Biomedical subjects

Andrea T Hüser

Publications and source records attributed to Andrea T Hüser.

7 recordsLinked to original sources

PubMed2007

The alternative sigma factor SigB of Corynebacterium glutamicum modulates global gene expression during transition from exponential growth to stationary phase.

BACKGROUND: Corynebacterium glutamicum is a gram-positive soil bacterium widely used for the industrial production of amino acids. There is great interest in the examination of the molecular mechanism of transcription control. One of these control mechanisms are sigma factors. C. glutamicum ATCC 13032 has seven putative sigma factor-encoding genes, including sigA and sigB. The sigA gene encodes the essential primary sigma factor of C. glutamicum and is responsible for promoter recognition of house-keeping genes. The sigB gene codes for the non-essential sigma factor SigB that has a proposed role in stress reponse. RESULTS: The sigB gene expression was highest at transition between exponential growth and stationary phase, when the amount of sigA mRNA was already decreasing. Genome-wide transcription profiles of the wild-type and the sigB mutant were recorded by comparative DNA microarray hybridizations. The data indicated that the mRNA levels of 111 genes are significantly changed in the sigB-proficient strain during the transition phase, whereas the expression profile of the sigB-deficient strain showed only minor changes (26 genes). The genes that are higher expressed during transition phase only in the sigB-proficient strain mainly belong to the functional categories amino acid metabolism, carbon metabolism, stress defense, membrane processes, and phosphorus metabolism. The transcription start points of six of these genes were determined and the deduced promoter sequences turned out to be indistinguishable from that of the consensus promoter recognized by SigA. Real-time reverse transcription PCR assays revealed that the expression profiles of these genes during growth were similar to that of the sigB gene itself. In the sigB mutant, however, the transcription profiles resembled that of the sigA gene encoding the house-keeping sigma factor. CONCLUSION: During transition phase, the sigB gene showed an enhanced expression, while simultaneously the sigA mRNA decreased in abundance. This might cause a replacement of SigA by SigB at the RNA polymerase core enzyme and in turn results in increased expression of genes relevant for the transition and the stationary phase, either to cope with nutrient limitation or with the accompanying oxidative stress. The increased expression of genes encoding anti-oxidative or protection functions also prepares the cell for upcoming limitations and environmental stresses.

Bacterial Proteins↗

PubMed2006

The DtxR protein acting as dual transcriptional regulator directs a global regulatory network involved in iron metabolism of Corynebacterium glutamicum.

BACKGROUND: The knowledge about complete bacterial genome sequences opens the way to reconstruct the qualitative topology and global connectivity of transcriptional regulatory networks. Since iron is essential for a variety of cellular processes but also poses problems in biological systems due to its high toxicity, bacteria have evolved complex transcriptional regulatory networks to achieve an effective iron homeostasis. Here, we apply a combination of transcriptomics, bioinformatics, in vitro assays, and comparative genomics to decipher the regulatory network of the iron-dependent transcriptional regulator DtxR of Corynebacterium glutamicum. RESULTS: A deletion of the dtxR gene of C. glutamicum ATCC 13032 led to the mutant strain C. glutamicum IB2103 that was able to grow in minimal medium only under low-iron conditions. By performing genome-wide DNA microarray hybridizations, differentially expressed genes involved in iron metabolism of C. glutamicum were detected in the dtxR mutant. Bioinformatics analysis of the genome sequence identified a common 19-bp motif within the upstream region of 31 genes, whose differential expression in C. glutamicum IB2103 was verified by real-time reverse transcription PCR. Binding of a His-tagged DtxR protein to oligonucleotides containing the 19-bp motifs was demonstrated in vitro by DNA band shift assays. At least 64 genes encoding a variety of physiological functions in iron transport and utilization, in central carbohydrate metabolism and in transcriptional regulation are controlled directly by the DtxR protein. A comparison with the bioinformatically predicted networks of C. efficiens, C. diphtheriae and C. jeikeium identified evolutionary conserved elements of the DtxR network. CONCLUSION: This work adds considerably to our currrent understanding of the transcriptional regulatory network of C. glutamicum genes that are controlled by DtxR. The DtxR protein has a major role in controlling the expression of genes involved in iron metabolism and exerts a dual regulatory function as repressor of genes participating in iron uptake and utilization and as activator of genes responsible for iron storage and DNA protection. The data suggest that the DtxR protein acts as global regulator by controlling the expression of other regulatory proteins that might take care of an iron-dependent regulation of a broader transcriptional network of C. glutamicum genes.

Bacterial Proteins↗

PubMed2005

The transcriptional regulator SsuR activates expression of the Corynebacterium glutamicum sulphonate utilization genes in the absence of sulphate.

