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Introduction: Helicobacter pylori is a bacterium that colonizes the gastric epithelium, which affects millions of people worldwide. H. pylori infection can lead to various gastrointestinal diseases, including gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Conventional antibiotic therapies face challenges due to increasing antibiotic resistance and patient non-compliance, necessitating the exploration of alternative treatment approaches. In this study, we focused on Hp0231 (DsbK), an essential component of the H. pylori Dsb (disulfide bond) oxidative pathway, and investigated peptide-based inhibition as a potential therapeutic strategy. Methods: Three inhibitory peptides designed by computational modeling were evaluated for their effectiveness using a time-resolved fluorescence assay. We also examined the binding affinity between Hp0231 and the peptides using microscale thermophoresis. Results and discussion: Our findings demonstrate that in silico -designed synthetic peptides can effectively inhibit Hp0231-mediated peptide oxidation. Targeting Hp0231 oxidase activity could attenuate H. pylori virulence without compromising bacterial viability. Therefore, peptide-based inhibitors of Hp0231 could be candidates for the development of new targeted strategy, which does not influence the composition of the natural human microbiome, but deprive the bacterium of its pathogenic properties., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MKD declared a shared parent affiliation with the authors MP and WB to the handling editor at the time of review., (Copyright © 2024 Roszczenko-Jasińska, Giełdoń, Mazur, Spodzieja, Plichta, Czaplewski, Bal, Jagusztyn-Krynicka and Bartosik.)

The cold-adapted Psychrobacter sp. strain DAB_AL62B, isolated from ornithogenic deposits on the Arctic island of Spitsbergen, harbors a 34.5 kb plasmid, pP62BP1, which carries a genetic SLF module predicted to enable the host bacterium to metabolize alkyl sulfates including sodium dodecyl sulfate (SDS), a common anionic surfactant. In this work, we experimentally confirmed that the pP62BP1-harboring strain is capable of SDS degradation. The slfCHSL genes were shown to form an operon whose main promoter, P slfC , is negatively regulated by the product of the slfR gene in the absence of potential substrates. We showed that lauryl aldehyde acts as an inducer of the operon. The analysis of the draft genome sequence of the DAB_AL62B strain revealed that the crucial enzyme of the SDS degradation pathway-an alkyl sulfatase-is encoded only within the plasmid. The SLF module is flanked by two restriction-modification systems, which were shown to exhibit the same sequence specificity. We hypothesize that the maintenance of pP62BP1 may be dependent on this unique genetic organization.

The overuse of antibiotics has led to a rise in infections caused by multidrug-resistant bacteria, resulting in a need for new antibacterial compounds with different modes of action. In this paper, we describe a new class of compounds called lipooligoureas, which are foldamer-based mimetics of antimicrobial lipopeptides. The lipooligoureas consist of an acyl chain connected to the N-terminus of an oligourea head group that exhibits a well-defined 2.5-helix secondary structure, which is further stabilized by the attachment of the lipophilic chain to the oligourea moiety. These compounds meet the established criteria for membranolytic compounds by possessing an amphiphilic structure that promotes the internalization and partitioning of the molecules into the lipid membrane. The presence of positively charged urea residues promotes electrostatic interactions with the negatively charged bacterial membrane. The subtle structural differences in oligourea head group influence the compounds' aggregation behavior, with the number and position of positively charged urea residues correlating with their aggregation ability. The biological activity of these compounds in inhibiting bacterial growth is correlated with their ability to aggregate, with stronger antibacterial properties exhibited by those that aggregate more easily. However, the concentration inhibiting bacterial growth is significantly lower than the critical aggregation concentration values, suggesting that the mechanism of action involves the monomeric forms of lipooligoureas. Nonetheless, a mechanism based on membrane-induced aggregation cannot be ruled out. The lipooligoureas exhibit higher activity towards Gram-positive bacteria than against Gram-negative bacteria, which is indicative of certain selectivity of these compounds. It is also demonstrated that lipooligoureas exhibit increased stability against proteolytic degradation in human blood serum., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Slawomir Sek has patent Lipooligomoczniki, kompozycja farmaceutyczna, oraz lipooligomoczniki do zastosowania jako lek pending to University of Warsaw., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The rapidly emerging phenomenon of antibiotic resistance threatens to substantially reduce the efficacy of available antibacterial therapies. Dissemination of resistance, even between phylogenetically distant bacterial species, is mediated mainly by mobile genetic elements, considered to be natural vectors of horizontal gene transfer. Transposable elements (TEs) play a major role in this process-due to their highly recombinogenic nature they can mobilize adjacent genes and can introduce them into the pool of mobile DNA. Studies investigating this phenomenon usually focus on the genetic load of transposons and the molecular basis of their mobility. However, genes introduced into evolutionarily distant hosts are not necessarily expressed. As a result, bacterial genomes contain a reservoir of transcriptionally silent genetic information that can be activated by various transposon-related recombination events. The TEs themselves along with processes associated with their transposition can introduce promoters into random genomic locations. Thus, similarly to integrons, they have the potential to convert dormant genes into fully functional antibiotic resistance determinants. In this review, we describe the genetic basis of such events and by extension the mechanisms promoting the emergence of new drug-resistant bacterial strains.

