Vol. 43 No. 3

SCIENCE IN CHINA (Series C)

June 2000

Cloning and characterization of the glutamate dehydrogenase gene in Bacillus licheniformis ZHU Bing (朱 冰), YU Guanqiao (俞冠翘), ZHU Jiabi (朱家璧) & SHEN Shanjiong (San Chium Shen, 沈善炯) Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, Shanghai 200032, China Correspondence should be addressed to Zhu Jiabi (email: [email protected]) Received August 27, 1999

Abstract The gdhA genes of IRC-3 GDH strain and IRC-8 GDH+ strain were cloned, and they both successfully complemented the nutritional lesion of an E. coli glutamate auxotroph, Q100 － GDH . However, the gdhA gene from the mutant IRC-8 GDH+ strain failed to complement the glutamate deficiency of the wild type strain IRC-3. The gdhA genes of the wild type and mutant origin were sequenced separately. No nucleotide difference was detected between them. Further investigations indicated that the gdhA genes were actively expressed in both the wild type and the mutant. Additionally, no GDH inhibitor was found in the wild type strain IRC-3. It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of the gdhA expression. Examination of the deduced amino acid sequence of Bacillus licheniformis GDH revealed the presence of the motifs characteristic of the familyⅠ-type hexameric protein, while the GDH of Bacillus subtilis belongs to family II. －

Keywords: Bacillus licheniformis, glutamate dehydrogenase, gdhA gene. ＋

NADP -dependent glutamate dehydrogenase (GDH, EC 1.4.1.2-4) catalyzes the reaction: NH4+ +α-ketoglutarate + NAD(P)H ⇔ glutamate + NAD(P)+. It stimulates the synthesis of glutamate, while NAD+-dependent GDH is the enzyme for glutamate catabolism[1]. GDHs have been isolated and sequenced from various sources and fall into two oligomeric classes. Most GDHs have six identical subunits, with a subunit mass between 48 and 55 ku. The hexameric GDHs are encoded by gdhA genes. Tetrameric GDHs have been reported in some fungi, with four identical subunits of 115 ku. Tetrameric GDHs are encoded by gdhB genes[2]. Bacillus licheniformis wild type strain IRC-3, formerly wrong identified as Bacillus subtilis, －

－

is deficient in the activity of GDH (GDH ) and glutamate synthase (GOGAT ). It fails to grow in the minimal medium, unless glutamate is supplied, so it appears as the glutamate auxotroph. A mutant strain IRC-8, obtained by nitrous acid mutagenesis, acquired the GDH activity and hence the ability of growing in minimal medium[3,4]1) .

－

The gdhA genes of the wild type IRC-3 GDH

and the mutant IRC-8 GDH+ were cloned and sequenced respectively. This is the first report on cloning and sequencing of the gdhA gene in Bacilli. So far the sequence of Bacilli gdhA gene 1) Unpublished data, Zhu Bing’s PhD thesis.

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available in GenBank was actually deduced from the GDH encoding open reading frame obtained by genome sequencing of Bacillus subtilis. In comparison of the gdhA of wild type origin with －

that of the mutant origin, we found that the gdhA genes from the wild type IRC-3 GDH

and the

+

mutant IRC-8 GDH exhibit the identical nucleotide sequence and the same function. Furthermore, －

the gdhA gene originated from the wild type IRC-3 GDH

has been proved to be actively ex-

pressed when cloned to the wild type and the mutant cells. It thus suggests that a mutation may be involved at the post-translational level of gdhA expression, presumably related to the covalence regulation of the GDH. Based upon the sequencing data of DNA and the deduced amino acids of gdhA genes, the evolution of GDH has been discussed. 1 1.1

Materials and methods Strains and plasmids Bacterial strains and plasmids used in this work are listed in table 1. Table 1 Bacterial strains and plasmids Strains and plasmids Strains Bacillus licheniformis IRC-3a) IRC-8a) Escherichia coli JM105 Q100 Plasmids pRB381 pGBL1 pGBL2 pGBL3 pGBL35 pGBL85 pGBL37 pGBL87

