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U Gerischer

Publications and source records attributed to U Gerischer.

8 recordsLinked to original sources
PubMed1998

Transcriptional regulation of Alcaligenes eutrophus hydrogenase genes.

Alcaligenes eutrophus H16 produces a soluble hydrogenase (SH) and a membrane-bound hydrogenase (MBH) which catalyze the oxidation of H2, supplying the organism with energy for autotrophic growth. The promoters of the structural genes for the SH and the MBH, PSH and PMBH, respectively, were identified by means of the primer extension technique. Both promoters were active in vivo under hydrogenase-derepressing conditions but directed only low levels of transcription under condition which repressed hydrogenase synthesis. The cellular pools of SH and MBH transcripts under the different growth conditions correlated with the activities of the respective promoters. Also, an immediate and drastic increase in transcript pool levels occurred upon derepression of the hydrogenase system. Both promoters were dependent on the minor sigma factor sigma 54 and on the hydrogenase regulator HoxA in vivo. PSH was stronger than PMBH under both heterotrophic and autotrophic growth conditions. The two promoters were induced at approximately the same rates upon derepression of the hydrogenase system in diauxic cultures. The response regulator HoxA mediated low-level activation of PSH and PMBH in a heterologous system.

Alcaligenes↗
PubMed1998

PcaU, a transcriptional activator of genes for protocatechuate utilization in Acinetobacter.

The Acinetobacter pcaIJFBDKCHG operon encodes the six enzymes that convert protocatechuate to citric acid cycle intermediates. Directly downstream from the operon are qui and pob genes encoding sets of enzymes that convert quinate and p-hydroxybenzoate, respectively, to protocatechuate. Prior to this investigation, the only known regulatory gene in the pca-qui-pob cluster was pobR, which encodes a transcriptional activator that responds to p-hydroxybenzoate and activates transcription of pobA. The pca and qui genes were known to be expressed in response to protocatechuate, but a protein that mediated this induction had not been identified. This study was initiated by characterization of a spontaneous mutation that mapped upstream from pcaI and prevented expression of the pca genes. Sequencing of wild-type DNA extending from the translational start of pcaI through and beyond the location of the mutation revealed a 282-bp intergenic region and a divergently transcribed open reading frame, designated pcaU. Downstream from pcaU are two open reading frames encoding proteins similar in amino acid sequence to those associated with the oxidation of acyl thioesters. Inactivation of pcaU reduced the induced expression of pca structural genes by about 90% and impeded but did not completely prevent growth of the mutant cells with protocatechuate. PcaU was expressed in Escherichia coli and shown to bind to a portion of the pcaI-pcaU intergenic region containing a sequence identical in 16 of 19 nucleotide residues to a segment of the pob operator. Further similarity of the two regulatory systems is indicated by 54% amino acid sequence identity in the aligned primary structures of PobR and PcaU. The pob and pca systems were shown to differ, however, in the relative orientations of transcriptional starts with respect to the site where the activator binds to DNA, the size of the intergenic region, and the tightness of transcriptional control. The spontaneous mutation blocking pca gene expression was located in the promoter for the pca operon. The 19-nucleotide residue operator sequences were shown to be parts of a consensus associated with transcriptional activation of genes associated with protocatechuate catabolism. Two different binding sites for Pseudomonas putida PcaR differ from the consensus in only a single nucleotide residue, and DNA directly downstream from Acinetobacter pcaU contains a 19-bp segment differing from the consensus in only two residues. PcaU was shown to bind to DNA containing this segment as well as to the DNA in the pcaU-pcaI intergenic region.

Acetyl-CoA C-Acyltransferase↗
PubMed1996

IS1236, a newly discovered member of the IS3 family, exhibits varied patterns of insertion into the Acinetobacter calcoaceticus chromosome.

Analysis of spontaneous mutations in Acinetobacter calcoaceticus revealed a 1237 bp insertion sequence named IS1236 and possessing a nucleotide sequence resembling those of members of the lS3 family. The chromosome of A. calcoaceticus strain ADP1 contains seven copies of IS1236 which appears to insert preferentially into pobR, the transcriptional activator of the structural gene for p-hydroxybenzoate hydroxylase. IS1236 creates tandem 3 bp DNA duplications flanking the sites of its insertion in pobR. Different duplication patterns are found following insertion of IS1236 into pcaH, a structural gene for protocatechuate 3,4-dioxygenase. Therefore the insertion of properties of IS1236 appear to be influenced by its DNA target. Amino acid sequences associated with the apparent transposase function have been conserved in ORFB of IS1236 whereas the presumed DNA-binding helix-turn-helix region of IS1236 ORFA exhibits substantial amino acid sequence divergence from its IS3 counterparts. IS1236 ORFA and ORFB coding sequences overlap considerably, and sequence evidence indicates mechanisms for ORFB expression in IS1236 may resemble those employed by other members of the IS3 family. Portions of the IS1236 terminal repeats exhibit substantial sequence divergence from other members of the IS3 family, but evolution appears to have conserved a mechanism preventing expression of the insertion sequence genes as a consequence of transcriptional readthrough.

