Biomedical subjects

Gabriele Klug

Publications and source records attributed to Gabriele Klug.

31 records · Page 2Linked to original sources

PubMed2004

Responses of the Rhodobacter sphaeroides transcriptome to blue light under semiaerobic conditions.

Exposure to blue light of the facultative phototrophic proteobacterium Rhodobacter sphaeroides grown semiaerobically results in repression of the puc and puf operons involved in photosystem formation. To reveal the genome-wide effects of blue light on gene expression and the underlying photosensory mechanisms, transcriptome profiles of R. sphaeroides during blue-light irradiation (for 5 to 135 min) were analyzed. Expression of most photosystem genes was repressed upon irradiation. Downregulation of photosystem development may be used to prevent photooxidative damage occurring when the photosystem, oxygen, and high-intensity light are present simultaneously. The photoreceptor of the BLUF-domain family, AppA, which belongs to the AppA-PpsR antirepressor-repressor system, is essential for maintenance of repression upon prolonged irradiation (S. Braatsch et al., Mol. Microbiol. 45:827-836, 2002). Transcriptome data suggest that the onset of repression is also mediated by the AppA-PpsR system, albeit via an apparently different sensory mechanism. Expression of several genes, whose products may participate in photooxidative damage defense, including deoxypyrimidine photolyase, glutathione peroxidase, and quinol oxidoreductases, was increased. Among the genes upregulated were genes encoding two sigma factors: sigmaE and sigma38. The consensus promoter sequences for these sigma factors were predicted in the upstream sequences of numerous upregulated genes, suggesting that coordinated action of sigmaE and sigma38 is responsible for the upregulation. Based on the dynamics of upregulation, the anti-sigmaE factor ChrR or its putative upstream partner is proposed to be the primary sensor. The identified transcriptome responses provided a framework for deciphering blue-light-dependent signal transduction pathways in R. sphaeroides.

Bacterial Proteins↗

PubMed2004

Composition and activity of the Rhodobacter capsulatus degradosome vary under different oxygen concentrations.

The influence of changes in temperature or oxygen tension during growth of Rhodobacter capsulatus on the composition and activity of the degradosome, an RNA-processing protein complex, was investigated. Only minor differences in the amount of specific proteins of the complex were observed after a decrease or increase of the temperature, but dramatic variations were detectable during growth at different oxygen concentrations. In particular, the amount of the transcription factor Rho, which was previously shown to be associated with the R. capsulatus degradosome, was strongly increased under aerobic conditions. Remarkably, oxygen tension oppositely affected the levels of the two helicases associated with the degradosome. RNase E and the degradosome from aerobically grown cultures degraded a transcript which represents part of the puf operon encoding proteins of the photosynthetic apparatus faster than did the degradosome from semiaerobically grown cultures.

Operon↗

PubMed2003

Expression of the trxA gene for thioredoxin 1 in Rhodobacter sphaeroides during oxidative stress.

Expression of the thioredoxin ( trxA) gene of Rhodobacter sphaeroides is regulated by oxidative stress at the transcriptional and post-transcriptional levels. All oxidative stress agents tested resulted in a moderate or strong increase of trxA mRNA levels, which was not due to increased mRNA stability. While the kinetics of increased trxA mRNA and of sodB mRNA, encoding superoxide dismutase, were similar after addition of tert-butyl hydroperoxide ( t-BOOH) or hydrogen peroxide (H(2)O(2)), different kinetics were observed after addition of diamide or paraquat, indicating the involvement of different stress responses in the regulation of these genes. The level of TrxA did not increase to the same extent as trxA mRNA levels. Furthermore, the addition of H(2)O(2) or t-BOOH led to increased turnover of the protein. Apparently, increased txA transcription compensated, at least in part, for the reduced stability of the protein. A strain expressing lower levels of thioredoxin 1 showed decreased resistance to diamide and H(2)O(2) but increased resistance to paraquat and t-BOOH compared to the wild-type. These data implicate the involvement of various systems in the response to different types of oxidative stress and the participation of thioredoxin 1 in the defense against oxidative stress caused by diamide or H(2)O(2).

Bacterial Proteins↗

PubMed2003

An exosome-like complex in Sulfolobus solfataricus.

We present the first experimental evidence for the existence of an exosome-like protein complex in Archaea. In Eukarya, the exosome is essential for many pathways of RNA processing and degradation. Co-immunoprecipitation with antibodies directed against the previously predicted Sulfolobus solfataricus orthologue of the exosome subunit ribosomal-RNA-processing protein 41 (Rrp41) led to the purification of a 250-kDa protein complex from S. solfataricus. Approximately half of the complex cosediments with ribosomal subunits. It comprises four previously predicted orthologues of the core exosome subunits from yeast (Rrp41, Rrp42, Rrp4 and Csl4 (cepl synthetic lethality 4; an RNA-binding protein and exosome sub-unit)), whereas other predicted subunits were not found. Surprisingly, the archaeal homologue of the bacterial DNA primase DnaG was tightly associated with the complex. This suggests an RNA-related function for the archaeal DnaG-like proteins. Comparison of experimental data from different organisms shows that the minimal core of the exosome consists of at least one phosphate-dependent ribonuclease PH homologue, and of Rrp4 and Csl4. Such a protein complex was probably present in the last common ancestor of Archaea and Eukarya.