Relevant characteristics

－

－

wild type, GDH , GOGAT , glutamate auxotroph GDH+ mutant derived from IRC-3

Source or reference

lab collection lab collection

lac pro thi strA endA sbc15 hsdR4 (F′traD36 proAB lacIq lacZ△M15) thr-1 leuB6 gdh-1 hisG1 gltB31 argH1 thi-1 ara-14 lacY1 gal-6 malA1 xyl-7 mtl-2 rpsL9 tonA2 supE44 hsdR2

ref. [5]

shuttle vector for E. coli and Bacilli, Kmr (in Bacilli), Apr (in E. coli) 476 bp PCR fragment in pGEM-T vector 2.2 kb PCR fragment in pGEM-T vector 1.0 kb PCR fragment in pGEM-T vector lacZ gene under the control of IRC-3 gdhA promoter, RBS and translational start codon, using pRB381 as vector lacZ gene under the control of IRC-8 gdhA promoter, RBS and translational start codon, using pRB381 as vector intact IRC-3 gdhA gene cloned in pRB381 intact IRC-8 gdhA gene cloned in pRB381

ref. [7] this study this study this study

ref. [6]

this study this study this study this study

a) Formerly identified as Bacillus subtilis.

1.2

PCR primers PCR primers were synthesized by the National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology. Primer 1: 5′ -GGNCCNTAYAARGGNGG-3′ ； Primer 2: 5′ -TAYTGNGCNACRTTNCC-3′ ； Primer 3: 5′ -CATCCTTCTGTAAATGCGAG-3′ ； Primer 4: 5′ -ATGCCGCCTTTATACGGACC-3′ ；

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Primer 5: 5′ -AACCGTCGACTTCCCTGGTGGAGGTTGCGG-3′ ； Primer 6: 5′ -CCACAGCTGTTACGGTTAAACGACTCCCT-3′ ； Primer 7: 5′ -ATGCGGATCCTGCATCTTTCTTCAGCTCCC-3′ ； Primer 8: 5′ -ATGCATACGCTAGAAAAAATGG-3′ N=A or T or G or C，Y=C or T，R=G or A. 1.3

PCR Formal PCR reactions were carried out according to Sambrook et al.[8]. The reaction condi-

tion of Single Primer PCR 1) was as follows: 93℃ denaturing 2 min, 40℃ annealing 30 s, 72℃ extension 1 min, for 1 cycle; 93℃ denaturing 30 min, 60℃ annealing 30 s, 72℃ extension 1 min, for 40 cycles; 72℃ incubation 8 min. TaKaRa Ex Taq was used for Single Primer PCR, and the Boehringle Mannheim ExpandTM High Fidelity PCR system was used in other PCR reactions followed with further cloning steps. 1.4

Recombinant DNA techniques and cloning procedures Standard DNA methods according to Sambrook et al.[8] were used unless otherwise stated. DNA fragments were extracted from 1% (w/v) low-gelling-temperature agarose with phenol/chloroform. Restriction endonucleases and DNA polymerase (Klenow fragment), etc. from Promega (USA) and MBI Fermentas (Lithuania) were used according to the companies’ instructions. DNA sequencing was done by TaKaRa Biotech. 1.5

Northern blot analysis Isolation of total RNA: 200 mL overnight culture was pelleted by centrifugation. The pellet was suspended in 20 mL SET buffer (20% sucrose, 50 mmol/L EDTA , 50 mmol/L Tris-HCl pH 7.5), and lysozyme was added to the final concentration of 2 mg/mL. The mixture was incubated for 10 min at 37 ℃. The soft cells were pelleted by centrifugation and resuspended in 10 mL Trizol (Sangon product). After incubation for 10 min at room temperature, 2 mL chloroform was added to the bacterial lysate; after being mixed well the aqueous phase was separated by centrifugation. Total RNA was then precipitated by adding 5 mL isopropanol. RNA formaldehyde-agarose electrophoresis, blotting and hybridization were done according to Sambrook et al.[8]. HybondTM-C filters were obtained from Amersham. A 1.4 kb DNA fragment containing the IRC-8 gdhA gene coding region was amplified using primer 6 and primer 8, and was labelled by α-32P-dATP as the probe for hybridization. Nick translation system was obtained from Promega. α -32P-dATP was obtained from Amersham. 1.6

PEG/protoplast-mediated transformation of Bacillus licheniformis Chang’s method was used[9], except that the kanamycin final concentration in DMP media

1) See the footnote on page 254.

