scholarly journals The Arginine Regulatory Protein Mediates Repression by Arginine of the Operons Encoding Glutamate Synthase and Anabolic Glutamate Dehydrogenase in Pseudomonas aeruginosa

2004 ◽  
Vol 186 (12) ◽  
pp. 3848-3854 ◽  
Author(s):  
Shehab Hashim ◽  
Dong-Hyeon Kwon ◽  
Ahmed Abdelal ◽  
Chung-Dar Lu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Glutamate Dehydrogenase ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Regulatory Protein ◽  
Glutamate Synthase ◽  
Promoter Regions ◽  
Cell Extracts ◽  
Dnase I Footprinting ◽  
Glutamate Biosynthesis

ABSTRACT The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD+-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in α-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of β-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.

Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism

1981 ◽  
Vol 1 (2) ◽  
pp. 158-164
Author(s):  
N S Dunn-Coleman ◽  
E A Robey ◽  
A B Tomsett ◽  
R H Garrett
Keyword(s):  
Neurospora Crassa ◽  
Glutamate Dehydrogenase ◽  
Nitrogen Metabolism ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Glutamate Synthase ◽  
Wild Type ◽  
Mutant Strains ◽  
Synthase Regulation ◽  
Glutamate Biosynthesis ◽  
Glutamine Limitation

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.

scholarly journals Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism.

1981 ◽  
Vol 1 (2) ◽  
pp. 158-164 ◽  
Author(s):  
N S Dunn-Coleman ◽  
E A Robey ◽  
A B Tomsett ◽  
R H Garrett
Keyword(s):  
Neurospora Crassa ◽  
Glutamate Dehydrogenase ◽  
Nitrogen Metabolism ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Glutamate Synthase ◽  
Wild Type ◽  
Mutant Strains ◽  
Synthase Regulation ◽  
Glutamate Biosynthesis ◽  
Glutamine Limitation

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.

scholarly journals DesT Coordinates the Expression of Anaerobic and Aerobic Pathways for Unsaturated Fatty Acid Biosynthesis in Pseudomonas aeruginosa

2009 ◽  
Vol 192 (1) ◽  
pp. 280-285 ◽  
Author(s):  
Chitra Subramanian ◽  
Charles O. Rock ◽  
Yong-Mei Zhang
Keyword(s):  
Fatty Acids ◽  
Pseudomonas Aeruginosa ◽  
Fatty Acid ◽  
Dna Sequences ◽  
Unsaturated Fatty Acid ◽  
Unsaturated Fatty Acids ◽  
Fatty Acid Biosynthesis ◽  
Acid Synthesis ◽  
Cell Extracts ◽  
Translational Start

ABSTRACT The fabA and fabB genes are responsible for anaerobic unsaturated fatty acid formation in Pseudomonas aeruginosa. Expression of the fabAB operon was repressed by exogenous unsaturated fatty acids, and DNA sequences upstream of the translational start site were used to affinity purify DesT. The single protein interaction with the fabAB promoter detected in wild-type cell extracts was absent in the desT deletion strain, as was the repression of fabAB expression by unsaturated fatty acids. Thus, DesT senses the overall composition of the acyl-coenzyme A pool to coordinate the expression of the operons for the anaerobic (fabAB) and aerobic (desCB) pathways for unsaturated fatty acid synthesis.

scholarly journals RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Chen Li ◽  
Xinqiang Liu ◽  
Chao Lei ◽  
Han Yan ◽  
Zhihui Shao ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Biosynthetic Gene Cluster ◽  
High Yield ◽  
Biosynthetic Gene ◽  
Amycolatopsis Mediterranei ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Content Type

ABSTRACT Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium leprae. Although the biosynthetic pathway of rifamycin has been extensively studied in Amycolatopsis mediterranei, little is known about the regulation in rifamycin biosynthesis. Here, an in vivo transposon system was employed to identify genes involved in the regulation of rifamycin production in A. mediterranei U32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted in AMED_0655 (rifZ, encoding a LuxR family regulator). The rifZ gene was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster (rif cluster) was remarkably reduced in the rifZ null mutant. Based on the cotranscription assay results, genes within the rif cluster were grouped into 10 operons, sharing six promoter regions. By use of electrophoretic mobility shift assay and DNase I footprinting assay, RifZ was proved to specially bind to all six promoter regions, which was consistent with the fact that RifZ regulated the transcription of the whole rif cluster. The binding consensus sequence was further characterized through alignment using the RifZ-protected DNA sequences. By use of bionformatic analysis, another five promoters containing the RifZ box (CTACC-N8-GGATG) were identified, among which the binding of RifZ to the promoter regions of both rifK and orf18 (AMED_0645) was further verified. As RifZ directly regulates the transcription of all operons within the rif cluster, we propose that RifZ is a pathway-specific regulator for the rif cluster. IMPORTANCE To this day, rifamycin and its derivatives are still the first-line antituberculosis drugs. The biosynthesis of rifamycin has been extensively studied, and most biosynthetic processes have been characterized. However, little is known about the regulation of the transcription of the rifamycin biosynthetic gene cluster (rif cluster), and no regulator has been characterized. Through the employment of transposon screening, we here characterized a LuxR family regulator, RifZ, as a direct transcriptional activator for the rif cluster. As RifZ directly regulates the transcription of the entire rif cluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation of rif cluster transcription, RifZ may provide a new clue for further engineering of high-yield industrial strains.

scholarly journals Mapping of a replication origin within the promoter region of two unlinked, abundantly transcribed actin genes of Physarum polycephalum.

