scholarly journals ArnR, a Novel Transcriptional Regulator, Represses Expression of the narKGHJI Operon in Corynebacterium glutamicum

2008 ◽  
Vol 190 (9) ◽  
pp. 3264-3273 ◽  
Author(s):  
Taku Nishimura ◽  
Haruhiko Teramoto ◽  
Alain A. Vertès ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Nitrate Reductase ◽  
Corynebacterium Glutamicum ◽  
Consensus Sequence ◽  
Transcriptional Regulator ◽  
Anaerobic Growth ◽  
Mrna Levels ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Terminal Electron Acceptor ◽  
Aerobic Conditions

ABSTRACT The narKGHJI operon that comprises putative nitrate/nitrite transporter (narK) and nitrate reductase (narGHJI) genes is required for the anaerobic growth of Corynebacterium glutamicum with nitrate as a terminal electron acceptor. In this study, we identified a gene, arnR, which encodes a transcriptional regulator that represses the expression of the narKGHJI operon in C. glutamicum cells under aerobic conditions. Disruption of arnR induced nitrate reductase activities of C. glutamicum cells and increased narKGHJI mRNA levels under aerobic growth conditions. DNA microarray analyses revealed that besides the narKGHJI operon, the hmp gene, which encodes flavohemoglobin, is negatively regulated by ArnR under aerobic conditions. Promoter-reporter assays indicated that arnR gene expression was positively autoregulated by its gene product, ArnR, under both aerobic and anaerobic conditions. Electrophoretic mobility shift assay experiments showed that purified hexahistidyl-tagged ArnR protein specifically binds to promoter regions of the narKGHJI operon and the hmp and arnR genes. A consensus sequence, TA(A/T)TTAA(A/T)TA, found in the promoter regions of these genes was demonstrated to be involved in the binding of ArnR. Effects on LacZ activity by deletion of the ArnR binding sites within the promoter regions fused to the reporter gene were consistent with the view that the expression of the narKGHJI operon is repressed by the ArnR protein under aerobic conditions, whereas the expression of the arnR gene is autoinduced by ArnR.

scholarly journals The Transcriptional Regulator RovS Controls the Attachment of Streptococcus agalactiae to Human Epithelial Cells and the Expression of Virulence Genes

2006 ◽  
Vol 74 (10) ◽  
pp. 5625-5635 ◽  
Author(s):  
Ulrike M. Samen ◽  
Bernhard J. Eikmanns ◽  
Dieter J. Reinscheid
Keyword(s):  
Gene Expression ◽  
Streptococcus Agalactiae ◽  
Bacterial Infections ◽  
Virulence Genes ◽  
Consensus Sequence ◽  
Transcriptional Regulator ◽  
Epithelial Cell Line ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Virulence Gene Expression

ABSTRACT Streptococcus agalactiae is part of the normal flora of the human gastrointestinal tract and also the leading cause of bacterial infections in human newborns and immunocompromised adults. The colonization and infection of different regions within the human host require a regulatory network in S. agalactiae that senses environmental stimuli and controls the formation of specific virulence factors. In the present study, we characterized an Rgg-like transcriptional regulator, designated RovS (regulator of virulence in Streptococcus agalactiae). Deletion of the rovS gene in the genome of S. agalactiae resulted in strain 6313 ΔrovS, which exhibited an increased attachment to immobilized fibrinogen and a significant increase in adherence to the eukaryotic lung epithelial cell line A549. Quantification of expression levels of known and putative S. agalactiae virulence genes by real-time PCR revealed that RovS influences the expression of fbsA, gbs0230, sodA, rogB, and the cyl operon. The altered gene expression in mutant 6313 ΔrovS was restored by plasmid-mediated expression of rovS, confirming the RovS deficiency as the cause for the observed changes in virulence gene expression in S. agalactiae. DNA electrophoretic mobility shift assays showed that RovS specifically binds to the promoter regions of fbsA, gbs0230, sodA, and the cyl operon, indicating that RovS directly regulates their expression. Deletion and mutation studies in the promoter region of fbsA, encoding the main fibrinogen receptor in S. agalactiae, identified a RovS DNA motif. Similar motifs were also found in the promoter regions of gbs0230, sodA, and the cyl operon, and alignments allowed us to propose a consensus sequence for the DNA-binding site of RovS.

scholarly journals Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 11-21 ◽  
Author(s):  
Mónica Barriuso-Iglesias ◽  
Carlos Barreiro ◽  
Fabio Flechoso ◽  
Juan F. Martín
Keyword(s):  
Corynebacterium Glutamicum ◽  
Consensus Sequence ◽  
Transcriptional Analysis ◽  
Alkaline Ph ◽  
Promoter Regions ◽  
Stress Response Genes ◽  
Promoter Probe ◽  
Promoter Probe Vector ◽  
Reduced Rate ◽  
Extension Analysis