Partitioning systems ensure the stable inheritance of bacterial low-copy-number replicons, such as chromosomes, chromids, and megaplasmids. These loci consist of two genes encoding partition proteins A and B, and at least one parS centromere-like sequence. In chromids and megaplasmids, partitioning systems are often located in the vicinity of replication systems. An extreme example of this co-localization are alphaproteobacterial repABC replicons, where the partition ( repAB ) and replication ( repC ) genes form a single operon, with parS sequences usually positioned in close proximity to these genes. In this study, we characterized a more complex repABC system found in Paracoccus aminophilus ( Rhodobacterales ) megaplasmid pAMI4 (438 kb). Besides the repABC operon with a single parS site, this replicon has a 2-kb non-coding locus positioned 11.5 kb downstream of repC , which contains three additional parS repeats ( 3parS ). We demonstrated that 3parS is bound by partition protein B in vitro and is essential for proper pAMI4 partitioning in vivo . In search of similar loci, we conducted a comparative analysis of parS distribution in other repABC replicons. This revealed different patterns of parS localization in Rhodobacterales and Rhizobiales . However, in both these taxonomic orders, parS sites are almost always located inside or close to the repABC operon. No other 3parS -like loci were found in the closest relatives of pAMI4. Another evolutionarily-independent example of such a locus was identified as a conserved feature in chromosome 2 of Allorhizobium vitis and related replicons. IMPORTANCE The repABC replication/partitioning loci are widespread in extrachromosomal replicons of Alphaproteobacteria . They are evolutionarily diverse, subject to multi-layer self-regulation, and are responsible for the maintenance of different types of replicons, such as plasmids (e.g., Agrobacterium pTi and pRi tumorigenic and rhizogenic plasmids), megaplasmids (e.g., Sinorhizobium pSymA and pSymB) and essential chromids (e.g., secondary chromosomes of Agrobacterium , Brucella and Rhodobacter ). In this study, we functionally analyzed an atypical partition-related component of repABC systems, the 3parS locus, found in the P. aminophilus megaplasmid pAMI4. We also identified parS centromere-like site distribution patterns in different groups of repABC replicons and found other unrelated 3parS -like loci, which had been overlooked. Our findings raise questions concerning the biological reasons for differential parS distribution, which may reflect variations in repABC operon regulation as well as different replication and partition modes of replicons belonging to the repABC family.

Bacteria of the genus Listeria (phylum Firmicutes ) include both human and animal pathogens, as well as saprophytic strains. A common component of Listeria spp. genomes are plasmids, i.e., extrachromosomal replicons that contribute to gene flux in bacteria. This study provides an in-depth insight into the structure, diversity and evolution of plasmids occurring in Listeria strains inhabiting various environments under different anthropogenic pressures. Apart from the components of the conserved plasmid backbone (providing replication, stable maintenance and conjugational transfer functions), these replicons contain numerous adaptive genes possibly involved in: (i) resistance to antibiotics, heavy metals, metalloids and sanitizers, and (ii) responses to heat, oxidative, acid and high salinity stressors. Their genomes are also enriched by numerous transposable elements, which have influenced the plasmid architecture. The plasmidome of Listeria is dominated by a group of related replicons encoding the RepA replication initiation protein. Detailed comparative analyses provide valuable data on the level of conservation of these replicons and their role in shaping the structure of the Listeria pangenome, as well as their relationship to plasmids of other genera of Firmicutes , which demonstrates the range and direction of flow of genetic information in this important group of bacteria.

Phenotypic and genotypic resistance to benzalkonium chloride (BC), cadmium and arsenic was tested (by susceptibility assays and molecular methods) in 287 Listeria monocytogenes strains isolated from fish and fish products, and food-producing factories in Poland. Overall, 40% of the isolates were resistant to BC, 56% to cadmium and 41% to arsenic (57% displayed resistance to more than one of the tested compounds). Among BC-resistant isolates, the most commonly detected resistance determinant was the qacH gene (83%). Three distinct types of cadA gene determining resistance to cadmium were detected, with the cadA1 variant predominant (88%), while most arsenic-resistant isolates (86%) harbored the arsA gene associated with a Tn554-like transposon (one strain harbored two copies of arsA in different arsenic resistance cassettes). 53% of all tested isolates contained plasmids (from 4 kb to > 90 kb in size), which were classified into 11 groups (p1-p11) based on their restriction patterns. Interestingly, 12 isolates harbored the small mobilizable pLMST6-like plasmid pLIS3 encoding multidrug efflux pump EmrC. Clustering analysis of PFGE patterns revealed that these isolates represent several diverse bacterial populations, which strongly suggests mobility of the pLMST6-like plasmids among L. monocytogenes strains and their role in dissemination of BC resistance., (Copyright © 2021. Published by Elsevier Ltd.)

The highly efficient bioelectrodes based on single layer graphene (SLG) functionalized with pyrene self-assembled monolayer and novel cytochromec 553 (cytc 553 )peptide linker variants were rationally designed to optimize the direct electron transfer (DET) between SLG and the heme group of cyt. Through a combination of photoelectrochemical and quantum mechanical (QM/MM) approaches we show that the specific amino acid sequence of a short peptide genetically inserted between the cytc 553 holoprotein and thesurface anchoring C-terminal His 6 -tag plays a crucial role in ensuring the optimal orientation and distance of the heme group with respect to the SLG surface. Consequently, efficient DET occurring between graphene and cyt c 553 leads to a 20-fold enhancement of the cathodic photocurrent output compared to the previously reported devices of a similar type. The QM/MM modeling implies that a perpendicular or parallel orientation of the heme group with respect to the SLG surface is detrimental to DET, whereas the tilted orientation favors the cathodic photocurrent generation. Our work confirms the possibility of fine-tuning the electronic communication within complex bio-organic nanoarchitectures and interfaces due to optimization of the tilt angle of the heme group, its distance from the SLG surface and optimal HOMO/LUMO levels of the interacting redox centers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The increasing resistance of bacteria to available antibiotics has stimulated the search for new antimicrobial compounds with less specific mechanisms of action. These include the ability to disrupt the structure of the cell membrane, which in turn leads to its damage. In this context, amphiphilic lipopeptides belong to the class of the compounds which may fulfill this requirement. In this paper, we describe two linear analogues of battacin with modified acyl chains to tune the balance between the hydrophilic and hydrophobic portion of lipopeptides. We demonstrate that both compounds display antimicrobial activity with the lowest values of minimum inhibitory concentrations found for Gram-positive pathogens. Therefore, their mechanism of action was evaluated on a molecular level using model lipid films mimicking the membrane of Gram-positive bacteria. The surface pressure measurements revealed that both lipopeptides show ability to bind and incorporate into the lipid monolayers, resulting in decreased ordering of lipids and membrane fluidization. Atomic force microscopy (AFM) imaging demonstrated that the exposure of the model bilayers to lipopeptides leads to a transition from the ordered gel phase to disordered liquid crystalline phase. This observation was confirmed by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) results, which revealed that lipopeptide action causes a substantial increase in the average tilt angle of lipid acyl chains with respect to the surface normal to compensate for lipopeptide insertion into the membrane. Moreover, the peptide moieties in both molecules do not adopt any well-defined secondary structure upon binding with the lipid membrane. It was also observed that a small difference in the structure of a lipophilic chain, altering the balance between hydrophobic and hydrophilic portion of the molecules, results in different insertion depth of the active compounds.

