- Email: [email protected]
Organization of the genes involved in the ribulose monophosphate pathway in an obligate methylotrophic bacterium, Methylomonas aminofaciens 77a
FEMS Microbiology Letters 176 (1999) 125^130
Organization of the genes involved in the ribulose monophosphate pathway in an obligate methylotrophic bacterium, Methylomonas aminofaciens 77a Yasuyoshi Sakai a , Ryoji Mitsui a , Yumiko Katayama a , Hideshi Yanase b , Nobuo Kato a; * a
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan b Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680-8552, Japan Received 18 March 1999 ; received in revised form 30 April 1999; accepted 4 May 1999
Abstract The 4.4-kb PstI fragment harboring the gene encoding 3-hexulose-6-phosphate synthase, rmpA, which was previously cloned from the chromosome of an obligate methylotroph, Methylomonas aminofaciens 77a, was investigated in detail. In addition to the rmpA gene, the fragment contained three open reading frames encoding transaldolase (rmpD), IS10-R (rmpI), and 6phospho-3-hexuloisomerase (PHI) (rmpB). The rmpB gene product was overproduced in Escherichia coli cells, purified to homogeneity, and then enzymatically identified as PHI. The gene organization of the ribulose monophosphate pathway enzymes together with a transposon, IS10-R, is discussed from both evolutionary and regulatory aspects. ß 1999 Published by Elsevier Science B.V. All rights reserved. Keywords : 3-Hexulose-6-phosphate synthase; 6-Phospho-3-hexuloisomerase ; IS10-R; Ribulose monophosphate pathway; Methylomonas aminofaciens 77a
1. Introduction The ribulose monophosphate (RuMP) pathway for formaldehyde ¢xation exists in a wide range of bacteria which can grow on C1 compounds such as methane, methanol, and methylated amines. Bacteria possessing this pathway have been reported to make a signi¢cant contribution to the ecological carbon cycle [1]. The RuMP pathway begins with a reaction
* Corresponding author. Tel./Fax: +81 (75) 753 6385; E-mail: [email protected]
catalyzed by two unique enzymes, 3-hexulose-6phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) [1], and is postulated to involve several enzymes from the pentose phosphate pathway and the Entner-Doudoro¡ pathway. This information was obtained from the biochemical analysis of RuMP pathway bacteria. However, a lack of genetic studies has prevented further insights into the physiological and evolutionary aspects of this pathway. To obtain genetic information on the RuMP pathway, we cloned a 4.4-kb PstI fragment harboring the gene encoding HPS from the chromosomal DNA of an obligate methylotrophic bacterium, Methylomo-
0378-1097 / 99 / $20.00 ß 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 2 8 - 1
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nas aminofaciens 77a, using partial amino acid sequences of the puri¢ed HPS [2]. Here, we further analyze and characterize this 4.4-kb PstI fragment. This is the ¢rst description of the RuMP pathway gene cluster, i.e. the genetic organization of the RuMP pathway enzymes.
