The genome of Halobacterium strain 63‐R2 was recently reported and provides the opportunity to resolve long‐standing issues regarding the source of two widely used model strains of Halobacterium ...salinarum, NRC‐1 and R1. Strain 63‐R2 was isolated in 1934 from a salted buffalo hide (epithet “cutirubra”), along with another strain from a salted cow hide (91‐R6T, epithet “salinaria,” the type strain of Hbt. salinarum). Both strains belong to the same species according to genome‐based taxonomy analysis (TYGS), with chromosome sequences showing 99.64% identity over 1.85 Mb. The chromosome of strain 63‐R2 is 99.99% identical to the two laboratory strains NRC‐1 and R1, with only five indels, excluding the mobilome. The two reported plasmids of strain 63‐R2 share their architecture with plasmids of strain R1 (pHcu43/pHS4, 99.89% identity; pHcu235/pHS3, 100.0% identity). We detected and assembled additional plasmids using PacBio reads deposited at the SRA database, further corroborating that strain differences are minimal. One plasmid, pHcu190 (190,816 bp) corresponds to pHS1 (strain R1) but is even more similar in architecture to pNRC100 (strain NRC‐1). Another plasmid, pHcu229, assembled partially and completed in silico (229,124 bp), shares most of its architecture with pHS2 (strain R1). In deviating regions, it corresponds to pNRC200 (strain NRC‐1). Further architectural differences between the laboratory strain plasmids are not unique, but are present in strain 63‐R2, which contains characteristics from both of them. Based on these observations, it is proposed that the early twentieth‐century isolate 63‐R2 is the immediate ancestor of the twin laboratory strains NRC‐1 and R1.
The complete genomes of four Halobacterium salinarum strains were compared in detail. Two strains (91‐R6T and 63‐R2) were isolated in 1934 by Lochhead from cow and buffalo hides. From the results of these comparisons, we conclude that strain 63‐R2 is the immediate ancestor of the two, widely used laboratory strains NRC‐1 and R1.
Halophilic archaea of the class
are the most salt-requiring prokaryotes within the domain
. In 1997, minimal standards for the description of new taxa in the order
were proposed. From then on, the ...taxonomy of the class
provides an excellent example of how changing concepts on prokaryote taxonomy and the development of new methods were implemented. The last decades have witnessed a rapid expansion of the number of described taxa within the class
coinciding with the era of genome sequencing development. The current members of the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of
propose these revisions to the recommended minimal standards and encourage the use of advanced technologies in the taxonomic description of members of the
. Most previously required and some recommended minimal standards for the description of new taxa in the class
were retained in the present revision, but changes have been proposed in line with the new methodologies. In addition to the 16S rRNA gene, the
gene is an important molecular marker for the identification of members of the
. Phylogenomic analysis based on concatenated conserved, single-copy marker genes is required to infer the taxonomic status of new taxa. The overall genome relatedness indexes have proven to be determinative in the classification of the taxa within the class
. Average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values should be calculated for rigorous comparison among close relatives.
Haloquadratum walsbyi commonly dominates the microbial flora of hypersaline waters. Its cells are extremely fragile squares requiring >14%(w/v) salt for growth, properties that should limit its ...dispersal and promote geographical isolation and divergence. To assess this, the genome sequences of two isolates recovered from sites at near maximum distance on Earth, were compared.
Both chromosomes are 3.1 MB in size, and 84% of each sequence was highly similar to the other (98.6% identity), comprising the core sequence. ORFs of this shared sequence were completely synteneic (conserved in genomic orientation and order), without inversion or rearrangement. Strain-specific insertions/deletions could be precisely mapped, often allowing the genetic events to be inferred. Many inferred deletions were associated with short direct repeats (4-20 bp). Deletion-coupled insertions are frequent, producing different sequences at identical positions. In cases where the inserted and deleted sequences are homologous, this leads to variant genes in a common synteneic background (as already described by others). Cas/CRISPR systems are present in C23(T) but have been lost in HBSQ001 except for a few spacer remnants. Numerous types of mobile genetic elements occur in both strains, most of which appear to be active, and with some specifically targetting others. Strain C23(T) carries two ∼6 kb plasmids that show similarity to halovirus His1 and to sequences nearby halovirus/plasmid gene clusters commonly found in haloarchaea.
