Halobacterium salinarum R1 is an extremely halophilic archaeon, able to attach to the surface and to form characteristic biofilm structures under physiological conditions. However, the effect of environmental stress factors like heavy metals on biofilms was still unknown. Here, we report on the first insights into H. salinarum biofilm formation when exposed to copper, nickel and zinc and describe the effects of metal ions on the architecture of mature biofilms. We also studied the effects on gene expression in planktonic cells. Investigation of planktonic growth and cell adhesion in the presence of sub-lethal metal concentrations yielded an up to 60% reduced adhesion in case of copper and a significantly enhanced adhesion in case of zinc, whereas nickel treatment had no effect on adhesion. A PMA-qPCR assay was developed to quantify live/dead cells in planktonic cultures and mature biofilms, enabling the investigation of cell vitality after metal exposure. An increased resistance was observed in biofilms with up to 80% in case of copper- and up to 50% in case of zinc exposure compared to planktonic cells. However, nickel-treated biofilms showed no significant increase of cell survival. Microscopic investigation of the architecture of mature biofilms exposed to lethal metal concentrations demonstrated an increased detachment and the formation of large microcolonies after copper treatment, whereas the number of adherent cells increased strongly in nickel-exposed biofilms. In contrast, zinc exposed-biofilms showed no differences compared to the control. Analysis of the expression of genes encoding putative metal transporters by qRT-PCR revealed specific changes upon treatment of the cells with heavy metals. Our results demonstrate diverse effects of heavy metal ions on H. salinarum and imply a metal-specific protective response of cells in biofilms.
Halobacterium salinarum R1 is an extremely halophilic archaeon capable of adhesion and forming biofilms, allowing it to adjust to a range of growth conditions. We have recently shown that living in biofilms facilitates its survival under Cu 2+ and Ni 2+ stress, with specific rearrangements of the biofilm architecture observed following exposition. In this study, quantitative analyses were performed by SWATH mass spectrometry to determine the respective proteomes of planktonic and biofilm cells after exposition to Cu 2+ and Ni 2+ .Quantitative data for 1180 proteins were obtained, corresponding to 46% of the predicted proteome. In planktonic cells, 234 of 1180 proteins showed significant abundance changes after metal ion treatment, of which 47% occurred in Cu 2+ and Ni 2+ treated samples. In biofilms, significant changes were detected for 52 proteins. Only three proteins changed under both conditions, suggesting metal-specific stress responses in biofilms. Deletion strains were generated to assess the potential role of selected target genes. Strongest effects were observed for OE5245F and OE2816F strains which exhibited increased and decreased biofilm mass after Ni 2+ exposure, respectively. Moreover, EPS obviously plays a crucial role in H. salinarum metal ion resistance. Further efforts are required to elucidate the molecular basis and interplay of additional resistance mechanisms.
Porous calcium phosphate (CaP) materials as bone graft substitutes can be prepared from Ca carbonate biomineral structures by hydrothermal conversion into pseudomorphic CaP scaffolds. The present study aims at furnishing such phosphatized Ca carbonate biomineral (PCCB) materials with antibacterial Ag ions in order to avoid perisurgical wound infections. Prior to this study, PCCB materials with Mg and/or Sr ions incorporated for stimulating bone formation were prepared from coral skeletons and sea urchin spines as starting materials. The porous PCCB materials were treated with aqueous solutions of Ag nitrate with concentrations of 10 or 100 mmol/L, resulting in the formation of Ag phosphate nanoparticles on the sample surfaces through a replacement reaction. The materials were characterized using scanning electron microscopy (SEM) energy-dispersive X-ray spectroscopy (EDS) and X-ray diffractometry (XRD). In contact with Ringer`s solution, the Ag phosphate nanoparticles dissolved and released Ag ions with concentrations up to 0.51 mg/L, as shown by atomic absorption spectroscopy (AAS) analyses. In tests against Pseudomonas aeruginosa and Staphylococcus aureus on agar plates, antibacterial properties were similar for both types of Ag-modified PCCB materials. Concerning the antibacterial performance, the treatment with AgNO3 solutions with 10 mmol/L was almost as effective as with 100 mmol/L.
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