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was changed to 0.5 mg/mL. 1.7

GDH activity assay GDH activity was measured spectrophotometrically by recording the increase in absorbance at 340 nm due to the production of NADPH. The reaction mixture was referred to Phibbs’s method[10]. Cell lysates were prepared by sonicating freeze-thawed cells resuspended in 50 mmol/L Tris-HCl (pH8)-10 mmol/L mercaptoethanol buffer. These cells were centrifuged in the cold for 15 min. The supernatant was assayed for activity. Protein concentration was determined by the Bradford assay. 1.8

β-galactosidase activity assay β-galactosidase activity was determined according to Miller’s method[11].

2

Results

2.1 Cloning, sequencing and functional test of the gdhA genes from the wild type IRC-3 and the mutant IRC-8 2.1.1 Cloning of the gdhA gene from IRC-3. Two highly degenerate oligo primers, primer 1 and primer 2, were synthesized referring to the consensus amino acid sequences Pro-Tyr-LysGly-Gly and Gly-Asn-Val-Ala-Tyr, the most conserved region of the known NADP-dependent GDHs . These primers were used for PCR amplification in the presence of the restricted genomic DNA of IRC-3 as the template. A 476 bp fragment thus obtained was ligated to pGEM-T vector to form the recombinant plasmid pGBL1 and was sequenced. According to the sequence of the 5′ end and 3′end of this fragment, the oligos, primer 3 and primer 4 were correspondingly synthesized for Single Primer PCR. Two amplified fragments were obtained. One is a 2.2 kb fragment downstream of the 476 bp fragment, and the other 1.0 kb fragment upstream of the fragment. These amplified fragments were then ligated respectively to pGEM-T vector to give rise to two recombinant plasmids pGBL2 and pGBL3. Sequencing of these two amplified fragments together with that of the 476 bp fragment revealed the tentative sequence of the IRC-3 gdhA gene. Now based upon the sequence data of IRC-3 gdhA gene shown in fig.1, two primers, primers 5 and 6 were synthesized, and subjected to amplification in the presence of the restricted IRC-3 genomic DNA to directly clone the gdhA gene. The amplified fragment of 2 kb in length contains the promoter and coding regions of gdhA gene. After being restricted with Sal I and Pvu II, it was ligated with the EcoR I and Sal I restricted pRB381 plasmid DNA to form the recombinant plasmid pGB37. The sequence data of the fragment were consistent with that shown in fig. 1. So it was proved to be the cloned IRC-3 gdhA gene. 2.1.2 Cloning of the IRC-8 gdhA gene and comparison of the nucleotide sequence of IRC-8 gdhA with that of IRC-3 gdhA gene. The gdhA gene of the mutant IRC-8 was cloned by using primer 5 and primer 6 just the same way as was the gdhA gene of the wild type IRC-3. The PCRamplified fragment which contains the IRC-8 gdhA gene was ligated with pBR381. The recombi-

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Fig. 1. Nucleotide sequence of Bacillus licheniformis gdhA gene. The sequence data have been accepted by GenBank under the accession number of AF092086. RBS: ribosome binding site.

nant was named pGBL87 and sequenced. No nucleotide sequence difference was observed between the IRC-8 gdhA and the IRC-3 gdhA genes, indicating the identical structure of gdhA genes in the wild type IRC-3 and the mutant IRC-8.

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2.1.3 Functional tests of the gdhA gene. The recombinant plasmids pGBL37 and pGBL 87, bearing the IRC-3 gdhA gene and the IRC-8 gdhA gene respectively, were transferred to E. coli -