Short modified oligonucleotides that bind in a sequence-specific way to messenger RNA essential for bacterial growth could be useful to fight bacterial infections. One such promising oligonucleotide is peptide nucleic acid (PNA), a synthetic DNA analog with a peptide-like backbone. However, the limitation precluding the use of oligonucleotides, including PNA, is that bacteria do not import them from the environment. We have shown that vitamin B 12 , which most bacteria need to take up for growth, delivers PNAs to Escherichia coli cells when covalently linked with PNAs. Vitamin B 12 enters E. coli via a TonB-dependent transport system and is recognized by the outer-membrane vitamin B 12 -specific BtuB receptor. We engineered the E. coli ΔbtuB mutant and found that transport of the vitamin B 12 -PNA conjugate requires BtuB. Thus, the conjugate follows the same route through the outer membrane as taken by free vitamin B 12 . From enhanced sampling all-atom molecular dynamics simulations, we determined the mechanism of conjugate permeation through BtuB. BtuB is a β-barrel occluded by its luminal domain. The potential of mean force shows that conjugate passage is unidirectional and its movement into the BtuB β-barrel is energetically favorable upon luminal domain unfolding. Inside BtuB, PNA extends making its permeation mechanically feasible. BtuB extracellular loops are actively involved in transport through an induced-fit mechanism. We prove that the vitamin B 12 transport system can be hijacked to enable PNA delivery to E. coli cells., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Listeria monocytogenes is a pathogen responsible for severe cases of food poisoning. Listeria spp. strains occurring in soil and water environments may serve as a reservoir of resistance determinants for pathogenic L. monocytogenes strains. A large collection of Listeria spp. strains (155) isolated from natural, agricultural, and urban areas was screened for resistance to heavy metals and metalloids, and the presence of resistance determinants and extrachromosomal replicons. Of the tested strains, 35% were resistant to cadmium and 17% to arsenic. Sequence analysis of resistance plasmids isolated from strains of Listeria seeligeri and Listeria ivanovii , and the chromosome of L. seeligeri strain Sr73, identified a novel variant of the cadAC cadmium resistance efflux system, cadA6 , that was functional in L. monocytogenes cells. The cadA6 cassette was detected in four Listeria species, including strains of L. monocytogenes , isolated from various countries and sources-environmental, food-associated, and clinical samples. This resistance cassette is harbored by four novel composite or non-composite transposons, which increases its potential for horizontal transmission. Since some cadAC cassettes may influence virulence and biofilm formation, it is important to monitor their presence in Listeria spp. strains inhabiting different environments.

We have designed and synthesized new short lipopeptides composed of tetrapeptide conjugated to fatty acids with different chain lengths. The amino acid sequence of the peptide moiety included d-phenylalanine, two residues of l-2,4-diaminobutyric acid and l-leucine. To explore the possible mechanism of lipopeptide action, we have provided a physicochemical characterization of their interactions with artificial lipid membranes. For this purpose, we have used monolayers and bilayers composed of lipids representative of Gram-negative and Gram-positive bacterial membranes. Using surface pressure measurements and atomic force microscopy, we were able to monitor the changes occurring within the films upon exposure to lipopeptides. Our experiments revealed that all lipopeptides can penetrate the lipid membranes and affect their molecular ordering. The latter results in membrane thinning and fluidization. However, the effect is stronger in the lipid films mimicking Gram-positive bacterial membranes. The results of the physicochemical characterization were compared with the biological activity of lipopeptides. The effect of lipopeptides on bacterial growth was tested on several strains of bacteria. It was revealed that lipopeptides show stronger antimicrobial activity against Gram-positive bacteria. At the same time, all tested compounds display relatively low hemolytic activity.

The identification of novel targets for antimicrobial agents is crucial for combating infectious diseases caused by evolving bacterial pathogens. Components of bacterial toxin-antitoxin (TA) systems have been recognized as promising therapeutic targets. These widespread genetic modules are usually composed of two genes that encode a toxic protein targeting an essential cellular process and an antitoxin that counteracts the activity of the toxin. Uncontrolled toxin expression may elicit a bactericidal effect, so they may be considered "intracellular molecular bombs" that can lead to elimination of their host cells. Based on the molecular nature of antitoxins and their mode of interaction with toxins, TA systems have been classified into six groups. The most prevalent are type II TA systems. Due to their ubiquity among clinical isolates of pathogenic bacteria and the essential processes targeted, they are promising candidates for the development of novel antimicrobial strategies. In this review, we describe the distribution of type II TA systems in clinically relevant human pathogens, examine how these systems could be developed as the targets for novel antibacterials, and discuss possible undesirable effects of such therapeutic intervention, such as the induction of persister cells, biofilm formation and toxicity to eukaryotic cells.

