High-coverage long-read sequencing of the
type strain (91-R6) revealed a 2.17-Mb chromosome and two large plasmids (148 and 102 kb). Population heterogeneity and long repeats were observed. Strain ...91-R6 and laboratory strain R1 showed 99.63% sequence identity in common chromosomal regions and only 38 strain-specific segments. This information resolves the previously uncertain relationship between type and laboratory strains.
Plasmids PL6A and PL6B are both carried by the C23
strain of the square archaeon
, and are closely related (76% nucleotide identity), circular, about 6 kb in size, and display the same gene synteny. ...They are unrelated to other known plasmids and all of the predicted proteins are cryptic in function. Here we describe two additional PL6-related plasmids, pBAJ9-6 and pLT53-7, each carried by distinct isolates of
that were recovered from hypersaline waters in Australia. A third PL6-like plasmid, pLTMV-6, was assembled from metavirome data from Lake Tyrell, a salt-lake in Victoria, Australia. Comparison of all five plasmids revealed a distinct plasmid family with strong conservation of gene content and synteny, an average size of 6.2 kb (range 5.8-7.0 kb) and pairwise similarities between 61-79%. One protein (F3) was closely similar to a protein carried by betapleolipoviruses while another (R6) was similar to a predicted AAA-ATPase of His 1 halovirus (His1V_gp16). Plasmid pLT53-7 carried a gene for a FkbM family methyltransferase that was not present in any of the other plasmids. Comparative analysis of all PL6-like plasmids provided better resolution of conserved sequences and coding regions, confirmed the strong link to haloviruses, and showed that their sequences are highly conserved among examples from
isolates and metagenomic data that collectively cover geographically distant locations, indicating that these genetic elements are widespread.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many ...bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.
Background: CRISPR/Cas systems allow archaea and bacteria to resist invasion by foreign nucleic acids.
Results: The CRISPR/Cas system in Haloferax recognized six different PAM sequences that could trigger a defense response.
Conclusion: The PAM sequence specificity of the defense response in type I CRISPR systems is more relaxed than previously thought.
Significance: The PAM sequence requirements for interference and adaptation appear to differ markedly.
Summary
The Cas/CRISPR system of prokaryotes is a recently recognized defence system that protects cells from invasion by foreign nucleic acids, such as plasmids or bacteriophages. Using the archaeon ...Sulfolobus, Gudbergsdottir et al. have demonstrated that when foreign DNA is both a target of the CRISPR system and also carries a gene that is essential for cell growth, surviving cells display mutations in the CRISPR system that eliminate the target directed spacer. This simulates natural scenarios where the invading DNA cannot be readily destroyed, such as a temperate bacteriophage that manages to evade CRISPR attack and integrate into the genome, so becoming ‘self’. CRISPR deletions ranged from precise removal of a single spacer up to elimination of an entire CRISPR array but the mechanism involved is not understood. Another surprising feature was that from one to two base mutations in the same target protospacer did not affect recognition by the Sulfolobus CRISPR system, something unexpected from studies in bacteria where spacer and target protospacer must match exactly. This study shows that even though the CRISPR system is a powerful weapon it can also be a dangerous one for the cell that carries it.
We report the genome sequence of a monophasic
subsp
Typhimurium strain (TW-Stm6) isolated in Australia that is similar to epidemic multidrug-resistant strains from Europe and elsewhere. This strain ...carries additional antibiotic and heavy-metal resistance genes on a large (275-kb) IncHI2 plasmid.
Gene expression in Haloarcula hispanica cells infected with the gammapleolipovirus His2 was studied using a custom DNA microarray. Total RNA from cells sampled at 0, 1, 2, 3, and 4.5 hr postinfection ...was reverse‐transcribed into labeled cDNA and hybridized to microarrays, revealing temporal and differential expression in both host and viral genes. His2 gene expression occurred in three main phases (early, middle, and late), and by 4.5 hr p.i. the majority of genes were actively transcribed, including those encoding the major structural proteins. Eighty host genes were differentially regulated ≥twofold postinfection, with most of them predicted to be involved in transport, translation, and metabolism. Differentially expressed host genes could also be grouped into early‐, middle‐, and late‐expressed genes based on the timing of their up‐ and downregulation postinfection. The altered host transcriptional pattern suggests regulation by His2 infection, which may reprogram host metabolism to facilitate its own DNA replication and propagation. This study enhances the characterization of many hypothetical viral genes and provides insights into the interaction between His2 and its host.