Corynebacterium glutamicum, a soil Gram-positive bacterium used for industrial amino acid production, was found to grow optimally at pH 7·0–9·0 when incubated in 5 litre fermenters under pH-controlled conditions. The highest biomass was accumulated at pH 9·0. Growth still occurred at pH 9·5 but at a reduced rate. The expression of the pH-regulated F0F1 ATPase operon (containing the eight genes atpBEFHAGDC) was induced at alkaline pH. A 7·5 kb transcript, corresponding to the eight-gene operon, was optimally expressed at pH 9·0. The same occurred with a 1·2 kb transcript corresponding to the atpB gene. RT-PCR studies confirmed the alkaline pH induction of the F0F1 operon and the existence of the atpI gene. The atpI gene, located upstream of the F0F1 operon, was expressed at a lower level than the polycistronic 7·5 kb mRNA, from a separate promoter (P-atp1). Expression of the major promoter of the F0F1 operon, designated P-atp2, and the P-atp1 promoter was quantified by coupling them to the pET2 promoter-probe vector. Both P-atp1 and P-atp2 were functional in C. glutamicum and Escherichia coli. Primer extension analysis identified one transcription start point inside each of the two promoter regions. The P-atp1 promoter fitted the consensus sequence of promoters recognized by the vegetative σ factor of C. glutamicum, whereas the −35 and −10 boxes of P-atp2 fitted the consensus sequence for σ H-recognized Mycobacterium tuberculosis promoters CC/GGGA/GAC 17–22 nt C/GGTTC/G, known to be involved in expression of heat-shock and other stress-response genes. These results suggest that the F0F1 operon is highly expressed at alkaline pH, probably using a σ H RNA polymerase.

scholarly journals Compensatory periplasmic nitrate reductase activity supports anaerobic growth ofPseudomonas aeruginosaPAO1 in the absence of membrane nitrate reductase

2009 ◽  
Vol 55 (10) ◽  
pp. 1133-1144 ◽  
Author(s):  
Nadine E. Van Alst ◽  
Lani A. Sherrill ◽  
Barbara H. Iglewski ◽  
Constantine G. Haidaris
Keyword(s):  
Nitrate Reductase ◽  
Response Regulator ◽  
Reductase Activity ◽  
Transcriptional Start Site ◽  
Atp Synthesis ◽  
Anaerobic Growth ◽  
Start Site ◽  
Terminal Electron Acceptor ◽  
Periplasmic Nitrate Reductase ◽  
Growth Recovery

Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa . Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. The inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon, and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The roles of the 2 dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expressions, were examined by use of a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 h. In contrast, the nitrate sensor-response regulator mutant ΔnarXL displayed growth arrest initially, but resumed growth after 72 h and reached the early stationary phase in liquid culture after 120 h. Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild-type P. aeruginosa PAO1, the nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase.

scholarly journals AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions

2015 ◽  
Vol 197 (24) ◽  
pp. 3788-3796 ◽  
Author(s):  
Takayuki Kuge ◽  
Haruhiko Teramoto ◽  
Masayuki Inui
Keyword(s):  
Corynebacterium Glutamicum ◽  
Binding Sites ◽  
Large Scale ◽  
Transcriptional Regulator ◽  
Scale Production ◽  
Primary Role ◽  
Promoter Regions ◽  
Independent Manner ◽  
Content Type

ABSTRACTInCorynebacterium glutamicumATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression ofl-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDAandgalM-araR) and two (BSE1and BSE2) upstream ofaraE.l-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSBmutation resulted in derepression of botharaBDAandgalM-araRoperons. The effects of BSE1and/or BSE2mutation onaraEexpression revealed that the two sites independently function as theciselements, but BSE1plays the primary role. However, AraR was shown to bind to these sites with almost the same affinityin vitro. Taken together, the expression ofaraBDAandaraEis strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the −10 or −35 region of thegalM-araRandaraEpromoters is less effective in repression. Furthermore, downregulation ofaraBDAandaraEdependent onl-arabinose catabolism observed in the BSBmutant and the AraR-independentaraRpromoter identified withingalM-araRadd complexity to regulation of the AraR regulon derepressed byl-arabinose.IMPORTANCECorynebacterium glutamicumhas a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. MostC. glutamicumstrains are unable to use a pentose sugarl-arabinose as a carbon source. However, genes forl-arabinose utilization and its regulation have been recently identified inC. glutamicumATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the derepression byl-arabinose and thereby highlights the complex regulatory feedback loops in combination withl-arabinose catabolism-dependent repression of the AraR regulon in an AraR-independent manner.

scholarly journals A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor.

