APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1992, p. 3165-3169
0099-2240/92/093165-05$02.00/0
Vol. 58, No. 9
Copyright X 1992, American Society for Microbiology
Organization and Nucleotide Sequence of the Glutamine Synthetase (ginA) Gene from Lactobacillus delbrueckii subsp. bulgaricus YOSHIZUMI ISHINO,1t* PHAIK MORGENTHALER,2 HERBERT HOTTINGER,2 AND DIETER SOLL1 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511,1 and Nestle Research Centre, Nestle Ltd., BP353, CH-1800 Vevey, Switzerland2 Received 13 January 1992/Accepted 10 June 1992
A 3.3-kb BamHI fragment of Lactobacilus delbrueckii subsp. bulgaricus DNA was cloned and sequenced. It complements an Escherichia coli ginA deletion strain and hybridizes strongly to a DNA containing the Bacilus subtilis glnA gene. DNA sequence analysis of the L. delbrueckii subsp. bulgaricus DNA showed it to contain the ginA gene encoding class I glutamine synthetase, as judged by extensive homology with other prokaryotic ginA genes. The sequence suggests that the enzyme encoded in this gene is not controlled by adenylylation. Based on a comparison of glutamine synthetase sequences, L. delbrueckii subsp. bulgaricus is much closer to gram-positive eubacteria, especially Clostridium acetobutylicum, than to gram-negative eubacteria and archaebacteria. The fragment contains another open reading frame encoding a protein of unknown function consisting of 306 amino acids (ORF306), which is also present upstream of g1nA of Bacilus cereus. In B. cereus, a repressor gene, gInR, is found between the open reading frame andgnA. Two proteins encoded by the L. delbrueckii subsp. bulgaricus gene were identified by the maxicell method; the sizes of these proteins are consistent with those of the open reading frames of ORF306 and gin4. The lack of a glnR gene in the L. delbrueckii subsp. bulgaricus DNA in this position may indicate a gene rearrangement or a different mechanism of glnA gene expression.
Lactobacillus delbrueckii subsp. bulgancus is a member of the large, economically and physiologically important genus Lactobacillus (17) and is used in the dairy industry for starter cultures for cheese, yogurts, and other dairy product fermentations. Though it grows well in milk, it is a fastidious organism to cultivate in the laboratory. Growth is achieved only in rich or milk-based medium. Whether the enzymes involved in the synthesis of different amino acids are present in the organism is not clear. In our efforts to study the amino acid biosynthetic pathways of L. bulgaricus, we isolated the glutamine synthetase gene of this organism by complementation of an Eschenchia coli mutant. Several glnA genes, the structural genes for glutamine synthetase, have been cloned from prokaryotes and eukaryotes and sequenced. The derived amino acid sequences have significant similarities (22, 27, 34). In this report, we describe the isolation and characterization of the glnA gene from L. delbrueckii subsp. bulgaricus and its expression in E. coli. L. delbrueckii subsp. bulgaricus ATCC 11842 was grown in MRS medium (Difco) supplemented with 2% glucose at 42°C without aeration. Total cellular DNA of L. delbrueckii subsp. bulgaricus was prepared as described previously (14). In vitro manipulations of DNA were done by standard procedures (20). The DNA was digested with 10 different restriction enzymes, resolved by electrophoresis on a 0.7% agarose gel, and transferred to nitrocellulose paper (Millipore Corp.). The 1.2-kbp glnA gene fragment of Bacillus subtilis isolated from pSF9 (10) was used as the hybridization probe. The probe was prepared by random priming with [a-32P]dATP as previously described (9). Hybridization was done at 55°C for 10 h in a solution containing 6x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH 7.0]),
1% sodium dodacyl sulfate (SDS), 5 x Denhardt's solution, 100 ,ug of calf thymus DNA per ml, and 107 cpm of labeled probe without formamide (20). Washings were carried out in lx SSC-0.1% SDS for 1 h at 50°C. This genomic Southern hybridization generated several positive bands of various sizes (data not shown). A single hybridization band that was obtained with BamHI digestion was selected because the size of the fragment, 3.5 kb, was expected to be large enough for the entire glnA structural gene. To clone the BamHIdigested positive fragment, fragments in the 3- to 4-kb range deriving from the BamHI digest were separated by sucrose density gradient as described previously (20), ligated into the BamHI site of pACYC184 (5), and used for transformation of YMC11 (2), which is a glnA deletion mutant (endAl thi-1 hsdRl 7 supE44 AlacUl69 hUtCklebs AglnALG2000). Transformants on the LB plates (20) containing 20 ,±g of chloramphenicol per ml were harvested, and the plasmid DNAs were prepared like the gene library. YMC11 was transformed again with the gene library DNA, and the transformants that grew on the M9 minimal medium plate (21) were selected. Two tiny colonies were found on the plate after incubation for 3 days. Plasmids from these colonies were prepared and used to retransform YMC11. The transformation efficiency of YMC11 to Gln+ was related to the efficiency of transformation from chloramphenicol sensitivity to chloramphenicol resistance (the number of colonies on an LB plate containing chloramphenicol and a M9 plate were the same). Further analysis of these transformants showed that they harbor plasmids with a 3.3-kbp BamHI fragment. The fragment that was contained in one of these plasmids, pIS201, hybridized to the glnA fragment of B. subtilis that was used as the probe in the genomic Southern hybridization (data not shown). The L. delbrueckii subsp. bulgaricus DNA fragment (3.3 kbp) contained in pIS201 was sequenced by dideoxynucleotide-chain termination essentially as described by Sanger et al. (29). Restriction fragments were subcloned into M13mpl8
* Corresponding author. t Present address: Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Otsu, Shiga 520-21, Japan.
