Interplay between desiccation and oxidative stress responses in iron-oxidizing acidophilic bacteria.

Variations in water availability represent a foremost stress factor affecting the growth and survival of microorganisms. Acidophilic bioleaching bacteria are industrially applied for releasing metals from mineral sulphides, and they are considered extremely tolerant to oxidative conditions prevailing in acidic bioleaching environments. Such processes usually are performed in heaps and thus these microorganisms are also exposed to intermittent desiccations or high osmolarity periods that reduce the water availability. However, the tolerance to water stress and the molecular basis of adaptation to it are still largely unknown. The aim of this work was to determine the cellular response to desiccation stress and establish its relationship to oxidative stress response in the acidophilic iron-oxidizing bacteria Acidithiobacillus ferrooxidans ATCC 23270 and Leptospirillum ferriphilum DSM 14647. Results showed that the exposure of cell cultures to desiccation (0-120 min) led to a significant reduction in cell growth, and to an increase in content in reactive oxygen species in both bacteria. However, Leptospirillum ferriphilum turned out to be more tolerant than Acidithiobacillus ferrooxidans. In addition, the pre-treatment of the cell cultures with compatible solutes (trehalose and ectoine), and antioxidants (glutathione and cobalamin) restored all stress parameters to levels exhibited by the control cultures. To evaluate the role of the osmotic and redox homeostasis mechanisms in coping with desiccation stress, the relative expression of a set of selected genes was approached by RT-qPCR experiments in cells exposed to desiccation for 30 min. Results showed a generalized upregulation of genes that code for mechanosensitive channels, and enzymes related to the biosynthesis of compatible solutes and oxidative stress response in both bacteria. These data suggest that acidophiles show variable tolerance to desiccation and allow to establish that water stress can trigger oxidative stress, and thus anti-oxidative protection capability can be a relevant mechanism when cells are challenged by desiccation or other anhydrobiosis states.

Insights into Systems for Iron-Sulfur Cluster Biosynthesis in Acidophilic Microorganisms.

Fe-S clusters are versatile and essential cofactors that participate in multiple and fundamental biological processes. In Escherichia coli, the biogenesis of these cofactors requires either the housekeeping Isc pathway, or the stress-induced Suf pathway which plays a general role under conditions of oxidative stress or iron limitation. In the present work, the Fe-S cluster assembly Isc and Suf systems of acidophilic Bacteria and Archaea, which thrive in highly oxidative environments, were studied. This analysis revealed that acidophilic microorganisms have a complete set of genes encoding for a single system (either Suf or Isc). In acidophilic Proteobacteria and Nitrospirae, a complete set of isc genes (iscRSUAX-hscBA-fdx), but not genes coding for the Suf system, was detected. The activity of the Isc system was studied in Leptospirillum sp. CF-1 (Nitrospirae). RT-PCR experiments showed that eight candidate genes were co-transcribed and conform the isc operon in this strain. Additionally, RT-qPCR assays showed that the expression of the iscS gene was significantly up-regulated in cells exposed to oxidative stress imposed by 260 mM Fe2(SO4)3 for 1 h or iron starvation for 3 h. The activity of cysteine desulfurase (IscS) in CF-1 cell extracts was also up-regulated under such conditions. Thus, the Isc system from Leptospirillum sp. CF-1 seems to play an active role in stressful environments. These results contribute to a better understanding of the distribution and role of Fe-S cluster protein biogenesis systems in organisms that thrive in extreme environmental conditions.

A novel gene from the acidophilic bacterium Leptospirillum sp. CF-1 and its role in oxidative stress and chromate tolerance.

BACKGROUND: Acidophilic microorganisms like Leptospirillum sp. CF-1 thrive in environments with extremely low pH and high concentrations of dissolved heavy metals that can induce the generation of reactive oxygen species (ROS). Several hypothetical genes and proteins from Leptospirillum sp. CF-1 are known to be up-regulated under oxidative stress conditions. RESULTS: In the present work, the function of hypothetical gene ABH19_09590 from Leptospirillum sp. CF-1 was studied. Heterologous expression of this gene in Escherichia coli led to an increase in the ability to grow under oxidant conditions with 5 mM K2CrO4 or 5 mM H2O2. Similarly, a significant reduction in ROS production in E. coli transformed with a plasmid carrying ABH19_09590 was observed after exposure to these oxidative stress elicitors for 30 min, compared to a strain complemented with the empty vector. A co-transcriptional study using RT-PCR showed that ABH19_09590 is contained in an operon, here named the "och" operon, that also contains ABH19_09585, ABH19_09595 and ABH19_09600 genes. The expression of the och operon was significantly up-regulated in Leptospirillum sp. CF-1 exposed to 5 mM K2CrO4 for 15 and 30 min. Genes of this operon potentially encode a NADH:ubiquinone oxidoreductase, a CXXC motif-containing protein likely involved in thiol/disulfide exchange, a hypothetical protein, and a di-hydroxy-acid dehydratase. A comparative genomic analysis revealed that the och operon is a characteristic genetic determinant of the Leptospirillum genus that is not present in other acidophiles. CONCLUSIONS: Altogether, these results suggest that the och operon plays a protective role against chromate and hydrogen peroxide and is an important mechanism required to face polyextremophilic conditions in acid environments.