Analysis of the Genome and Mobilome of a Dissimilatory Arsenate Reducing Aeromonas sp. O23A Reveals Multiple Mechanisms for Heavy Metal Resistance and Metabolism

Aeromonas spp. are among the most ubiquitous microorganisms, as they have been isolated from different environmental niches including waters, soil, as well as wounds and digestive tracts of poikilothermic animals and humans. Although much attention has been paid to the pathogenicity of Aeromonads, the role of these bacteria in environmentally important processes, such as transformation of heavy metals, remains to be discovered. Therefore, the aim of this study was a detailed genomic characterization of Aeromonas sp. O23A, the first representative of this genus capable of dissimilatory arsenate reduction. The strain was isolated from microbial mats from the Zloty Stok mine (SW Poland), an environment strongly contaminated with arsenic. Previous physiological studies indicated that O23A may be involved in both mobilization and immobilization of this metalloid in the environment. To discover the molecular basis of the mechanisms behind the observed abilities, the genome of O23A (∼5.0 Mbp) was sequenced and annotated, and genes for arsenic respiration, heavy metal resistance (hmr) and other phenotypic traits, including siderophore production, were identified. The functionality of the indicated gene modules was assessed in a series of minimal inhibitory concentration analyses for various metals and metalloids, as well as mineral dissolution experiments. Interestingly, comparative analyses revealed that O23A is related to a fish pathogen Aeromonas salmonicida subsp. salmonicida A449 which, however, does not carry genes for arsenic respiration. This indicates that the dissimilatory arsenate reduction ability may have been lost during genome reduction in pathogenic strains, or acquired through horizontal gene transfer. Therefore, particular emphasis was placed upon the mobilome of O23A, consisting of four plasmids, a phage, and numerous transposable elements, which may play a role in the dissemination of hmr and arsenic metabolism genes in the environment. The obtained results indicate that Aeromonas sp. O23A is well-adapted to the extreme environmental conditions occurring in the Zloty Stok mine. The analysis of genome encoded traits allowed for a better understanding of the mechanisms of adaptation of the strain, also with respect to its presumable role in colonization and remediation of arsenic-contaminated waters, which may never have been discovered based on physiological analyses alone

mRNA:guanine-N7 cap methyltransferases: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence-structure-function relationships

BACKGROUND: The 5'-terminal cap structure plays an important role in many aspects of mRNA metabolism. Capping enzymes encoded by viruses and pathogenic fungi are attractive targets for specific inhibitors. There is a large body of experimental data on viral and cellular methyltransferases (MTases) that carry out guanine-N7 (cap 0) methylation, including results of extensive mutagenesis. However, a crystal structure is not available and cap 0 MTases are too diverged from other MTases of known structure to allow straightforward homology-based interpretation of these data. RESULTS: We report a 3D model of cap 0 MTase, developed using sequence-to-structure threading and comparative modeling based on coordinates of the glycine N-methyltransferase. Analysis of the predicted structural features in the phylogenetic context of the cap 0 MTase family allows us to rationalize most of the experimental data available and to propose potential binding sites. We identified a case of correlated mutations in the cofactor-binding site of viral MTases that may be important for the rational drug design. Furthermore, database searches and phylogenetic analysis revealed a novel subfamily of hypothetical MTases from plants, distinct from "orthodox" cap 0 MTases. CONCLUSIONS: Computational methods were used to infer the evolutionary relationships and predict the structure of Eukaryotic cap MTase. Identification of novel cap MTase homologs suggests candidates for cloning and biochemical characterization, while the structural model will be useful in designing new experiments to better understand the molecular function of cap MTases

Novel non-specific DNA adenine methyltransferases

The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N6-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N6-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N6-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential ‘sequence specificity’ could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation

Structural basis of the methylation specificity of R.DpnI

R.DpnI consists of N-terminal catalytic and C-terminal winged helix domains that are separately specific for the Gm6ATC sequences in Dam-methylated DNA. Here we present a crystal structure of R.DpnI with oligoduplexes bound to the catalytic and winged helix domains and identify the catalytic domain residues that are involved in interactions with the substrate methyl groups. We show that these methyl groups in the Gm6ATC target sequence are positioned very close to each other. We further show that the presence of the two methyl groups requires a deviation from B-DNA conformation to avoid steric conflict. The methylation compatible DNA conformation is complementary with binding sites of both R.DpnI domains. This indirect readout of methylation adds to the specificity mediated by direct favorable interactions with the methyl groups and solvation/desolvation effects. We also present hydrogen/deuterium exchange data that support ‘crosstalk’ between the two domains in the identification of methylated DNA, which should further enhance R.DpnI methylation specificity

Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes - Characterization of a Novel Phage Helper-Satellite System.

Two novel prophages ФAH14a and ФAH14b of a psychrotolerant Antarctic bacterium Pseudomonas sp. ANT_H14 have been characterized. They were simultaneously induced with mitomycin C and packed into capsids of the same size and protein composition. The genome sequences of ФAH14a and ФAH14b have been determined. ФAH14b, the phage with a smaller genome (16,812 bp) seems to parasitize ФAH14a (55,060 bp) and utilizes its capsids, as only the latter encodes a complete set of structural proteins. Both viruses probably constitute a phage helper-satellite system, analogous to the P2-P4 duo. This study describes the architecture and function of the ФAH14a and ФAH14b genomes. Moreover, a functional analysis of a ФAH14a-encoded lytic enzyme and a DNA methyltransferase was performed. In silico analysis revealed the presence of the homologs of ФAH14a and ФAH14b in other Pseudomonas genomes, which may suggest that helper-satellite systems related to the one described in this work are common in pseudomonads

Characteristics and Comparative Genomic Analysis of a Novel Virus, VarioGold, the First Bacteriophage of <i>Variovorax</i>

Variovorax represents a widespread and ecologically significant genus of soil bacteria. Despite the ecological importance of these bacteria, our knowledge about the viruses infecting Variovorax spp. is quite poor. This study describes the isolation and characterization of the mitomycin-induced phage, named VarioGold. To the best of our knowledge, VarioGold represents the first characterized virus for this genus. Comparative genomic analyses suggested that VarioGold is distinct from currently known bacteriophages at both the nucleotide and protein levels; thus, it could be considered a new virus genus. In addition, another 37 prophages were distinguished in silico within the complete genomic sequences of Variovorax spp. that are available in public databases. The similarity networking analysis highlighted their general high diversity, which, despite clustering with previously described phages, shows their unique genetic load. Therefore, the novelty of Variovorax phages warrants the great enrichment of databases, which could, in turn, improve bioinformatic strategies for finding (pro)phages