2. Materials and methods
(Applied Biosystem, model 373A) and an ABI Prism1 dye terminator cycle sequencing kit (Perkin-Elmer). The nucleotide sequence data reported in this paper appear in the DDBJ/EMBL/GenBank nucleotide sequence database under accession number AB026428. Sequence data were analyzed with the BLAST program against GenBank, EMBL, and Swiss-Prot. 2.4. Northern blot analysis
2.1. Strains and culture conditions M. aminofaciens 77a was grown on the methanol medium described previously [2]. Escherichia coli JM109, used as the host for plasmid propagation, was grown on Luria-Bertani broth in the presence of ampicillin (10 Wg ml31 ). pUH1 has a 4.4-kb insert containing the open reading frame (ORF) for HPS from M. aminofaciens 77a on pUC118 [2]. 2.2. DNA manipulation Restriction enzymes, ExTaq DNA polymerase, and a deletion kit were purchased from Takara Shuzo Co., Ltd. [K-32 P]dCTP was purchased from Amersham Pharmacia Corp. The DNA methods were as described previously [2]. pUH1 was double-digested with BamHI/KpnI or BstXI/BglII prior to ExoIII deletion. To express rmpB found on the pUH1 insert in E. coli (see below), the coding region was PCR-ampli¢ed using pUH1 as the template. Upstream and downstream primers were designed from the obtained sequence, N-terminal side, 5P-GGAATTCCTATTTAAGGTGAATGAAC-3P, and C-terminal, 5P-GGAATTCCTTACTCGAGGTTAGCATGAAT-3P. The PCR product was puri¢ed and cloned into the EcoRI site of pKK223-3, the resultant plasmid being designated pKP1, and then transformed into E. coli JM109. 2.3. Nucleotide sequencing DNA sequencing was performed by the dideoxy chain termination method using a DNA sequencer
Total RNAs were extracted by the AGPC method using Isogen (Nippon Gene Co., Ltd.), and RNA samples (20 Wg per lane) were separated on a 1.0% agarose gel containing 20 mM MOPS bu¡er containing 1 mM EDTA and 2.2 M formaldehyde. Electrophoresis was performed in 20 mM MOPS bu¡er. The hybridization probes were separated from subcloned plasmids containing rmpA- or rmpB-containing regions ampli¢ed by PCR (see below), and then labeled with a random primed DNA labeling kit (Boehringer Mannheim). 2.5. Protein manipulation The activities of HPS and PHI were assayed according to the methods of Strom et al. [3]. 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 the native enzyme was determined by gel ¢ltration with a fast protein liquid chromatography system, on a Superdex 200 column (Pharmacia Biotech). The recombinant PHI (rmpB product) was puri¢ed from a cell-free extract of E. coli JM109 [pKP1] by DEAE-Sepharose column chromatography. Elution was carried out with a linear gradient between 10 and 100 mM Tris-HCl (pH 8.2) containing 1 mM DTT, 5 mM MgCl2 , and 0.15 mM phenylmethylsulfonyl £uoride. The N-terminal amino acid sequence was determined with a protein sequencer (Perkin Elmer, type 476A).
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3. Results 3.1. Nucleotide sequence and structural analysis of the pUH1 insert Determination of the total nucleotides of the 4.4kb PstI insert on pUH1 revealed four ORFs in the
127
same direction: from the 5P-end, rmpD, rmpA, rmpI, and rmpB (Fig. 1). The deduced amino acid sequence of rmpD encodes 317 amino acid residues with a theoretical molecular mass of 34 850 Da. BLAST analysis revealed that the deduced amino acid sequence of rmpD was signi¢cantly similar to those of the transaldolases from Haemophilus in£uenzae
Fig. 1. Nucleotide sequence of the 4451-bp PstI fragment containing the rmp ORFs of M. aminofaciens 77a. Solid and dotted lines under the sequences indicate putative SD and promoter sequences, respectively. The target sequences of IS10-R are shown in reversed boxes. Arrows indicate inverted repeat sequences, as putative transcriptional terminators.
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Fig. 2. Deletion analysis of the 4.4-kb PstI fragment from pUH1, and the enzyme activities of HPS and PHI. Relative activity (%) was calculated on the basis that the activities of HPS and PHI were 5.9 Wmol mg31 min31 and 159 Wmol mg31 min31 , respectively.
