Acute toxicity of organoarsenic chemical warfare agents to Danio rerio embryos.

During the 20th century, thousands of tons of munitions containing organoarsenic chemical warfare agents (CWAs) were dumped into oceans, seas and inland waters around the world. As a result, organoarsenic CWAs continue to leak from corroding munitions into sediments and their environmental concentrations are expected to peak over the next few decades. There remains, however, a lack of knowledge about their potential toxicity to aquatic vertebrates, such as fish. The aim of this study was to fill in this gap in research, by investigating the acute toxicity of organoarsenic CWAs on fish embryos, using the model species, Danio rerio. To estimate the acute toxicity thresholds of organoarsenic CWAs (Clark I, Adamsite, PDCA), a CWA-related compound (TPA), as well as four organoarsenic CWA degradation products (Clark I[ox], Adamsite[ox], PDCA[ox], TPA[ox]), standardized tests were performed following the OECD no. 236 Fish Embryo Acute Toxicity Test guidelines. Additionally, the detoxification response in D. rerio embryos was investigated by analysing the mRNA expression of five genes encoding antioxidant enzymes (CAT, SOD, GPx, GR and GST). During the 96 h of exposure, organoarsenic CWAs induced lethal effects in D. rerio embryos at very low concentrations (classified as 1st category pollutants according to GHS categorization), and were therefore deemed to be serious environmental hazards. Although TPA and the four CWA degradation products caused no acute toxicity even at their maximum solubility, the transcription of antioxidant-related genes was altered upon exposure to these compounds, indicating the need for further testing for chronic toxicity. Incorporating the results of this study into ecological risk assessments will provide a more accurate prediction of the environmental hazards posed by CWA-related organoarsenicals.

Structure and functions of a multireplicon genome of Antarctic Psychrobacter sp. ANT_H3: characterization of the genetic modules suitable for the construction of the plasmid-vectors for cold-active bacteria.

Among Psychrobacter spp., there are several multireplicon strains, carrying more than two plasmids. Psychrobacter sp. ANT_H3 carries as many as 11 extrachromosomal replicons, which is the highest number in Psychrobacter spp. Plasmids of this strain were subjected to detailed genomic analysis, which enables an insight into the structure and functioning of this multireplicon genome. The replication and conjugal transfer modules of ANT_H3 plasmids were analyzed functionally to discover their potential for being used as building blocks for the construction of novel plasmid-vectors for cold-active bacteria. It was shown that two plasmids have a narrow host range as they were not able to replicate in species other than Psychrobacter, while remaining plasmids had a wider host range and were functional in various Alpha- and Gammaproteobacteria. Moreover, it was confirmed that mobilization modules of seven plasmids were functional, i.e., could be mobilized for conjugal transfer by the RK2 conjugation system. Auxiliary genes were also distinguished in ANT_H3 plasmids, including these encoding putative DNA-protecting protein DprA, multidrug efflux SMR transporter of EmrE family, glycine cleavage system T protein, MscS small-conductance mechanosensitive channel protein, and two type II restriction-modification systems. Finally, all genome-retrieved plasmids of Psychrobacter spp. were subjected to complex genome- and proteome-based comparative analyses showing that Antarctic replicons are significantly different from plasmids from other locations.

Characteristics and Comparative Genomic Analysis of a Novel Virus, VarioGold, the First Bacteriophage of Variovorax.

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.

Characterization of Three Novel Virulent Aeromonas Phages Provides Insights into the Diversity of the Autographiviridae Family.