3165
3166
NOTES
and 19 (37), or several deletion clones were generated by the procedure of Henikoff (12). Sequenase version 2.0 (U.S. Biochemical Corp.) was used for the DNA chain elongation reactions. The DNA was radiolabelled with [oa-35S]dATP (>1,000 Ci/mmol). The reactions were done with the nucleotide analog dITP to help elimination of compressions of the bands on the sequencing gels. Sequences were analyzed by using the program DNASIS (Hitachi Software Engineering Co., Ltd.). The G+C content of the L. delbrueckii subsp. bulganicus fragment was 49.1 mol%, compared with the reported value of 50 to 51 mol% for genomic L. delbrueckii subsp. bulgaricus DNA (19). An open reading frame (ORF) encoding a protein of 445 amino acids was found. A consensus sequence for ribosome-binding sites was found upstream of this ORF. The deduced amino acid sequence and the polypeptide length from this ORF were highly homologous with those of the prokaryotic class I glutamine synthetase (Fig. 1). Therefore, this ORF can be considered to be the glnA structural gene. Upstream of this ORF, another ORF of 918 nucleotides was found. The deduced amino acid sequence of 306 redidues was not homologous with B. subtilis and Bacillus cereus GlnRs (23, 31), which are the products of glnR genes located upstream of glnA genes and consist of operons in both Bacillus strains. However, the derived amino acid sequence of ORF306 was significantly homologous to that of an ORF that lies upstream of the glnRA operon in B. cereus (Fig. 2). The B. cereus ORF extends upstream beyond the sequenced region (22), and the gene products have not been identified. The upstream sequence of B. subtilis is not yet available. The ginA and ORF306 gene products were identified by the maxicell method (28). CSR603 cells containing plasmids were irradiated with UV light and incubated overnight in the presence of cycloserine (200 jig/ml). The cells were labeled with [35S]methionine for 120 min after they had been starved of amino acids for 120 min. Proteins in the cell lysates were separated by electrophoresis on a 10% SDS-polyacrylamide gel and visualized by fluorography. Two proteins with approximate molecular weights of 49,000 and 33,000 were observed in the maxicells carrying the pIS201. These proteins were not detected in the cells carrying pACYC184 (Fig. 3). The sizes of these two proteins correspond to those of the products from the two ORFs. This result strongly supports the hypothesis that the two ORFs on the BamHI 3.3-kb fragment were actually translated in E. coli. Plasmid pIS201 was digested with PvuI (which cuts in the structural gene of ginA), blunted, and religated. This new plasmid, designated pIS202, was introduced into YMC11 to determine its ability to transform glutamine auxotrophy. Moreover, pIS201 was digested with Sal, which cuts in ORF306 and in the pACYC184 vector. An isolated SalI fragment containing the entire glnA and a part of ORF306 was reinserted into pACYC184; the new plasmid, designated pIS203, was introduced into YMC11. Neither pIS202 nor pIS203 could compliment the glutamine auxotrophy of YMC11 any more. It is not known whether the promoter that is recognized by E. coli RNA polymerase is located between ORF306 and glnA.
APPL. ENVIRON. MICROBIOL.