(identity 70.2%) and E. coli (66.1%). Transaldolase has been assumed to be responsible for the regeneration of ribulose 5-phosphate, the formaldehyde acceptor in the RuMP pathway. Our previous study showed that rmpA (formerly designated hps) encoded HPS [2]. The third ORF, rmpI, which encodes a putative product of 401 amino acids (theoretical molecular mass, 46 025 Da), and the surrounding region completely matched a transposable element, IS10-R [4]. The target sequences, TACATAGCT, was detected in both the 5- and 3-neighboring regions. rmpB encodes a putative product of 206 amino acids. A putative Shine-Dalgarno (SD) sequence is located 5^11 bases upstream of the ATG triplet on the gene. A long inverted repeat sequence was found downstream of the transcriptional termination triplet (TAA). This putative rmpB product did not exhibit any signi¢cant similarity to previously known proteins in the databases. rmpB was shown to encode PHI, as described below. 3.2. Deletion analysis A cell-free extract of E. coli [pUH1] was found to have signi¢cant PHI as well as HPS activity. Since
rmpB was the only putative PHI-encoding gene in our structural analysis, a series of deletion clones derived from the 4.4-kb PstI insert were constructed, and introduced into E. coli JM109, and then the activities of HPS and PHI in each cell-free extract were determined (Fig. 2). As expected, no detectable HPS or PHI activity was found in the transformant carrying pUH1d3P-37 or pUH1d5P-25. Since pUH1d5P-29, pUH1d5P-46, and pUH1d5P-4 exhibited detectable PHI activity, rmpB was thought to encode PHI. During the course of this study, partial deletions of the rmpI region were found to have a dramatic e¡ect on the expression levels of rmpA and rmpB in E. coli. Southern blot analysis con¢rmed that the changes in expression levels were not due to changes in the copy numbers of the plasmids (data not shown). When the rmpI region was completely deleted (pUH1d3P-36), the rmpA expression level increased to 5.7-fold, compared to the level in pUH1 (Fig. 2). In contrast, partial deletion of rmpI resulted in a dramatic decrease in rmpB expression. A transformant carrying only the rmpB-coding region (pUH1d5P-4) showed very low PHI activity. When the rmpI region was present, the E. coli transformant
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showed considerable PHI activity. These experiments showed that rmpI has negative and positive e¡ects on the expression of rmpA and rmpB, respectively. 3.3. Characterization of the rmpB product expressed in E. coli In order to characterize the rmpB product, the rmpB-coding region was PCR-ampli¢ed and introduced into a high-expression vector, pKK223-3. A cell-free extract of the E. coli transformant with the constructed plasmid, pKP1, exhibited high-level PHI activity and gave an intensive protein band at the 20kDa position on SDS-PAGE. The rmpB product was puri¢ed to homogeneity by one-step column chromatography on DEAE-Sepharose. The N-terminal amino acid sequence (20 residues) was identical to that of the hypothetical rmpB product. The puri¢ed protein exhibited PHI activity of 15 400 Wmol min31 mg protein31 . The apparent molecular mass of the puri¢ed enzyme was 20 kDa on SDS-PAGE and 45 kDa on gel ¢ltration, suggesting that the enzyme is dimeric. The subunit molecular mass is in close agreement with the theoretical value for the deduced amino acid sequence of rmpB. Judging from these ¢ndings, the PHI produced in E. coli is composed of two identical subunits. 3.4. Northern blot analysis A DNA fragment of rmpA or rmpB was used as the probe against total RNA from M. aminofaciens 77a (data not shown). Hybridized bands were detected at di¡erent positions to 0.6 and 0.55 kb, for rmpA and rmpB, respectively, which correspond to the sizes of their translation products. This suggested that each gene is monocistronically transcribed.
4. Discussion We described here for the ¢rst time the gene cluster of the RuMP pathway in a methylotrophic bacterium. Two key enzymes in the RuMP pathway, HPS (rmpA) and PHI (rmpB), an enzyme in the pentose-phosphate pathway, transaldolase (rmpD), and a transposon, IS10-R (rmpI), are included. This gene cluster has several interesting features and provides