1996 ◽  
Vol 16 (3) ◽  
pp. 968-976 ◽  
Author(s):  
M Bénard ◽  
C Lagnel ◽  
D Pallotta ◽  
G Pierron
Keyword(s):  
Dna Sequences ◽  
Promoter Region ◽  
Physarum Polycephalum ◽  
Consensus Sequence ◽  
S Phase ◽  
Computer Search ◽  
Replication Origins ◽  
Promoter Regions ◽  
Autonomously Replicating Sequence ◽  
Actin Genes

We analyzed the replication of two unlinked actin genes, ardB and ardC , which are abundantly transcribed in the naturally synchronous plasmodium of the slime mold Physarum polycephalum. Detection and size measurements of single-stranded nascent replication intermediates (RIs) demonstrate that these two genes are concomitantly replicated at the onset of the 3-h S phase and tightly linked to replication origins. Appearance of RIs on neutral-neutral two-dimensional gels at specific time points in early S phase and analysis of their structure confirmed these results and further established that, in both cases, an efficient, site-specific, bidirectional origin of replication is localized within the promoter region of the gene. We also determined similar elongation rates for the divergent replication forks of the ardC gene replicon. Finally, taking advantage of a restriction fragment length polymorphism, we studied allelic replicons and demonstrate similar localizations and a simultaneous firing of allelic replication origins. Computer search revealed a low level of homology between the promoters of ardB and ardC and, most notably, the absence of DNA sequences similar to the yeast autonomously replicating sequence consensus sequence in these Physarum origin regions. Our results with the ardB and ardC actin genes support the model of early replicating origins located within the promoter regions of abundantly transcribed genes in P. polycephalum.

scholarly journals Two Roles for the Leucine-Responsive Regulatory Protein in Expression of the Alanine Catabolic Operon (dadAB) inKlebsiella aerogenes

1999 ◽  
Vol 181 (3) ◽  
pp. 1054-1058 ◽  
Author(s):  
Brian K. Janes ◽  
Robert A. Bender
Keyword(s):  
Glutamate Dehydrogenase ◽  
Dna Sequence ◽  
Regulatory Protein ◽  
Glutamate Synthase ◽  
Klebsiella Aerogenes

ABSTRACT The lrp gene, which codes for the leucine-responsive regulatory protein (Lrp), was cloned from Klebsiella aerogenes W70. The DNA sequence was determined, and the clone was used to create a disruption of the lrp gene. The lack of functional Lrp led to an increased expression of the alanine catabolic operon (dad) in the absence of the inducerl-alanine but also to a decreased expression of the operon in the presence of l-alanine. Thus, Lrp is both a repressor and activator of dad expression. Lrp is also necessary for glutamate synthase formation but not for the formation of two other enzymes controlled by the nitrogen regulatory (Ntr) system, glutamate dehydrogenase and histidase.

scholarly journals Molecular Characterization and Regulation of an Operon Encoding a System for Transport of Arginine and Ornithine and the ArgR Regulatory Protein in Pseudomonas aeruginosa

1998 ◽  
Vol 180 (21) ◽  
pp. 5559-5566 ◽  
Author(s):  
Takayuki Nishijyo ◽  
Seung-Moon Park ◽  
Chung-Dar Lu ◽  
Yoshifumi Itoh ◽  
Ahmed T. Abdelal
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Consensus Sequence ◽  
Regulatory Protein ◽  
Enteric Bacteria ◽  
Open Reading Frames ◽  
Significant Similarity ◽  
S1 Nuclease ◽  
Bacteria Transport ◽  
Transport Studies ◽  
Upstream Promoter