In this study, we isolated and characterized three novel virulent Autographiviridae bacteriophages, vB_AspA_Bolek, vB_AspA_Lolek, and vB_AspA_Tola, which infect different Aeromonas strains. These three host-pathogen pairs were derived from the same sampling location-the arsenic-containing microbial mats of the Zloty Stok gold mine. Functional analysis showed they are psychrotolerant (4-25 °C), albeit with a much wider temperature range of propagation for the hosts (≤37 °C). Comparative genomic analyses revealed a high nucleotide and amino acid sequence similarity of vB_AspA_Bolek and vB_AspA_Lolek, with significant differences exclusively in the C-terminal region of their tail fibers, which might explain their host range discrimination. The protein-based phage network, together with a phylogenetic analysis of the marker proteins, allowed us to assign vB_AspA_Bolek and vB_AspA_Lolek to the Beijerinckvirinae and vB_AspA_Tola to the Colwellvirinae subfamilies, but as three novel species, due to their low nucleotide sequence coverage and identity with other known phage genomes. Global comparative analysis showed that the studied phages are also markedly different from most of the 24 Aeromonas autographiviruses known so far. Finally, this study provides in-depth insight into the diversity of the Autographiviridae phages and reveals genomic similarities between selected groups of this family as well as between autographiviruses and their relatives of other Caudoviricetes families.

Metagenomic Analysis of the Gastrointestinal Microbiota of Gadus morhua callarias L. Originating from a Chemical Munition Dump Site.

Several hundred thousand tonnes of munitions containing chemical warfare agents (CWAs) are lying on the seafloor worldwide. CWAs have started leaking from corroded munitions, and their presence in the environment and in organisms inhabiting dump sites has been detected. The presence of CWAs in the water negatively affects fish, macrobenthos and free-living bacteria. It can be expected that the presence of CWAs would also affect the gut-associated bacteria in fish, which are vital for their condition. The main aim of this study was to test if the microbiota of cod collected in the Baltic Bornholm Deep (highly polluted with CWAs) is dysregulated. To investigate this, we conducted metagenomic studies based on 16S rRNA gene sequencing. We found that the microbiota of cod inhabiting the dump site was significantly less taxonomically diverse compared to those from a non-polluted reference site. Moreover, taxa associated with fish diseases (e.g., Vibrionaceae, Aeromonadaceae) were more prevalent, and probiotic taxa (e.g., Actinobacteriota, Rhodobacteraceae) were less frequent in the guts of individuals from the dump site, than those from the reference site. The differences in vulnerability of various bacterial taxa inhabiting cod gastrointestinal tracts to CWAs were hypothesised to be responsible for the observed microbiota dysregulation.

Genome Study of a Novel Virulent Phage vB_SspS_KASIA and Mu-like Prophages of Shewanella sp. M16 Provides Insights into the Genetic Diversity of the Shewanella Virome.

Shewanella is a ubiquitous bacterial genus of aquatic ecosystems, and its bacteriophages are also isolated from aquatic environments (oceans, lakes, ice, and wastewater). In this study, the isolation and characterization of a novel virulent Shewanella phage vB_SspS_KASIA and the identification of three prophages of its host, Shewanella sp. M16, including a mitomycin-inducible Mu-like siphovirus, vB_SspS_MuM16-1, became the starting point for comparative analyses of phages infecting Shewanella spp. and the determination of their position among the known bacterial viruses. A similarity networking analysis revealed the high diversity of Shewanella phages in general, with vB_SspS_KASIA clustering exclusively with Colwellia phage 9A, with which it forms a single viral cluster composed of two separate viral subclusters. Furthermore, vB_SspS_MuM16-1 presented itself as being significantly different from the phages deposited in public databases, expanding the diversity of the known Mu-like phages and giving potential molecular markers for the identification of Mu-like prophages in bacterial genomes. Moreover, the functional analysis performed for vB_SspS_KASIA suggested that, despite the KASIA host, the M16 strain grows better in a rich medium and at 30 °C the phage replication cycle seems to be optimal in restrictive culture conditions mimicking their natural environment, the Zloty Stok gold and arsenic mine.

Identification, Characterization, and Genomic Analysis of Novel Serratia Temperate Phages from a Gold Mine.

