Project description:Halophilic microorganisms have long been known to survive within the brine inclusions of salt crystals, as evidenced by their pigmentation. 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 present methods to resolve these two technical challenges. In addition, we apply these 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. Low quantities of RNA from halite brine inclusions corroborate the hypothesis of low transcriptional and translational activities. 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 inclusions microenvironment. The methods and hypotheses presented here enable future studies of the survival of halophiles in both culture model and natural halite systems.
Project description:In hypersaline brines, biodegradation of recalcitrant plant polymers can be inhibited by salt-induced microbial stress and/or caused by inadequate metabolic capabilities of extremely halophilic microbes. Therefore, woody materials can be well-preserved even in NaCl brines that are less biologically hostile than most other brines. Here, we considered whether the nanohaloarchaea, that live alongside (the related) haloarchaea, ever partake in the degradation of xylan, a major hemicellulose component of wood. Samples were taken from natural evaporitic brines and anthropogenic solar salterns located in various parts of Europe and Asia. We recently demonstrated that nanohaloarchaeon Ca. Nanohalobium constans lives as an ectosymbiont associated with the chitinolytic haloarchaeon Halomicrobium. Here, we describe an extremely halophilic xylan-degrading consortium with three members, where nanohaloarchaea act as ectosymbionts of Haloferax lucertensis, which in turn acts as a scavenger of xylan-degradation products, produced by a primary xylan hydrolytic Halorhabdus species. The two corresponding binary associations of nanohaloarchaea, Candidatus Nanohalococcus occultus SVXNc and Candidatus Nanohalovita haloferacivicina BNXNv and their hosts were obtained, stably cultivated and characterized. In contrast to the previously described association of chitinolytic haloarchaeon Halomicrobium and its amylolytic symbiont Ca. Nanohalobium, the host haloarchaea within the xylan-degrading consortium could metabolize α-glucans (glycogen and starch), and, thus, obtained no obvious trophic benefit from ectosymbionts. The current study has broadened the range of culturable ectosymbiontic nanohaloarchaea and demonstrates that they are an important ecophysiological component of polysaccharide-degrading halophilic microbial communities and can be readily isolated in binary co-cultures by using the appropriate enrichment strategy.