Further transcriptional analyses are required to understand the genetic organization of the ORF and the relationship between these two genes. The absence of a glnR gene upstream of the gInA gene in L. delbrueckii subsp. bulgaricus may indicate the gene rearrangement, or Lactobacillus species may have some different mechanism of ginA gene expression. Comparison of the amino acid sequence of ORF306 with those in the protein data bases shows very low homology (28.6% identity in a 49-amino-acid overlap) with human protein-glutamine -y-glutamyltransferase (24), which catalyzes the transfer of a glutamate residue of the glutamyl peptide to an amino acid. The product of this ORF may interact with glutamate residues. The nucleotide sequence of the gInA structural gene of L. delbrueckii subsp. bulgaricus was compared with those of the glnA genes of B. subtilis (33), Clostridium acetobutylicum (16), and E. coli (6). The B. subtilis glnA gene was more similar to that of L. bulgaricus (50% match) than to the C. acetobutylicum (44%) or the E. coli (34%) glnA gene. It is not known whether the clostridial or E. coli glnA fragment can be used as the hybridization probe, but it would be difficult to determine proper conditions for hybridization and washing when these less homologous probes are used. The codon usages of the glutamine synthetase and ORF306 of L. delbrueckii subsp. bulgaricus were not significantly different from those of the expressed lactose-metabolizing genes of L. delbrueckii subsp. bulgaricus (18, 30; data not shown). The amino acid sequence deduced from the glnA gene was compared with the published sequences of the class I glutamine synthetases from 13 prokaryotes, including eubacteria, archaebacteria, and cyanophyta, all of which are available except for that of Bacteroides fragilis. The glutamine synthetase sequences compared were from the following: B. subtilis (33), B. cereus (22), E. coli (6), Salmonella typhimurium (15), C. acetobutylicum (16), Streptomyces coelicolor (36), Thiobacillus ferrooxidans (26), Methanococcus voltae (25), Rhizobium leguminosarum (7), Azospirillum brasilense Sp7 (3), Methylococcus capsulatus (4), Anabaena sp. strain 7210 (35), and Sulfolobus solfataricus (27). The glnA product of B. fiagilis is substantially larger (792 amino acids) than the others, and its amino acid sequence is quite dissimilar to that of the others (13). The glutamine synthetase of L. delbrueckii subsp. bulgaricus was highly homologous to the other prokaryotic class I glutamine synthetases. Based on the percentage of homology, the L. delbrueckii subsp. bulgaricus glutamine synthetase resembles glutamine synthetases from the gram-positive bacteria C. acetobutylicum, B. subtilis, and B. cereus more closely than it resembles those from gram-negative bacteria. The amino acid sequences of the glutamine synthetase from those bacteria were aligned by using a computer (Fig. 1). Clustal V multiple sequence alignments (from D. Higgins, European Molecular Biology Laboratory) was used for sequence comparison. The five regions, which are conserved in both prokaryotic and eukaryotic glutamine synthetases (25) and are associated with the proposed active site (1), are also shown. The tryptophan residue of region I, which is
FIG. 1. Alignment of the deduced amino acid sequences of glnA gene products of prokaryotes. The positions of identical or similar amino acids are indicated by asterisks and dots, respectively. The numbering is based on the ungapped L. delbrueckii subsp. bulgaricus sequence. Five regions of high similarity are underlined. Important residues described in the text for comparison are boxed. Abbreviations: LBU, L. delbrueckii subsp. bulgaricus; CAC, C. acetobutylicum (16); BSU, B. subtilis (33); BCE, B. cereus (22); MVO, M. voltae (25); STY, S. typhymurium (15); ECO, E. coli (6); MCA, M. capsulatus (4); TFE, T. ferrooxidans (26); RLE, R. leguminosarum (7); ANA, Anabaena sp. strain 7210 (35); ABR, A. brasilense (3); SCO, S. coelicolor (36); SSO, S. solfataricus (27).
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B.c. V A Y V G G L T Y S H V K I A I C S I D A IJ EE KEL L T I S FIG. 2. Comparison of the deduced amino acid sequences between ORF306 and the B. cereus ORF upstream of glnR. The asterisks indicate that the amino acids of the two sequences are identical. The B. cereus ORF extends beyond the published nucleotide sequence toward the N terminus.
thought to complete the active site formed between adjacent subunits (1) of the gram-negative bacteria, is substituted by phenylalanine in all of the gram-positive bacteria, including L. delbrueckii subsp. bulgaricus. The histidine residue of region III, which is though to be included in the active site of the enzyme because of the oxidative inactivation of that residue (8), was conserved in all of the bacteria. The amino acid sequences around the site of glutamine synthetase adenylylation (11, 32) were also compared; L. delbrueckii subsp. bulgaricus glutamine synthetase contained phenylalanine, as did C. acetobutylicum and M. voltae glutamine synthetases, but the glutamine synthetases from all other bacteria have tyrosine at the adenylylation site. The sequence in the vicinity of the tyrosine residue was highly conserved in E. coli, S. typhimurium, and T. ferrooxidans, in which the activity of glutamine synthetase is regulated by adenylylation, but the glutamine synthetases of bacteria that do not have this regulation contain some diverse sequence. It is not known whether L. delbrueckii subsp. bulgancus GS