129
us with an evolutionary insight into the RuMP pathway. Also this report is the ¢rst description of homogeneously puri¢ed PHI. Two genes, rmpA and rmpB, encoding two key enzymes in the pathway, are adjacent to and separated by IS10-R. The presence of the RuMP gene cluster together with IS10-R re£ects the possible transposition of the RuMP pathway gene cluster during the evolution of methylotrophs. Indeed, homologous genes for the RuMP pathway enzymes are present in non-methylotrophs [5]. Interestingly, another gene involved in the RuMP pathway, rmpD, encoding transaldolase, was found in the cloned fragment. This gene organization supports the previous biochemical ¢nding that a transaldolase-catalyzed reaction in the pentose-phosphate pathway is involved in the assimilation of methanol in RuMP pathway bacteria. Our deletion analysis revealed that IS10-R acts like a regulatory element as to the expression of HPS and PHI in E. coli. In certain cases, the insertion of an IS element can transcriptionally trigger the expression of an adjacent gene, that is, orientationdependent turn-on of distal genes by class I insertion elements IS2 and IS3 [4]. In IS10-R, two promoters (pIN and pOUT) are located in the upstream region and IS10-R negatively regulates the expression of its own transposase protein at the transcriptional level [6,7]. A third promoter sequence (pIII) exists close to the inside end of IS10-R [8]. The expression of rmpA was increased by deletion of pIII and further enhanced by the existence of another two promoters. The expression of rmpB was completely repressed on deletion of the pIN and pOUT regions. These ¢ndings and the transcriptional directions of these promoters suggest that pOUT is in con£ict with the native rmpA promoter, and that pIN and pIII cause triggering of rmpB expression. Although our results were obtained with an E. coli transformant, it is quite possible that regulation by IS10-R occurs in the same Gram-negative methylotroph, M. aminofaciens, and has a regulatory function in the pertinent C1 metabolism, since (1) the gene expression in E. coli depended on the originally cloned promoters, (2) both rmpA and rmpB were monocistronic in M. aminofaciens, (3) expression of PHI did not occur in the transformant carrying pUH1-derived plasmids lacking IS10-R, and (4) the
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activity ratio of HPS to PHI (1:27) in the pUH1 transformant of E. coli was close to that in M. aminofaciens (1:20). Recently, we found a second gene cluster encoding rmpA, rmpI, and rmpB in the chromosome of M. aminofaciens 77a. Further work is in progress to elucidate the function of the RuMP pathway gene clusters in this organism.
[3]
[4] [5]
[6]
References [1] Quayle, J.R. and Ferenci, T. (1978) Evolutionary aspects of autotrophy. Microbiol. Rev. 42, 251^273. [2] Yanase, H., Ikeyama, K., Mitsui, R., Ra, S., Kita, K., Sakai, Y. and Kato, N. (1996) Cloning and sequence of the gene encoding 3-hexulose-6-phosphate synthase from the methylotrophic bacterium, Methylomonas aminofaciens 77a, and its
[7] [8]
expression in Escherichia coli. FEMS Microbiol. Lett. 135, 201^205. Strom, T., Ferenci, T. and Quayle, J.R. (1974) The carbon assimilation pathways of Methylococcus capsulatus, Pseudomonas methanica and Methylococcus trichosporium (OB3B) during growth on methane. Biochem. J. 144, 465^476. Kleckner, N. (1981) Transposable elements in prokaryotes. Annu. Rev. Genet. 15, 341^404. Reizer, J., Reizer, A. and Saier, M.H. (1997) Is the ribulose monophosphate pathway widely distributed in bacteria ? Microbiology 143, 2519^2520. Davis, M.A., Simons, R.W. and Kleckner, N. (1985) Tn10 protects itself at two levels from fortuitous activation by external promoters. Cell 43, 379^387. Simons, R.W. and Kleckner, N. (1983) Translational control of IS10 transposition. Cell 34, 683^691. Simons, R.W., Hoopes, B.C., McClure, W.R. and Kleckner, N. (1983) Three promoters near the termini of IS10 : pIN, pOUT, and pIII. Cell 34, 673^682.
