Gabriele Klug

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.

The archaeal exosome core is a hexameric ring structure with three catalytic subunits

The exosome is a 3′ → 5′ exoribonuclease complex involved in RNA processing. We report the crystal structure of the RNase PH core complex of the Sulfolobus solfataricus exosome determined at a resolution of 2.8 Å. The structure reveals a hexameric ring-like arrangement of three Rrp41–Rrp42 heterodimers, where both subunits adopt the RNase PH fold common to phosphorolytic exoribonucleases. Structure-guided mutagenesis reveals that the activity of the complex resides within the active sites of the Rrp41 subunits, all three of which face the same side of the hexameric structure. The Rrp42 subunit is inactive but contributes to the structuring of the Rrp41 active site. The high sequence similarity of this archaeal exosome to eukaryotic exosomes and its high structural similarity to the bacterial mRNA–degrading PNPase support a common basis for RNA-degrading machineries in all three domains of life.

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.

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 (cep1 synthetic lethality 4; an RNA‐binding protein and exosome subunit)), 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.

Thioredoxins in bacteria: functions in oxidative stress response and regulation of thioredoxin genes

Thioredoxins fulfill a number of different important cellular functions in all living organisms. In bacteria, thioredoxin genes are often regulated by external factors. In turn, thioredoxins influence the expression of many other genes. The multiple and important functions of thioredoxins in cells necessitate to appropriately adjust their level. This review outlines different strategies that have evolved for the regulation of bacterial thioredoxin genes. It also summarizes effects of thioredoxins on gene regulation and presents a recent model for a redox-dependent gene regulation that is mediated by thioredoxins.

RNA polyadenylation in Archaea : not observed in Haloferax while the exosome polynucleotidylates RNA in Sulfolobus

The addition of poly(A) tails to RNA is a phenomenon common to all organisms examined so far. No homologues of the known polyadenylating enzymes are found in Archaea and little is known concerning the mechanisms of messenger RNA degradation in these organisms. Hyperthermophiles of the genus Sulfolobus contain a protein complex with high similarity to the exosome, which is known to degrade RNA in eukaryotes. Halophilic Archaea, however, do not encode homologues of these eukaryotic exosome components. In this work, we analysed RNA polyadenylation and degradation in the archaea Sulfolobus solfataricus and Haloferax volcanii. No RNA polyadenylation was detected in the halophilic archaeon H. volcanii. However, RNA polynucleotidylation occurred in hyperthermophiles of the genus Sulfolobus and was mediated by the archaea exosome complex. Together, our results identify the first organism without RNA polyadenylation and show a polyadenylation activity of the archaea exosome.

Photooxidative stress-induced and abundant small RNAs in Rhodobacter sphaeroides.

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen (1O2) in Rhodobacter sphaeroides. Our study reports the genome‐wide search for small RNAs (sRNAs) involved in the regulatory response to 1O2. By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with 1O2 or superoxide (O–2). One sRNA was specifically induced by 1O2 and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by 1O2 and O–2 were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoHI and RpoHII. The most abundant sRNA was processed in the presence of 1O2 but not by O–2. From this and a second sRNA a conserved 3′‐segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half‐lives, abundance and processing of 1O2‐affected sRNAs. Orthologues of three sRNA genes are present in different alpha‐proteobacteria, but the majority was unique to R. sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by 1O2 exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.

The role of mRNA degradation in the regulated expression of bacterial photosynthesis genes

Regulation of gene expression in bacteria, as in eukaryotic cells, is often achieved by variation of mRNA levels. Since the steady state levels of mRNA depend on both the rate of synthesis and the rate of decay, both mechanisms are important for gene regulation. After considerable effort undertaken over many years to understand the regulation of transcription, mRNA degradation has recently gained Increasing attention as an important step in the regulation of some bacterial genes, and many investigations have addressed the mechanisms involved in mRNA decay. The puf mRNA of Rhodobacter capsulatus encoding pigment binding proteins has become a model system to study decay of a polycistronic mRNA and the role of mRNA degradation in gene expression. Individual segments of the polycistronic puf mRNA display extremely different half‐lives. These differences in stability of mRNA segments are involved in the differential expression of puf encoded genes and consequently contribute to the stoichiometry of light‐harvesting I and reaction centre complexes that results in optimal growth. In addition, control of mRNA stability is involved in the oxygen‐dependent regulation of photosynthesis genes. High oxygen tension results in decreased stability of the reaction‐centre specific puf mRNA segment, most likely by affecting the rate of endonucleolytic cleavage within the reaction centre coding region. The results obtained from studying puf mRNA degradation in R. capsulatus and Escherichia coli suggest that a specific distribution of decay promoting and decay impeding mRNA elements along the polycistronic mRNA is responsible for the different half‐lives of individual puf segments.

Singlet Oxygen Stress in Microorganisms

Singlet oxygen is the primary agent of photooxidative stress in microorganisms. In photosynthetic microorganisms, sensitized generation by pigments of the photosystems is the main source of singlet oxygen and, in nonphotosynthetic microorganisms, cellular cofactors such as flavins, rhodopsins, quinones, and porphyrins serve as photosensitizer. Singlet oxygen rapidly reacts with a wide range of cellular macromolecules including proteins, lipids, DNA, and RNA, and thereby further reactive substances including organic peroxides and sulfoxides are formed. Microorganisms that face high light intensities or exhibit potent photosensitizers have evolved specific mechanisms to prevent photooxidative stress. These mechanisms include the use of quenchers, such as carotenoids, which interact either with excited photosensitizer molecules or singlet oxygen itself to prevent damage of cellular molecules. Scavengers like glutathione react with singlet oxygen. Despite those protection mechanisms, damage by reactions with singlet oxygen on cellular macromolecules disturbs cellular functions. Microorganisms that regularly face photooxidative stress have evolved specific systems to sense singlet oxygen and tightly control the removal of singlet oxygen reaction products. Responses to photooxidative stress have been investigated in a range of photosynthetic and nonphotosynthetic microorganisms. However, detailed knowledge on the regulation of this response has only been obtained for the phototrophic alpha-proteobacterium Rhodobacter sphaeroides. In this organism and in related proteobacteria, the extracytoplasmic function (ECF) sigma factor RpoE is released from the cognate antisigma factor ChrR in the presence of singlet oxygen and triggers the expression of genes providing protection against photooxidative stress. Recent experiments show that singlet oxygen acts as a signal, which is sensed by yet unknown components and leads to proteolysis of ChrR. RpoE induces expression of a second alternative sigma factor, RpoH(II), which controls a large set of genes that partially overlaps with the heat-shock response controlled by RpoH(I). In addition to the transcriptional control of gene regulation by alternative sigma factors, a set of noncoding small RNAs (sRNAs) appear to affect the synthesis of several proteins involved in the response to photooxidative stress. The interaction of mRNA targets with those sRNAs is usually mediated by the RNA chaperone Hfq. Deletion of the gene encoding Hfq leads to a singlet oxygen-sensitive phenotype, which underlines the control of gene regulation on the posttranscriptional level by sRNAs in R. sphaeroides. Hence, a complex network of different regulatory components controls the defense against photooxidative stress in anoxygenic photosynthetic bacteria.

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.