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is subject to adenylylation. The sequence of L. delbrueckii subsp. bulganicus suggests that the substrate specificity of the glutamine synthetase adenylyltransferase of L. delbrueckii subsp. bulganicus must be different if the glutamine synthetase is adenylylated. Further analyses are required to see whether the activity of L. delbrueckii subsp. bulganicus glutamine synthetase is regulated by adenylylation in vivo. Nucleotide sequence accession number. The nucleotide sequence of this work (accession number D10020) has been deposited with GenBank EMBL and DDBJ. We thank R. A. Bender for his thoughtful comments. We thank B. Magasanik for providing us E. coli YMC11, A. L. Sonenshein for the plasmid pSF9, and S. Ishino, M. Kitagawa, and H. Iwasaki for technical assistances. This work was supported in part by a Public Health Service grant from the National Institutes of Health to D.S. REFERENCES 1. Almassy, R. J., C. A. Janson, R. Hamlin, N. H. Xuong, and D. Eisenberg. 1986. Novel subunit-subunit interactions in the structure of glutamine synthetase. Nature (London) 323:304-309. 2. Backman, K., Y.-M. Chen, and B. Magasanik. 1981. Physical and genetic characterization of the ginA-ginG region of the Eschenichia coli chromosome. Proc. Natl. Acad. Sci. USA 78:3743-3747. 3. Bozouklian, H., and C. Elmerich. 1986. Nucleotide sequence of the Azospirillum brasilense Sp7 glutamine synthetase structural gene. Biochimie 68:1181-1187. 4. Cardy, D. L. N., and J. C. Murrell. 1990. Cloning, sequencing and expression of the glutamine synthetase structural gene (glnA) from the obligate methanotroph Methylococcus capsulatus (Bath). J. Gen. Microbiol. 136:343-352. 5. Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P1SA cryptic miniplasmid. J. Bacteriol. 134: 1141-1156. 6. Colombo, G., and J. J. Villafranca. 1986. Amino acid sequence of Escherichia coli glutamine synthetase deduced from the DNA nucleotide sequence. J. Biol. Chem. 261:10587-10591. 7. Colonna-Romano, S., A. Riccio, M. Guida, R. Defez, A. Lamberti, M. laccarino, W. Arnold, U. Priefer, and A. Puhler. 1987. Tight linkage of glnA and a putative regulatory gene in Rhizobium leguminosarum. Nucleic Acids Res. 15:1951-1963. 8. Farber, J. M., and R. L. Levine. 1986. Sequence of a peptide susceptible to mixed-function oxidation: probable cation binding site in glutamine synthetase. J. Biol. Chem. 261:4574-4578. 9. Feinberg, A. P., and B. Vogelstein. 1983. A technique for
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19. 20. 21. 22.
23.
radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. Fisher, S. H., M. S. Rosenkrantz, and A. L. Sonenshein. 1984. Glutamine synthetase gene of Bacillus subtilis. Gene 32:427438. Heinrikson, R. L., and H. S. Kongdon. 1971. Primary structure of Escherichia coli glutamine synthetase: the complete amino acid sequence of a tryptic heneicosapeptide containing covalently bound adenylic acid. J. Biol. Chem. 246:1099-1106. Henikoff, S. 1984. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351359. Hill, R. T., J. R. Parker, H. J. K. Goodman, D. T. Jones, and D. R. Woods. 1989. Molecular analysis of a novel glutamine synthetase of the anaerobe Bacteroides fragilis. J. Gen. Microbiol. 135:3271-3279. Hottinger, H., T. Ohgi, M.-C. Zwahlen, S. Dhamija, and D. S1ll. 1987. Allele-specific complementation of an Escherichia coli leuB mutation by a Lactobacillus bulgaricus tRNA gene. Gene 60:75-83. Janson, C. A., P. S. Kayne, R. J. Almassy, M. Grunstein, and D. Eisenberg. 1986. Sequence of glutamine synthetase from Salmonella typhimurium and implications for the protein structure. Gene 46:297-300. Janssen, P. J., W. A. Jones, D. T. Jones, and D. R. Woods. 1988. Molecular analysis and regulation of the glnA gene of the gram-positive anaerobe Clostridium acetobutylicum. J. Bacteriol. 170:400-408. Kandler, D., and N. Weiss. 1986. Regular, nonsporing Grampositive rods, p. 1208-1260. In J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. Leong-Morgenthaler, P., M. C. Zwahlen, and H. Hottinger. 1991. Lactose metabolism in Lactobacillus bulgaricus: analysis of the primary structure and expression of the genes involved. J. Bacteriol. 173:1951-1957. London, J. 1976. The ecology and taxonomic status of the lactobacilli. Annu. Rev. Microbiol. 30:279-301. Maniatis, T., E. F. Fritsch, and J. Sambrook 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Nakano, Y., C. Kato, E. Tanaka, K. Kimura, and K. Horikoshi. 1989. Nucleotide sequence of the glutamine synthetase gene (glnA) and its upstream region from Bacillus cereus. J. Biochem. 106:209-215. Nakano, Y., and K. Kimura. 1991. Purification and characterization of a repressor for the Bacillus cereus glnRA operon. J. Biochem. 109:223-228.
