DNA binding by Corynebacterium glutamicum TetR-type transcription regulator AmtR

<p>Abstract</p> <p>Background</p> <p>The TetR family member AmtR is the central regulator of nitrogen starvation response in <it>Corynebacterium glutamicum</it>. While the AmtR regulon was physiologically characterized in great detail up to now, mechanistic questions of AmtR binding were not addressed. This study presents a characterization of functionally important amino acids in the DNA binding domain of AmtR and of crucial nucleotides in the AmtR recognition motif.</p> <p>Results</p> <p>Site-directed mutagenesis, the characterization of corresponding mutant proteins by gel retardation assays and surface plasmon resonance and molecular modelling revealed several amino acids, which are directly involved in DNA binding, while others have more structural function. Furthermore, we could show that the spacing of the binding motif half sites is crucial for repression of transcription by AmtR.</p> <p>Conclusion</p> <p>Although the DNA binding domain of TetR-type repressors is highly conserved and a core binding motif was identified for AmtR and TetR(D), the AmtR binding domain shows individual properties compared to other TetR proteins. Besides by distinct amino acids of AmtR, DNA binding is influenced by nucleotides not only of the conserved binding motif but also by spacing nucleotides in <it>C. glutamicum</it>.</p

Comparative Bioinformatics and Experimental Analysis of the Intergenic Regulatory Regions of Bacillus cereus hbl and nhe Enterotoxin Operons and the Impact of CodY on Virulence Heterogeneity

Bacillus cereus is a food contaminant with greatly varying enteropathogenic potential. Almost all known strains harbor the genes for at least one of the three enterotoxins Nhe, Hbl, and CytK. While some strains show no cytotoxicity, others have caused outbreaks, in rare cases even with lethal outcome. The reason for these differences in cytotoxicity is unknown. To gain insight into the origin of enterotoxin expression heterogeneity in different strains, the architecture and role of 5' intergenic regions (5' IGRs) upstream of the nhe and hbl operons was investigated. In silico comparison of 142 strains of all seven phylogenetic groups of B. cereus sensu lato proved the presence of long 5' IGRs upstream of the nheABC and hblCDAB operons, which harbor recognition sites for several transcriptional regulators, including the virulence regulator PIcR, redox regulators ResD and Fnr, the nutrient-sensitive regulator CodY as well as the master regulator for biofilm formation SinR. By determining transcription start sites, unusually long 5' untranslated regions (5' UTRs) upstream of the nhe and hbl start codons were identified, which are not present upstream of cytK-1 and cytK-2. Promoter fusions lacking various parts of the nhe and hbl 5' UTR in B. cereus INRA C3 showed that the entire 331 bp 5' UTR of nhe is necessary for full promoter activity, while the presence of the complete 606 bp hbl 5' UTR lowers promoter activity. Repression was caused by a 268 bp sequence directly upstream of the hbl transcription start. Luciferase activity of reporter strains containing nhe and hbl 5' IGR lux fusions provided evidence that toxin gene transcription is upregulated by the depletion of free amino acids. Electrophoretic mobility shift assays showed that the branched-chain amino acid sensing regulator CodY binds to both nhe and hbl 5' UTR downstream of the promoter, potentially acting as a nutrient responsive roadblock repressor of toxin gene transcription. PIcR binding sites are highly conserved among all B. cereus sensu lato strains, indicating that this regulator does not significantly contribute to the heterogeneity in virulence potentials. The CodY recognition sites are far less conserved, perhaps conferring varying strengths of CodY binding, which might modulate toxin synthesis in a strain-specific manner

The Mutation Glu151Asp in the B-Component of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) Leads to a Diverging Reactivity in Antibody-Based Detection Systems