Macromolecular Substances↗

PubMed2003

Thioredoxin 2 is involved in oxidative stress defence and redox-dependent expression of photosynthesis genes in Rhodobacter capsulatus.

Thioredoxins are small ubiquitous proteins that display different functions mainly via redox-mediated processes. The facultatively photosynthetic bacterium Rhodobacter capsulatus harbours at least two genes for thioredoxin 1 and 2, trxA and trxC. It is demonstrated that thioredoxin 2 of R. capsulatus can partially replace the thioredoxin 1 function as a hydrogen donor for methionine sulfoxide reductase but cannot replace thioredoxin 1 as a subunit of phage T7 DNA polymerase. By inactivating the trxC gene in R. capsulatus, it is shown that thioredoxin 2 is involved in resistance against oxidative stress. As thioredoxin 1 of Rhodobacter sphaeroides, R. capsulatus thioredoxin 2 affects the oxygen-dependent expression of photosynthesis genes, albeit in an opposite way. The trxC mutant of R. capsulatus shows a stronger increase in photosynthesis gene expression after a decrease in oxygen tension than the isogenic wild-type strain. The expression of the trxC gene is downregulated by oxygen.

Bacteriochlorophylls↗

PubMed2002

Individual gvp transcript segments in Haloferax mediterranei exhibit varying half-lives, which are differentially affected by salt concentration and growth phase.

The mc-gvp genes for gas vesicle formation in Haloferax mediterranei are transcribed from two promoters located in front of the mc-gvpA and mc-gvpD genes. The different transcripts originating from both promoters show different abundances dependent on salt concentration in the medium and growth phase. Here we show that the half-lives of these transcripts differ significantly and that the small gvp transcripts exhibit higher stabilities than the larger gvp transcripts. While the stability of most gvp transcripts is independent of the salt concentration in the medium, the gvpA mRNA decays about twice as fast in cultures grown at 18% salt compared to cultures grown at 25% salt. The stability of the 0.45 kb transcript population derived from the 5' part of the gvpD gene depends on the growth phase of the culture. Thus, differences in mRNA stability contribute to the salt-dependent and growth phase-dependent abundance of gvp transcripts. This implies that, like in bacteria and eukarya, mRNA processing contributes to regulated gene expression in archaea.

Blotting, Northern↗

PubMed2002

Dehydrogenases from all three domains of life cleave RNA.

Specific interactions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with RNA have been reported both in vitro and in vivo. We show that eukaryotic and bacterial GAPDH and two proteins from the hyperthermophilic archaeon Sulfolobus solfataricus, which are annotated as dehydrogenases, cleave RNA producing similar degradation patterns. RNA cleavage is most efficient at 60 degrees C, at MgCl(2) concentrations up to 5 mm, and takes place between pyrimidine and adenosine. The RNase active center of the putative aspartate semialdehyde dehydrogenase from S. solfataricus is located within the N-terminal 73 amino acids, which comprise the first mononucleotide-binding site of the predicted Rossmann fold. Thus, RNA cleavage has to be taken into account in the ongoing discussion of the possible biological function of RNA binding by dehydrogenases.

Archaeal Proteins↗

PubMed2002

One functional subunit is sufficient for catalytic activity and substrate specificity of Escherichia coli endoribonuclease III artificial heterodimers.

To study the intersubunit communication required for the activity of the normally homodimeric enzyme endoribonuclease III of Escherichia coli we have constructed and analysed an artificial heterodimer. This heterodimer is composed of one wild-type and one catalytically inactive subunit. The inactive subunit has one amino acid exchanged (E117K, rnc70 mutant) which abolishes cleavage activity but still allows substrate binding of a rnc70-homodimer. Our results show that one functional active site is sufficient for cleavage activity of the heterodimer.

Base Sequence↗

PubMed2002

BLUF: a novel FAD-binding domain involved in sensory transduction in microorganisms.

A novel FAD-binding domain, BLUF, exemplified by the N-terminus of the AppA protein from Rhodobacter sphaeroides, is present in various proteins, primarily from Bacteria. The BLUF domain is involved in sensing blue-light (and possibly redox) using FAD and is similar to the flavin-binding PAS domains and cryptochromes. The predicted secondary structure reveals that the BLUF domain is a novel FAD-binding fold.

Amino Acid Sequence↗

PubMed2002

A single flavoprotein, AppA, integrates both redox and light signals in Rhodobacter sphaeroides.

