP gene cluster are necessary to elucidate the mechanism of transcriptional regulation in facultative methylotrophic bacteria. Considerable HPS and PHI activities were present in ethanol-grown cells, and the transcription of the RuMP operon responded to ethanol. In methylotrophic Bacillus C1, NADþ -dependent methanol dehydrogenase, which could catalyze the oxidation of methanol and ethanol, is induced in methanol and ethanol medium [12]. On the other hand, HPS and PHI are assumed to be speci¢c enzymes for methanol assimilation but not for ethanol assimilation. The physiological signi¢cance of induction of the RuMP operon by ethanol remains unclear.
Acknowledgements This study was partly supported by a grant to Y.S. from the Ministry of Education, Culture, Sports, Science and Technology.
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