Gene expression in Haloarcula hispanica cells infected with the gammapleolipovirus His2 was studied using a custom DNA microarray. Temporal and differential expression in both host and viral genes was observed. This study facilitates the characterization of many hypothetical viral genes and provides insights into the interaction between His2 and its host.
Strains C23T and HBSQ001 were isolated from solar salterns and are novel square-shaped, aerobic, extremely halophilic members of the domain Archaea and family Halobacteriaceae. Cells stained ...Gram-negative and grew optimally in media containing 18 % salts at around neutral pH. Mg2+ is not required. The DNA G+C content of both isolates was 46.9 mol% and DNA-DNA cross-hybridization showed a relatedness of 80 %. Their 16S rRNA gene sequences showed only 2 nucleotide differences (99.9 % identity) and phylogenetic tree reconstructions with other recognized members of the Halobacteriaceae indicated that they formed a distinct clade, with the closest relative being Halogeometricum borinquense PR 3T (91.2 % sequence identity). The major polar glycolipid of both isolates was the sulfated diglycosyl diether lipid S-DGD-1. Electron cryomicrosopy of whole cells revealed similar internal structures, such as gas vesicles and polyhydroxyalkanoate granules, but the cell wall of isolate HBSQ001 displayed a more complex S-layer compared with that of isolate C23T. The phenotypic characterization and phylogenetic data support the placement of isolates C23T and HBSQ001 in a novel species in a new genus within the Halobacteriaceae, for which we propose the name Haloquadratum walsbyi gen. nov., sp. nov. The type strain of Haloquadratum walsbyi is C23T (=JCM 12705T=DSM 16854T).
Cultivation of Walsby’s square haloarchaeon Burns, David G.; Camakaris, Helen M.; Janssen, Peter H. ...
FEMS microbiology letters,
09/2004, Letnik:
238, Številka:
2
Journal Article
Recenzirano
The
square
haloarchaea
of
Walsby (SHOW group) dominate hypersaline microbial communities but have not been cultured since their discovery 25 years ago. We show that natural water dilution cultures ...can be used to isolate members of this group and, once in pure culture, they can be grown in standard halobacterial media. Cells display a square morphology and contain gas vesicles and poly-β-hydroxybutyrate (PHB) granules. The 16S rRNA gene sequence was >99% identical to other SHOW group sequences. They prefer high salinities (23–30%), and can grow with a doubling time of 1–2 days in rich media. The ability to culture SHOW group organisms makes it possible to study, in a comprehensive way, the microbial ecology of salt lakes.
Hypersaline environments are the source of many viruses infecting different species of halophilic euryarchaea. Information on infection mechanisms of archaeal viruses is scarce, due to the lack of ...genetically accessible virus-host models. Recently, a new archaeal siphovirus, Haloferax tailed virus 1 (HFTV1), was isolated together with its host belonging to the genus
, but it is not infectious on the widely used model euryarcheon
. To gain more insight into the biology of HFTV1 host strain LR2-5, we studied characteristics that might play a role in its virus susceptibility: growth-dependent motility, surface layer, filamentous surface structures, and cell shape. Its genome sequence showed that LR2-5 is a new strain of
. LR2-5 lacks obvious viral defense systems, such as CRISPR-Cas, and the composition of its cell surface is different from
, which might explain the different viral host range. This work provides first deep insights into the relationship between the host of halovirus HFTV1 and other members of the genus
. Given the close relationship to the genetically accessible
, LR2-5 has high potential as a new model for virus-host studies in euryarchaea.
Haloquadratum walsbyi is frequently a dominant member of the microbial communities in hypersaline waters. 16S rRNA gene sequences indicate that divergence within this species is very low but ...relatively few sites have been examined, particularly in the southern hemisphere. The diversity of Haloquadratum was examined in three coastal, but geographically distant saltern crystallizer ponds in Australia, using both culture-independent and culture-dependent methods. Two 97%-OTU, comprising Haloquadratum- and Halorubrum-related sequences, were shared by all three sites, with the former OTU representing about 40% of the sequences recovered at each site. Sequences 99.5% identical to that of Hqr. walsbyi C23T were present at all three sites and, overall, 98% of the Haloquadratum-related sequences displayed ≤2% divergence from that of the type strain. While haloarchaeal diversity at each site was relatively low (9-16 OTUs), seven phylogroups (clones and/or isolates) and 4 different clones showed ≤90% sequence identity to classified taxa, and appear to represent novel genera. Six of these branched together in phylogenetic tree reconstructions, forming a clade (MSP8-clade) whose members were only distantly related to classified taxa. Such sequences have only rarely been previously detected but were found at all three Australian crystallizers.
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