The sacQ gene of Bacillus subtilis, a pleiotropic gene affecting the expression of a number of secreted gene products, has been identified as a small 46-amino-acid polypeptide. The increased expression of this polypeptide in strains carrying the sacQ36 allele, or in strains carrying the sacQ gene on a high copy plasmid, appears to be responsible for the phenotype of higher levels of proteases seen in these strains. A deletion of the sacQ gene had no apparent phenotype, indicating that it is not an essential gene.A number of mutations have been isolated in the sucrose metabolism of Bacillus subtilis that cause the hyperproduction of the secreted enzyme levansucrase (20). Two loci, sacU and sacQ, were at first regarded as regulatory genes which played a specific role in the control of the levansucrase gene, sacB (20,21). However the strains carrying sacU and sacQ mutations are affected in the production of other extracellular enzymes, including amylase and proteases (19). Indeed, a number of mutations such as pap and amyB, which were defined on the basis of the overproduction of secreted amylase or protease, were shown to be identical to the sacU mutation (37).These mutations affect the production of genes which are regulated in very different fashions. For example, levansucrase is induced by growth in the presence of sucrose and is only produced during vegetative growth (11); however, amylases and the proteases are produced during postexponential growth (31). The only common feature of the genes whose production is affected by these pleiotropic mutations is that they are all secreted gene products, and an attractive theory is that they affect some component of the secretion system. Although a great deal of attention has focused on the ability of Bacillus species to secrete large quantities of industrial enzymes, very few studies have focused on the secretion process itself (8,9,31). Recently the sacU gene of B. subtilis has been isolated and shown to be a 46-kilodalton membrane protein (3). This report describes the isolation of the sacQ gene of B. subtilis, identifies its gene product as a 46-amino-acid polypeptide, and shows by deletion of the gene that it is nonessential for growth or secretion. MATERIALS AND METHODSBacterial strains, plasmids, and media. Escherichia coli MM294 (F-supE44 endAl thi-J hsdR4) was used as a host for plasmid constructions (4). Strain GM48 (dam-), received from H Boyer, was used to prepare plasmids that were subsequently digested with BclI. The B. subtilis strains used in this study are listed in Table 1. Amylase production was tested by growing colonies overnight on a nutrient broth plate containing 1% starch and staining the plate with iodine (34).Colonies were tested for P-galactosidase expression on minimal medium plates (36) containing 0.5% glucose, 0.1% casein hydrolysate and 50 p,g of 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside per ml. Plasmids pBR322 (7), pJF751 * Corresponding author. t Present address: Genencor, Inc., South San Francisco, CA 94080.(13), pC194 (17), pJ...

The gene for the catabolic NAD-linked glutamate dehydrogenase of Peptostreptococcus asaccharolyticus was cloned by selection in Escherichia coli for complementation of a biosynthetic defect. Cloned fragments containing the gene and the P. asaccharolyticus transcription and translation signals are very highly expressed in E. coli. The nucleotide sequence of the cloned gene was determined. It codes for a polypeptide of 421 amino acids, the sequence of which is similar to those of the NADP-accepting glutamate dehydrogenases. The sequence similarity of this protein to the mammalian glutamate dehydrogenases, which accept both NADP and NAD, is greater than its similarity to the bacterial NADP-specific dehydrogenases, suggesting that this NAD-specific bacterial glutamate dehydrogenase and the NADP-specific bacterial dehydrogenases diverged separately from the line leading to the dual-specificity mammalian glutamate dehydrogenases.As part of a program to develop live cell catalysts for the manufacture of amino acids by transatination of the corresponding alpha-keto acids, we cloned in Escherichia coli the gene for the NAD-dependent glutamate dehydrogenase of an anaerobic gram-positive coccus, Peptostreptococcus asaccharolyticus. This glutamate dehydrogenase is one of the few bacterial NAD-dependent glutamate dehydrogenases that have been purified and characterized (14,16,18,33). In P. asaccharolyticus, it acts in the hydroxyglutarate fermentation of glutamate, in which its normal role is the degradation of glutamate (15). We selected for the reverse of this reaction to obtain the clone. In this paper, we report the isolation, expression, and nucleotide sequence analysis of the P. asaccharolyticus gene for glutamate dehydrogenase. We compare the amino acid sequence of the translated gene with those of other glutamate dehydrogenases to show that this glutamate dehydrogenase is more closely related to the dual-specificity (NAD-and NADP-accepting) mammalian glutamate dehydrogenases than to the NADP-linked glutamate dehydrogenase of E. coli. MATERIALS AND METHODSBacterial strains. The strains used in this work are listed in Table 1. To construct strain Q100, we moved a host restriction mutation into a glutamate auxotroph by cotransduction with a nearby TnJO insertion. Strain PA340 was transduced with P1 phage prepared from strain LCK8, and the Tetr transductants were scored for the retention of the hsdR2 locus by comparing the efficiency of plaque formation when host-modified and unmodified P1 phages were plated on each transductant. Selection for the loss of the TnJO insertion was carried out by the method of Bochner and coworkers (2). DNA isolation. P. asaccharolyticus cells were grown anaerobically in Difco thiol broth. Cycloserine (6 ,ug/ml) was added just before the cells entered stationary phase in order to make them more susceptible to lysozyme action. The harvested cells were washed in 0.15 M NaCl and were then * Corresponding author. resuspended in 18.75% sucrose-37.5 mM Tris-HCl buffer (pH 8) containing 5 mg of lys...