Anoxygenic photosynthetic proteobacteria exhibit various light responses, including changing levels of expression of photosynthesis genes. However, the underlying mechanisms are largely unknown. We show that expression of the puf and puc operons encoding structural proteins of the photosynthetic complexes is strongly repressed by blue light under semi-aerobic growth in Rhodobacter sphaeroides but not in the related species Rhodobacter capsulatus. At very low oxygen tension, puf and puc expression is independent of blue light in both species. Photosynthetic electron transport does not mediate the blue light repression, implying the existence of specific photoreceptors. Here, we show that the flavoprotein AppA is likely to act as the photoreceptor for blue light-dependent repression during continuous illumination. The FAD cofactor of AppA is essential for the blue light-dependent sensory transduction of this response. AppA, which is present in R. sphaeroides but not in R. capsulatus, is known to participate in the redox-dependent control of photosynthesis gene expression. Thus, AppA is the first example of a protein with dual sensing capabilities that integrates both redox and light signals.

Bacterial Proteins↗

PubMed2002

Atypical processing in domain III of 23S rRNA of Rhizobium leguminosarum ATCC 10004(T) at a position homologous to an rRNA fragmentation site in protozoa.

For still unknown reasons, the 23S rRNA of many alpha-Proteobacteria shows a unique fragmentation pattern compared to other bacteria. The 23S rRNA processing involves RNase III and additional, yet unidentified enzymes. The alpha-proteobacterium Rhizobium leguminosarum ATCC 10004(T) possesses two fragmentation sites in its 23S rRNA. The first one harbors an intervening sequence in helix 9 which is cleaved by RNase III. We demonstrate that the mature 5' end of the resulting 2.6-kb rRNA fragment is generated by additional removal of helix 10. A fraction of the 2.6-kb rRNA is further processed in domain III, giving rise to two 1.3-kb rRNA fragments. We mapped the domain III fragmentation site and found it to be at a position which has only been reported for trypanosomatid protozoa. This fragmentation site is also unique in that it lacks an intervening sequence. We found that the simultaneous occurrence of 2.6-kb and 1.3-kb rRNA fragments is not due to interoperonal sequence differences but rather reflects slow processing. The different characteristics of the two fragmentation sites in the 23S rRNA suggest that they are processed by different mechanisms. Interestingly, the amount of 2.6-kb rRNA varies during culture growth. We observed a transient increase in the relative amount of 2.6-kb rRNA fragments during the first hours after inoculation, which points to changes in the ratio of rRNA synthesis rate to domain III processing rate during the growth of a culture.

Animals↗

PubMed2002

Oxygen-regulated expression of genes for pigment binding proteins in Rhodobacter capsulatus.

Oxygen is the major external factor affecting the expression of photosynthesis genes in facultatively photosynthetic bacteria. Many investigations over the last years mainly carried out on the closely related species Rhodobacter capsulatus and Rhodobacter sphaeroides have identified a number of proteins involved in the oxygen-regulated signal pathway, in which the RegB/RegA two component system plays a central role. While the RegB/RegA system activates photosynthesis genes under low oxygen tension other proteins like CrtJ and PPBP have a repressing function under high oxygen tension. Additional DNA binding proteins like the integration host factor can modulate the expression of photosynthesis genes. The role of alternative sigma factors in this signal pathway is still unclear.

Gene Expression Regulation, Bacterial↗

PubMed1997

Thioredoxin is essential for Rhodobacter sphaeroides growth by aerobic and anaerobic respiration.

To investigate the biological role of thioredoxin in the facultative photosynthetic bacterium Rhodobacter sphaeroides, attempts were made to construct a thioredoxin-deficient mutant by site-specific mutagenesis, using the Tn903 kanamycin resistance gene for selection. In situ and Southern hybridization analyses have demonstrated that the TrxA- mutation is lethal for R. sphaeroides growth under anaerobic conditions with DMSO as terminal electron acceptor and under aerobic conditions. In addition, the DNA region upstream of the trxA initiation codon is essential for aerobic growth of R. sphaeroides. An ORF of unknown function was identified in this region and is suggested to encode a product essential for aerobic metabolism of R. sphaeroides. The mechanism of thioredoxin action was also analysed by using the procedure for gene replacement to introduce a Cys33 to Ser mutation into the trxA chromosomal copy. The strain carrying this mutation produced a thioredoxin impaired in its protein-disulfide reductase activity and was also not viable. These data suggest that the physiological function of R. sphaeroides thioredoxin is redox-dependent. Thioredoxin purified from R. sphaeroides was shown to have a glutathione-disulfide oxidoreductase activity typical of glutaredoxins. This unexpected finding suggests that R. sphaeroides thioredoxin, in contrast to Escherichia coli thioredoxin, has the potential to act in GSH-dependent processes. Thus, the fundamental role of R. sphaeroides thioredoxin in cell growth probably originates from the multiple functions it can serve in vivo.

Aerobiosis↗