Bacteria of the genus Serratia inhabit a variety of ecological niches like water, soil, and the bodies of animals, and have a wide range of lifestyles. Currently, the complete genome sequences of 25 Serratia phages are available in the NCBI database. All of them were isolated from nutrient-rich environments like sewage, with the use of clinical Serratia strains as hosts. In this study, we identified a novel Serratia myovirus named vB_SspM_BZS1. Both the phage and its host Serratia sp. OS31 were isolated from the same oligotrophic environment, namely, an abandoned gold mine (Zloty Stok, Poland). The BZS1 phage was thoroughly characterized here in terms of its genomics, morphology, and infection kinetics. We also demonstrated that Serratia sp. OS31 was lysogenized by mitomycin-inducible siphovirus vB_SspS_OS31. Comparative analyses revealed that vB_SspM_BZS1 and vB_SspS_OS31 were remote from the known Serratia phages. Moreover, vB_SspM_BZS1 was only distantly related to other viruses. However, we discovered similar prophage sequences in genomes of various bacteria here. Additionally, a protein-based similarity network showed a high diversity of Serratia phages in general, as they were scattered across nineteen different clusters. In summary, this work broadened our knowledge on the diverse relationships of Serratia phages.

Identification and Characterization of the First Virulent Phages, Including a Novel Jumbo Virus, Infecting Ochrobactrum spp.

The Ochrobactrum genus consists of an extensive repertoire of biotechnologically valuable bacterial strains but also opportunistic pathogens. In our previous study, a novel strain, Ochrobactrum sp. POC9, which enhances biogas production in wastewater treatment plants (WWTPs) was identified and thoroughly characterized. Despite an insightful analysis of that bacterium, its susceptibility to bacteriophages present in WWTPs has not been evaluated. Using raw sewage sample from WWTP and applying the enrichment method, two virulent phages, vB_OspM_OC and vB_OspP_OH, which infect the POC9 strain, were isolated. These are the first virulent phages infecting Ochrobactrum spp. identified so far. Both phages were subjected to thorough functional and genomic analyses, which allowed classification of the vB_OspM_OC virus as a novel jumbo phage, with a genome size of over 227 kb. This phage encodes DNA methyltransferase, which mimics the specificity of cell cycle regulated CcrM methylase, a component of the epigenetic regulatory circuits in Alphaproteobacteria. In this study, an analysis of the overall diversity of Ochrobactrum-specific (pro)phages retrieved from databases and extracted in silico from bacterial genomes was also performed. Complex genome mining allowed us to build similarity networks to compare 281 Ochrobactrum-specific viruses. Analyses of the obtained networks revealed a high diversity of Ochrobactrum phages and their dissimilarity to the viruses infecting other bacteria.

Characterization of the virome of Paracoccus spp. (Alphaproteobacteria) by combined in silico and in vivo approaches.

Bacteria of the genus Paracoccus inhabit various pristine and anthropologically-shaped environments. Many Paracoccus spp. have biotechnological value and several are opportunistic human pathogens. Despite extensive knowledge of their metabolic potential and genome architecture, little is known about viruses of Paracoccus spp. So far, only three active phages infecting these bacteria have been identified. In this study, 16 Paracoccus strains were screened for the presence of active temperate phages, which resulted in the identification of five novel viruses. Mitomycin C-induced prophages were isolated, visualized and their genomes sequenced and thoroughly analyzed, including functional validation of their toxin-antitoxin systems. This led to the identification of the first active Myoviridae phage in Paracoccus spp. and four novel Siphoviridae phages. In addition, another 53 prophages were distinguished in silico within genomic sequences of Paracoccus spp. available in public databases. Thus, the Paracoccus virome was defined as being composed of 66 (pro)phages. Comparative analyses revealed the diversity and mosaicism of the (pro)phage genomes. Moreover, similarity networking analysis highlighted the uniqueness of Paracoccus (pro)phages among known bacterial viruses.

Genomic Analysis of Shewanella sp. O23S-The Natural Host of the pSheB Plasmid Carrying Genes for Arsenic Resistance and Dissimilatory Reduction.

