scholarly journals Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor.

1995 ◽  
Vol 177 (16) ◽  
pp. 4820-4824 ◽  
Author(s):  
P J Pomposiello ◽  
R A Bender
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Nitrogen Assimilation ◽  
Klebsiella Aerogenes ◽  
Control Protein ◽  
Lacz Promoter ◽  
Lysr Family

scholarly journals Genetic Analysis, Using P22 Challenge Phage, of the Nitrogen Activator Protein DNA-Binding Site in the Klebsiella aerogenes put Operon

1998 ◽  
Vol 180 (3) ◽  
pp. 571-577 ◽  
Author(s):  
Li-Mei Chen ◽  
Thomas J. Goss ◽  
Robert A. Bender ◽  
Simon Swift ◽  
Stanley Maloy
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Genetic Analysis ◽  
Nitrogen Assimilation ◽  
Regulatory Protein ◽  
Klebsiella Aerogenes ◽  
Dna Binding Site ◽  
Control Protein

ABSTRACT The nac gene product is a LysR regulatory protein required for nitrogen regulation of several operons fromKlebsiella aerogenes and Escherichia coli. We used P22 challenge phage carrying the put control region from K. aerogenes to identify the nucleotide residues important for nitrogen assimilation control protein (NAC) binding in vivo. Mutations in an asymmetric 30-bp region prevented DNA binding by NAC. Gel retardation experiments confirmed that NAC specifically binds to this sequence in vitro, but NAC does not bind to the corresponding region from the put operon of Salmonella typhimurium, which is not regulated by NAC.

scholarly journals The Amino-Terminal 100 Residues of the Nitrogen Assimilation Control Protein (NAC) Encode All Known Properties of NAC from Klebsiella aerogenes and Escherichia coli

1999 ◽  
Vol 181 (3) ◽  
pp. 934-940 ◽  
Author(s):  
Wilson B. Muse ◽  
Robert A. Bender
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Nitrogen Assimilation ◽  
Factor Xa ◽  
Klebsiella Aerogenes ◽  
Reading Frame ◽  
Amino Terminal ◽  
Control Protein

ABSTRACT The nitrogen assimilation control protein (NAC) fromKlebsiella aerogenes or Escherichia coli(NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within thenac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NACK and NACE fragments with 100 or more amino acids (wild-type NACK and NACE both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NACK and NACE were isolated as chimeric proteins with the maltose-binding protein (MBP). NACK and NACE released from such chimeras were able to activatehut transcription in a purified system in vitro, as were NACK129 and NACE100 (a NACKfragment of 129 amino acids and a NACE fragment of 100 amino acids) released from comparable chimeras. A set of NACE and NACK fragments carrying nickel-binding histidine tags (his6) at their C termini were also generated. All such constructs derived from NACE were insoluble, as was NACE itself. Of the his6-tagged constructs derived from NACK, NACK100 was inactive, but NACK120 was active. Several NAC fragments were tested for dimerization. NACK120-his6 and NACK100-his6 were dimers in solution. MBP-NACK and MBP-NACK129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NACE was readily cleaved by factor Xa, but the resulting NACE was also degraded by the protease. However, MBP-NACE-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein.

scholarly journals Nitrogen Regulation of the codBA (Cytosine Deaminase) Operon from Escherichia coli by the Nitrogen Assimilation Control Protein, NAC

2003 ◽  
Vol 185 (9) ◽  
pp. 2920-2926 ◽  
Author(s):  
Wilson B. Muse ◽  
Christopher J. Rosario ◽  
Robert A. Bender
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Assimilation ◽  
Cytosine Deaminase ◽  
In Vitro Transcription ◽  
Dependent Manner ◽  
E Coli ◽  
Different Response ◽  
Control Protein

ABSTRACT Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. β-Galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position −59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions −120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions −83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.

scholarly journals Alanine Catabolism in Klebsiella aerogenes: Molecular Characterization of the dadAB Operon and Its Regulation by the Nitrogen Assimilation Control Protein

1998 ◽  
Vol 180 (3) ◽  
pp. 563-570 ◽  
Author(s):  
Brian K. Janes ◽  
Robert A. Bender
Keyword(s):  
Amino Acid ◽  
Binding Sites ◽  
Nitrogen Assimilation ◽  
Regulatory Protein ◽  
Klebsiella Aerogenes ◽  
Sequence Matching ◽  
Acid Dehydrogenase ◽  
Amino Acid Dehydrogenase ◽  
Glutamate Production ◽  
Control Protein

ABSTRACT Klebsiella aerogenes strains with reduced levels ofd-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of Klebsiella aerogenes,dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon ofSalmonella typhimurium and the dadAX operon ofEscherichia coli, each of which codes for the smaller subunit of d-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position −44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.

scholarly journals Isolation of a Negative Control Mutant of the Nitrogen Assimilation Control Protein, NAC, in Klebsiella aerogenes

2003 ◽  
Vol 185 (2) ◽  
pp. 688-692 ◽  
Author(s):  
Brian K. Janes ◽  
Christopher J. Rosario ◽  
Robert A. Bender
Keyword(s):  
Nitrogen Assimilation ◽  
Negative Control ◽  
Klebsiella Aerogenes ◽  
Complex Structures ◽  
Wild Type ◽  
Control Protein

ABSTRACT A negative control mutant of the nitrogen assimilation control protein, NAC, has been isolated. Mutants with the leucine at position 111 changed to a nonhydrophobic residue activate transcription from hut and ure promoters, but fail to repress gdhA expression. This failure does not result from failure to bind to either of the two sites required for gdhA repression, but the binding at those sites is altered in the mutant. It appears that the NAC negative control mutants fail to form the complex structures (probably tetramers) formed by wild-type NAC at the gdhA promoter.

scholarly journals Repression of Glutamate Dehydrogenase Formation in Klebsiella aerogenes Requires Two Binding Sites for the Nitrogen Assimilation Control Protein, NAC

2002 ◽  
Vol 184 (24) ◽  
pp. 6966-6975 ◽  
Author(s):  
Thomas J. Goss ◽  
Brian K. Janes ◽  
Robert A. Bender
Keyword(s):  
Binding Site ◽  
Glutamate Dehydrogenase ◽  
Binding Sites ◽  
Nitrogen Limitation ◽  
Nitrogen Assimilation ◽  
Point Mutations ◽  
Klebsiella Aerogenes ◽  
Transcriptional Fusion ◽  
The Face ◽  
Control Protein

ABSTRACT In Klebsiella aerogenes, the gdhA gene codes for glutamate dehydrogenase, one of the enzymes responsible for assimilating ammonia into glutamate. Expression of a gdhAp-lacZ transcriptional fusion was strongly repressed by the nitrogen assimilation control protein, NAC. This strong repression (>50-fold under conditions of severe nitrogen limitation) required the presence of two separate NAC binding sites centered at −89 and +57 relative to the start of gdhA transcription. Mutants lacking either or both of these sites lost the strong repression. The distance between the two sites was less important than the face of the helix on which they lay. Insertion or deletion of 10 bp between the sites had little effect on the strong repression, but insertion of 5 bp or deletion of either 5 or 15 bp decreased the repression significantly. We propose that the strong repression of gdhAp-lacZ expression requires an interaction between the NAC molecules bound at the two sites. A weaker repression of gdhAp-lacZ expression (about threefold) required only the NAC site centered at −89. This weaker repression appears to result from NAC's ability to prevent the action of a positive effector the target of which overlaps the NAC binding site centered at −89. Point mutations and deletions of this region result in the same threefold reduction in gdhAp-lacZ expression as the presence of NAC at this site.

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