Dataset Information

The nac (nitrogen assimilation control) gene from Escherichia coli.

ABSTRACT: The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from the Klebsiella aerogenes nac (nacK) gene. The E. coli nac gene (nacE) was isolated from a cosmid clone by complementation of a nac mutation in K. aerogenes. nacE was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between nacE and nacK, with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NAC(E) is 305 amino acids, the same as for NAC(K). A null mutation, nac-28, was generated by reverse genetics. Mutants bearing nac-28 have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nitrogen regulation of histidase formation, nac-28 mutants also showed a loss of a weak repression of glutamate dehydrogenase formation. This repression was unexpected because it is balanced by a NAC-independent activation of glutamate dehydrogenase formation during nitrogen-limited growth. Attempts to purify NAC(E) by using methods established for NAC(K) failed, and NAC(E) appears to be degraded with a half-life at 30 degrees C as short as 15 min during inhibition of protein synthesis.

SUBMITTER: Muse WB

PROVIDER: S-EPMC107004 | biostudies-literature | 1998 Mar

REPOSITORIES: biostudies-literature

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The nac (nitrogen assimilation control) gene from Escherichia coli.

Muse W B WB Bender R A RA

Journal of bacteriology 19980301 5

The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from the Klebsiella aerogenes nac (nacK) gene. The E. coli nac gene (nacE) was isolated from a cosmid clone by complementation of a nac mutation in K. aerogenes. nacE was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between nacE and nacK, with a predicted amino acid sequence identity of only 79% ...[more]

PMID: 9495755

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Project description:Escherichia coli serine hydroxymethyltransferase (GlyA) converts serine to glycine, and glyA mutants are auxotrophic for glycine. CycA is a transporter that mediates glycine uptake. Deleting glyA in E. coli strain W3110 led to activation of CysB, which was related to novobiocin (NOV) susceptibility. Moreover, deleting glyA resulted in increased sensitivity to NOV, and this could be reversed by high concentrations of glycine. Reverse mutants of ΔglyA were selected and one of them had a mutation in yrdC, the gene encoding threonylcarbamoyl-AMP synthase. Subsequent proteome analysis showed that deleting glyA led to increased expression of TcyP and TdcB, making this bacterium dependent on CycA for glycine assimilation. Furthermore, deleting cycA in a ΔglyA background caused a severe growth defect on Luria-Bertani medium, which could be complemented by high concentrations of exogenous glycine. Mutation of yrdC led to decreased expression of TdcB but increased expression of ThrA/B/C and LtaE, which favored the conversion of threonine to glycine and thus avoided the dependence on CycA. Correspondingly, deleting of tcyP, tdcB, or gshA could reverse the NOV-sensitive phenotype of ΔglyA mutants. Overexpression of cycA resulted in increased sensitivity to NOV, whereas deleting this gene caused NOV resistance. Moreover, overexpression of cycA led to increased accumulation of NOV upon drug treatment. Therefore, inactivation of glyA in E. coli led to CycA-dependent glycine assimilation, which enhanced the accumulation of NOV and then made the bacterium more sensitive to this drug. These findings broaden our understanding of glycine metabolism and mechanisms of NOV susceptibility. IMPORTANCE Novobiocin (NOV) has been used in clinical practice as an ATPase inhibitor for decades. However, because it has been withdrawn from the market, pharmaceutical companies are searching for other ATPase inhibitors. Thus, probing the mechanisms of susceptibility to NOV will be beneficial to those efforts. In this study, we showed that inactivation of glyA in E. coli led to CycA-dependent glycine assimilation, which accompanied the accumulation of NOV and thereby increased the sensitivity to this drug. To date, this is the first report demonstrating the linkage between glycine assimilation and NOV susceptibility, and it is also the first report showing that YrdC is able to modulate the metabolic flux of threonine.

Dataset Information

The nac (nitrogen assimilation control) gene from Escherichia coli.

Publications

The nac (nitrogen assimilation control) gene from Escherichia coli.

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