FEMSLE 8821 17-6-99
Organization of the genes involved in the ribulose monophosphate pathway in an obligate methylotrophic bacterium, Methylomonas aminofaciens 77a Yasuyoshi Sakai a , Ryoji Mitsui a , Yumiko Katayama a , Hideshi Yanase b , Nobuo Kato a; * a
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan b Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680-8552, Japan Received 18 March 1999 ; received in revised form 30 April 1999; accepted 4 May 1999
Abstract The 4.4-kb PstI fragment harboring the gene encoding 3-hexulose-6-phosphate synthase, rmpA, which was previously cloned from the chromosome of an obligate methylotroph, Methylomonas aminofaciens 77a, was investigated in detail. In addition to the rmpA gene, the fragment contained three open reading frames encoding transaldolase (rmpD), IS10-R (rmpI), and 6phospho-3-hexuloisomerase (PHI) (rmpB). The rmpB gene product was overproduced in Escherichia coli cells, purified to homogeneity, and then enzymatically identified as PHI. The gene organization of the ribulose monophosphate pathway enzymes together with a transposon, IS10-R, is discussed from both evolutionary and regulatory aspects. ß 1999 Published by Elsevier Science B.V. All rights reserved. Keywords : 3-Hexulose-6-phosphate synthase; 6-Phospho-3-hexuloisomerase ; IS10-R; Ribulose monophosphate pathway; Methylomonas aminofaciens 77a
1. Introduction The ribulose monophosphate (RuMP) pathway for formaldehyde ¢xation exists in a wide range of bacteria which can grow on C1 compounds such as methane, methanol, and methylated amines. Bacteria possessing this pathway have been reported to make a signi¢cant contribution to the ecological carbon cycle [1]. The RuMP pathway begins with a reaction
* Corresponding author. Tel./Fax: +81 (75) 753 6385; E-mail: [email protected]
catalyzed by two unique enzymes, 3-hexulose-6phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) [1], and is postulated to involve several enzymes from the pentose phosphate pathway and the Entner-Doudoro¡ pathway. This information was obtained from the biochemical analysis of RuMP pathway bacteria. However, a lack of genetic studies has prevented further insights into the physiological and evolutionary aspects of this pathway. To obtain genetic information on the RuMP pathway, we cloned a 4.4-kb PstI fragment harboring the gene encoding HPS from the chromosomal DNA of an obligate methylotrophic bacterium, Methylomo-
0378-1097 / 99 / $20.00 ß 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 2 8 - 1
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Y. Sakai et al. / FEMS Microbiology Letters 176 (1999) 125^130
nas aminofaciens 77a, using partial amino acid sequences of the puri¢ed HPS [2]. Here, we further analyze and characterize this 4.4-kb PstI fragment. This is the ¢rst description of the RuMP pathway gene cluster, i.e. the genetic organization of the RuMP pathway enzymes.
2. Materials and methods
(Applied Biosystem, model 373A) and an ABI Prism1 dye terminator cycle sequencing kit (Perkin-Elmer). The nucleotide sequence data reported in this paper appear in the DDBJ/EMBL/GenBank nucleotide sequence database under accession number AB026428. Sequence data were analyzed with the BLAST program against GenBank, EMBL, and Swiss-Prot. 2.4. Northern blot analysis
2.1. Strains and culture conditions M. aminofaciens 77a was grown on the methanol medium described previously [2]. Escherichia coli JM109, used as the host for plasmid propagation, was grown on Luria-Bertani broth in the presence of ampicillin (10 Wg ml31 ). pUH1 has a 4.4-kb insert containing the open reading frame (ORF) for HPS from M. aminofaciens 77a on pUC118 [2]. 2.2. DNA manipulation Restriction enzymes, ExTaq DNA polymerase, and a deletion kit were purchased from Takara Shuzo Co., Ltd. [K-32 P]dCTP was purchased from Amersham Pharmacia Corp. The DNA methods were as described previously [2]. pUH1 was double-digested with BamHI/KpnI or BstXI/BglII prior to ExoIII deletion. To express rmpB found on the pUH1 insert in E. coli (see below), the coding region was PCR-ampli¢ed using pUH1 as the template. Upstream and downstream primers were designed from the obtained sequence, N-terminal side, 5P-GGAATTCCTATTTAAGGTGAATGAAC-3P, and C-terminal, 5P-GGAATTCCTTACTCGAGGTTAGCATGAAT-3P. The PCR product was puri¢ed and cloned into the EcoRI site of pKK223-3, the resultant plasmid being designated pKP1, and then transformed into E. coli JM109. 2.3. Nucleotide sequencing DNA sequencing was performed by the dideoxy chain termination method using a DNA sequencer
Total RNAs were extracted by the AGPC method using Isogen (Nippon Gene Co., Ltd.), and RNA samples (20 Wg per lane) were separated on a 1.0% agarose gel containing 20 mM MOPS bu¡er containing 1 mM EDTA and 2.2 M formaldehyde. Electrophoresis was performed in 20 mM MOPS bu¡er. The hybridization probes were separated from subcloned plasmids containing rmpA- or rmpB-containing regions ampli¢ed by PCR (see below), and then labeled with a random primed DNA labeling kit (Boehringer Mannheim). 2.5. Protein manipulation The activities of HPS and PHI were assayed according to the methods of Strom et al. [3]. 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 the native enzyme was determined by gel ¢ltration with a fast protein liquid chromatography system, on a Superdex 200 column (Pharmacia Biotech). The recombinant PHI (rmpB product) was puri¢ed from a cell-free extract of E. coli JM109 [pKP1] by DEAE-Sepharose column chromatography. Elution was carried out with a linear gradient between 10 and 100 mM Tris-HCl (pH 8.2) containing 1 mM DTT, 5 mM MgCl2 , and 0.15 mM phenylmethylsulfonyl £uoride. The N-terminal amino acid sequence was determined with a protein sequencer (Perkin Elmer, type 476A).
