Search Results (13)

The CRISPR-Cas system represents an adaptive immune mechanism found across diverse Archaea and Bacteria, allowing them to defend against invading genetic elements such as viruses and plasmids. Despite its broad distribution, the prevalence and complexity of CRISPR-Cas systems differ significantly between these domains. This study aimed to characterize and compare the genomic distribution, structural features, and functional implications of CRISPR-Cas systems and associated antibiotic resistance genes in 30 archaeal and 30 bacterial genomes. Through bioinformatic analyses of CRISPR arrays, cas gene architectures, direct repeats (DRs), and thermodynamic properties, we observed that Archaea exhibit a higher number and greater complexity of CRISPR loci, with more diverse cas gene subtypes exclusively of Class 1. Bacteria, in contrast, showed fewer CRISPR loci, comprising a mix of Class 1 and Class 2 systems, with Class 1 representing the majority (~75%) of the detected systems. Notably, Bacteria lacking CRISPR-Cas systems displayed a higher prevalence of antibiotic resistance genes, suggesting a possible inverse correlation between the presence of these immune systems and the acquisition of such genes. Phylogenetic and thermodynamic analyses further highlighted domain-specific adaptations and conservation patterns. These findings support the hypothesis that CRISPR-Cas systems play a dual role: first, as a defense mechanism preventing the integration of foreign genetic material—reflected in the higher complexity and diversity of CRISPR loci in Archaea—and second, as a regulator of horizontal gene transfer, evidenced by the lower frequency of antibiotic resistance genes in organisms with active CRISPR-Cas systems. Together, these results underscore the evolutionary and functional diversification of CRISPR-Cas systems in response to environmental and selective pressures. Full article

Phage φ29 and related bacteriophages are currently the smallest known tailed viruses infecting various representatives of both Gram-positive and Gram-negative bacteria. They are characterised by genomic content features and distinctive properties that are unique among known tailed phages; their characteristics include protein primer-driven replication and a packaging process characteristic of this group. Searches conducted using public genomic databases revealed in excess of 2000 entries, including bacteriophages, phage plasmids and sequences identified as being archaeal that share the characteristic features of phage φ29. An analysis of predicted proteins, however, indicated that the metagenomic sequences attributed as archaeal appear to be misclassified and belong to bacteriophages. An analysis of the translated polypeptides of major capsid proteins (MCPs) of φ29-related phages indicated the dissimilarity of MCP sequences to those of almost all other known Caudoviricetes groups and a possible distant relationship to MCPs of T7-like (Autographiviridae) phages. Sequence searches conducted using HMM revealed the relatedness between the main structural proteins of φ29-like phages and an unusual lactococcal phage, KSY1 (Chopinvirus KSY1), whose genome contains two genes of RNA polymerase that are similar to the RNA polymerases of phages of the Autographiviridae and Schitoviridae (N4-like) families. An analysis of the tail tube proteins of φ29-like phages indicated their dissimilarity of the lower collar protein to tail proteins of all other viral groups, but revealed its possible distant relatedness with proteins of toxin translocation complexes. The combination of the unique features and distinctive origin of φ29-related phages suggests the categorisation of this vast group in a new order or as a new taxon of a higher rank. Full article

Even though viruses and plasmids are both drivers of horizontal gene transfer, they differ fundamentally in their mode of transfer. Virus genomes are enclosed in virus capsids and are not dependent on cell-to-cell contacts for their dissemination. In contrast, the transfer of plasmids most often requires physical contact between cells. However, plasmid pR1SE of Halorubrum lacusprofundi is disseminated between cells, independent of cell-cell contacts, in specialized membrane vesicles that contain plasmid proteins. In this study, we searched for pR1SE-like elements in public databases and a metagenomics dataset from Australian salt lakes and identified 40 additional pR1SE-like elements in hypersaline environments worldwide. Herein, these elements are named apHPVs (archaeal plasmids of haloarchaea potentially transferred in plasmid vesicles). They share two sets of closely related proteins with conserved synteny, strongly indicating an organization into different functional clusters. We find that apHPVs, besides transferring themselves, have the potential to transfer large fragments of DNA between host cells, including virus defense systems. Most interestingly, apHPVs likely play an important role in the evolution of viruses and plasmids in haloarchaea, as they appear to recombine with both of them. This further supports the idea that plasmids and viruses are not distinct but closely related mobile genetic elements. Full article