Q100 glutamate auxotroph (GDH ) separately. The transformants obtained all grew well in the minimal medium using NH4+ as the sole nitrogen source for growth, indicating that gdhA genes from the IRC-3 and IRC-8 were all capable of complementing the nutritional lesion of the re-

cipient bacteria. However, when the wild type IRC-3 (GDH ) of B. licheniformis transformed with the plasmid pGBL87 which carries the IRC-8 (GDH+) gdhA gene, the transformants generated all failed to grow in the minimal medium. These results summarized in table 2 illustrated clearly that the gdhA genes from the respective strains IRC-3 (GDH ) and IRC-8 (GDH+) were -

all active in producing GDH in E. coli, and that the gdhA gene, whatever it origins, produced the active GDH only in the mutant IRC-8, not in the wild type IRC-3 of B. licheniformis. Table 2

The function of the IRC-3 and IRC-8 gdhA genes

Strains/plasmids

Minimum media

B. licheniformis IRC-8 IRC-3 IRC-3/pGBL87 (carrying IRC-8 gdhA gene) E. coli Q100 Q100/pRB381 Q100/pGBL37 (carrying IRC-3 gdhA gene) Q100/pGBL87 (carrying IRC-8 gdhA gene) M9 media with 20 mmol/L NH4Cl were used as the minimum media[8].

＋ － －

GDH activity/IU· (g protein)-1 12.2 0 0

0 － 0 － 20.8 ＋ 26.1 ＋ －, Not able to grow; ＋, grow well.

The results of GDH activity assay of the transformants were consistent with the result of complementation tests. As shown in table 2, the NADP-dependent GDH activity was only demonstrated in the transformants capable of growing in the minimal medium with NH4+ as the nitrogen source for bacterial growth. 2.1.4 B. licheniformis gdhA gene exists as a mono-cistron. A 27 bp reverse repeat was found in the 1 981－2 007 region of B. licheniformis gdhA gene. It adjoins downstreamly with the two in frame stop codens of gdhA coding region. As shown in fig. 2, in this region, the synthesized mRNA will form a stable stem-loop structure followed by several uracils, a typical structure motif for transcription terminators. Since the cloned gdhA gene fragment covers the inFig. 2. Transcription terminator structure of B. lichetact promoter and transcription terminator, no more niformis gdhA gene. Note: UAA, two in frame stop open reading frame has been found in the transcrip- codens for B. licheniformis gdhA gene. tion unit, so the B. licheniformis gdhA gene exists as a mono-cistron.

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2.2

Expression of gdhA gene Northern blot analysis and gdh-lacZ fusion experiments were performed to investigate the expression of the gdhA gene in IRC-3 and IRC-8. 2.2.1 gdhA transcripts exist in both IRC-3 and IRC-8.

Total RNA was isolated from the cul-

tures of the wild type IRC-3 (GDH ) and mutant IRC-8 (GDH+) respectively. Each RNA sample -

was subjected to the formaldehyde-agarose electrophoresis and then transferred to nitrocellulose filter. A 1.4 kb DNA fragment containing the IRC-8 (GDH+) gdhA gene coding region was amplified by using primer 6 and primer 8. Then this 1.4 kb DNA fragment labelled with α-32P-dATP was used as probe for hybridization. As shown in fig. 3, approximately the same sized IRC-3 and IRC-8 gdhA transcripts were visualized. It indicates that the gdhA genes were transcriptionally -

expressed in both the wild type IRC-3 (GDH ) and the mutant IRC-8 (GDH+) cells. 2.2.2 Expression of gdhA-lacZ fusions in IRC-3 and IRC-8. A 0.6 kb PCR product was obtained using primer 5 and primer 7 with IRC-3 (GDH ) or IRC-8 (GDH+) DNA as tem-

plate for amplification. This amplified fragment containing the promoter region of IRC-3 gdhA gene or IRC-8 gdhA gene was restricted by Sal I Fig. 3. Northern analysis of the gdhA gene expression in IRC-3 and IRC-8. 1, IRC-3 total RNA (10 μg); 2, IRC-3 total and BamH I, and then ligated the same restricted RNA (30 μg); 3, IRC-8 total RNA (10 μg); 4, IRC-8 total plasmid pRB381 which carries the promoterless RNA (30 μg). α-32P-dATP-labelled 1.4 kb IRC-8 gdhA gene lacZ gene. The recombinant plasmids, carrying fragment was used as probe for hybridization. the IRC-3 gdhA-lacZ or IRC-8 gdhA-lacZ fusion designated as pGBL35 or pGBL85 were transferred separately to the IRC-3 and IRC-8 protoplasts. The generated transformants as shown in table 3 all exhibited the high activity of β-galactosidase, -

demonstrating the expression of the gdhA originated from the wild type IRC-3 (GDH ) and the mutant IRC-8 (GDH+) as well. Table 3 β-galactosidase activity assay of the B. licheniformis transformants carrying gdhA-lacZ fusions Strains IRC-3 IRC-8 －, No activity.

