The NAD(P)H-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

Abstract

Previous studies have suggested that regulation of the enzymes of ammonia assimilation in human colonicBacteroides species is coordinated differently than in other eubacteria. The gene encoding an NAD(P)Hdependent glutamate dehydrogenase (gdhA) in Bacteroides thetaiotaomicron was cloned and expressed in Escherichia coli by mutant complementation from the recombinant plasmid pANS100. Examination of the predicted GdhA amino acid sequence revealed that this enzyme possesses motifs typical of the family I-type hexameric GDH proteins. Northern blot analysis with a gdhA-specific probe indicated that a single transcript with an electrophoretic mobility of ϳ1.6 kb was produced in both B. thetaiotaomicron and E. coli gdhA ؉ transformants. Although gdhA transcription was unaffected, no GdhA enzyme activity could be detected in E. coli transformants when smaller DNA fragments from pANS100, which contained the entire gdhA gene, were analyzed. Enzyme activity was restored if these E. coli strains were cotransformed with a second plasmid, which contained a 3-kb segment of DNA located downstream of the gdhA coding region. Frameshift mutagenesis within the DNA downstream of gdhA in pANS100 also resulted in the loss of GdhA enzyme activity. Collectively, these results are interpreted as evidence for the role of an additional gene product(s) in modulating the activity of GDH enzyme activity. Insertional mutagenesis experiments which led to disruption of the gdhA gene on the B. thetaiotaomicron chromosome indicated that gdhA mutants were not glutamate auxotrophs, but attempts to isolate similar mutants with insertion mutations in the region downstream of the gdhA gene were unsuccessful.Bacteroidaceae are gram-negative obligate anaerobes and are predominant members of the microflora associated with the oral cavity and digestive tract of humans and herbivorous animals. The human colonic species have been particularly well studied in relation to the genetics and biochemistry of antibiotic resistance (35, 37), gene transfer (42,43,48,50), and other virulence factors (21). Significant progress has also been made in dissecting the enzymology and regulation of polysaccharide utilization in colonic Bacteroides species (2,10,12,23,36,38). Most species of human colonic Bacteroides are incapable of utilizing amino acids as a nitrogen source, and growth with oligopeptides is restricted to only a few species. Therefore, pathways of ammonia assimilation are critical to the growth and development of these bacteria in the large intestine. Although current understanding of the pathways of nitrogen assimilation and nitrogen regulation in these bacteria is limited, studies which have demonstrated that the synthesis and secretion of specific proteases, which play a role in pathogenicity (17,21), are subject to nitrogen regulation (28) underscore the importance of further investigation. Moreover, a new precedent in terms of the structure of glutamine synthetases (GS) was established from studies with Bacteroides fragilis (46), and when compared to its...

The Medically Important Bacteroides spp. in Health and Disease

“…This suggests that control of activity may be the primary mechanism used to regulate GdhA, and a candidate for this inactivation activity is encoded by the gdhX gene, which is co-transcribed with gdhA in B. thetaiotaomicron. Overall, however, the role for GdhA is not entirely clear because the growth of a B. thetaiotaomicron mutant lacking an intact gdhA gene was not affected on any medium tested (Baggio and Morrision, 1996). That is, there was no obvious phenotype for the B. thetaiotaomicron gdhA mutant, but similar studies have not yet been performed in B. fragilis.…”

“…Bacteroides thetaiotaomicron also has glutamine synthetase activity; no pyridine nucleotidelinked glutamate synthase activity was detected in whole cell assays (Baggio and Morrision, 1996). The glutamine synthetase from B. fragilis is novel when compared to those of other eubacterial species.…”

“…Also, when cells are grown in the presence of high concentrations of peptide nitrogen, the activity of GdhA is low but increases if ammonia is added to the culture (Abrahams et al, 2001). There have been no transcriptional studies of gdhA in B. fragilis, but in B. thetaiotaomicron, levels of message for different growth conditions did not change (Baggio and Morrision, 1996). This suggests that control of activity may be the primary mechanism used to regulate GdhA, and a candidate for this inactivation activity is encoded by the gdhX gene, which is co-transcribed with gdhA in B. thetaiotaomicron.…”

Hetero-oligomeric glutamate dehydrogenase from Thermus thermophilus

“…1) (Smith et al, 1975), providing a major pathway for the interconversion of a-amino acids and 2-oxo acids. GDH is classified into three groups by coenzyme specificity: NAD + -dependent GDH (EC 1.4.1.2) (Camardella et al, 2002;Veronese et al, 1974), NAD(P) + -dependent GDH (EC 1.4.1.3) (Baggio & Morrison, 1996;Wen & Morrison, 1996) and NADP + -dependent GDH (EC 1.4.1.4) (Aalén et al, 1997;Kobayashi et al, 1995). NAD + -dependent GDHs are further classified into three subgroups according to the molecular size of a single subunit: GDH50s, GDH115s and GDH180s (Miñambres et al, 2000).…”

Expression of glutamine synthetase and glutamate dehydrogenase by marine bacterioplankton: Assay optimizations and efficacy for assessing nitrogen to carbon metabolic balance in situ

“…Like GS types, there are different regulatory motifs of gene expression for GDH isozymes, but in contrast to GS, expression of GDH isozymes is increased when N substrates are in excess of organic C substrates. These conditions include high (mM) NH 4 + or N-rich amino acids like arginine, ornthine, or histidine (Smith et al 1975;Hudson and Daniel 1993;Baggio and Morrison 1996). Excess organic C and energy substrates like glucose will repress GDH, particularly catabolic GDH types (e.g., Bonete et al 1996;Belitsky et al 2004).…”