Shewanella sp. O23S is a dissimilatory arsenate reducing bacterial strain involved in arsenic transformations within the abandoned gold mine in Zloty Stok (SW Poland). Previous physiological studies revealed that O23S may not only release arsenic from minerals, but also facilitate its immobilization through co-precipitation with reduced sulfur species. Given these uncommon, complementary characteristics and the application potential of the strain in arsenic-removal technologies, its genome (~5.3 Mbp), consisting of a single chromosome, two large plasmids (pSheA and pSheB) and three small plasmid-like phages (pSheC-E) was sequenced and annotated. Genes encoding putative proteins involved in heavy metal transformations, antibiotic resistance and other phenotypic traits were identified. An in-depth comparative analysis of arsenic respiration (arr) and resistance (ars) genes and their genetic context was also performed, revealing that pSheB carries the only copy of the arr genes, and a complete ars operon. The plasmid pSheB is therefore a unique natural vector of these genes, providing the host cells arsenic respiration and resistance abilities. The functionality of the identified genes was determined based on the results of the previous and additional physiological studies, including: the assessment of heavy metal and antibiotic resistance under various conditions, adhesion-biofilm formation assay and BiologTM metabolic preferences test. This combined genetic and physiological approach shed a new light on the capabilities of O23S and their molecular basis, and helped to confirm the biosafety of the strain in relation to its application in bioremediation technologies.

Characterization of Sinorhizobium sp. LM21 Prophages and Virus-Encoded DNA Methyltransferases in the Light of Comparative Genomic Analyses of the Sinorhizobial Virome.

The genus Sinorhizobium/Ensifer mostly groups nitrogen-fixing bacteria that create root or stem nodules on leguminous plants and transform atmospheric nitrogen into ammonia, which improves the productivity of the plants. Although these biotechnologically-important bacteria are commonly found in various soil environments, little is known about their phages. In this study, the genome of Sinorhizobium sp. LM21 isolated from a heavy-metal-contaminated copper mine in Poland was investigated for the presence of prophages and DNA methyltransferase-encoding genes. In addition to the previously identified temperate phage, ΦLM21, and the phage-plasmid, pLM21S1, the analysis revealed the presence of three prophage regions. Moreover, four novel phage-encoded DNA methyltransferase (MTase) genes were identified and the enzymes were characterized. It was shown that two of the identified viral MTases methylated the same target sequence (GANTC) as cell cycle-regulated methyltransferase (CcrM) of the bacterial host strain, LM21. This discovery was recognized as an example of the evolutionary convergence between enzymes of sinorhizobial viruses and their host, which may play an important role in virus cycle. In the last part of the study, thorough comparative analyses of 31 sinorhizobial (pro)phages (including active sinorhizobial phages and novel putative prophages retrieved and manually re-annotated from Sinorhizobium spp. genomes) were performed. The networking analysis revealed the presence of highly conserved proteins (e.g., holins and endolysins) and a high diversity of viral integrases. The analysis also revealed a large number of viral DNA MTases, whose genes were frequently located within the predicted replication modules of analyzed prophages, which may suggest their important regulatory role. Summarizing, complex analysis of the phage protein similarity network enabled a new insight into overall sinorhizobial virome diversity.

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.

Molecular characterization of the pSinB plasmid of the arsenite oxidizing, metallotolerant Sinorhizobium sp. M14 - insight into the heavy metal resistome of sinorhizobial extrachromosomal replicons.