Background: Carotenoids are natural tetraterpene pigments widely utilized in the food, pharmaceutical and cosmetic industries. Currently, chemical synthesis of these compounds outperforms their production in Escherichia coli or yeast due to the limited efficiency of the latter. The use of natural microbial carotenoid producers, such as bacteria of the genus Paracoccus (Alphaproteobacteria), may help to optimize this process. In order to couple the ability to synthesize these pigments with the metabolic versatility of this genus, we explored the possibility of introducing carotenoid synthesis genes into strains capable of efficient growth on simple low-cost media., Results: We constructed two carotenoid-producing strains of Paracoccus carrying a new plasmid, pCRT01, which contains the carotenoid synthesis gene locus crt from Paracoccus marcusii OS22. The plasmid was created in vivo via illegitimate recombination between crt-carrying vector pABW1 and a natural "paracoccal" plasmid pAMI2. Consequently, the obtained fusion replicon is stably maintained in the bacterial population without the need for antibiotic selection. The introduction of pCRT01 into fast-growing "colorless" strains of Paracoccus aminophilus and Paracoccus kondratievae converted them into efficient producers of a range of both carotenes and xanthophylls. The exact profile of the produced pigments was dependent on the strain genetic background. To reduce the cost of carotenoid production in this system, we tested the growth and pigment synthesis efficiency of the two strains on various simple media, including raw industrial effluent (coal-fired power plant flue gas desulfurization wastewater) supplemented with molasses, an industrial by-product rich in sucrose., Conclusions: We demonstrated a new approach for the construction of carotenoid-producing bacterial strains which relies on a single plasmid-mediated transfer of a pigment synthesis gene locus between Paracoccus strains. This strategy facilitates screening for producer strains in terms of synthesis efficiency, pigment profile and ability to grow on low-cost industrial waste-based media, which should increase the cost-effectiveness of microbial production of carotenoids.

Background: Gene overlapping is a frequent phenomenon in microbial genomes. Excluding so-called "trivial overlapping", there are significant implications of such genetic arrangements, including regulation of gene expression and modification of protein activity. It is also postulated that, besides gene duplication, the appearance of overlapping genes (OGs) is one of the most important factors promoting a genome's novelty and evolution. OGs coding for in-frame proteins with different functions are a particularly interesting case. In this study we identified and characterized two in-frame proteins encoded by OGs on plasmid pIGRK from Klebsiella pneumoniae, a representative of the newly distinguished pHW126 plasmid family., Results: A single repR locus located within the replication system of plasmid pIGRK encodes, in the same frame, two functional polypeptides: a full-length RepR protein and a RepR' protein (with N-terminal truncation) translated from an internal START codon. Both proteins form homodimers, and interact with diverse DNA regions within the plasmid replication origin and repR promoter operator. Interestingly, RepR and RepR' have opposing functions - RepR is crucial for initiation of pIGRK replication, while RepR' is a negative regulator of this process. Nevertheless, both proteins act cooperatively as negative transcriptional regulators of their own expression., Conclusions: Regulation of the initiation of pIGRK replication is a complex process in which a major role is played by two in-frame proteins with antagonistic functions. In-frame encoded Rep proteins are uncommon, having been described in only a few plasmids. This is the first description of such proteins in a plasmid of the pHW126 family.

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.

Psychrobacter sp. DAB_AL32B, originating from Spitsbergen island (Arctic), carries the large plasmid pP32BP2 (54,438 bp). Analysis of the pP32BP2 nucleotide sequence revealed the presence of three predicted phenotypic modules that comprise nearly 30% of the plasmid genome. These modules appear to be involved in fimbriae synthesis via the chaperone-usher pathway (FIM module) and the aerobic and anaerobic metabolism of carnitine (CAR and CAI modules, respectively). The FIM module was found to be functional in diverse hosts since it facilitated the attachment of bacterial cells to abiotic surfaces, enhancing biofilm formation. The CAI module did not show measurable activity in any of the tested strains. Interestingly, the CAR module enabled the enzymatic breakdown of carnitine, but this led to the formation of the toxic by-product trimethylamine, which inhibited bacterial growth. Thus, on the one hand, pP32BP2 can enhance biofilm formation, a highly advantageous feature in cold environments, while on the other, it may prevent bacterial growth under certain environmental conditions. The detrimental effect of harboring pP32BP2 (and its CAR module) seems to be conditional, since this replicon may also confer the ability to use carnitine as an alternative carbon source, although a pathway to utilize trimethylamine is most probably necessary to make this beneficial. Therefore, the phenotype determined by this CAR-containing plasmid depends on the metabolic background of the host strain.

The sixty-seven nonpathogenic Listeria spp. strains isolated from food and food processing environments in Poland were examined for the presence of benzalkonium chloride (BC) resistance cassette (bcrABC) and four different variants of cadmium resistance determinants (cadA1-cadA4). All the strains were phenotypically resistant to cadmium and 22 among them were also resistant to BC. PCR-based analysis revealed that bcrABC cassette was harbored by 95.5% of the strains phenotypically resistant to BC. All of them harbored also either cadA1 or cadA2 genes (none carried cadA3 or cadA4), which corresponded to the presence of plasmids with two restriction patterns. The strains resistant to cadmium but susceptible to BC harbored only the cadA1 gene variant. DNA-DNA hybridization analysis showed that all the identified bcrABC, cadA1 and cadA2 genes were located within plasmids, classified into 11 groups of RFLP profiles. Only one of the plasmids - pLIS1 of Listeria welshimeri (carrying bcrABC and cadA2) - was capable of efficient conjugal transfer from nonpathogenic Listeria isolates to a pathogenic Listeria monocytogenes strain. Analysis of the complete nucleotide sequence of pLIS1 (the first sequenced plasmid of L. welshimeri species) revealed the presence of genes involved in plasmid replication, stabilization and transfer as well as genes conferring resistance phenotypes. Comparative analysis showed that pLIS1 genome is highly similar to a group of plasmids originating from L. monocytogenes strains. A common feature of pLIS1 and its relatives, besides the presence of the resistance genes, is the presence of numerous transposable elements (TEs). The analysis revealed the important role of TEs in both promoting genetic rearrangements within Listeria spp. plasmids and the acquisition of resistance determinants., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Paracoccus denitrificans Pd 1222 is a model methylotrophic bacterium. Its methylotrophy is based on autotrophic growth (enabled by the Calvin cycle) supported by energy from the oxidation of methanol or methylamine. The growing availability of genome sequence data has made it possible to investigate methylotrophy in other Paracoccus species . The examination of a large number of Paracoccus spp. genomes reveals great variability in C1 metabolism, which have been shaped by different evolutionary mechanisms. Surprisingly, the methylotrophy schemes of many Paracoccus strains appear to have quite different genetic and biochemical bases. Besides the expected 'autotrophic methylotrophs', many strains of this genus possess another C1 assimilatory pathway, the serine cycle, which seems to have at least three independent origins. Analysis of the co-occurrence of different methylotrophic pathways indicates, on the one hand, evolutionary linkage between the Calvin cycle and the serine cycle, and, on the other hand, that genes encoding some C1 substrate-oxidizing enzymes occur more frequently in association with one or the other. This suggests that some genetic module combinations form more harmonious enzymatic sets, which act with greater efficiency in the methylotrophic process and thus undergo positive selection.

