Halobacterium salinarum is an extremely halophilic archaeon that is widely distributed in hypersaline environments and was originally isolated as a spoilage organism of salted fish and hides. The type strain 91-R6 (DSM 3754 T ) has seldom been studied and its genome sequence has only recently been determined by our group. The exact relationship between the type strain and two widely used model strains, NRC-1 and R1, has not been described before. The genome of Hbt. salinarum strain 91-R6 consists of a chromosome (2.17 Mb) and two large plasmids (148 and 102 kb, with 39,230 bp being duplicated). Cytosine residues are methylated ( m4 C) within CTAG motifs. The genomes of type and laboratory strains are closely related, their chromosomes sharing average nucleotide identity (ANIb) values of 98% and in silico DNA-DNA hybridization (DDH) values of 95%. The chromosomes are completely colinear, do not show genome rearrangement, and matching segments show <1% sequence difference. Among the strain-specific sequences are three large chromosomal replacement regions (>10 kb). The well-studied AT-rich island (61 kb) of the laboratory strains is replaced by a distinct AT-rich sequence (47 kb) in 91-R6. Another large replacement (91-R6: 78 kb, R1: 44 kb) codes for distinct homologs of proteins involved in motility and N-glycosylation. Most (107 kb) of plasmid pHSAL1 (91-R6) is very closely related to part of plasmid pHS3 (R1) and codes for essential genes (e.g. arginine-tRNA ligase and the pyrimidine biosynthesis enzyme aspartate carbamoyltransferase). Part of pHS3 (42.5 kb total) is closely related to the largest strain-specific sequence (164 kb)in the type strain chromosome. Genome sequencing unraveled the close relationship between the Hbt. salinarum type strain and two well-studied laboratory strains at the DNA and protein levels. Although an independent isolate, the type strain shows a remarkably low evolutionary difference to the laboratory strains. K E Y W O R D Scomparative genomics, genomic variability, haloarchaea, halobacteria, megaplasmid, type strain 2 of 44 | PFEIFFER Et al.

Early and mature biofilm formation in the extremely halophilic euryarchaeon Halobacterium salinarum strain R1 was characterized by SWATH-LC/MS/MS. Using a simple surfactant-assisted protein solubilization protocol and one-dimensional ultra-high performance nanoflow chromatography on the front end, 63.2 and 58.6% of the predicted H. salinarum R1 proteome could be detected and quantified, respectively. Analysis of biophysical protein properties, functional analysis and pathway mapping indicated comprehensive characterization of the proteome. Sixty point eight percent of the quantified proteins (or 34.5% of the predicted proteome) exhibited significant abundance changes between planktonic and sessile states, demonstrating that haloarchaeal biofilm formation represents a profound "lifestyle change" on the molecular level. Our results and analysis constitute the first comprehensive study to track molecular changes from planktonic cultures to initial and mature archaeal biofilms on the proteome level. Data are available via ProteomeXchange, identifier PXD003667. Proteins exemplifying different protein expression level profiles were selected, and their corresponding gene transcripts targeted by qRT-PCR to test the feasibility of establishing rapid PCR-based assays for archaeal biofilm formation.

It was recently shown that haloarchaeal strains of different genera are able to adhere to surfaces and form surface-attached biofilms. However, the surface structures mediating the adhesion were still unknown. We have identified a novel surface structure with Halobacterium salinarum strain R1, crucial for surface adhesion. Electron microscopic studies of surface-attached cells frequently showed pili-like surface structures of two different diameters that were irregularly distributed on the surface. The thinner filaments, 7–8 nm in diameter, represented a so far unobserved novel pili-like structure. Examination of the Hbt. salinarum R1 genome identified two putative gene loci (pil-1 and pil-2) encoding type IV pilus biogenesis complexes besides the archaellum encoding fla gene locus. Both pil-1 and pil-2 were expressed as transcriptional units, and the transcriptional start of pil-1 was identified. In silico analyses revealed that the pil-1 locus is present with other euryarchaeal genomes whereas the pil-2 is restricted to haloarchaea. Comparative real time qRT-PCR studies indicated that the general transcriptional activity was reduced in adherent vs. planktonic cells. In contrast, the transcription of pilB1 and pilB2, encoding putative type IV pilus assembly ATPases, was induced in comparison to the archaella assembly/motor ATPase (flaI) and the ferredoxin gene. Mutant strains were constructed that incurred a flaI deletion or flaI/pilB1 gene deletions. The absence of flaI caused the loss of the archaella while the additional absence of pilB1 led to loss of the novel pili-like surface structures. The ΔflaI/ΔpilB1 double mutants showed a 10-fold reduction in surface adhesion compared to the parental strain. Since surface adhesion was not reduced with the non-archaellated ΔflaI mutants, the pil-1 filaments have a distinct function in the adhesion process.

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.

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