The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic

Das GlnR-abhängige Netzwerk zur Stickstoffregulation in Mycobacterium smegmatis

Mycobacterium smegmatis is a saprophytic and fast-growing member of the genus Mycobacterium and is often used as a model organism for related pathogenic species such as Mycobacterium tuberculosis. Focus of interest in this study was nitrogen metabolism and especially transcriptional response to nitrogen limitation in M. smegmatis, while the regulatory protein GlnR was the focus of attention. It was shown that the bacterium is able to use different substrates (amino acids, bases and inorganic nitrogen compounds) as nitrogen sources. Analyses of a glnR deletion strain revealed that GlnR is substantially involved in the utilization of several of these substrates. Moreover, 125 new putative GlnR target genes were identified in transcriptome analyses. In subsequent experiments, a GlnR-dependent increase of transcripts of 32 of these genes was verified, including genes involved in uptake and assimilation of amino acids, peptides and inorganic nitrogen sources. This confirms the role of GlnR as global nitrogen regulator in M. smegmatis. Moreover, also genes not involved in nitrogen metabolism were identified as GlnR targets, suggesting a more global regulatory function of GlnR. A GlnR subordinate role in regulation of some genes involved in nitrogen metabolism was also monitored for AmtR. In addition to that, different methods for purification of GlnR were established and in vitro analyses of DNA-binding behavior of the purified protein were carried out. Direct binding to promoter regions of half of the identified target genes was monitored, leading to the conclusion that indirect GlnR-control via further transcriptional regulators or so far unknown mechanisms is also involved in gene expression. At least three GlnR binding sites were identified in the promoter region of amtB, supporting the idea of a “galloping” DNA binding model which is described for E. coli regulator OmpR. As a member of the OmpR-family of transcriptional regulators, GlnR is supposed to be phosphorylated by a corresponding sensor histidine kinase. Although this kinase could not be identified within this study, highly conserved aspartate residues were distinguished as phosphorylation sites of GlnR. First experiments with M. smegmatis and M. tuberculosis GlnR pointed to distinct differences in DNA binding and activation of nitrogen response, although the two GlnR proteins share identical C-terminal DNA binding and N-terminal phosphorylation domains. Nevertheless, the model of nitrogen control in M. smegmatis developed in this study can indeed be used as a first pattern for similar processes in M. tuberculosis.Mycobacterium smegmatis ist ein saprophytisch lebender und schnell wachsender Vertreter der Gattung Mycobacterium und wird oft als Modellorganismus für verwandte pathogene Spezies wie Mycobacterium tuberculosis verwendet. Zentrales Thema dieser Arbeit war der Stickstoffmetabolismus in M. smegmatis, insbesondere die Antwort auf Stickstoffmangel auf transkriptioneller Ebene, wobei der Regulator GlnR im Mittelpunkt der Betrachtungen stand. Es wurde gezeigt, dass das Bakterium verschiedenste Substrate (Aminosäuren, Basen und anorganische Stickstoffverbindungen) als Stickstoffquellen nutzen kann. Analysen eines glnR Deletionsstammes verdeutlichten, dass GlnR maßgeblich an der Verwertung einiger dieser Substrate beteiligt ist. Darüber hinaus wurden durch Transkriptomanalysen 125 neue putative GlnR-Zielgene identifiziert. In darauffolgenden Experimenten wurde für 32 dieser Gene eine GlnR-abhängige Steigerung der Transkriptmenge bestätigt, darunter Gene, die an der Aufnahme sowie Assimilation von Aminosäuren, Peptiden und anorganischen Stickstoffverbindungen beteiligt sind. Dies verdeutlicht die Rolle von GlnR als globalen Stickstoffregulator in M. smegmatis. Weiterhin standen auch Gene, die nicht dem Stickstoffmetabolismus angehören, unter GlnR-Kontrolle, was auf eine umfassendere Funktion des Regulators hindeutet. Außerdem wurde eine untergeordnete Rolle von AmtR in der Regulation einiger stickstoffrelevanter Gene beobachtet. Zusätzlich wurden verschiedene Methoden zur Reinigung von GlnR etabliert und in vitro Untersuchungen zum DNA-Bindeverhalten des gereinigten Proteins durchgeführt, wobei eine direkte Bindung an Promotorfragmente der Hälfte aller identifizierten Zielgene beobachtet wurde. Dies führte zu dem Schluss, dass auch indirekte GlnR-Kontrolle über weitere Transkriptionsregulatoren oder bisher unbekannte Mechanismen eine Rolle für die Expression einiger Gene spielt. Außerdem wurden in der Promotorregion von amtB wenigstens drei GlnR-Bindestellen ausgemacht, was das Modell der „galoppierenden“ DNA-Bindung von OmpR aus E. coli unterstützt. Als ein Vertreter der OmpR-Familie von Transkriptionsregulatoren soll GlnR durch eine zugehörige Sensorhistidinkinase phosphoryliert werden. Obwohl diese im Rahmen dieser Arbeit nicht ermittelt werden konnte, wurden hochkonservierte Aspartatreste als Phosphorylierungsstellen von GlnR identifiziert. Erste Experimente mit M. smegmatis und M. tuberculosis GlnR deuteten trotz der identischen C-terminalen DNA-Bindedomänen und N-terminalen Phosphorylierungsdomänen der beiden Proteine auf klare Unterschiede in der DNA-Bindung und der Aktivierung der Stickstoffantwort hin. Dennoch kann das in dieser Arbeit entwickelte Modell der Stickstoffkontrolle in M. smegmatis durchaus als ein erstes Schema für M. tuberculosis verwendet werden

FarR, a putative regulator of amino acid metabolism in Corynebacterium glutamicum

Haenssler E, Mueller T, Jessberger N, et al. FarR, a putative regulator of amino acid metabolism in Corynebacterium glutamicum. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 2007;76(3):625-632.With the publication of the Corynebacterium glutamicum genome sequence, a global characterization of genes controlled by functionally uncharacterized transcriptional regulators became possible. We used DNA microarrays in combination with gel retardation experiments to study gene regulation by FarR, a HutC/FarR-type regulator of the GntR family. Based on our results, FarR seems to be involved in the regulation of amino acid biosynthesis in C. glutamicum. Especially, transcript levels of the arg cluster and the gdh gene are influenced by deletion of farR