N-glycosylation is a post-translational modification of proteins that occurs across all three domains of life. In Archaea, N-glycosylation is crucial for cell stability and motility, but importantly also has significant implications for virus–host interactions. While some archaeal viruses present glycosylated proteins or interact with glycosylated host proteins, the direct influence of N-glycosylation on archaeal virus–host interactions remains to be elucidated. In this study, we generated an N-glycosylation-deficient mutant of Halorubrum lacusprofundi, a halophilic archaeon commonly used to study cold adaptation, and examined the impact of compromised N-glycosylation on the infection dynamics of two very diverse viruses. While compromised N-glycosylation had no influence on the life cycle of the head-tailed virus HRTV-DL1, we observed a significant effect on membrane-containing virus HFPV-1. Both intracellular genome numbers and extracellular virus particle numbers of HFPV-1 were increased in the mutant strain, which we attribute to instability of the surface-layer which builds the protein envelope of the cell. When testing the impact of compromised N-glycosylation on the life cycle of plasmid vesicles, specialized membrane vesicles that transfer a plasmid between host cells, we determined that plasmid vesicle stability is strongly dependent on the host glycosylation machinery. Our study thus provides important insight into the role of N-glycosylation in virus–host interactions in Archaea, while pointing to how this influence strongly differs amongst various viruses and virus-like elements. Full article

Coastal sediments in the proximity of wastewater and emergency outfalls are often sinks of pharmaceutical compounds and other organic and inorganic contaminants that are likely to affect the microbial community. The metabolites of these contaminants affect microbial diversity and their metabolic processes, resulting in undesirable effects on ecosystem functioning, thus necessitating the need to understand their composition and functions. In the present investigation, we studied the metagenomes of 12 coastal surface sediments through whole genome shot-gun sequencing. Taxonomic binning of the genes predicted about 86% as bacteria, 1% as archaea, >0.001% as viruses and Eukaryota, and 12% as other communities. The dominant bacterial, archaeal, and fungal genera were Woeseia, Nitrosopumilus, and Rhizophagus, respectively. The most prevalent viral families were Myoviridae and Siphoviridae, and the T4 virus was the most dominant bacteriophage. The unigenes further aligned to 26 clusters of orthologous genes (COGs) and five carbohydrate-active enzymes (CAZy) classes. Glycoside hydrolases (GH) and glycoside transferase (GT) were the highest-recorded CAzymes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) level 3 functions were subjugated by purine metabolism > ABC transporters > oxidative phosphorylation > two-component system > pyrimidine metabolism > pyruvate metabolism > quorum sensing > carbon fixation pathways > ribosomes > and glyoxalate and dicarboxylate metabolism. Sequences allying with plasmids, integrons, insertion sequences and antibiotic-resistance genes were also observed. Both the taxonomies and functional abundances exhibited variation in relative abundances, with limited spatial variability (ANOVA p > 0.05; ANOSIM-0.05, p > 0.05). This study underlines the dominant microbial communities and functional genes in the marine sediments of Kuwait as a baseline for future biomonitoring programs. Full article