The search for new, non-standard targets is currently a high priority in the design of new antibacterial compounds. Bacterial toxin-antitoxin systems (TAs) are genetic modules that encode a toxin protein that causes growth arrest by interfering with essential cellular processes, and a cognate antitoxin, which neutralizes the toxin activity. TAs have no human analogs, are highly abundant in bacterial genomes, and therefore represent attractive alternative targets for antimicrobial drugs. This study demonstrates how artificial activation of Escherichia coli mazEF and hipBA toxin-antitoxin systems using sequence-specific antisense peptide nucleic acid oligomers is an innovative antibacterial strategy. The growth arrest observed in E. coli resulted from the inhibition of translation of the antitoxins by the antisense oligomers. Furthermore, two other targets, related to the activities of mazEF and hipBA , were identified as promising sites of action for antibacterials. These results show that TAs are susceptible to sequence-specific antisense agents and provide a proof-of-concept for their further exploitation in antimicrobial strategies.

Bacteria of the genus Paracoccus are common components of the microbiomes of many naturally- and anthropogenically shaped environments. One species, Paracoccus yeei , is unique within the genus because it is associated with opportunistic human infections. Therefore, strains of P. yeei may serve as an interesting model to study the transition from a saprophytic to a pathogenic lifestyle in environmental bacteria. Unfortunately, knowledge concerning the biology, genetics and genomic content of P. yeei is fragmentary; also the mechanisms of pathogenicity of this bacterium remain unclear. In this study we provide the first insight into the genome composition and metabolic potential of a clinical isolate, P. yeei CCUG 32053. This strain has a multipartite genome (4,632,079 bp) composed of a circular chromosome plus eight extrachromosomal replicons pYEE1-8: 3 chromids and 5 plasmids, with a total size of 1,247,173 bp. The genome has been significantly shaped by the acquisition of genomic islands, prophages ( Myoviridae and Siphoviridae phage families) and numerous insertion sequences (ISs) representing seven IS families. Detailed comparative analysis with other complete genomic sequences of Paracoccus spp. (including P. yeei FDAARGOS_252 and TT13, as well as non-pathogenic strains of other species in this genus) enabled us to identify P. yeei species-specific genes and to predict putative determinants of virulence. This is the first attempt to identify pathoadaptive genetic information of P. yeei and to estimate the role of the mobilome in the evolution of pathogenicity in this species.

Cold-active bacteria of the genus Polaromonas (class Betaproteobacteria ) are important components of glacial microbiomes. In this study, extrachromosomal replicons of 26 psychrotolerant Polaromonas strains, isolated from Arctic and Antarctic glaciers, were identified, sequenced, and characterized. The plasmidome of these strains consists of 13 replicons, ranging in size from 3,378 to 101,077 bp. In silico sequence analyses identified the conserved backbones of these plasmids, composed of genes required for plasmid replication, stable maintenance, and conjugal transfer. Host range analysis revealed that all of the identified plasmids are narrow-host-range replicons, only able to replicate in bacteria of closely related genera ( Polaromonas and Variovorax ) of the Comamonadaceae family. Special attention was paid to the identification of plasmid auxiliary genetic information, which may contribute to the adaptation of bacteria to environmental conditions occurring in glaciers. Detailed analysis revealed the presence of genes encoding proteins potentially involved in (i) protection against reactive oxygen species, ultraviolet radiation, and low temperatures; (ii) transport and metabolism of organic compounds; (iii) transport of metal ions; and (iv) resistance to heavy metals. Some of the plasmids also carry genes required for the molecular assembly of iron-sulfur [Fe-S] clusters. Functional analysis of the predicted heavy metal resistance determinants demonstrated that their activity varies, depending on the host strain. This study provides the first molecular insight into the mobile DNA of Polaromonas spp. inhabiting polar glaciers. It has generated valuable data on the structure and properties of a pool of plasmids and highlighted their role in the biology of psychrotolerant Polaromonas strains and their adaptation to the environmental conditions of Arctic and Antarctic glaciers.

Psychrobacter sp. DAB_AL43B, isolated from ornithogenic soil collected on the Arctic island of Spitsbergen, is a newly sequenced psychrophilic strain susceptible to conjugation and electrotransformation. Its genome consists of a circular chromosome (3.3 Mb) and four plasmids (4.4-6.4kb). In silico genome mining and microarray-based phenotypic analysis were performed to describe the metabolic potential of this strain and identify possible biotechnological applications. Metabolic reconstruction indicated that DAB_AL43B prefers low-molecular-weight carboxylates and amino acids as carbon and energy sources. Genetic determinants of heavy-metal resistance, anthracene degradation and possible aerobic denitrification were also identified. Comparative analyses revealed a relatively close relationship between DAB_AL43B and other sequenced Psychrobacter species. In addition, the plasmids of this strain were used as the basis for the construction of Escherichia coli-Psychrobacter spp. shuttle vectors. Taken together, the results of this work suggest that DAB_AL43B is a promising candidate as a new model strain for studies on Psychrobacter spp., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Horizontal gene transfer (HGT) contributes greatly to the plasticity and evolution of prokaryotic and eukaryotic genomes. The main carriers of foreign DNA in HGT are mobile genetic elements (MGEs) that have extremely diverse genetic structures and properties. Various strategies are used for the maintenance and spread of MGEs, including (i) vegetative replication, (ii) transposition (and other types of recombination), and (iii) conjugal transfer. In many MGEs, all of these processes are dependent on rolling-circle replication (RCR). RCR is one of the most well characterized models of DNA replication. Although many studies have focused on describing its mechanism, the role of replication initiator proteins has only recently been subject to in-depth analysis, which indicates their involvement in multiple biological process associated with RCR. In this review, we present a general overview of RCR and its impact in HGT. We focus on the molecular characteristics of RCR initiator proteins belonging to the HUH and Rep_ trans protein families. Despite analogous mechanisms of action these are distinct groups of proteins with different catalytic domain structures. This is the first review describing the multifunctional character of various types of RCR initiator proteins, including the latest discoveries in the field. Recent reports provide evidence that (i) proteins initiating vegetative replication (Rep) or mobilization for conjugal transfer (Mob) may also have integrase (Int) activity, (ii) some Mob proteins are capable of initiating vegetative replication (Rep activity), and (iii) some Rep proteins can act like Mob proteins to mobilize plasmid DNA for conjugal transfer. These findings have significant consequences for our understanding of the role of RCR, not only in DNA metabolism but also in the biology of many MGEs.

