Zinc oxide, titanium dioxide and C60 fullerene nanoparticles, alone and in mixture, differently affect biomarker responses and proteome in the clam Ruditapes philippinarum.

Continuous release of nanoparticles (NPs) into marine coastal environments results in an increased risk of exposure to complex NP mixtures for marine organisms. However, to date, the information on the effects at molecular and biochemical levels induced by the exposure to NPs, singly and as a mixture, is still scant. The present work aimed at exploring the independent and combined effects and the mechanism(s) of action induced by 7-days exposure to 1 µg/L nZnO, 1 µg/L nTiO2 and 1 µg/L FC60 fullerene in the Manila clam Ruditapes philippinarum, using a battery of immunological and oxidative stress biomarkers in haemolymph, gills and digestive gland. In addition, proteomics analyses were performed in gills and the digestive gland, where NP bioaccumulation was also assessed. Increased bioaccumulation of single NPs and the mixture was linked with increased oxidative stress and higher damage to proteins, lipids and DNA in all tissues analysed. The proteomics approach highlighted protein modulation in terms of abundance and damage (higher redox-thiol and carbonylated groups content). In particular, the modulated proteins (16 in gills and 18 in digestive gland) were mostly related to cytoskeleton and energetic metabolism. The digestive gland was the tissue more affected. For all biomarkers measured, increased detrimental effects were observed in the mixture compared to single NP exposures.

Regulation of the hydrogenase-4 operon of Escherichia coli by the sigma(54)-dependent transcriptional activators FhlA and HyfR.

The hyf locus (hyfABCDEFGHIJ-hyfR-focB) of Escherichia coli encodes a putative 10-subunit hydrogenase complex (hydrogenase-4 [Hyf]); a potential sigma(54)-dependent transcriptional activator, HyfR (related to FhlA); and a putative formate transporter, FocB (related to FocA). In order to gain insight into the physiological role of the Hyf system, we investigated hyf expression by using a hyfA-lacZ transcriptional fusion. This work revealed that hyf is induced under fermentative conditions by formate at a low pH and in an FhlA-dependent fashion. Expression was sigma(54) dependent and was inhibited by HycA, the negative transcriptional regulator of the formate regulon. Thus, hyf expression resembles that of the hyc operon. Primer extension analysis identified a transcriptional start site 30 bp upstream of the hyfA structural gene, with appropriately located -24 and -12 boxes indicative of a sigma(54)-dependent promoter. No reverse transcriptase PCR product could be detected for hyfJ-hyfR, suggesting that hyfR-focB may be independently transcribed from the rest of the hyf operon. Expression of hyf was strongly induced ( approximately 1,000-fold) in the presence of a multicopy plasmid expressing hyfR from a heterologous promoter. This induction was dependent on low pH, anaerobiosis, and postexponential growth and was weakly enhanced by formate. The hyfR-expressing plasmid increased fdhF-lacZ transcription just twofold but did not influence the expression of hycB-lacZ. Interestingly, inactivation of the chromosomal hyfR gene had no effect on hyfA-lacZ expression. Purified HyfR was found to specifically interact with the hyf promoter/operator region. Inactivation of the hyf operon had no discernible effect on growth under the range of conditions tested. No Hyf-derived hydrogenase or formate dehydrogenase activity could be detected, and no Ni-containing protein corresponding to HyfG was observed.