Osmolyte Signatures for the Protection of Aspergillus sydowii Cells under Halophilic Conditions and Osmotic Shock

Aspergillus sydowii is a moderate halophile fungus extensively studied for its biotechnological potential and halophile responses, which has also been reported as a coral reef pathogen. In a recent publication, the transcriptomic analysis of this fungus, when growing on wheat straw, showed that genes related to cell wall modification and cation transporters were upregulated under hypersaline conditions but not under 0.5 M NaCl, the optimal salinity for growth in this strain. This led us to study osmolyte accumulation as a mechanism to withstand moderate salinity. In this work, we show that A. sydowii accumulates trehalose, arabitol, mannitol, and glycerol with different temporal dynamics, which depend on whether the fungus is exposed to hypo- or hyperosmotic stress. The transcripts coding for enzymes responsible for polyalcohol synthesis were regulated in a stress-dependent manner. Interestingly, A. sydowii contains three homologs (Hog1, Hog2 and MpkC) of the Hog1 MAPK, the master regulator of hyperosmotic stress response in S. cerevisiae and other fungi. We show a differential regulation of these MAPKs under different salinity conditions, including sustained basal Hog1/Hog2 phosphorylation levels in the absence of NaCl or in the presence of 2.0 M NaCl, in contrast to what is observed in S. cerevisiae. These findings indicate that halophilic fungi such as A. sydowii utilize different osmoadaptation mechanisms to hypersaline conditions.

A Novel pH and Thermo-Tolerant Halophilic Alpha-amylase From Moderate Halophile Nesterenkonia Sp.Strain F: Gene Analysis, Molecular Cloning, Heterologous Expression and Biochemical Characterization

A novel pH and thermo-tolerate halophilic alpha-amylase from moderately halophilic bacterium, Nesterenkonia sp.strain F was cloned and expressed in Escherichia coli. 16S rRNA sequence of the strain shared 99.46 % similarities with closely related type species. Also, the genome sequence shared ANI values below 92 % and dDDH values below 52 % with the closely related type species. Consequently, it is proposed that strain F represents a novel species. The AmyF gene was 1390 bp long and encodes an alpha-amylase of 463 amino acid residues with pI of 4.62. The deduced AmyF shared very low sequence similarity (<24%) with functionally characterized recombinant halophilic alpha- amylases. The recombinant alpha-amylase was successfully purified from Ni-NTA columns with a molecular mass of about 52 KDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active over a wide range of temperature (25–75 °C) and pH (4–9) with optimum activity at 45 °C and 7.5, respectively. Also, although it was active over a various concentrations of NaCl and KCl (0–4 M), increasing activity of the enzyme was observed with increasing concentration of these salts. Low concentrations of Ca2+ ion had no activating effect, but high concentrations of the ion (40 - 200 mM) enhanced activity of AmyF. The enzyme activity was increased by increasing concentrations of Mg2+, Zn2+, Hg2+ and Fe3+. However, it was inhibited only at very high concentrations of these metal ions. Cu2+ did not decrease the amylase activity and the highest activity was observed at 100 mM of the ion. These properties indicate wide potential applications of this recombinant enzyme in starch processing industries. This is the first isolation, cloning and characterization of a gene encoding alpha-amylase from Nesternkonia genus.

Bioreduction of Cr(VI) by Viable Cells and Cell-Free Extract of a Moderate Halophile, Halomonas Smyrnensis KS802 and Evaluation of Their in Vitro Efficacy in Tannery Effluent

The present study was aimed to characterize the chromate reducing ability of cells and cell-free extract (CFE) of Halomonas smyrnensis KS802 (GenBank Accession No. KU982965) and evaluate their effectiveness in tannery effluents. Viable cells of the strain reduced 200 µM Cr(VI) in basal medium for halophiles (MH) in 10 h and was inversely proportional to Cr(VI) concentrations. The rapid reduction by cells (10⁹ cells/mL) was achieved with 7.5% NaCl, at pH 7 and 37°C which increased with increasing cell density (10¹° cells/mL). While acetate, Cu³⁺, Fe³⁺, SO₄²⁻, and CO₃²⁻ were stimulating the reduction, the inhibitors retarded the process significantly. The NADH-dependent chromate reduction of the CFE was found to be constitutive with Km and Vmax values of 56.58 µM and 3.37 µM/min/mg protein respectively. The optimal reductase activity of the CFE was evident at 200 µM Cr(VI), 10% NaCl, pH 8.0 and at 45°C. A higher concentration of CFE and electron donors increased the enzyme activity but was impacted negatively by toxic metals and anions. Both the cells and CFE were capable of reducing Cr(VI) remarkably from tannery effluent. FTIR and XRD spectra of chromate reducing cells confirmed possible complexation of reduced Cr-species with functional groups on cell surface.

Draft Genome Sequence of Alkalicoccus halolimnae BZ-SZ-XJ29T, a Moderately Halophilic Bacterium Isolated from a Salt Lake

ABSTRACT The moderate halophile Alkalicoccus halolimnae BZ-SZ-XJ29T grows optimally in a relative broad range of 8.3% to 12.3% (wt/vol) NaCl. The draft genome consists of approximately 3.66 Mb and contains 3,534 putative genes. Various genes involved in osmotic stress were predicted, providing pertinent insights into specific adaptations to the hypersaline environment.