Molecular stress responses to nano-sized zero-valent iron (nZVI) particles in the soil bacterium Pseudomonas stutzeri.

Nanotoxicological studies were performed in vitro using the common soil bacterium Pseudomonas stutzeri to assess the potentially toxic impact of commercial nano-sized zero-valent iron (nZVI) particles, which are currently used for environmental remediation projects. The phenotypic response of P. stutzeri to nZVI toxicity includes an initial insult to the cell wall, as evidenced by TEM micrographs. Transcriptional analyses using genes of particular relevance in cellular activity revealed that no significant changes occurred among the relative expression ratios of narG, nirS, pykA or gyrA following nZVI exposure; however, a significant increase in katB expression was indicative of nZVI-induced oxidative stress in P. stutzeri. A proteomic approach identified two major defence mechanisms that occurred in response to nZVI exposure: a downregulation of membrane proteins and an upregulation of proteins involved in reducing intracellular oxidative stress. These biomarkers served as early indicators of nZVI response in this soil bacterium, and may provide relevant information for environmental hazard assessment

TEM micrographs showing <i>P. stutzeri</i> control cells (A) and cells exposed to 5 g L⁻¹ nZVI (B).

<p>TEM micrographs showing <i>P. stutzeri</i> control cells (A) and cells exposed to 5 g L⁻¹ nZVI (B).</p

Characteristics of the set of RT-qPCR primers used in this study.

<p>Characteristics of the set of RT-qPCR primers used in this study.</p

Differentially expressed proteins identified in <i>P. stutzeri</i> specifically upregulated by nZVI treatment.

<p>Differentially expressed proteins identified in <i>P. stutzeri</i> specifically upregulated by nZVI treatment.</p

Gene expression fold change in <i>P. stutzeri</i> cells treated with 5 g L⁻¹ nZVI.

<p>Gene expression fold change in <i>P. stutzeri</i> cells treated with 5 g L⁻¹ nZVI.</p

Protein profiles of control and nZVI-treated bacteria are shown in the gels.

<p>Proteins excised from the gels for identification have been highlighted in white.</p

Methodologies to generate, extract, purify and fractionate yeast ECM for analytical use in proteomics and glycomics

In a multicellular organism, the extracellular matrix (ECM) provides a cell-supporting scaffold and helps maintaining the biophysical integrity of tissues and organs. At the same time it plays crucial roles in cellular communication and signalling, with implications in spatial organisation, motility and differentiation. Similarly, the presence of an ECM-like extracellular polymeric substance is known to support and protect bacterial and fungal multicellular aggregates, such as biofilms or colonies. However, the roles and composition of this microbial ECM are still poorly understood.Authors would like to acknowledge Joana Tulha for assistance on yeasts overlay photographs, and to Rui Armada for C. albicans ECM SDS-PAGE experiment. The proteomic analysis was carried out at the proteomics facility UCM-PCM, a member of the ProteoRed network. The polysaccharide analysis was performed at the Laboratory of Glycoconjugates Biochemistry and Cellular Biology, UFRJ, Brazil. Fabio Faria-Oliveira was supported by a PhD scholarship SFRH/BD/45368/2008 from FCT (Fundacao para a Ciencia e a Tecnologia). This work was funded by Marie Curie Initial Training Network GLYCOPHARM (PITN-GA-2012-317297), and by FCT/MEC through Portuguese funds (PIDDAC) - PEst-OE/BIA/UI4050/2014. The authors would also like to acknowledge Hugh S. Johnson for critical reading of the manuscript regarding English usage