Plasmids play an important role in the adaptation of bacteria to changeable environmental conditions. As the main vectors of horizontal gene transfer, they can spread genetic information among bacteria, sometimes even across taxonomic boundaries. Some plasmids carry genes involved in the utilization of particular carbon compounds, which can provide a competitive advantage to their hosts in particular ecological niches. Analysis of the multireplicon genome of the soil bacterium P. aminovorans JCM 7685 revealed the presence of an extrachromosomal replicon pAMV1 (185 kb) with a unique structure and properties. This lifestyle-determining plasmid carries genes facilitating the metabolism of many different carbon compounds including sugars, short-chain organic acids and C1 compounds. Plasmid pAMV1 contains a large methylotrophy island (MEI) that is essential not only for the utilization of particular C1 compounds but also for the central methylotrophic metabolism required for the assimilation of C1 units (serine cycle). We demonstrate that the expression of the main serine cycle genes is induced in the presence of C1 compounds by the transcriptional regulator ScyR. The extrachromosomal localization of the MEI and the distribution of related genes in Paracoccus spp. indicate that it could have been acquired by HGT by an ancestor of P. aminovorans., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Short modified oligonucleotides targeted at bacterial DNA or RNA could serve as antibacterial agents provided that they are efficiently taken up by bacterial cells. However, the uptake of such oligonucleotides is hindered by the bacterial cell wall. To overcome this problem, oligomers have been attached to cell-penetrating peptides, but the efficiency of delivery remains poor. Thus, we have investigated the ability of vitamin B 12 to transport peptide nucleic acid (PNA) oligomers into cells of Escherichia coli and Salmonella Typhimurium. Vitamin B 12 was covalently linked to a PNA oligomer targeted at the mRNA of a reporter gene expressing Red Fluorescent Protein. Cu-catalyzed 1,3-dipolar cycloaddition was employed for the synthesis of PNA-vitamin B 12 conjugates; namely the vitamin B 12 azide was reacted with PNA possessing the terminal alkyne group. Different types of linkers and spacers between vitamin B 12 and PNA were tested, including a disulfide bond. We found that vitamin B 12 transports antisense PNA into E. coli cells more efficiently than the most widely used cell-penetrating peptide (KFF) 3 K. We also determined that the structure of the linker impacts the antisense effect. The results of this study provide the foundation for developing vitamin B 12 as a carrier of PNA oligonucleotides into bacterial cells.

Pseudomonas strains isolated from the heavily contaminated Lubin copper mine and Zelazny Most post-flotation waste reservoir in Poland were screened for the presence of integrons. This analysis revealed that two strains carried homologous DNA regions composed of a gene encoding a DNA_BRE_C domain-containing tyrosine recombinase (with no significant sequence similarity to other integrases of integrons) plus a three-component array of putative integron gene cassettes. The predicted gene cassettes encode three putative polypeptides with homology to (i) transmembrane proteins, (ii) GCN5 family acetyltransferases, and (iii) hypothetical proteins of unknown function (homologous proteins are encoded by the gene cassettes of several class 1 integrons). Comparative sequence analyses identified three structural variants of these novel integron-like elements within the sequenced bacterial genomes. Analysis of their distribution revealed that they are found exclusively in strains of the genus Pseudomonas .

The Svalbard archipelago (Spitsbergen Island) is the northernmost landmass in the European Arctic and has a variety of small- and medium-sized glaciers. The plasmidome of eleven psychrophilic strains of Variovorax spp. isolated from the ice surface of Hans and Werenskiold Glaciers of Spitsbergen Island, was defined. This analysis revealed the presence of six plasmids whose nucleotide sequences have been determined. Four of them, exhibiting high reciprocal sequence similarity, possess unique structures, since their genomes lack any recognized genes. These miniature replicons, not exceeding 1 kb in size, include pHW69V1 (746 bp), which is the smallest autonomous replicon so far identified in free-living bacteria. The miniature plasmids share no similarity with known sequences present in the databases. In silico and experimental analyses identified conserved DNA regions essential for the initiation of replication of these replicons., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Odontomas constitute a developmental defect of hard dental tissues and are classified as benign odontogenic tumours. They are composed of all dental structures and tissues: enamel, dentin, cementum and pulp. As regards histomorphological features, two types of odontomas have been differentiated: complex and compound. Odontomas represent from 4.7% to 76% of odontogenic tumours. Their aetiology has not been fully understood, yet injury, infection and genetic factors are often named among the causes. Odontomas are usually detected by chance in radiographic images taken in relation to disrupted eruption or mislocation of teeth., Aim of Study: To present the case study of an erupting compound odontoma on the right side of the mandible in an 11-year-old girl., Material, Methods and Results: The analysis covered medical documentation of the patient, diagnostic casts, orthopantomographs and cone beam computed tomography scans. The case study has been complemented with the review of up-to-date literature. A lesion composed of 30 odontoids was removed during one-day surgery without subsequent augmentation with bone substitute material. There were no post-surgery complications. A follow-up orthopantomograph taken 3 months later showed that the wound had been healing correctly, which made it possible to plan subsequent orthodontic treatment with a fixed appliance., Conclusions: 1. Odontomas are benign lesions which can be removed during one-day surgery without the absolute need of augmentation with xenogenic or allogenic material. 2. The pressure exerted by the plate of a removable appliance is very likely to initiate the eruption of the odontoma.