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24. Phillips, M. A., B. E. Stewart, Q. Qin, R. Chakravarty, E. E. Floyd, A. M. Jetten, and R. H. Rice. 1990. Primary structure of keratinocyte transglutaminase. Proc. Natl. Acad. Sci. USA 87:9333-9337. 25. Possot, O., L. Sibold, and J.-P. Aubert. 1989. Nucleotide sequence and expression of the glutamine synthetase structural gene, glnA, of the archaebacterium Methanococcus voltae. Res. Microbiol. 140:355-371. 26. Rawlings, D. E., W. A. Jones, E. G. O'Neill, and D. R. Woods. 1987. Nucleotide sequence of the glutamine synthetase gene and its controlling region from the acidophilic autotroph Thiobacillus ferrooxidans. Gene 53:211-217. 27. Sanangelantoni, A. M., D. Barbarini, G. D. Pasquale, P. Cammarano, and 0. Tiboni. 1990. Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: phylogenetic implications. Mol. Gen. Genet. 221:187-194. 28. Sancar, A., R. P. Wharton, S. Seltzer, B. M. Kacinski, N. D. Clarke, and W. D. Rupp. 1981. Identification of the uvrA gene product. J. Mol. Biol. 148:45-62. 29. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 30. Schmidt, B. F., R. M. Adams, C. Requadt, S. Power, and S. E. Mainzer. 1989. Expression and nucleotide sequence of the Lactobacillus bulgaricus 3-galactosidase gene cloned in Escherichia coli. J. Bacteriol. 171:625-635. 31. Schreier, H. J., S. W. Brown, K. D. Hirschi, J. F. Nomellini, and A. L. Sonenshein. 1989. Regulation of Bacillus subtilis glutamine synthetase gene expression by the product of the glnR gene. J. Mol. Biol. 210:51-63. 32. Shapiro, B. M., and E. R. Stadtman. 1968. 5'-Adenylyl-Otyrosine, the novel phosphodiester of adenylylated glutamine synthetasefrom Escherichia coli. J. Biol. Chem. 243:3769-3771. 33. Strauch, M. A., A. I. Aronson, S. W. Brown, H. J. Schreier, and A. L. Sonenshein. 1988. Sequence of the Bacillus subtilis glutamine synthetase gene region. Gene 71:257-265. 34. Tischer, E., S. Dassarma, and H. M. Goodman. 1986. Nucleotide sequence of an alfalfa glutamine synthetase gene. Mol. Gen. Genet. 203:221-229. 35. Tumer, N. E., S. J. Robinson, and R. Haselkorn. 1983. Different promoters for the Anabaena glutamine synthetase gene during growth using molecular or fixed nitrogen. Nature (London) 306:337-342. 36. Wray, L. V., Jr., and S. H. Fisher. 1988. Cloning and nucleotide sequence of the Streptomyces coelicolor gene encoding glutamine synthetase. Gene 71:247-256. 37. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mpl8 and pUC19 vectors. Gene 33:103-119.
0099-2240/92/093165-05$02.00/0
Vol. 58, No. 9
Copyright X 1992, American Society for Microbiology
Organization and Nucleotide Sequence of the Glutamine Synthetase (ginA) Gene from Lactobacillus delbrueckii subsp. bulgaricus YOSHIZUMI ISHINO,1t* PHAIK MORGENTHALER,2 HERBERT HOTTINGER,2 AND DIETER SOLL1 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511,1 and Nestle Research Centre, Nestle Ltd., BP353, CH-1800 Vevey, Switzerland2 Received 13 January 1992/Accepted 10 June 1992
A 3.3-kb BamHI fragment of Lactobacilus delbrueckii subsp. bulgaricus DNA was cloned and sequenced. It complements an Escherichia coli ginA deletion strain and hybridizes strongly to a DNA containing the Bacilus subtilis glnA gene. DNA sequence analysis of the L. delbrueckii subsp. bulgaricus DNA showed it to contain the ginA gene encoding class I glutamine synthetase, as judged by extensive homology with other prokaryotic ginA genes. The sequence suggests that the enzyme encoded in this gene is not controlled by adenylylation. Based on a comparison of glutamine synthetase sequences, L. delbrueckii subsp. bulgaricus is much closer to gram-positive eubacteria, especially Clostridium acetobutylicum, than to gram-negative eubacteria and archaebacteria. The fragment contains another open reading frame encoding a protein of unknown function consisting of 306 amino acids (ORF306), which is also present upstream of g1nA of Bacilus cereus. In B. cereus, a repressor gene, gInR, is found between the open reading frame andgnA. Two proteins encoded by the L. delbrueckii subsp. bulgaricus gene were identified by the maxicell method; the sizes of these proteins are consistent with those of the open reading frames of ORF306 and gin4. The lack of a glnR gene in the L. delbrueckii subsp. bulgaricus DNA in this position may indicate a gene rearrangement or a different mechanism of glnA gene expression.