Acinetobacter calcoaceticus↗
PubMed1995

Spontaneous mutations in pcaH and -G, structural genes for protocatechuate 3,4-dioxygenase in Acinetobacter calcoaceticus.

Bacteria containing spontaneous null mutations in pcaH and -G, structural genes for protocatechuate 3,4-dioxygenase, were selected by exposure of an Acinetobacter calcoaceticus strain to physiological conditions in which expression of the genes prevents growth. The parental bacterial strain exhibits high competence for natural transformation, and this procedure was used to characterize 94 independently isolated spontaneous mutations. Four of the mutations were caused by integration of a newly identified insertion sequence, IS1236. Many (22 of 94) of the mutations were lengthy deletions, the largest of which appeared to eliminate at least 17 kb of DNA containing most of the pca-qui-pob supraoperonic gene cluster. DNA sequence determination revealed that the endpoints of four smaller deletions (74 to 440 bp in length) contained DNA sequence repetitions aligned imprecisely with the sites of mutation. Analysis of direct and inverted DNA sequence repetitions associated with the sites of mutation suggested the existence of DNA slippage structures that make unhybridized nucleotides particularly susceptible to mutation.

Acinetobacter calcoaceticus↗
PubMed1992

mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis.

By using primer extension analysis, we located the transcription start point of the acetoacetate decarboxylase (adc) gene of Clostridium acetobutylicum 90 nucleotides upstream from the initiation codon with A as the first transcribed nucleotide. From this site the promoter structure TTTACT(18 bp)TATAAT was identified; it shows high homology to the consensus sequences of gram-positive bacteria and Escherichia coli. Northern blot experiments revealed a length of 850 bases for the transcript of the adc gene. It thus represents a monocistronic operon. Transcription of adc was induced by conditions necessary for the onset of solvent formation. Induction occurred long before the respective fermentation product (acetone) could be detected in the medium. Transcription of the operon containing the genes for acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase (designated ctf) downstream of the adc gene but divergently transcribed is also induced by conditions necessary for the onset of solvent formation. The length of the respective RNA transcript, 4.1 kb, indicates additional coding capacity, since the genes for the two subunits of the coenzyme A transferase cover only approximately 1.5 kb. No distinct transcripts for the other open reading frames of the adc gene region, ORF1 and ORF2, could be detected. Computer analysis indicated that ORF1, which showed significant similarity to the alpha-amylase gene of Bacillus subtilis (U. Gerischer and P. Dürre, J. Bacteriol. 172:6907-6918, 1990), probably is indeed a coding region. ORF2, however, does not seem to have a coding function.

Amino Acid Sequence↗
PubMed1990

Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum.

Acetoacetate decarboxylase (ADC) (EC4.1.1.4) of Clostridium acetobutylicum DSM 792 was purified to homogeneity, and its first 25 N-terminal amino acids were determined. Oligonucleotide probes deduced from this sequence were used to detect positive clones in partial gene banks derived from Sau3A and HaeIII digests with following ligation into the vector pUC9. In Escherichia coli, the 2.1-kbp HaeIII clones expressed high levels of ADC activity. The expression was independent of the orientation of the insert with respect to the lac promoter of the vector and also of the addition of isopropyl-beta-D-thiogalactopyranoside, thus indicating that sequences located on the clostridial DNA controlled transcription and translation. From the E. coli clone with the recombinant plasmid pUG93 containing the 2.1-kbp HaeIII fragment, the ADC protein was purified and compared with the native enzyme. Both were indistinguishable with respect to the molecular mass of subunits and native protein as well as to activity stain. The 2.9-kbp Sau3A fragment could be shown to contain the amino terminus of the acetoacetate decarboxylase (adc) gene but did not express enzyme activity. It partially overlapped with the HaeIII fragment, spanning together 4,053 bp of the clostridial genome that were completely sequenced. Four open reading frames (ORFs) could be detected, one of which was unambiguously assigned to the acetoacetate decarboxylase (adc) gene. Amino acid sequences of the N terminus and the catalytic center as deduced from the nucleotide sequence were identical to sequences obtained from direct analysis of the protein. Typical procaryotic transcriptional and translational start and stop signals could be found in the DNA sequence. Together with these regulatory sequences, the adc gene formed a single operon. The carboxyl terminus of the enzyme proved to be rather hydrophobic. In vitro transcription-translation assays resulted in formation of ADC and ORF3 gene product; the other two ORFs were not expressed. Whereas no homology of the adc gene and ORF2 could be detected with sequences available in the EMBL or GenBank data bases, the obviously truncated ORF1 showed significant similarity to alpha-amylase of Bacillus subtilis. The restriction pattern and N-terminal amino acid sequence (as deduced from the nucleotide sequence) of ORF3 proved to be identical to those of the large subunit of acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase.

Amino Acid Sequence↗