1994 ◽  
Vol 14 (2) ◽  
pp. 1383-1394 ◽  
Author(s):  
Y H Lee ◽  
M Yano ◽  
S Y Liu ◽  
E Matsunaga ◽  
P F Johnson ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Ternary Complex ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Mrna Levels ◽  
Binding Studies ◽  
Mobility Shift ◽  
Specific Expression ◽  
Nuclear Extracts

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

scholarly journals Identification of RamA, a Novel LuxR-Type Transcriptional Regulator of Genes Involved in Acetate Metabolism of Corynebacterium glutamicum

2006 ◽  
Vol 188 (7) ◽  
pp. 2554-2567 ◽  
Author(s):  
Annette Cramer ◽  
Robert Gerstmeir ◽  
Steffen Schaffer ◽  
Michael Bott ◽  
Bernhard J. Eikmanns
Keyword(s):  
Corynebacterium Glutamicum ◽  
Isocitrate Lyase ◽  
Glyoxylate Cycle ◽  
Transcriptional Regulator ◽  
Malate Synthase ◽  
Acetate Kinase ◽  
Acetate Metabolism ◽  
Promoter Regions ◽  
Cell Extracts ◽  
Peptide Mass

ABSTRACT In Corynebacterium glutamicum, the acetate-activating enzymes phosphotransacetylase and acetate kinase and the glyoxylate cycle enzymes isocitrate lyase and malate synthase are coordinately up-regulated in the presence of acetate in the growth medium. This regulation is due to transcriptional control of the respective pta-ack operon and the aceA and aceB genes, brought about at least partly by the action of the negative transcriptional regulator RamB. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, mass spectrometry, and peptide mass fingerprinting, we identified a LuxR-type transcriptional regulator, designated RamA, which binds to the pta-ack and aceA/aceB promoter regions. Inactivation of the ramA gene in the genome of C. glutamicum resulted in mutant RG2. This mutant was unable to grow on acetate as the sole carbon and energy source and, in comparison to the wild type of C. glutamicum, showed very low specific activities of phosphotransacetylase, acetate kinase, isocitrate lyase, and malate synthase, irrespective of the presence of acetate in the medium. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. By electrophoretic mobility shift analysis, purified His-tagged RamA protein was shown to bind specifically to the pta-ack and the aceA/aceB promoter regions, and deletion and mutation studies revealed in both regions two binding motifs each consisting of tandem A/C/TG4-6T/C or AC4-5A/G/T stretches separated by four or five arbitrary nucleotides. Our data indicate that RamA represents a novel LuxR-type transcriptional activator of genes involved in acetate metabolism of C. glutamicum.

scholarly journals Identification of a Repressor for the Two iol Operons Required for Inositol Catabolism in Geobacillus Kaustophilus

2020 ◽  
Author(s):  
Ken-ichi Yoshida ◽  
Yusuke Shirae ◽  
Ryo Nishimura ◽  
Kaho Fukui ◽  
Shu Ishikawa
Keyword(s):  
Dna Binding ◽  
Gene Cluster ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Binding Property ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Geobacillus Kaustophilus ◽  
Inositol Dehydrogenase

Abstract BackgroundGeobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, grows on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis. The iol gene cluster consists of two tandem operons induced in the presence of inositol; however, the mechanism underlying the induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding a scyllo-inositol dehydrogenase, and its homolog in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and termed as iolQ in G. kaustophilus.ResultsWhen iolQ was inactivated, not only the myo-inositol dehydrogenase activity in the cell due to the expression of gk1899 but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal His-tag fusion in Escherichia coli and subjected to the in vitro gel mobility shift assay to examine its DNA binding property. It was observed that IolQ bound to the DNA fragments containing each of the two iol promoter regions, and its DNA binding was antagonized by myo-inositol. Moreover, DNase I footprint analyses were conducted to determine the two binding sites of IolQ within each of the iol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to be a palindrome of 5′-RGWAAGCGCTTSCY-3′ (where R = A or G, W = A or T, S = G or C, and Y = C or T).ConclusionIolQ functions as a transcriptional repressor regulating the induction of the two iol operons responding to myo-inositol.

scholarly journals High-yield production of L-serine through a novel identified exporter combined with synthetic pathway in Corynebacterium glutamicum