Lactobacillus delbrueckii subsp. bulgancus is a member of the large, economically and physiologically important genus Lactobacillus (17) and is used in the dairy industry for starter cultures for cheese, yogurts, and other dairy product fermentations. Though it grows well in milk, it is a fastidious organism to cultivate in the laboratory. Growth is achieved only in rich or milk-based medium. Whether the enzymes involved in the synthesis of different amino acids are present in the organism is not clear. In our efforts to study the amino acid biosynthetic pathways of L. bulgaricus, we isolated the glutamine synthetase gene of this organism by complementation of an Eschenchia coli mutant. Several glnA genes, the structural genes for glutamine synthetase, have been cloned from prokaryotes and eukaryotes and sequenced. The derived amino acid sequences have significant similarities (22, 27, 34). In this report, we describe the isolation and characterization of the glnA gene from L. delbrueckii subsp. bulgaricus and its expression in E. coli. L. delbrueckii subsp. bulgaricus ATCC 11842 was grown in MRS medium (Difco) supplemented with 2% glucose at 42°C without aeration. Total cellular DNA of L. delbrueckii subsp. bulgaricus was prepared as described previously (14). In vitro manipulations of DNA were done by standard procedures (20). The DNA was digested with 10 different restriction enzymes, resolved by electrophoresis on a 0.7% agarose gel, and transferred to nitrocellulose paper (Millipore Corp.). The 1.2-kbp glnA gene fragment of Bacillus subtilis isolated from pSF9 (10) was used as the hybridization probe. The probe was prepared by random priming with [a-32P]dATP as previously described (9). Hybridization was done at 55°C for 10 h in a solution containing 6x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH 7.0]),
1% sodium dodacyl sulfate (SDS), 5 x Denhardt's solution, 100 ,ug of calf thymus DNA per ml, and 107 cpm of labeled probe without formamide (20). Washings were carried out in lx SSC-0.1% SDS for 1 h at 50°C. This genomic Southern hybridization generated several positive bands of various sizes (data not shown). A single hybridization band that was obtained with BamHI digestion was selected because the size of the fragment, 3.5 kb, was expected to be large enough for the entire glnA structural gene. To clone the BamHIdigested positive fragment, fragments in the 3- to 4-kb range deriving from the BamHI digest were separated by sucrose density gradient as described previously (20), ligated into the BamHI site of pACYC184 (5), and used for transformation of YMC11 (2), which is a glnA deletion mutant (endAl thi-1 hsdRl 7 supE44 AlacUl69 hUtCklebs AglnALG2000). Transformants on the LB plates (20) containing 20 ,±g of chloramphenicol per ml were harvested, and the plasmid DNAs were prepared like the gene library. YMC11 was transformed again with the gene library DNA, and the transformants that grew on the M9 minimal medium plate (21) were selected. Two tiny colonies were found on the plate after incubation for 3 days. Plasmids from these colonies were prepared and used to retransform YMC11. The transformation efficiency of YMC11 to Gln+ was related to the efficiency of transformation from chloramphenicol sensitivity to chloramphenicol resistance (the number of colonies on an LB plate containing chloramphenicol and a M9 plate were the same). Further analysis of these transformants showed that they harbor plasmids with a 3.3-kbp BamHI fragment. The fragment that was contained in one of these plasmids, pIS201, hybridized to the glnA fragment of B. subtilis that was used as the probe in the genomic Southern hybridization (data not shown). The L. delbrueckii subsp. bulgaricus DNA fragment (3.3 kbp) contained in pIS201 was sequenced by dideoxynucleotide-chain termination essentially as described by Sanger et al. (29). Restriction fragments were subcloned into M13mpl8
* Corresponding author. t Present address: Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Otsu, Shiga 520-21, Japan.
3165
3166
NOTES
and 19 (37), or several deletion clones were generated by the procedure of Henikoff (12). Sequenase version 2.0 (U.S. Biochemical Corp.) was used for the DNA chain elongation reactions. The DNA was radiolabelled with [oa-35S]dATP (>1,000 Ci/mmol). The reactions were done with the nucleotide analog dITP to help elimination of compressions of the bands on the sequencing gels. Sequences were analyzed by using the program DNASIS (Hitachi Software Engineering Co., Ltd.). The G+C content of the L. delbrueckii subsp. bulganicus fragment was 49.1 mol%, compared with the reported value of 50 to 51 mol% for genomic L. delbrueckii subsp. bulgaricus DNA (19). An open reading frame (ORF) encoding a protein of 445 amino acids was found. A consensus sequence for ribosome-binding sites was found upstream of this ORF. The deduced amino acid sequence and the polypeptide length from this ORF were highly homologous with those of the prokaryotic class I glutamine synthetase (Fig. 1). Therefore, this ORF can be considered to be the glnA structural gene. Upstream of this ORF, another ORF of 918 nucleotides was found. The deduced amino acid sequence of 306 redidues was not homologous with B. subtilis and Bacillus cereus GlnRs (23, 31), which are the products of glnR genes located upstream of glnA genes and consist of operons in both Bacillus strains. However, the derived amino acid sequence of ORF306 was significantly homologous to that of an ORF that lies upstream of the glnRA operon in B. cereus (Fig. 2). The B. cereus ORF extends upstream beyond the sequenced region (22), and the gene products have not been identified. The upstream sequence of B. subtilis is not yet available. The ginA and ORF306 gene products were identified by the maxicell method (28). CSR603 cells containing plasmids were irradiated with UV light and incubated overnight in the presence of cycloserine (200 jig/ml). The cells were labeled with [35S]methionine for 120 min after they had been starved of amino acids for 120 min. Proteins in the cell lysates were separated by electrophoresis on a 10% SDS-polyacrylamide gel and visualized by fluorography. Two proteins with approximate molecular weights of 49,000 and 33,000 were observed in the maxicells carrying the pIS201. These proteins were not detected in the cells carrying pACYC184 (Fig. 3). The sizes of these two proteins correspond to those of the products from the two ORFs. This result strongly supports the hypothesis that the two ORFs on the BamHI 3.3-kb fragment were actually translated in E. coli. Plasmid pIS201 was digested with PvuI (which cuts in the structural gene of ginA), blunted, and religated. This new plasmid, designated pIS202, was introduced into YMC11 to determine its ability to transform glutamine auxotrophy. Moreover, pIS201 was digested with Sal, which cuts in ORF306 and in the pACYC184 vector. An isolated SalI fragment containing the entire glnA and a part of ORF306 was reinserted into pACYC184; the new plasmid, designated pIS203, was introduced into YMC11. Neither pIS202 nor pIS203 could compliment the glutamine auxotrophy of YMC11 any more. It is not known whether the promoter that is recognized by E. coli RNA polymerase is located between ORF306 and glnA.