2.3

β-galactosidase activity (Miller unit) pGBL35 IRC-3 gdhA-lacZ － 268±77 － 159±30 All the data are the average of three independent repeats. pRB381

pGBL85 IRC-8 gdhA-lacZ 250±37 107±22

B. licheniformis GDH as the member of hexameric GDH family I Amino acid sequence analysis was done using PC-GENE program for 30 known hexameric GDHs, including B. licheniformis GDH and B. subtilis GDH. Phylogenetic tree derived from the analysis is diagrammed in fig. 4. The results have shown that the hexameric GDHs fall into two

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Fig. 4. Phylogenetic tree derived from analysis of the GDHs.

families, family I and family II, which is consistent with Benachenhou’s result [12]. Furthermore, analysis of the amino acid sequencing data of the 15 known hexameric GDHs revealed that B. licheniformis apparently exhibits high homology to the member of family I, such as E. coli, and less homology to the member of family II, so it should be classified to the family I, whereas B. subtilis GDH shows high homology to the member of family II, and less homology to the member of family I, thus it belongs to family II (table 4 and fig. 5). 3

Discussion As shown in our investigations, B. licheniformis gdhA gene exists as a mono-cistron. The

gdhA genes from the wild type IRC-3 GDH strain and the mutant IRC-8 GDH+ strain gave identi-

cal nucleotide sequence. Therefore, the GDH phenotype difference between the wild type and the

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mutant is not the result of the gdhA gene mutation, nor the possibility of polar effect that happened in the cistron. Table 4

Amino acids sequence homology among 15 different GDHs

GDH source

Family I GDHs

Family II GDHs

Escherichia coli Salmonella typhimurium Bacillus licheniformis Clostridium symbiosum Giardia lambia Chlorella sorokiniana Neurospora crassa Saccharomyces cerevisiae Clostridium difficile Bacillus subtilis Thermococcus litoralis Zea mays (Corn) Vitis vinifera (Grape) Human Mouse

Amino acid identity (%) for GDH Bacillus licheniformis Bacillus subtilis 60 25.1 59.6 23 100 30.5 52.4 27.2 54.3 23 53.7 27 48.6 21.4 48.1 24.4 30.2 49.4 30.5 100 27.9 51.3 25.1 42.3 27.7 42.3 17.4 31.2 17.4 32.2

Expression of the gdhA genes was demonstrated in both the wild type IRC-3 and the mutant IRC-8 by Northern analysis and gdhA-lacZ fusion test. According to our previous finding that GDH cross reacting materials were detected in the wild type IRC-3 GDH strain[13], gdhA gene is -

expressed in the wild type IRC-3, though its product is inactive. Therefore, we exclude the possibility of causing the GDH phenotype difference between the wild type IRC-3 and the mutant IRC-8 at the transcriptional or translational level of gdhA gene expression. Since no GDH inhibitor was found in the wild type strain IRC-3 (data not shown), we believe that the failure of producing the active GDH in the wild type IRC-3 is the result of the impairment at the post-translational regulation of the gdhA product. As we know, the activity of the tetrameric GDHs of Candida utilis and Saccharomyces cerevisiae is regulated by a reversible conversion between phosphorylated form and dephosphorylated form at the post-translational level. However, the mechanism of the post-translational regulation of the activity of hexameric GDHs is not clear. The conversion of the inactive gdhA gene product to the active GDH, associated with other gene product(s), was shown in Bacteroides thetaiotaomicron[14]. Lin et al.[15] reported that E. coli GDH is phosphorylated in a histidine residue. Moreover, they found that the extent of phosphorylation of GDH is distinct at different stage of bacterial growth and is the result of the overall activity of GDH kinase and phosphatase. They suggested that a reversible phosphorylation-dephosphorylation process is responsible for regulation of the activity of GDH. In B. licheniformis, we assume, the wild type strain IRC-3 might be a cryptic mutant of the GDH+ ancestor strain and the mutant IRC-8 GDH+ derived from it through back mutation. The mutation happened not in gdhA gene, but in another gene related to the post-translational regulation of the activity of GDH. Studies on the mechanism of the