Paracoccus aminophilus JCM 7686 (Alphaproteobacteria) is a facultative, heterotrophic methylotroph capable of utilizing a wide range of C1 compounds as sole carbon and energy sources. Analysis of the JCM 7686 genome revealed the presence of genes involved in the oxidation of methanol, methylamine, dimethylamine, trimethylamine, N,N-dimethylformamide, and formamide, as well as the serine cycle, which appears to be the only C1 assimilatory pathway in this strain. Many of these genes are located in different extrachromosomal replicons and are not present in the genomes of most members of the genus Paracoccus, which strongly suggests that they have been horizontally acquired. When compared with Paracoccus denitrificans Pd1222 (type strain of the genus Paracoccus), P. aminophilus JCM 7686 has many additional methylotrophic capabilities (oxidation of dimethylamine, trimethylamine, N,N-dimethylformamide, the serine cycle), which are determined by the presence of three separate gene clusters. Interestingly, related clusters form compact methylotrophy islands within the genomes of Paracoccus sp. N5 and many marine bacteria of the Roseobacter clade.

Methanogenic Archaea produce approximately one billion tons of methane annually, but their biology remains largely unknown. This is partially due to the large phylogenetic and phenotypic diversity of this group of organisms, which inhabit various anoxic environments including peatlands, freshwater sediments, landfills, anaerobic digesters and the intestinal tracts of ruminants. Research is also hampered by the inability to cultivate methanogenic Archaea. Therefore, biodiversity studies have relied on the use of 16S rRNA and mcrA [encoding the α subunit of the methyl coenzyme M (methyl-CoM) reductase] genes as molecular markers for the detection and phylogenetic analysis of methanogens. Here, we describe four novel molecular markers that should prove useful in the detailed analysis of methanogenic consortia, with a special focus on methylotrophic methanogens. We have developed and validated sets of degenerate PCR primers for the amplification of genes encoding key enzymes involved in methanogenesis: mcrB and mcrG (encoding β and γ subunits of the methyl-CoM reductase, involved in the conversion of methyl-CoM to methane), mtaB (encoding methanol-5-hydroxybenzimidazolylcobamide Co-methyltransferase, catalyzing the conversion of methanol to methyl-CoM) and mtbA (encoding methylated [methylamine-specific corrinoid protein]:coenzyme M methyltransferase, involved in the conversion of mono-, di- and trimethylamine into methyl-CoM). The sensitivity of these primers was verified by high-throughput sequencing of PCR products amplified from DNA isolated from microorganisms present in anaerobic digesters. The selectivity of the markers was analyzed using phylogenetic methods. Our results indicate that the selected markers and the PCR primer sets can be used as specific tools for in-depth diversity analyses of methanogenic consortia.

Paracoccus kondratievae NCIMB 13773(T), isolated from the maize rhizosphere, carries a large (95,049 bp) plasmid pKON1, whose structure has been significantly influenced by transposition. Almost 30% of the plasmid genome is composed of complete or truncated insertion sequences (ISs), representing seven IS families. The ISs are accompanied by numerous genes and gene clusters commonly found in bacterial chromosomes, encoding, among others, (i) a putative type III secretion system of the Rhizobiales-T3SS family, (ii) a type I restriction-modification system associated with the anti-codon nuclease (ACNase) gene prrC and (iii) OstA and OstB proteins involved in trehalose synthesis. The backbone of pKON1 is composed of replication and partitioning modules conserved in several large alphaproteobacterial replicons, including secondary chromid pAMI6 of Paracoccus aminophilus JCM 7686 and chromosome 2 (chromid) of Rhodobacter sphaeroides 2.4.1. pKON1 also contains a toxin-antitoxin system of the hipAB family, whose presence precludes removal of the plasmid from bacterial cells. This system, unlike two other related hipAB-family loci originating from plasmid pAMI8 and the chromosome of Paracoccus aminophilus JCM 7686, is highly efficient and permits very stable maintenance of a heterologous replicon in various hosts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The Lubin underground mine, is one of three mining divisions in the Lubin-Glogow Copper District in Lower Silesia province (Poland). It is the source of polymetallic ore that is rich in copper, silver and several heavy metals. Black shale is also significantly enriched in fossil organic matter in the form of long-chain hydrocarbons, polycyclic aromatic hydrocarbons, organic acids, esters, thiophenes and metalloporphyrins. Biological analyses have revealed that this environment is inhabited by extremophilic bacteria and fungi. Kupfershiefer black shale and samples of water, bottom and mineral sediments from the underground (below 600 m) Lubin mine were taken and 20 bacterial strains were isolated and characterized. All exhibited multi-resistant and hypertolerant phenotypes to heavy metals. We analyzed the plasmidome of these strains in order to evaluate the diversity and role of mobile DNA in adaptation to the harsh conditions of the mine environment. Experimental and bioinformatic analyses of 11 extrachromosomal replicons were performed. Three plasmids, including a broad-host-range replicon containing a Tn3 family transposon, carried genes conferring resistance to arsenic, cadmium, cobalt, mercury and zinc. Functional analysis revealed that the resistance modules exhibit host specificity, i.e., they may increase or decrease tolerance to toxic ions depending on the host strain. The other identified replicons showed diverse features. Among them we identified a catabolic plasmid encoding enzymes involved in the utilization of histidine and vanillate, a putative plasmid-like prophage carrying genes responsible for NAD biosynthesis, and two repABC-type plasmids containing virulence-associated genes. These findings provide an unique molecular insight into the pool of extrachromosomal replicons and highlight their role in the biology and adaptation of extremophilic bacteria inhabiting terrestrial deep subsurface.

