Co-regulation of the nitrogen-assimilatory gene cluster in Clostridium saccharobutylicum

Abstract

Nitrogen assimilation is important during solvent production by Clostridium saccharobutylicum NCP262, as acetone and butanol yields are significantly affected by the nitrogen source supplied. Growth of this bacterium was dependent on the concentration of organic nitrogen supplied and the expression of the assimilatory enzymes, glutamine synthetase (GS) and glutamate synthase (GOGAT), was shown to be induced in nitrogen-limiting conditions. The regions flanking the gene encoding GS, glnA, were isolated from C. saccharobutylicum genomic DNA, and DNA sequencing revealed that the structural genes encoding the GS (glnA) and GOGAT (gltA and gltB) enzymes were clustered together with the nitR gene in the order glnA-nitR-gltAB. RNA analysis showed that the glnA-nitR and the gltAB genes were co-transcribed on 2.3 and 6.2 kb RNA transcripts respectively, and that all four genes were induced under the same nitrogen-limiting conditions. Complementation of an Escherichia coli gltD mutant, lacking a GOGAT small subunit, was achieved only when both the C. saccharobutylicum gltA and gltB genes were expressed together under anaerobic conditions. This is believed to be the first functional analysis of a gene cluster encoding the key enzymes of nitrogen assimilation, GS and GOGAT. A similar gene arrangement is seen in Clostridium beijerinckii NCIMB 8052, and based on the common regulatory features of the promoter regions upstream of the glnA operons in both species, we suggest a model for their co-ordinated regulation by an antitermination mechanism as well as antisense RNA.

Role of GlnR in Acid-Mediated Repression of Genes Encoding Proteins Involved in Glutamine and Glutamate Metabolism inStreptococcus mutans

“…It is conceivable that GlnR acquires DNA-binding affinity through conformational changes under low pH, as shown for L. lactis GadR (49). Furthermore, the arrangement of glnRA and gltAD in S. mutans is identical to the glnA-nitR and gltAB genes of Clostridium saccharobutylicum and Clostridium beijerinckii (55). However, this arrangement is not found in other streptococci.…”

“…However, this arrangement is not found in other streptococci. The clustering and coordinated regulation of glnA and gltAB in C. saccharobutylicum and C. beijerinckii appear to be a simple and efficient mean of ensuring that glutamine synthetase (GS) and glutamate synthase are available at the same time (55). Our microarray data show that the expression of gltD was slightly reduced (0.41-fold) at acidic pH (see Table S3 in the supplemental material), suggesting that the expression of glnA and gltAD genes was regulated coordinately in S. mutans.…”

Glutamate and histidine improve both solvent yields and the acid tolerance response of Clostridium beijerinckii NCP 260

“…In times of nitrogen limitation, solventogenic Clostridium saccharobutylicum and Clostridium beijerinckii have been shown to assimilate inorganic ammonia into organic nitrogen by the biosynthesis of the amino acids, glutamate and glutamine (Stutz et al . ). Clostridium saccharobutylicum and Cl.…”

“…beijerinckii have nitrogen assimilation gene clusters consisting of the glnA ‐ nitR ‐ gltAB genes which encode glutamine synthetase (GS), the nitrogen regulator (NitR) and the two subunits of the glutamate synthase (GOGAT) respectively (Reid and Stutz ; Stutz et al . ). The cluster also contains an RNA‐binding regulatory protein, NitR, which belongs to the AmiR and NasR transcription antitermination regulator (ANTAR) family (Shu and Zhulin ).…”

“…During high nitrogen conditions, asRNA encoded in the glnA region, binds to the ribosomal binding sites of the two operons and downregulates intracellular GS‐GOGAT enzyme levels (Stutz et al . ). This gene arrangement, seen in both Cl.…”