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3. Results 3.1. Nucleotide sequence and structural analysis of the pUH1 insert Determination of the total nucleotides of the 4.4kb PstI insert on pUH1 revealed four ORFs in the
127
same direction: from the 5P-end, rmpD, rmpA, rmpI, and rmpB (Fig. 1). The deduced amino acid sequence of rmpD encodes 317 amino acid residues with a theoretical molecular mass of 34 850 Da. BLAST analysis revealed that the deduced amino acid sequence of rmpD was signi¢cantly similar to those of the transaldolases from Haemophilus in£uenzae
Fig. 1. Nucleotide sequence of the 4451-bp PstI fragment containing the rmp ORFs of M. aminofaciens 77a. Solid and dotted lines under the sequences indicate putative SD and promoter sequences, respectively. The target sequences of IS10-R are shown in reversed boxes. Arrows indicate inverted repeat sequences, as putative transcriptional terminators.
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Fig. 2. Deletion analysis of the 4.4-kb PstI fragment from pUH1, and the enzyme activities of HPS and PHI. Relative activity (%) was calculated on the basis that the activities of HPS and PHI were 5.9 Wmol mg31 min31 and 159 Wmol mg31 min31 , respectively.
(identity 70.2%) and E. coli (66.1%). Transaldolase has been assumed to be responsible for the regeneration of ribulose 5-phosphate, the formaldehyde acceptor in the RuMP pathway. Our previous study showed that rmpA (formerly designated hps) encoded HPS [2]. The third ORF, rmpI, which encodes a putative product of 401 amino acids (theoretical molecular mass, 46 025 Da), and the surrounding region completely matched a transposable element, IS10-R [4]. The target sequences, TACATAGCT, was detected in both the 5- and 3-neighboring regions. rmpB encodes a putative product of 206 amino acids. A putative Shine-Dalgarno (SD) sequence is located 5^11 bases upstream of the ATG triplet on the gene. A long inverted repeat sequence was found downstream of the transcriptional termination triplet (TAA). This putative rmpB product did not exhibit any signi¢cant similarity to previously known proteins in the databases. rmpB was shown to encode PHI, as described below. 3.2. Deletion analysis A cell-free extract of E. coli [pUH1] was found to have signi¢cant PHI as well as HPS activity. Since
rmpB was the only putative PHI-encoding gene in our structural analysis, a series of deletion clones derived from the 4.4-kb PstI insert were constructed, and introduced into E. coli JM109, and then the activities of HPS and PHI in each cell-free extract were determined (Fig. 2). As expected, no detectable HPS or PHI activity was found in the transformant carrying pUH1d3P-37 or pUH1d5P-25. Since pUH1d5P-29, pUH1d5P-46, and pUH1d5P-4 exhibited detectable PHI activity, rmpB was thought to encode PHI. During the course of this study, partial deletions of the rmpI region were found to have a dramatic e¡ect on the expression levels of rmpA and rmpB in E. coli. Southern blot analysis con¢rmed that the changes in expression levels were not due to changes in the copy numbers of the plasmids (data not shown). When the rmpI region was completely deleted (pUH1d3P-36), the rmpA expression level increased to 5.7-fold, compared to the level in pUH1 (Fig. 2). In contrast, partial deletion of rmpI resulted in a dramatic decrease in rmpB expression. A transformant carrying only the rmpB-coding region (pUH1d5P-4) showed very low PHI activity. When the rmpI region was present, the E. coli transformant