Cytochrome P450 monooxygenases (CYPs/P450s) and their redox partners, ferredoxins, are ubiquitous in organisms. P450s have been studied in biology for over six decades owing to their distinct catalytic activities, including their role in drug metabolism. Ferredoxins are ancient proteins involved in oxidation-reduction reactions, such as transferring electrons to P450s. The evolution and diversification of P450s in various organisms have received little attention and no information is available for archaea. This study is aimed at addressing this research gap. Genome-wide analysis revealed 1204 P450s belonging to 34 P450 families and 112 P450 subfamilies, where some families and subfamilies are expanded in archaea. We also identified 353 ferredoxins belonging to the four types 2Fe-2S, 3Fe-4S, 7Fe-4S and 2[4Fe-4S] in 40 archaeal species. We found that bacteria and archaea shared the CYP109, CYP147 and CYP197 families, as well as several ferredoxin subtypes, and that these genes are co-present on archaeal plasmids and chromosomes, implying the plasmid-mediated lateral transfer of these genes from bacteria to archaea. The absence of ferredoxins and ferredoxin reductases in the P450 operons suggests that the lateral transfer of these genes is independent. We present different scenarios for the evolution and diversification of P450s and ferredoxins in archaea. Based on the phylogenetic analysis and high affinity to diverged P450s, we propose that archaeal P450s could have diverged from CYP109, CYP147 and CYP197. Based on this study’s results, we propose that all archaeal P450s are bacterial in origin and that the original archaea had no P450s. Full article

Due to their role in methane production, methanoarchaea are of high ecological relevance and genetic systems have been ever more established in the last two decades. The system for protein expression in Methanosarcina using a comprehensive shuttle vector is established; however, details about its replication mechanism in methanoarchaea remain unknown. Here, we report on a significant optimisation of the rather large shuttle vector pWM321 (8.9 kbp) generated by Metcalf through a decrease in its size by about 35% by means of the deletion of several non-coding regions and the ssrA gene. The resulting plasmid (pRS1595) still stably replicates in M. mazei and—most likely due to its reduced size—shows a significantly higher transformation efficiency compared to pWM321. In addition, we investigate the essential gene repA, coding for a rep type protein. RepA was heterologously expressed in Escherichia coli, purified and characterised, demonstrating the significant binding and nicking activity of supercoiled plasmid DNA. Based on our findings we propose that the optimised shuttle vector replicates via a rolling circle mechanism with RepA as the initial replication protein in Methanosarcina. On the basis of bioinformatic comparisons, we propose the presence and location of a double-strand and a single-strand origin, which need to be further verified. Full article

The genus of Curtobacterium, belonging to the Microbacteriaceae family of the Actinomycetales order, includes economically significant pathogenic bacteria of soybeans and other agricultural crops. Thorough phylogenetic and full-genome analysis using the latest genomic data has demonstrated a complex and contradictory taxonomic picture within the group of organisms classified as the Curtobacterium species. Based on these data, it is possible to delineate about 50 new species and to reclassify a substantial part of the Curtobacterium strains. It is suggested that 53 strains, including most of the Curtobacterium flaccumfaciens pathovars, can compose a monophyletic group classified as C. flaccumfaciens. A genomic analysis using the most recent inventory of bacterial chromosomal and plasmid genomes deposited to GenBank confirmed the possible role of Microbacteriaceae plasmids in pathogenicity and demonstrated the existence of a group of related plasmids carrying virulence factors and possessing a gene distantly related to DNA polymerase found in bacteriophages and archaeal and eukaryotic viruses. A PCR diagnostic assay specific to the genus Curtobacterium was developed and tested. The presented results assist in the understanding of the evolutionary relations within the genus and can lay the foundation for further taxonomic updates. Full article

A protocol was designed for plasmid curing using a novel counter-selectable marker, named pylS^ZK-pylT, in Escherichia coli. The pylS^ZK-pylT marker consists of the archaeal pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNA^pyl) with modification, and incorporates an unnatural amino acid (Uaa), N^ε-benzyloxycarbonyl-l-lysine (ZK), at a sense codon in ribosomally synthesized proteins, resulting in bacterial growth inhibition or killing. Plasmid curing is performed by exerting toxicity on pylS^ZK-pylT located on the target plasmid, and selecting only proliferative bacteria. All tested bacteria obtained using this protocol had lost the target plasmid (64/64), suggesting that plasmid curing was successful. Next, we attempted to exchange plasmids with the identical replication origin and an antibiotic resistance gene without plasmid curing using a modified protocol, assuming substitution of plasmids complementing genomic essential genes. All randomly selected bacteria after screening had only the substitute plasmid and no target plasmid (25/25), suggesting that plasmid exchange was also accomplished. Counter-selectable markers based on PylRS-tRNA^pyl, such as pylS^ZK-pylT, may be scalable in application due to their independence from the host genotype, applicability to a wide range of species, and high tunability due to the freedom of choice of target codons and Uaa’s to be incorporated. Full article