In a recent study, the putative regulatory gene cg0012 was shown to belong to the regulon of McbR, a global transcriptional regulator of sulphur metabolism in Corynebacterium glutamicum ATCC 13032. A deletion of cg0012, now designated ssuR (sulphonate sulphur utilization regulator), led to the mutant strain C. glutamicum DK100, which was shown to be blocked in the utilization of sulphonates as sulphur sources. According to DNA microarray hybridizations, transcription of the ssu and seu genes, encoding the sulphonate utilization system of C. glutamicum, was considerably decreased in C. glutamicum DK100 when compared with the wild-type strain. Electrophoretic mobility shift assays with purified SsuR protein demonstrated that the upstream regions of ssuI, seuABC, ssuD2 and ssuD1CBA contain SsuR binding sites. A nucleotide sequence alignment of the four DNA fragments containing the SsuR binding sites revealed a common 21 bp motif consisting of T-, GC- and A-rich domains. Mapping of the transcriptional start sites in front of ssuI, seuABC, ssuD2 and ssuD1CBA indicated that the SsuR binding sites are located directly upstream of identified promoter sequences and that the ssu genes are expressed by leaderless transcripts. Binding of the SsuR protein to its operator was shown to be diminished in vitro by the effector substance sulphate and its direct assimilation products adenosine 5'-phosphosulphate, sulphite and sulphide. Real-time reverse transcription polymerase chain reaction experiments verified that the expression of the ssu and seu genes was also repressed in vivo by the presence of sulphate or sulphite. Therefore, the regulatory protein SsuR activates the expression of the ssu and seu genes in C. glutamicum in the absence of the preferred sulphur source sulphate.

Bacterial Proteins↗

PubMed2005

Adaptation of Corynebacterium glutamicum to ammonium limitation: a global analysis using transcriptome and proteome techniques.

Theresponse of Corynebacterium glutamicum to ammonium limitation was studied by transcriptional and proteome profiling of cells grown in a chemostat. Our results show that ammonium-limited growth of C. glutamicum results in a rearrangement of the cellular transport capacity, changes in metabolic pathways for nitrogen assimilation, amino acid biosynthesis, and carbon metabolism, as well as a decreased cell division. Since transcription at different growth rates was studied, it was possible to distinguish specific responses to ammonium limitation and more general, growth rate-dependent alterations in gene expression. The latter include a number of genes encoding ribosomal proteins and genes for F(o)F(1)-ATP synthase subunits.

Adaptation, Physiological↗

PubMed2005

Rational design of a Corynebacterium glutamicum pantothenate production strain and its characterization by metabolic flux analysis and genome-wide transcriptional profiling.

A "second-generation" production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, DL-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity.

Bacterial Proteins↗

PubMed2003

Development of a Corynebacterium glutamicum DNA microarray and validation by genome-wide expression profiling during growth with propionate as carbon source.

A DNA microarray was developed to analyse global gene expression of the amino acid-producing bacterium Corynebacterium glutamicum. PCR products representing 93.4% of the predicted C. glutamicum genes were prepared and spotted in quadruplicate onto 3-aminopropyltrimethoxysilane-coated glass slides. The applicability of the C. glutamicum DNA microarray was demonstrated by co-hybridisation with fluorescently labelled cDNA probes. Analysis of the technical variance revealed that C. glutamicum genes detected with different intensities resulting in ratios greater than 1.52 or smaller than -1.52 can be regarded as differentially expressed with a confidence level of greater than 95%. In a validation example, we measured changes of the mRNA levels during growth of C. glutamicum with acetate and propionate as carbon sources. Acetate-grown C. glutamicum cultures were used as reference. At the 95% confidence interval, 117 genes revealed increased transcript levels in the presence of propionate, while 43 genes showed a decreased expression compared with the acetate-grown culture. Global expression profiling confirmed the induction of the prpD2B2C2 gene cluster already known to be essential for propionate degradation via the 2-methylcitrate cycle. Besides many genes of unknown function, the paralogous prpD1B1C1 gene cluster as well as fasI-B (encoding fatty-acid synthase IB), dtsR1 and dtsR2 (components of acyl-CoA carboxylases), gluABCD (glutamate transport system), putP (proline transport system), and pyc (pyruvate carboxylase) showed significantly increased expression levels. Differential expression of these genes was confirmed by real-time reverse transcription (RT) PCR assays.

Carbon↗

PubMed2003

Bioinformatics support for high-throughput proteomics.

In the "post-genome" era, mass spectrometry (MS) has become an important method for the analysis of proteome data. The rapid advancement of this technique in combination with other methods used in proteomics results in an increasing number of high-throughput projects. This leads to an increasing amount of data that needs to be archived and analyzed. To cope with the need for automated data conversion, storage, and analysis in the field of proteomics, the open source system ProDB was developed. The system handles data conversion from different mass spectrometer software, automates data analysis, and allows the annotation of MS spectra (e.g. assign gene names, store data on protein modifications). The system is based on an extensible relational database to store the mass spectra together with the experimental setup. It also provides a graphical user interface (GUI) for managing the experimental steps which led to the MS data. Furthermore, it allows the integration of genome and proteome data. Data from an ongoing experiment was used to compare manual and automated analysis. First tests showed that the automation resulted in a significant saving of time. Furthermore, the quality and interpretability of the results was improved in all cases.

Algorithms↗