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Abstract Inteins are mobile genetic elements that invade conserved genes across all domains of life and viruses. In some instances, a single gene will have several intein insertion sites. In Haloarchaea, the minichromosome maintenance (MCM) protein at the core of replicative DNA helicase contains four intein insertion sites within close proximity, where two of these sites (MCM-a and MCM-d) are more likely to be invaded. A haloarchaeon that harbors both MCM-a and MCM-d inteins, Haloferax mediterranei, was studied in vivo to determine intein invasion dynamics and the interactions between neighboring inteins. Additionally, invasion frequencies and the conservation of insertion site sequences in 129 Haloferacales mcm homologs were analyzed to assess intein distribution across the order. We show that the inteins at MCM-a and MCM-d recognize and cleave their respective target sites and, in the event that only one empty intein invasion site is present, readily initiate homing (i.e. single homing). However, when two inteins are present co-homing into an intein-free target sequence is much less effective. The two inteins are more effective when invading alleles that already contain an intein at one of the two sites. Our in vivo and computational studies also support that having a proline in place of a serine as the first C-terminal extein residue of the MCM-d insertion site prevents successful intein splicing, but does not stop recognition of the insertion site by the intein's homing endonuclease. 

Agent-based models are composed of individual agents coded for traits, such as cooperation and cheating, that interact in a virtual world based on defined rules. Here, we describe the use of an agent-based model of homologous recombina- tion in bacteria playing a public goods game. We describe steps for software installation, setting model parameters, running and testing models, and visuali- zation and statistical analysis. This protocol is useful in analyses of horizontal gene transfer, bacterial sociobiology, and game theory. 

Inteins, often referred to as protein introns, are highly mobile genetic elements that invade conserved genes throughout the tree of life. Inteins have been found to invade a wide variety of key genes within actinophages. While in the process of conducting a survey of these inteins in actinophages, we discovered that one protein family of methylases contained a putative intein, and two other unique insertion elements. These methylases are known to occur commonly in phages as orphan methylases (possibly as a form of resistance to restriction–modification systems). We found that the methylase family is not conserved within phage clusters and has a disparate distribution across divergent phage groups. We determined that two of the three insertion elements have a patchy distribution within the methylase protein family. Additionally, we found that the third insertion element is likely a second homing endonuclease, and that all three elements (the intein, the homing endonuclease, and what we refer to as the ShiLan domain) have different insertion sites that are conserved in the methylase gene family. Furthermore, we find strong evidence that both the intein and ShiLan domain are partaking in long-distance horizontal gene transfer events between divergent methylases in disparate phage hosts within the already dispersed methylase distribution. The reticulate evolutionary history of methylases and their insertion elements reveals high rates of gene transfer and within-gene recombination in actinophages. 

Abstract Interest and controversy surrounding the evolutionary origins of extremely halophilic Archaea has increased in recent years, due to the discovery and characterization of the Nanohaloarchaea and the Methanonatronarchaeia. Initial attempts in explaining the evolutionary placement of the two new lineages in relation to the classical Halobacteria (also referred to as Haloarchaea) resulted in hypotheses that imply the new groups share a common ancestor with the Haloarchaea. However, more recent analyses have led to a shift: the Nanohaloarchaea have been largely accepted as being a member of the DPANN superphylum, outside of the euryarchaeota; whereas the Methanonatronarchaeia have been placed near the base of the Methanotecta (composed of the class II methanogens, the Halobacteriales, and Archaeoglobales). These opposing hypotheses have far-reaching implications on the concepts of convergent evolution (distantly related groups evolve similar strategies for survival), genome reduction, and gene transfer. In this work, we attempt to resolve these conflicts with phylogenetic and phylogenomic data. We provide a robust taxonomic sampling of Archaeal genomes that spans the Asgardarchaea, TACK Group, euryarchaeota, and the DPANN superphylum. In addition, we assembled draft genomes from seven new representatives of the Nanohaloarchaea from distinct geographic locations. Phylogenies derived from these data imply that the highly conserved ATP synthase catalytic/noncatalytic subunits of Nanohaloarchaea share a sisterhood relationship with the Haloarchaea. We also employ a novel gene family distance clustering strategy which shows this sisterhood relationship is not likely the result of a recent gene transfer. In addition, we present and evaluate data that argue for and against the monophyly of the DPANN superphylum, in particular, the inclusion of the Nanohaloarchaea in DPANN. 