Deletion-coupled insertions show that Hqr. walsbyi evolves by uptake and precise integration of foreign DNA, probably originating from close relatives. Change is also driven by mobile genetic elements but these do not by themselves explain the atypically low gene coding density found in this species. The remarkable genome conservation despite the presence of active systems for genome rearrangement implies both an efficient global dispersal system, and a high selective fitness for this species.
Biofilm formation by haloarchaea Fröls, Sabrina; Dyall-Smith, Mike; Pfeifer, Felicitas
Environmental microbiology,
December 2012, Letnik:
14, Številka:
12
Journal Article
Recenzirano
Summary
A fluorescence‐based live‐cell adhesion assay was used to examine biofilm formation by 20 different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel ...natural isolates from an Antarctic salt lake. Thirteen of the 20 tested strains significantly adhered (P‐value < 0.05) to a plastic surface. Examination of adherent cell layers on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet‐like, multi‐layered biofilms containing micro‐ and macrocolonies (up to 50 μm in height) were formed by strains of Halobacterium salinarum and the Antarctic isolate t‐ADL strain DL24. The second type of biofilm, characterized by large aggregates of cells adhering to surfaces, was formed by Haloferax volcanii DSM 3757T and Halorubrum lacusprofundi DL28. Staining of the biofilms formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymers, such as eDNA and glycoconjugates, substances previously shown to stabilize bacterial biofilms. For Hbt. salinarum DSM 3754T and Hfx. volcanii DSM 3757T, cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754T showed several types of cellular appendages that could be involved in the initial attachment. Our results show that biofilm formation occurs in a surprisingly wide variety of haloarchaeal species.
As a group, the halophilic archaea (class
Halobacteria
) are the most salt-requiring and salt-resistant microorganisms within the domain
Archaea
. Halophilic archaea flourish in thalassohaline and ...athalassohaline environments and require over 100–150 g/L NaCl for growth and structural stability. Natural hypersaline environments vary in salt concentration, chemical composition and pH, and occur in climates ranging from tropical to polar and even under-sea. Accordingly, their resident haloarchaeal species vary enormously, as do their individual population compositions and community structures. These diverse halophilic archaeal strains are precious resources for theoretical and applied research but assessing their taxonomic and metabolic novelty and diversity in natural environments has been technically difficult up until recently. Environmental DNA-based high-throughput sequencing technology has now matured sufficiently to allow inexpensive recovery of massive amounts of sequence data, revealing the distribution and community composition of halophilic archaea in different hypersaline environments. While cultivation of haloarchaea is slow and tedious, and only recovers a fraction of the natural diversity, it is the conventional means of describing new species, and provides strains for detailed study. As of the end of May 2020, the class
Halobacteria
contains 71 genera and 275 species, 49.8% of which were first isolated from the marine salt environment and 50.2% from the inland salt environment, indicating that both thalassohaline and athalassohaline environments contain diverse halophilic archaea. However, there remain taxa that have not yet been isolated in pure culture, such as the nanohaloarchaea, which are widespread in the salt environment and may be one of the hot spots in the field of halophilic archaea research in the future. In this review, we focus on the cultivation strategies that have been used to isolate extremely halophilic archaea and point out some of the pitfalls and challenges.
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 3754T) 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 (m4C) 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.
The genome of the Halobacterium salinarum type strain (91‐R6, DSM 3754) was compared at the DNA and protein levels to the genomes of two well‐studied laboratory strains, NRC‐1 and R1. The chromosomes and portions of the plasmids were very closely related. However, distinct homologs for proteins involved in motility and N‐glycosylation were encountered.
Summary
A new virus of halophilic Archaea is reported by Liu et al., and is remarkable in many ways. SNJ2 is the first temperate, pleomorphic virus (pleolipovirus) that integrates into the genome of ...its host. Analyses of the virus structure and its genome have provided an unexpected puzzle while at the same time solving another. On the one hand, the study shows a curious relationship exists between SNJ2 and an unrelated provirus (SNJ1) found as a plasmid in the same cell. The presence of SNJ1 appears to allow much higher levels of SNJ2 virus to be produced, although the mechanism involved remains unclear. On the other hand, the curious occurrence of a conserved cluster of pleolipovirus‐related genes found widely distributed among haloarchaeal genomes and known for almost 10 years, now appears to correspond to SNJ2‐related proviruses.