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Fig. 5. Amino acid sequence comparison of Bacillus licheniformis (BL) GDH with GDHs of E. coli (EC) and Bacillus subtilis (BS).

post-translational regulation of B. licheniformis GDH will be undertaken in this laboratory. Most of the gene products between B. licheniformis and B. subtilis, such as 16s rRNA, leucine dehydrogenase and 6-phosphogluconate dehydrogenase, reveal high homology among them. However, low homology has been demonstrated between the B. licheniformis GDH and the B.

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subtilis GDH. The former is the member of hexameric GDH family I, and the latter the member of the family II. We are curious to know the significance for divergence of the GDHs between two phylogenetically related Bacilli in evolution. Acknowledgements We are grateful to Prof. Xu Gengjun for helpful suggestions throughout the investigation. We thank Dr. Snedecor for providing the E. coli strain Q100, Dr. Bruckner for providing the plasmid pRB381.

References 1. Smith, E. L., Austen, B. M., Blumenthal, K. M. et al., Glutamate dehydrogenase, in The Enzymes (ed. Boyer, P.D.), Vol. XI, New York: Academic Press, 1975, 293. 2. Britton, K. L., Baker, P. J., Rice, D. W. et al., Structural relationship between the hexameric and tetrameric family of glutamate dehydrogenases, Eur. J. Biochem., 1992, 209: 851. 3. Hong, M. M., Shen, S.C., Braunstein, A. E., Distribution of L-alanine dehydrogenase and L-glutamate dehydrogenase in Bacilli, Biochim. Biophys. Acta, 1959, 36: 288. 4. Shen, S. C., Hong, M. M., Chen, W. C., Conversion of alanine dehydrogenase to glutamic dehydrogenase by nitrous acid induced mutation in Bacillus subtilis (I)——Similarity in certain properties of two enzymes, Acta Biochim. Biophys. Sinica, 1963, 3: 220. 5. Yanisch-Perron, C., Vieira, J., Messing, J., Improved M13 phage cloning vectors and host strains: Nucleotide sequences of M13mp18 and pUC19 vectors, Gene, 1985, 33: 103. 6. Snedecor, B., Chu, H., Chen, E., Selection, expression and nucleotide sequencing of the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus, J. Bacteriol., 1991, 173: 6162. 7. Bruckner, R., A series of shuttle vectors for Bacillus subtilis and Escherichia coli, Gene, 1992, 122: 187. 8. Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor laboratory Press, 1989. 9. Chang, S., Cohen, S. N., High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA, Mol. Gen. Genet., 1979, 168: 111. 10. Phibbs, P. V., Jr., Bernlohr, R. W., Purification, properties, and regulation of glutamic dehydrogenase of Bacillus licheniformis, J. Bacteriol., 1971, 106: 375. 11. Miller, J. H., Experiments in Molecular Genetics, New York: Cold Spring Harbor Laboratory Press, 1972. 12. Benachenhou, N., Forterre, P., Labedan, B., Evolution of glutamate dehydrogenase genes: evidence for two paralogous protein families and unusual branching patterns of the archaebacteria in the universal tree of life, J. Mol. Evol., 1993, 36: 335. 13. Shen, S. C., Chu, C. P., Hong, M. M., Conversion of alanine dehydrogenase to glutamic dehydrogenase by nitrous acid-induced mutation in Bacillus subtilis (III)——Evidence on the presence of a protein antigenically related to GDH in wild type strains, Acta Biochim. Biophys. Sinica, 1964, 4: 242. 14. Baggio, L., Morrison, M., The NAD(P)-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA, J. Bacteriol., 1996, 178: 7212. 15. Lin, H. P., Reeves, H. C., In vivo phosphorylation of NADP+ glutamate dehydrogenase in Escherichia coli, Curr. Microbiol., 1994, 28: 63.