Abstract Whole-genome comparisons based on average nucleotide identities (ANI) and the genome-to-genome distance calculator have risen to prominence in rapidly classifying prokaryotic taxa using whole-genome sequences. Some implementations have even been proposed as a new standard in species classification and have become a common technique for papers describing newly sequenced genomes. However, attempts to apply whole-genome divergence data to the delineation of higher taxonomic units and to phylogenetic inference have had difficulty matching those produced by more complex phylogenetic methods. We present a novel method for generating statistically supported phylogenies of archaeal and bacterial groups using a combined ANI and alignment fraction-based metric. For the test cases to which we applied the developed approach, we obtained results comparable with other methodologies up to at least the family level. The developed method uses nonparametric bootstrapping to gauge support for inferred groups. This method offers the opportunity to make use of whole-genome comparison data, that is already being generated, to quickly produce phylogenies including support for inferred groups. Additionally, the developed ANI methodology can assist the classification of higher taxonomic groups.[Average nucleotide identity (ANI); genome evolution; prokaryotic species delineation; taxonomy.] 

Abstract Horizontal gene transfer is a means by which bacteria, archaea, and eukaryotes are able to trade DNA within and between species. While there are a variety of mechanisms through which this genetic exchange can take place, one means prevalent in the archaeon Haloferax volcanii involves the transient formation of cytoplasmic bridges between cells and is referred to as mating. This process can result in the exchange of very large fragments of DNA between the participating cells. Genes governing the process of mating, including triggers to initiate mating, mechanisms of cell fusion, and DNA exchange, have yet to be characterized. We used a transcriptomic approach to gain a more detailed knowledge of how mating might transpire. By examining the differential expression of genes expressed in cells harvested from mating conditions on a filter over time and comparing them to those expressed in a shaking culture, we were able to identify genes and pathways potentially associated with mating. These analyses provide new insights into both the mechanisms and barriers of mating in Hfx. volcanii . 

ABSTRACT Isolated from Aran-Bidgol Lake in Iran, and reported here, Halorubrum ezzemoulense strain Fb21 represents the first complete genome from this archaeal species. Local recombination in this genome is in stark contrast to equidistant recombination events in bacteria. The genome’s GC bias, however, points to a genome architecture and origin that resemble those of a bacterium. Its availability, genome signatures, and frequent intragenomic recombination mean that Fb21 presents an attractive model organism for this species. 

Restriction–modification (RM) systems in bacteria are implicated in multiple biological roles ranging from defense against parasitic genetic elements, to selfish addiction cassettes, and barriers to gene transfer and lineage homogenization. In bacteria, DNA-methylation without cognate restriction also plays important roles in DNA replication, mismatch repair, protein expression, and in biasing DNA uptake. Little is known about archaeal RM systems and DNA methylation. To elucidate further understanding for the role of RM systems and DNA methylation in Archaea, we undertook a survey of the presence of RM system genes and related genes, including orphan DNA methylases, in the halophilic archaeal class Halobacteria. Our results reveal that some orphan DNA methyltransferase genes were highly conserved among lineages indicating an important functional constraint, whereas RM systems demonstrated patchy patterns of presence and absence. This irregular distribution is due to frequent horizontal gene transfer and gene loss, a finding suggesting that the evolution and life cycle of RM systems may be best described as that of a selfish genetic element. A putative target motif (CTAG) of one of the orphan methylases was underrepresented in all of the analyzed genomes, whereas another motif (GATC) was overrepresented in most of the haloarchaeal genomes, particularly in those that encoded the cognate orphan methylase.