The genomes of many haloarchaea (extremely halophilic Archaea) contain one or more copies of highly conserved gene clusters that encode capsid‐related proteins similar to those found in pleolipoviruses. Surrounding these gene modules there are usually other virus‐ or plasmid‐related genes but the nature of these odd chromosomal loci has been puzzling. The study by Liu et al. (….) reveals that many (perhaps most) of them are proviruses of a new type of temperate pleolipovirus, as exemplified by SNJ2.
Annotation ambiguities and annotation errors are a general challenge in genomics. While a reliable protein function assignment can be obtained by experimental characterization, this is expensive and ...time-consuming, and the number of such Gold Standard Proteins (GSP) with experimental support remains very low compared to proteins annotated by sequence homology, usually through automated pipelines. Even a GSP may give a misleading assignment when used as a reference: the homolog may be close enough to support isofunctionality, but the substrate of the GSP is absent from the species being annotated. In such cases, the enzymes cannot be isofunctional. Here, we examined a variety of such issues in halophilic archaea (class Halobacteria), with a strong focus on the model haloarchaeon
.
Annotated proteins of
were identified for which public databases tend to assign a function that is probably incorrect. In some cases, an alternative, probably correct, function can be predicted or inferred from the available evidence, but this has not been adopted by public databases because experimental validation is lacking. In other cases, a probably invalid specific function is predicted by homology, and while there is evidence that this assigned function is unlikely, the true function remains elusive. We listed 50 of those cases, each with detailed background information, so that a conclusion about the most likely biological function can be drawn. For reasons of brevity and comprehension, only the key aspects are listed in the main text, with detailed information being provided in a corresponding section of the Supplementary Materials.
Compiling, describing and summarizing these open annotation issues and functional predictions will benefit the scientific community in the general effort to improve the evaluation of protein function assignments and more thoroughly detail them. By highlighting the gaps and likely annotation errors currently in the databases, we hope this study will provide a framework for experimentalists to systematically confirm (or disprove) our function predictions or to uncover yet more unexpected functions.
NaCl-saturated brines such as saltern crystalliser ponds, inland salt lakes, deep-sea brines and liquids-of-deliquescence on halite are commonly regarded as a paradigm for the limit of life on Earth. ...There are, however, other habitats that are thermodynamically more extreme. Typically, NaCl-saturated environments contain all domains of life and perform complete biogeochemical cycling. Despite their reduced water activity, ∼0.755 at 5 M NaCl, some halophiles belonging to the Archaea and Bacteria exhibit optimum growth/metabolism in these brines. Furthermore, the recognised water-activity limit for microbial function, ∼0.585 for some strains of fungi, lies far below 0.755. Other biophysical constraints on the microbial biosphere (temperatures of >121°C; pH > 12; and high chaotropicity; e.g. ethanol at >18.9% w/v (24% v/v) and MgCl2 at >3.03 M) can prevent any cellular metabolism or ecosystem function. By contrast, NaCl-saturated environments contain biomass-dense, metabolically diverse, highly active and complex microbial ecosystems; and this underscores their moderate character. Here, we survey the evidence that NaCl-saturated brines are biologically permissive, fertile habitats that are thermodynamically mid-range rather than extreme. Indeed, were NaCl sufficiently soluble, some halophiles might grow at concentrations of up to 8 M. It may be that the finite solubility of NaCl has stabilised the genetic composition of halophile populations and limited the action of natural selection in driving halophile evolution towards greater xerophilicity. Further implications are considered for the origin(s) of life and other aspects of astrobiology.
Halovirus HF2 was the first member of the
genus to have its genome fully sequenced, which revealed two classes of intergenic repeat (IR) sequences: class I repeats of 58 bp in length, and class II ...repeats of 29 bp in length. Both classes of repeat contain AT-rich motifs that were conjectured to represent promoters. In the present study, nine IRs were cloned upstream of the
reporter gene, and all displayed promoter activity, providing experimental evidence for the previous conjecture. Comparative genomics showed that IR sequences and their relative genomic positions were strongly conserved among other members of the same virus genus. The transcription of HF2 was also examined by the reverse-transcriptase-PCR (RT-PCR) method, which demonstrated very long transcripts were produced that together covered most of the genome, and from both strands. The presence of long counter transcripts suggests a regulatory role or possibly unrecognized coding potential.
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