Molecular acclimation of Halobacterium salinarum to halite brine inclusions

Abstract

Halophilic microorganisms have long been known to survive within the brine inclusions of salt crystals, as evidenced by the change in color for salt crystals containing pigmented halophiles. However, the molecular mechanisms allowing this survival has remained an open question for decades. While protocols for the surface sterilization of halite (NaCl) have enabled isolation of cells and DNA from within halite brine inclusions, "-omics" based approaches have faced two main technical challenges: (1) removal of all contaminating organic biomolecules (including proteins) from halite surfaces, and (2) performing selective biomolecule extractions directly from cells contained within halite brine inclusions with sufficient speed to avoid modifications in gene expression during extraction. In this study, we tested different methods to resolve these two technical challenges. Following this method development, we then applied the optimized methods to perform the first examination of the early acclimation of a model haloarchaeon (Halobacterium salinarum NRC-1) to halite brine inclusions. Examinations of the proteome of Halobacterium cells two months post-evaporation revealed a high degree of similarity with stationary phase liquid cultures, but with a sharp down-regulation of ribosomal proteins. While proteins for central metabolism were part of the shared proteome between liquid cultures and halite brine inclusions, proteins involved in cell mobility (archaellum, gas vesicles) were either absent or less abundant in halite samples. Proteins unique to cells within brine inclusions included transporters, suggesting modified interactions between cells and the surrounding brine inclusion microenvironment. The methods and hypotheses presented here enable future studies of the survival of halophiles in both culture model and natural halite systems.

Keywords: Halobacterium; LC-MS; halite (NaCl); halophile; proteomics.

Figure 1

TNPA laboratory-grown halite crystals internally inoculated with Halobacterium salinarum cells observed after two months of incubation at 37°C for crystallization replicates 1–3 (A–C, respectively).

Figure 2

Laboratory-grown halite surface observation. (A–F) TNPA crystals without cells. (G–J) Internally inoculated TNPA crystals with Halobacterium salinarum cells incubated 2 months at 37°C. (K–N) Externally inoculated TNPA crystals with Halobacterium salinarum cells after ~1 week incubated at 37°C. (A, G, K) Schematic representation of halite crystals without cells, internally inoculated and externally inoculated, respectively. (B, H, L) Visual observation of crystals without cells, internally inoculated and externally inoculated, respectively. (C, E) Secondary electron images. (I, M) Backscattered images. (D, F, J, N) Elemental composition analysis by EDX spectroscopy correlated, respectively, to the SEM images in panels C, E, I, M. Carbon and potassium appear in red and yellow, respectively.

Figure 3

Surface-bounded protein removal by chemical treatments with active-spray washes. (A) Percentage of proteins removed after NaOCl-NaOH treatment with or without an additional HCl treatment step, and subsequent residual protein quantity. (B) Overview of optimal NaOCl-NaOH-HCl chemical treatment with active-spray washes.

Figure 4

Venn diagrams of proteins identified by mass spectrometry for brine inclusions and liquid stationary culture samples. (A) Comparison of biological replicates for liquid stationary culture extracts. (B) Comparison of biological replicates for brine inclusions extracts. Black circles in (A,B) represent “core” proteins shared by the four replicates in both cases. (C) Comparison of core proteins between brine inclusion extracts and liquid stationary culture cells.

Figure 5

Venn diagram of the 68 proteins quantified by iTRAQ^® with triple injection which exhibit significative differential expression in brine extracts compared to liquid stationary culture cells.

Figure 6

Schematic overview of targets cellular functions involved in early acclimation of Halobacterium salinarum through halite brine inclusions (for extended matched proteins, see Supplementary Table 6-5). Shared (*) indicates proteins found in all four brine inclusion extracts and all four liquid stationary culture extracts. Partially shared (**) indicates proteins found in both brine inclusions and stationary phase liquid cultures, but not for all replicate samples for each condition.