APPL. ENVIRON. MICROBIOL.
Further transcriptional analyses are required to understand the genetic organization of the ORF and the relationship between these two genes. The absence of a glnR gene upstream of the gInA gene in L. delbrueckii subsp. bulgaricus may indicate the gene rearrangement, or Lactobacillus species may have some different mechanism of ginA gene expression. Comparison of the amino acid sequence of ORF306 with those in the protein data bases shows very low homology (28.6% identity in a 49-amino-acid overlap) with human protein-glutamine -y-glutamyltransferase (24), which catalyzes the transfer of a glutamate residue of the glutamyl peptide to an amino acid. The product of this ORF may interact with glutamate residues. The nucleotide sequence of the gInA structural gene of L. delbrueckii subsp. bulgaricus was compared with those of the glnA genes of B. subtilis (33), Clostridium acetobutylicum (16), and E. coli (6). The B. subtilis glnA gene was more similar to that of L. bulgaricus (50% match) than to the C. acetobutylicum (44%) or the E. coli (34%) glnA gene. It is not known whether the clostridial or E. coli glnA fragment can be used as the hybridization probe, but it would be difficult to determine proper conditions for hybridization and washing when these less homologous probes are used. The codon usages of the glutamine synthetase and ORF306 of L. delbrueckii subsp. bulgaricus were not significantly different from those of the expressed lactose-metabolizing genes of L. delbrueckii subsp. bulgaricus (18, 30; data not shown). The amino acid sequence deduced from the glnA gene was compared with the published sequences of the class I glutamine synthetases from 13 prokaryotes, including eubacteria, archaebacteria, and cyanophyta, all of which are available except for that of Bacteroides fragilis. The glutamine synthetase sequences compared were from the following: B. subtilis (33), B. cereus (22), E. coli (6), Salmonella typhimurium (15), C. acetobutylicum (16), Streptomyces coelicolor (36), Thiobacillus ferrooxidans (26), Methanococcus voltae (25), Rhizobium leguminosarum (7), Azospirillum brasilense Sp7 (3), Methylococcus capsulatus (4), Anabaena sp. strain 7210 (35), and Sulfolobus solfataricus (27). The glnA product of B. fiagilis is substantially larger (792 amino acids) than the others, and its amino acid sequence is quite dissimilar to that of the others (13). The glutamine synthetase of L. delbrueckii subsp. bulgaricus was highly homologous to the other prokaryotic class I glutamine synthetases. Based on the percentage of homology, the L. delbrueckii subsp. bulgaricus glutamine synthetase resembles glutamine synthetases from the gram-positive bacteria C. acetobutylicum, B. subtilis, and B. cereus more closely than it resembles those from gram-negative bacteria. The amino acid sequences of the glutamine synthetase from those bacteria were aligned by using a computer (Fig. 1). Clustal V multiple sequence alignments (from D. Higgins, European Molecular Biology Laboratory) was used for sequence comparison. The five regions, which are conserved in both prokaryotic and eukaryotic glutamine synthetases (25) and are associated with the proposed active site (1), are also shown. The tryptophan residue of region I, which is
FIG. 1. Alignment of the deduced amino acid sequences of glnA gene products of prokaryotes. The positions of identical or similar amino acids are indicated by asterisks and dots, respectively. The numbering is based on the ungapped L. delbrueckii subsp. bulgaricus sequence. Five regions of high similarity are underlined. Important residues described in the text for comparison are boxed. Abbreviations: LBU, L. delbrueckii subsp. bulgaricus; CAC, C. acetobutylicum (16); BSU, B. subtilis (33); BCE, B. cereus (22); MVO, M. voltae (25); STY, S. typhymurium (15); ECO, E. coli (6); MCA, M. capsulatus (4); TFE, T. ferrooxidans (26); RLE, R. leguminosarum (7); ANA, Anabaena sp. strain 7210 (35); ABR, A. brasilense (3); SCO, S. coelicolor (36); SSO, S. solfataricus (27).