ABSTRACT The complete nucleotide sequence for the aot operon ofPseudomonas aeruginosa PAO1 was determined. This operon contains six open reading frames. The derived sequences for four of these, aotJ, aotQ, aotM, andaotP, show high similarity to those of components of the periplasmic binding protein-dependent ABC (ATP binding cassette) transporters of enteric bacteria. Transport studies with deletion derivatives established that these four genes function in arginine-inducible uptake of arginine and ornithine but not lysine. TheaotO gene, which encodes a polypeptide with no significant similarity to any known proteins, is not essential for arginine and ornithine uptake. The sixth and terminal gene in the operon encodes ArgR, which has been recently shown to function in regulation of arginine metabolism. Studies with anaotJ::lacZ translational fusion showed that expression of the aot operon is strongly induced by arginine and that this effect is mediated by ArgR. S1 nuclease and primer extension experiments showed the presence of two promoters, P1 and P2. The downstream promoter, P2, is induced by arginine and appears to be subject to carbon catabolite repression. The upstream promoter, P1, is induced by glutamate. Footprinting experiments established the presence of a 44-bp ArgR binding site that overlaps the −35 region for P2, as was shown to be the case for the arginine-inducible aru promoter, and the −10 region for P1, as was shown to be the case for arginine-repressible operons in P. aeruginosa. Sequence alignment confirms the architecture and the consensus sequence of the ArgR binding sites, as was previously reported.

scholarly journals Abh and AbrB Control of Bacillus subtilis Antimicrobial Gene Expression

2007 ◽  
Vol 189 (21) ◽  
pp. 7720-7732 ◽  
Author(s):  
Mark A. Strauch ◽  
Benjamin G. Bobay ◽  
John Cavanagh ◽  
Fude Yao ◽  
Angelo Wilson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Regulatory Protein ◽  
Regulatory System ◽  
Regulatory Role ◽  
Wild Type ◽  
Promoter Regions ◽  
Active Growth ◽  
Positive Role ◽  
Dnase I Footprinting

ABSTRACT The Bacillus subtilis abh gene encodes a protein whose N-terminal domain has 74% identity to the DNA-binding domain of the global regulatory protein AbrB. Strains with a mutation in abh showed alterations in the production of antimicrobial compounds directed against some other Bacillus species and gram-positive microbes. Relative to its wild-type parental strain, the abh mutant was found deficient, enhanced, or unaffected for the production of antimicrobial activity. Using lacZ fusions, we examined the effects of abh upon the expression of 10 promoters known to be regulated by AbrB, including five that transcribe well-characterized antimicrobial functions (SdpC, SkfA, TasA, sublancin, and subtilosin). For an otherwise wild-type background, the results show that Abh plays a negative regulatory role in the expression of four of the promoters, a positive role for the expression of three, and no apparent regulatory role in the expression of the other three promoters. Binding of AbrB and Abh to the promoter regions was examined using DNase I footprinting, and the results revealed significant differences. The transcription of abh is not autoregulated, but it is subject to a degree of AbrB-afforded negative regulation. The results indicate that Abh is part of the complex interconnected regulatory system that controls gene expression during the transition from active growth to stationary phase.

scholarly journals Dual Regulation of the Bacillus subtilis Regulon Comprising the lmrAB and yxaGH Operons and yxaF Gene by Two Transcriptional Repressors, LmrA and YxaF, in Response to Flavonoids

2007 ◽  
Vol 189 (14) ◽  
pp. 5170-5182 ◽  
Author(s):  
Kazutake Hirooka ◽  
Satoshi Kunikane ◽  
Hiroshi Matsuoka ◽  
Ken-Ichi Yoshida ◽  
Kanako Kumamoto ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Multidrug Resistance ◽  
Promoter Region ◽  
Consensus Sequence ◽  
Transcriptional Repressors ◽  
Promoter Regions ◽  
In Vivo Experiments ◽  
Dnase I Footprinting

ABSTRACT Bacillus subtilis LmrA is known to be a repressor that regulates the lmrAB and yxaGH operons; lmrB and yxaG encode a multidrug resistance pump and quercetin 2,3-dioxygenase, respectively. DNase I footprinting analysis revealed that LmrA and YxaF, which are paralogous to each other, bind specifically to almost the same cis sequences, LmrA/YxaF boxes, located in the promoter regions of the lmrAB operon, the yxaF gene, and the yxaGH operon for their repression and containing a consensus sequence of AWTATAtagaNYGgTCTA, where W, Y, and N stand for A or T, C or T, and any base, respectively (three-out-of-four match [in lowercase type]). Gel retardation analysis indicated that out of the eight flavonoids tested, quercetin, fisetin, and catechin are most inhibitory for LmrA to DNA binding, whereas quercetin, fisetin, tamarixetin, and galangin are most inhibitory for YxaF. Also, YxaF bound most tightly to the tandem LmrA/YxaF boxes in the yxaGH promoter region. The lacZ fusion experiments essentially supported the above-mentioned in vitro results, except that galangin did not activate the lmrAB and yxaGH promoters, probably due to its poor incorporation into cells. Thus, the LmrA/YxaF regulon presumably comprising the lmrAB operon, the yxaF gene, and the yxaGH operon is induced in response to certain flavonoids. The in vivo experiments to examine the regulation of the synthesis of the reporter β-galactosidase and quercetin 2,3-dioxgenase as well as that of multidrug resistance suggested that LmrA represses the lmrAB and yxaGH operons but that YxaF represses yxaGH more preferentially.

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