The Sulfolobales have provided good model organisms for studying CRISPR-Cas systems of the crenarchaeal kingdom of the archaea. These organisms are infected by a wide range of exceptional archaea-specific viruses and conjugative plasmids, and their CRISPR-Cas systems generally exhibit extensive structural and functional diversity. They carry large and multiple CRISPR loci and often multiple copies of diverse Type I and Type III interference modules as well as more homogeneous adaptation modules. These acidothermophilic organisms have recently provided seminal insights into both the adaptation process, the diverse modes of interference, and their modes of regulation. The functions of the adaptation and interference modules tend to be loosely coupled and the stringency of the crRNA-DNA sequence matching during DNA interference is relatively low, in contrast to some more streamlined CRISPR-Cas systems of bacteria. Despite this, there is evidence for a complex and differential regulation of expression of the diverse functional modules in response to viral infection. Recent work also supports critical roles for non-core Cas proteins, especially during Type III-directed interference, and this is consistent with these proteins tending to coevolve with core Cas proteins. Various novel aspects of CRISPR-Cas systems of the Sulfolobales are considered including an alternative spacer acquisition mechanism, reversible spacer acquisition, the formation and significance of antisense CRISPR RNAs, and a novel mechanism for avoidance of CRISPR-Cas defense. Finally, questions regarding the basis for the complexity, diversity, and apparent redundancy, of the intracellular CRISPR-Cas systems are discussed. Full article

The aim of this study was to explore the possibility of using an archaeal microorganism as a host system for expressing mammalian olfactory receptors (ORs). We have selected the archaeon Haloferax volcanii as a cell host system and one of the most extensively investigated OR, namely I7-OR, whose preferred ligands are short-chain aldehydes, such as octanal, heptanal, nonanal. A novel plasmid has been constructed to express the rat I7-OR, fused with a hexahistidine-tag for protein immunodetection. The presence of the recombinant receptor at a membrane level was demonstrated by immunoblot of the membranes isolated from the transgenic archaeal strain. In addition, the lipid composition of archaeonanosomes containing ORs has been characterized in detail by High-Performance Thin-Layer Chromatography (HPTLC) in combination with Matrix-Assisted Laser Desorption Ionization—Time-Of-Flight/Mass Spectrometry (MALDI-TOF/MS) analysis. Full article

Comparative genomics has revealed that variations in bacterial and archaeal genome DNA sequences cannot be explained by only neutral mutations. Virus resistance and plasmid distribution systems have resulted in changes in bacterial and archaeal genome sequences during evolution. The restriction-modification system, a virus resistance system, leads to avoidance of palindromic DNA sequences in genomes. Clustered, regularly interspaced, short palindromic repeats (CRISPRs) found in genomes represent yet another virus resistance system. Comparative genomics has shown that bacteria and archaea have failed to gain any DNA with GC content higher than the GC content of their chromosomes. Thus, horizontally transferred DNA regions have lower GC content than the host chromosomal DNA does. Some nucleoid-associated proteins bind DNA regions with low GC content and inhibit the expression of genes contained in those regions. This form of gene repression is another type of virus resistance system. On the other hand, bacteria and archaea have used plasmids to gain additional genes. Virus resistance systems influence plasmid distribution. Interestingly, the restriction-modification system and nucleoid-associated protein genes have been distributed via plasmids. Thus, GC content and genomic signatures do not reflect bacterial and archaeal evolutionary relationships. Full article