Monica Zobawa

Contribution of Hfq to photooxidative stress resistance and global regulation in Rhodobacter sphaeroides

The photosynthetic alphaproteobacterium Rhodobacter sphaeroides has to cope with photooxidative stress that is caused by the bacteriochlorophyll a‐mediated formation of singlet oxygen (1O2). Exposure to 1O2 induces the alternative sigma factors RpoE and RpoHII which then promote transcription of photooxidative stress‐related genes, including small RNAs (sRNAs). The ubiquitous RNA chaperone Hfq is well established to interact with and facilitate the base‐pairing of sRNAs and target mRNAs to influence mRNA stability and/or translation. Here we report on the pleiotropic phenotype of a Δhfq mutant of R. sphaeroides, which is less pigmented, produces minicells and is more sensitive to 1O2. The higher 1O2 sensitivity of the Δhfq mutant is paralleled by a reduced RpoE activity and a disordered induction of RpoHII‐dependent genes. We used co‐immunoprecipitation of FLAG‐tagged Hfq combined with RNA‐seq to identify association of at least 25 sRNAs and of mRNAs encoding cell division proteins and ribosomal proteins with Hfq. Remarkably, > 70% of the Hfq‐bound sRNAs are 1O2‐affected. Proteomics analysis of the Hfq‐deficient strain revealed an impact of Hfq on amino acid transport and metabolic functions. Our data demonstrate for the first time an involvement of Hfq in regulation of photosynthesis genes and in the photooxidative stress response.

Anoxygenic photosynthesis and photooxidative stress: a particular challenge for Roseobacter

Roseobacter clade aerobic anoxygenic phototrophic bacteria (AAnP) are abundant in photic zone environments of marine ecosystems. These bacteria form a photosynthetic apparatus at oxygen saturation, a situation expected to generate high levels of singlet oxygen (¹O₂) when light is present. Rhodobacter sphaeroides, an anaerobic anoxygenic phototroph, represses photosynthesis genes at high oxygen tension. Here we report that Roseobacter denitrificans showed higher sensitivity to ¹O₂ compared with Rhb. sphaeroides. While photosynthetic membranes of Rsb. denitrificans generated more ¹O₂ during light exposure, key regulator genes rpoE and rpoH(II) were more strongly induced in response to ¹O₂ stress compared with Rhb. sphaeroides. The regulon controlled by RpoE was different in Rsb. denitrificans and Rhb. sphaeroides. Patterns of synthesized soluble proteins strongly changed upon high light exposure in Rsb. denitrificans but not in Rhb. sphaeroides, and most changes were not further promoted by artificial ¹O₂ generation. The strong increase of small RNA RDs2461 levels by photooxidative stress implies a role for sRNAs in post-transcriptional regulation of the response to ¹O₂ in AAnPs. Our data reveal similarities but also significant differences in the response of Rsb. denitrificans and Rhb. sphaeroides to ¹O₂, most likely a consequence of their different lifestyles.

Systematic Comparative Protein Expression Profiling of Clear Cell Renal Cell Carcinoma A PILOT STUDY BASED ON THE SEPARATION OF TISSUE SPECIMENS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS

Proteome-based technologies represent powerful tools for the analysis of protein expression profiles, including the identification of potential cancer candidate biomarkers. Thus, here we provide a comprehensive protein expression map for clear cell renal cell carcinoma established by systematic comparative two-dimensional gel electrophoresis-based protein expression profiling of 16 paired tissue systems comprising clear cell renal cell carcinoma lesions and corresponding tumor-adjacent renal epithelium using overlapping narrow pH gradients. This approach led to the mapping of 348 distinct spots corresponding to 248 different protein identities. By implementing restriction criteria concerning their detection frequency and overall regulation mode, 28 up- and 56 down-regulated single target spots were considered as potential candidate biomarkers. Based on their gene ontology information, these differentially expressed proteins were classified into distinct functional groups and according to their cellular distribution. Moreover, three representative members of this group, namely calbindin, gelsolin, and heart fatty acid-binding protein, were selected, and their expression pattern was analyzed by immunohistochemistry using tissue microarrays. Thus, this pilot study provides a significant update of the current renal cell carcinoma map and defines a number of differentially expressed proteins, but both their potential as candidate biomarkers and clinical relevance has to be further explored in tissues and for body fluids like serum and urine.