Sinorhizobium sp. M14 is an As(III)-oxidizing, psychrotolerant strain, capable of growth in the presence of extremely high concentrations of arsenic and many other heavy metals. Metallotolerant abilities of the M14 strain depend upon the presence of two extrachromosomal replicons: pSinA (∼ 109 kb) and pSinB (∼ 300 kb). The latter was subjected to complex analysis. The performed analysis demonstrated that the plasmid pSinB is a narrow-host-range repABC-type replicon, which is fully stabilized by the phd-vapC-like toxin-antitoxin stabilizing system. In silico analysis showed that among the phenotypic gene clusters of the plasmid pSinB, eight modules are potentially involved in heavy metals resistance (HMR). These modules carry genes encoding efflux pumps, permeases, transporters and copper oxidases, which provide resistance to arsenic, cadmium, cobalt, copper, iron, mercury, nickel, silver and zinc. The functional analysis revealed that the HMR modules are active and have an effect on the minimal inhibitory concentration (MIC) values observed for the heterological host cells. The phenotype was manifested by an increase or decrease of the MICs of heavy metals and it was strain specific. The analysis of distribution of the heavy metal resistance genes, i.e. resistome, in Sinorhizobium spp. plasmids, revealed that the HMR modules are common in these replicons.

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.

Two novel temperate bacteriophages co-existing in Aeromonas sp. ARM81 - characterization of their genomes, proteomes and DNA methyltransferases.

Aeromonas species are causative agents of a wide spectrum of diseases in animals and humans. Although these bacteria are commonly found in various environments, little is known about their phages. Thus far, only one temperate Aeromonas phage has been characterized. Whole-genome sequencing of an Aeromonas sp. strain ARM81 revealed the presence of two prophage clusters. One of them is integrated into the chromosome and the other was maintained as an extrachromosomal, linear plasmid-like prophage encoding a protelomerase. Both prophages were artificially and spontaneously inducible. We separately isolated both phages and compared their genomes with other known viruses. The novel phages show no similarity to the previously characterized Aeromonas phages and might represent new evolutionary lineages of viruses infecting Aeromonadaceae. Apart from the comparative genomic analyses of these phages, complemented with their structural and molecular characterization, a functional analysis of four DNA methyltransferases encoded by these viruses was conducted. One of the investigated N6-adenine-modifying enzymes shares sequence specificity with a Dam-like methyltransferase of its bacterial host, while another one is non-specific, as it catalyzes adenine methylation in various sequence contexts. The presented results shed new light on the diversity of Aeromonas temperate phages.

Construction of the recombinant broad-host-range plasmids providing their bacterial hosts arsenic resistance and arsenite oxidation ability.

The plasmid pSinA of Sinorhizobium sp. M14 was used as a source of functional phenotypic modules, encoding proteins involved in arsenite oxidation and arsenic resistance, to obtain recombinant broad-host-range plasmids providing their bacterial hosts arsenic resistance and arsenite oxidative ability. An arsenite oxidation module was cloned into pBBR1MCS-2 vector yielding plasmid vector pAIO1, while an arsenic resistance module was cloned into pCM62 vector yielding plasmid pARS1. Both plasmid constructs were introduced (separately and together) into the cells of phylogenetically distant (representing Alpha-, Beta-, and Gammaproteobacteria) and physiologically diversified (unable to oxidize arsenite and susceptible/resistant to arsenite and arsenate) bacteria. Functional analysis of the modified strains showed that: (i) the plasmid pARS1 can be used for the construction of strains with an increased resistance to arsenite [up to 20mM of As(III), (ii) the presence of the plasmid pAIO1 in bacteria previously unable to oxidize As(III) to As(V), contributes to the acquisition of arsenite oxidation abilities by these cells, (iii) the highest arsenite utilization rate are observed in the culture of strains harbouring both the plasmids pAIO1 and pARS1, (iv) the strains harbouring the plasmid pAIO1 were able to grow on arsenic-contaminated mine waters (∼ 3.0 mg As L(-1)) without any supplementation.

Molecular characterization of a novel temperate sinorhizobium bacteriophage, ФLM21, encoding DNA methyltransferase with CcrM-like specificity.