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B.c. V A Y V G G L T Y S H V K I A I C S I D A IJ EE KEL L T I S FIG. 2. Comparison of the deduced amino acid sequences between ORF306 and the B. cereus ORF upstream of glnR. The asterisks indicate that the amino acids of the two sequences are identical. The B. cereus ORF extends beyond the published nucleotide sequence toward the N terminus.
thought to complete the active site formed between adjacent subunits (1) of the gram-negative bacteria, is substituted by phenylalanine in all of the gram-positive bacteria, including L. delbrueckii subsp. bulgaricus. The histidine residue of region III, which is though to be included in the active site of the enzyme because of the oxidative inactivation of that residue (8), was conserved in all of the bacteria. The amino acid sequences around the site of glutamine synthetase adenylylation (11, 32) were also compared; L. delbrueckii subsp. bulgaricus glutamine synthetase contained phenylalanine, as did C. acetobutylicum and M. voltae glutamine synthetases, but the glutamine synthetases from all other bacteria have tyrosine at the adenylylation site. The sequence in the vicinity of the tyrosine residue was highly conserved in E. coli, S. typhimurium, and T. ferrooxidans, in which the activity of glutamine synthetase is regulated by adenylylation, but the glutamine synthetases of bacteria that do not have this regulation contain some diverse sequence. It is not known whether L. delbrueckii subsp. bulgancus GS
(KDa) 200 92.5 69
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FIG. 3. Identification of the glnA and ORF306 products in maxicells. [35S]methionine-labeled proteins produced in CSR603 carrying plasmids were separated by electrophoresis on a 10% SDS-polyacrylamide gel and visualized by fluorography. Cells carrying plasmids pACYC184 (lane 2) and pIS201 (lane 3) and 14C-labeled proteins for molecular weight standards (Amersham International plc.) (lane 1) were loaded into lanes. The arrows indicate the positions of glutamine synthetase (a) and ORF306 products (b).
is subject to adenylylation. The sequence of L. delbrueckii subsp. bulganicus suggests that the substrate specificity of the glutamine synthetase adenylyltransferase of L. delbrueckii subsp. bulganicus must be different if the glutamine synthetase is adenylylated. Further analyses are required to see whether the activity of L. delbrueckii subsp. bulganicus glutamine synthetase is regulated by adenylylation in vivo. Nucleotide sequence accession number. The nucleotide sequence of this work (accession number D10020) has been deposited with GenBank EMBL and DDBJ. We thank R. A. Bender for his thoughtful comments. We thank B. Magasanik for providing us E. coli YMC11, A. L. Sonenshein for the plasmid pSF9, and S. Ishino, M. Kitagawa, and H. Iwasaki for technical assistances. This work was supported in part by a Public Health Service grant from the National Institutes of Health to D.S. REFERENCES 1. Almassy, R. J., C. A. Janson, R. Hamlin, N. H. Xuong, and D. Eisenberg. 1986. Novel subunit-subunit interactions in the structure of glutamine synthetase. Nature (London) 323:304-309. 2. Backman, K., Y.-M. Chen, and B. Magasanik. 1981. Physical and genetic characterization of the ginA-ginG region of the Eschenichia coli chromosome. Proc. Natl. Acad. Sci. USA 78:3743-3747. 3. Bozouklian, H., and C. Elmerich. 1986. Nucleotide sequence of the Azospirillum brasilense Sp7 glutamine synthetase structural gene. Biochimie 68:1181-1187. 4. Cardy, D. L. N., and J. C. Murrell. 1990. Cloning, sequencing and expression of the glutamine synthetase structural gene (glnA) from the obligate methanotroph Methylococcus capsulatus (Bath). J. Gen. Microbiol. 136:343-352. 5. Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P1SA cryptic miniplasmid. J. Bacteriol. 134: 1141-1156. 6. Colombo, G., and J. J. Villafranca. 1986. Amino acid sequence of Escherichia coli glutamine synthetase deduced from the DNA nucleotide sequence. J. Biol. Chem. 261:10587-10591. 7. Colonna-Romano, S., A. Riccio, M. Guida, R. Defez, A. Lamberti, M. laccarino, W. Arnold, U. Priefer, and A. Puhler. 1987. Tight linkage of glnA and a putative regulatory gene in Rhizobium leguminosarum. Nucleic Acids Res. 15:1951-1963. 8. Farber, J. M., and R. L. Levine. 1986. Sequence of a peptide susceptible to mixed-function oxidation: probable cation binding site in glutamine synthetase. J. Biol. Chem. 261:4574-4578. 9. Feinberg, A. P., and B. Vogelstein. 1983. A technique for
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