Molecular analysis and regulation of the glnA gene of the gram-positive anaerobe Clostridium acetobutylicum

Abstract

The nucleotide sequence of a 2.0-kilobase DNA segment containing the Clostridium acetobutylicum ginA gene was determined. The upstream region of the glnA gene contained two putative extended promoter consensus sequences (Pi and P2), characteristic of gram-positive bacteria. A third putative extended gram-positive promoter consensus sequence (P3), oriented towards the ginA gene, was detected downstream of the structural gene. The sequences containing the proposed promoter regions Pi and P2 or P3 were shown to have promoter activity by subcloning into promoter probe vectors. The complete amino acid sequence (444 residues) of the C. acetobutylicum glutamine synthetase (GS) was deduced, and comparisons were made with the reported amino acid sequences of GS from other organisms. To determine whether the putative promoter P3 and a downstream region with an extensive stretch of inverted repeat sequences were involved in regulation of C. acetobutylicum glnA gene expression by nitrogen in Escherichia coli, deletion plasmids were constructed lacking P3 and various downstream sequences. Deletion of the putative promoter P3 and downstream inverted repeat sequences affected the regulation of GS and reduced the levels of GS approximately fivefold under nitrogen-limiting conditions but did not affect the repression of GS levels in cells grown under nitrogen-excess conditions.In the nitrogen metabolism of bacteria, glutamine synthetase (GS) (EC 6.3.1.2) plays a central role, as it catalyzes one of the main reactions by which ammonia is assimilated (54):

Cloning and Expression ofMethylovorus maysNo. 9 Gene Encoding γ-Glutamylmethylamide Synthetase: An Enzyme Usable in Theanine Formation by Coupling with the Alcoholic Fermentation System of Baker’s Yeast

“…Figure 3 is also notable in that despite their overall diversity with GS I, GMAS conserved some of the catalytic amino acids residues (indicated by closed circles). 20) In addition, the five conserved regions (indicated by arrows I-V), key structural features of the -sheet involved in the active site, reported initially in GS I 34) and subsequently in GS II 35) and GS III N , 18) were also partially conserved in GMAS. GMAS was certainly homologous with other GSs in respect to conservation of the two regions for glutamate-binding (arrow III) and ATP-binding (arrow IV), 18) whereas it lacks a domain carrying Asp50 and Ser53, which has been proposed to be involved in ammonia-binding.…”

The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli

“…Although the GSs from different micro-organisms exhibit structural similarities, their activities are regulated by different mechanisms, such as divalent-cationinduced conformational changes, feedback inhibition by various end-products, including glutamine, and covalent modification of the enzyme subunits by an adenylylation-deadenylylation system (Tyler, 1978). In general, the GSs from enteric bacteria and Streptomyces (Reitzer & Magasanik, 1987;Streicher 8z Tyler, 1981 ;Fisher & Wray, 1989) are subject to adenylylation unlike other Gram-positive bacteria (Deuel & Prusiner, 1974;Janssen et al, 1988), cyanobacteria (Fisher et al, 1981), and the archaeote Methanobacterium ivanovi (Bhatnagar et al, 1986). The insensitivity of the T. maritima GS transferase activity to SVP treatment indicates that it is not regulated by an adenylylation-deadenylylation mechanism, a conclusion also supported by the low degree of sequence conservation (4 out of 18 amino acids) around the tyrosine residue which functions as the adenylylation site in E. coli.…”

“…i l e glu ser leu pro gly ser leu lys glu ala V a l glu glu leu lys lys asp asp Val ATA ATA GAT GCA CIG GGA GAA CAT ATA TIC GAA AAA TIT' i l e i l e asp ala leu gly glu his i l e phe glu lys phe va1 glu a l a ala glu lys asg trg lys glu phe ser t h r tyr va1 t h r asa trp glu leu gln ;~rel tyr leu tyr leu (Nakano et al, 1989), Clostridium acetobutylicum (Janssen et al, 1988) and Methanococcus voltae (Possot et al, 1989), but slightly shorter than GS subunits from E. coli (Colombo & Villafranca, 1986) and other Bacteria (Toukdarian et al, 1990). The identity of the 1320 bp ORF with the T .…”

Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli

“…These results would suggest that E. coli-like promoters and ribosome binding sites are widespread in C. acetobutylicum, as has been reported for a number of genes from other gram-positive bacteria (21,38,40). Indeed, such sequences have been identified in the glnA (30), cbgA and cbgR (the present study) and adhl (61) genes from C. acetobutylicum, but in the case of the adhl gene, a vector promoter was necessary for expression in E. coli. However, there are reports of other Clostridium genes that are not expressed in E. coli (19,21) despite the presence of the appropriate transcriptional and translational signals.…”