UNLABELLED: ΦLM21 is a temperate phage isolated from Sinorhizobium sp. strain LM21 (Alphaproteobacteria). Genomic analysis and electron microscopy suggested that ΦLM21 is a member of the family Siphoviridae. The phage has an isometric head and a long noncontractile tail. The genome of ΦLM21 has 50,827 bp of linear double-stranded DNA encoding 72 putative proteins, including proteins responsible for the assembly of the phage particles, DNA packaging, transcription, replication, and lysis. Virion proteins were characterized using mass spectrometry, leading to the identification of the major capsid and tail components, tape measure, and a putative portal protein. We have confirmed the activity of two gene products, a lytic enzyme (a putative chitinase) and a DNA methyltransferase, sharing sequence specificity with the cell cycle-regulating methyltransferase (CcrM) of the bacterial host. Interestingly, the genome of Sinorhizobium phage ΦLM21 shows very limited similarity to other known phage genome sequences and is thus considered unique. IMPORTANCE: Prophages are known to play an important role in the genomic diversification of bacteria via horizontal gene transfer. The influence of prophages on pathogenic bacteria is very well documented. However, our knowledge of the overall impact of prophages on the survival of their lysogenic, nonpathogenic bacterial hosts is still limited. In particular, information on prophages of the agronomically important Sinorhizobium species is scarce. In this study, we describe the isolation and molecular characterization of a novel temperate bacteriophage, ΦLM21, of Sinorhizobium sp. LM21. Since we have not found any similar sequences, we propose that this bacteriophage is a novel species. We conducted a functional analysis of selected proteins. We have demonstrated that the phage DNA methyltransferase has the same sequence specificity as the cell cycle-regulating methyltransferase CcrM of its host. We point out that this phenomenon of mimicking the host regulatory mechanisms by viruses is quite common in bacteriophages.

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.

Architecture and functions of a multipartite genome of the methylotrophic bacterium Paracoccus aminophilus JCM 7686, containing primary and secondary chromids.

BACKGROUND: Paracoccus aminophilus JCM 7686 is a methylotrophic α-Proteobacterium capable of utilizing reduced one-carbon compounds as sole carbon and energy source for growth, including toxic N,N-dimethylformamide, formamide, methanol, and methylamines, which are widely used in the industry. P. aminophilus JCM 7686, as many other Paracoccus spp., possesses a genome representing a multipartite structure, in which the genomic information is split between various replicons, including chromids, essential plasmid-like replicons, with properties of both chromosomes and plasmids. In this study, whole-genome sequencing and functional genomics approaches were applied to investigate P. aminophilus genome information. RESULTS: The P. aminophilus JCM 7686 genome has a multipartite structure, composed of a single circular chromosome and eight additional replicons ranging in size between 5.6 and 438.1 kb. Functional analyses revealed that two of the replicons, pAMI5 and pAMI6, are essential for host viability, therefore they should be considered as chromids. Both replicons carry housekeeping genes, e.g. responsible for de novo NAD biosynthesis and ammonium transport. Other mobile genetic elements have also been identified, including 20 insertion sequences, 4 transposons and 10 prophage regions, one of which represents a novel, functional serine recombinase-encoding bacteriophage, ϕPam-6. Moreover, in silico analyses allowed us to predict the transcription regulatory network of the JCM 7686 strain, as well as components of the stress response, recombination, repair and methylation machineries. Finally, comparative genomic analyses revealed that P. aminophilus JCM 7686 has a relatively distant relationship to other representatives of the genus Paracoccus. CONCLUSIONS: P. aminophilus genome exploration provided insights into the overall structure and functions of the genome, with a special focus on the chromids. Based on the obtained results we propose the classification of bacterial chromids into two types: "primary" chromids, which are indispensable for host viability and "secondary" chromids, which are essential, but only under some environmental conditions and which were probably formed quite recently in the course of evolution. Detailed genome investigation and its functional analysis, makes P. aminophilus JCM 7686 a suitable reference strain for the genus Paracoccus. Moreover, this study has increased knowledge on overall genome structure and composition of members within the class Alphaproteobacteria.

Novel non-specific DNA adenine methyltransferases.

The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N(6)-(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 N(6)-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N(6)-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.