2020 ◽  
Author(s):  
Xiaomei Zhang ◽  
Yujie Gao ◽  
Ziwei Chen ◽  
Guoqiang Xu ◽  
Xiaojuan Zhang ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Fluorescent Protein ◽  
Transcriptional Regulator ◽  
High Yield ◽  
Market Demand ◽  
Mobility Shift ◽  
Synthetic Pathway ◽  
Key Enzyme ◽  
Novel Target ◽  
Green Fluorescent

Abstract Background: L-Serine has wide and increasing applications in industries with fast-growing market demand. Although strategies for achieving and improving L-serine production in Corynebacterium glutamicum (C. glutamicum) have focused on inhibiting its degradation and enhancing its biosynthetic pathway, L-serine yield has remained relatively low. Exporters play an essential role in the fermentative production of amino acids. To achieve higher L-serine yield, L-serine export from the cell should be improved. In C. glutamicum, ThrE, which can export L-threonine and L-serine, is the only identified L-serine exporter so far.Results: In this study, a novel L-serine exporter NCgl0580 was identified and characterized in C. glutamicum ΔSSAAI (SSAAI), and named as SerE (encoded by serE). Deletion of serE in SSAAI led to a 56.5% decrease in L-serine titer, whereas overexpression of serE compensated for the lack of serE with respect to L-serine titer. A fusion protein with SerE and enhanced green fluorescent protein (EGFP) was constructed to confirm that SerE localized at the plasma membrane. The function of SerE was studied by peptide feeding approaches, and the results showed that SerE is a novel exporter for L-serine and L-threonine in C. glutamicum. Subsequently, the interaction of a known L-serine exporter ThrE and SerE was studied, and the results suggested that SerE is more important than ThrE in L-serine export in SSAAI. In addition, probe plasmid and electrophoretic mobility shift assays (EMSA) revealed NCgl0581 as the transcriptional regulator of SerE. Comparative transcriptomics between SSAAI and the NCgl0581 deletion strain showed that NCgl0581 is a positive regulator of NCgl0580. Finally, by overexpressing the novel exporter SerE, combined with L-serine synthetic pathway key enzyme serAΔ197, serC, and serB, the resulting strain presented an L-serine titer of 43.9 g/L with a yield of 0.44 g/g sucrose, which is the highest L-serine titer and yield reported so far in C. glutamicum. Conclusions: This study provides a novel target for L-serine and L-threonine export engineering as well as a new global transcriptional regulator NCgl0581 in C. glutamicum.

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 Phenylacetic Acid Catabolism and Its Transcriptional Regulation in Corynebacterium glutamicum

2012 ◽  
Vol 78 (16) ◽  
pp. 5796-5804 ◽  
Author(s):  
Xi Chen ◽  
Thomas A. Kohl ◽  
Christian Rückert ◽  
Dmitry A. Rodionov ◽  
Ling-Hao Li ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Phenylacetic Acid ◽  
Specific Binding ◽  
Genomic Analysis ◽  
Negative Regulator ◽  
Comparative Genomic ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Content Type ◽  
Transcription Regulator

ABSTRACTThe industrially important organismCorynebacterium glutamicumhas been characterized in recent years for its robust ability to assimilate aromatic compounds. In this study,C. glutamicumstrain AS 1.542 was investigated for its ability to catabolize phenylacetic acid (PAA). Thepaagenes were identified; they are organized as a continuouspaagene cluster. The type strain ofC. glutamicum, ATCC 13032, is not able to catabolize PAA, but the recombinant strain ATCC 13032/pEC-K18mob2::paagained the ability to grow on PAA. ThepaaRgene, encoding a TetR family transcription regulator, was studied in detail. Disruption ofpaaRin strain AS 1.542 resulted in transcriptional increases of allpaagenes. Transcription start sites and putative promoter regions were determined. An imperfect palindromic motif (5′-ACTNACCGNNCGNNCGGTNAGT-3′; 22 bp) was identified in the upstream regions ofpaagenes. Electrophoretic mobility shift assays (EMSA) demonstrated specific binding of PaaR to this motif, and phenylacetyl coenzyme A (PA-CoA) blocked binding. It was concluded that PaaR is the negative regulator of PAA degradation and that PA-CoA is the PaaR effector. In addition, GlxR binding sites were found, and binding to GlxR was confirmed. Therefore, PAA catabolism inC. glutamicumis regulated by the pathway-specific repressor PaaR, and also likely by the global transcription regulator GlxR. By comparative genomic analysis, we reconstructed orthologous PaaR regulons in 57 species, including species ofActinobacteria,Proteobacteria, andFlavobacteria, that carry PAA utilization genes and operate by conserved binding motifs, suggesting that PaaR-like regulation might commonly exist in these bacteria.

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