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showed considerable PHI activity. These experiments showed that rmpI has negative and positive e¡ects on the expression of rmpA and rmpB, respectively. 3.3. Characterization of the rmpB product expressed in E. coli In order to characterize the rmpB product, the rmpB-coding region was PCR-ampli¢ed and introduced into a high-expression vector, pKK223-3. A cell-free extract of the E. coli transformant with the constructed plasmid, pKP1, exhibited high-level PHI activity and gave an intensive protein band at the 20kDa position on SDS-PAGE. The rmpB product was puri¢ed to homogeneity by one-step column chromatography on DEAE-Sepharose. The N-terminal amino acid sequence (20 residues) was identical to that of the hypothetical rmpB product. The puri¢ed protein exhibited PHI activity of 15 400 Wmol min31 mg protein31 . The apparent molecular mass of the puri¢ed enzyme was 20 kDa on SDS-PAGE and 45 kDa on gel ¢ltration, suggesting that the enzyme is dimeric. The subunit molecular mass is in close agreement with the theoretical value for the deduced amino acid sequence of rmpB. Judging from these ¢ndings, the PHI produced in E. coli is composed of two identical subunits. 3.4. Northern blot analysis A DNA fragment of rmpA or rmpB was used as the probe against total RNA from M. aminofaciens 77a (data not shown). Hybridized bands were detected at di¡erent positions to 0.6 and 0.55 kb, for rmpA and rmpB, respectively, which correspond to the sizes of their translation products. This suggested that each gene is monocistronically transcribed.
4. Discussion We described here for the ¢rst time the gene cluster of the RuMP pathway in a methylotrophic bacterium. Two key enzymes in the RuMP pathway, HPS (rmpA) and PHI (rmpB), an enzyme in the pentose-phosphate pathway, transaldolase (rmpD), and a transposon, IS10-R (rmpI), are included. This gene cluster has several interesting features and provides
129
us with an evolutionary insight into the RuMP pathway. Also this report is the ¢rst description of homogeneously puri¢ed PHI. Two genes, rmpA and rmpB, encoding two key enzymes in the pathway, are adjacent to and separated by IS10-R. The presence of the RuMP gene cluster together with IS10-R re£ects the possible transposition of the RuMP pathway gene cluster during the evolution of methylotrophs. Indeed, homologous genes for the RuMP pathway enzymes are present in non-methylotrophs [5]. Interestingly, another gene involved in the RuMP pathway, rmpD, encoding transaldolase, was found in the cloned fragment. This gene organization supports the previous biochemical ¢nding that a transaldolase-catalyzed reaction in the pentose-phosphate pathway is involved in the assimilation of methanol in RuMP pathway bacteria. Our deletion analysis revealed that IS10-R acts like a regulatory element as to the expression of HPS and PHI in E. coli. In certain cases, the insertion of an IS element can transcriptionally trigger the expression of an adjacent gene, that is, orientationdependent turn-on of distal genes by class I insertion elements IS2 and IS3 [4]. In IS10-R, two promoters (pIN and pOUT) are located in the upstream region and IS10-R negatively regulates the expression of its own transposase protein at the transcriptional level [6,7]. A third promoter sequence (pIII) exists close to the inside end of IS10-R [8]. The expression of rmpA was increased by deletion of pIII and further enhanced by the existence of another two promoters. The expression of rmpB was completely repressed on deletion of the pIN and pOUT regions. These ¢ndings and the transcriptional directions of these promoters suggest that pOUT is in con£ict with the native rmpA promoter, and that pIN and pIII cause triggering of rmpB expression. Although our results were obtained with an E. coli transformant, it is quite possible that regulation by IS10-R occurs in the same Gram-negative methylotroph, M. aminofaciens, and has a regulatory function in the pertinent C1 metabolism, since (1) the gene expression in E. coli depended on the originally cloned promoters, (2) both rmpA and rmpB were monocistronic in M. aminofaciens, (3) expression of PHI did not occur in the transformant carrying pUH1-derived plasmids lacking IS10-R, and (4) the
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activity ratio of HPS to PHI (1:27) in the pUH1 transformant of E. coli was close to that in M. aminofaciens (1:20). Recently, we found a second gene cluster encoding rmpA, rmpI, and rmpB in the chromosome of M. aminofaciens 77a. Further work is in progress to elucidate the function of the RuMP pathway gene clusters in this organism.
[3]
[4] [5]
[6]
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