The Tn 3 family of transposons includes diverse elements that encode homologous transposases and contain conserved terminal inverted repeat sequences (IRs). The recent identification of non-autonomous elements, named TIMEs (Tn 3 -derived Inverted-repeat Miniature Elements), has shed new light on the diversity and evolution of this transposon family. A common feature of TIMEs and other members of this family is their ability to mobilize genomic DNA for transposition as part of composite transposons. These elements significantly influence the structure and properties of plasmids and other mobile genetic elements (MGEs). They may contain and move by transposition (i) plasmid replication systems, (ii) toxin-antitoxin systems and (iii) site-specific recombination modules that can resolve plasmid multimers. Some Tn 3 family elements may also transfer large segments of chromosomal DNA into plasmids, which increases the pool of mobile DNA that can take part in horizontal gene transfer.

Bacterial genomic information can be divided between various replicons, including chromosomes, plasmids, and chromids (essential plasmid-like replicons with properties of both chromosomes and plasmids). Comparative analyses of bacterial plasmids, including homology searches, phylogenetic and phylogenomic analyses, as well as network construction for the characterization of their relationships, are good starting points for the identification of chromids. Chromids possess several chromosome-like genetic features (e.g., codon usage, GC content), but most significantly, they carry housekeeping genes, which make them indispensable for cell viability. However, it is important to confirm in silico predictions experimentally. The essential nature of a predicted chromid is usually verified by the application of a target-oriented replicon curing technique, based on the incompatibility phenomenon. Further tests examining growth in various media are used to distinguish secondary chromids from plasmids, and mutational analysis (e.g., using the yeast FLP/FRT recombination system) is employed to identify essential genes carried by particular chromids.

Antibiotic resistance determinants are frequently associated with plasmids and other mobile genetic elements, which simplifies their horizontal transmission. Several groups of plasmids (including replicons of the IncL/M incompatibility group) were found to play an important role in the dissemination of resistance genes encoding β-lactamases. The IncL/M plasmids are large, broad host range, and self-transmissible replicons. We have identified and characterized two novel members of this group: pARM26 (isolated from bacteria inhabiting activated sludge from a wastewater treatment plant) and pIGT15 (originating from a clinical strain of Escherichia coli). This instigated a detailed comparative analysis of all available sequences of IncL/M plasmids encoding β-lactamases. The core genome of these plasmids is comprised of 20 genes with conserved synteny. Phylogenetic analyses of these core genes allowed clustering of the plasmids into four separate groups, which reflect their antibiotic resistance profiles. Examination of the biogeography of the IncL/M plasmids revealed that they are most frequently found in bacteria of the family Enterobacteriaceae originating from the Mediterranean region and Western Europe and that they are able to persist in various ecological niches even in the absence of direct antibiotic selection pressure.

Extremely cold environments are a challenge for all organisms. They are mostly inhabited by psychrophilic and psychrotolerant bacteria, which employ various strategies to cope with the cold. Such harsh environments are often highly vulnerable to the influence of external factors and may undergo frequent dynamic changes. The rapid adjustment of bacteria to changing environmental conditions is crucial for their survival. Such "short-term" evolution is often enabled by plasmids-extrachromosomal replicons that represent major players in horizontal gene transfer. The genomic sequences of thousands of microorganisms, including those of many cold-active bacteria have been obtained over the last decade, but the collected data have yet to be thoroughly analyzed. This report describes the results of a meta-analysis of the NCBI sequence databases to identify and characterize plasmids of psychrophilic and psychrotolerant bacteria. We have performed in-depth analyses of 66 plasmids, almost half of which are cryptic replicons not exceeding 10 kb in size. Our analyses of the larger plasmids revealed the presence of numerous genes, which may increase the phenotypic flexibility of their host strains. These genes encode enzymes possibly involved in (i) protection against cold and ultraviolet radiation, (ii) scavenging of reactive oxygen species, (iii) metabolism of amino acids, carbohydrates, nucleotides and lipids, (iv) energy production and conversion, (v) utilization of toxic organic compounds (e.g., naphthalene), and (vi) resistance to heavy metals, metalloids and antibiotics. Some of the plasmids also contain type II restriction-modification systems, which are involved in both plasmid stabilization and protection against foreign DNA. Moreover, approx. 50% of the analyzed plasmids carry genetic modules responsible for conjugal transfer or mobilization for transfer, which may facilitate the spread of these replicons among various bacteria, including across species boundaries.

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Functional transposable elements (TEs) of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons--Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element "captured" with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.

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.

Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria.

Pseudomonas sp. GLE121 (a psychrophilic Antarctic strain) carries three plasmids: pGLE121P1 (6899 bp), pGLE121P2 (8330 bp) and pGLE121P3 (39,583 bp). Plasmids pGLE121P1 and pGLE121P2 show significant sequence similarity to members of the IncP-9 and IncP-7 incompatibility groups, respectively, while the largest replicon, pGLE121P3, is highly related to plasmid pNCPPB880-40 of Pseudomonas syringae pathovar tomato NCPPB880. All three plasmids have a narrow host range, limited to members of the genus Pseudomonas. Plasmid pGLE121P3 encodes a conjugal transfer system, while pGLE121P1 carries only a putative MOB module, conserved in many mobilizable plasmids. Plasmid pGLE121P3 contains an additional load of genetic information, including a pair of genes with homology to the rulAB operon, responsible for ultraviolet radiation (UVR) tolerance. Given the increasing UV exposure in Antarctic regions, the expression of these genes is likely to be an important adaptive response., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Six strains of Psychrobacter spp. isolated from guano of little auks collected on Spitsbergen island (Arctic) carried nine plasmids that were fully sequenced. These replicons (ranging in size from 2917 to 14924 bp) contained either repA (ColE2-type) or repB (iteron-type) replication systems of a relatively narrow host range, limited to Psychrobacter spp. All but one of the plasmids carried predicted mobilization for conjugal transfer systems, encoding relaxases of the MOBQ, MOBV or MOBP families. The plasmids also contained diverse additional genetic load, including a type II restriction-modification system and a gene encoding a putative subunit C of alkyl hydroperoxide reductase (AhpC)-an antioxidant enzyme and major scavenger of reactive oxygen species. Detailed comparative sequence analyses, extended to all plasmids identified so far in psychrophilic bacteria, distinguished groups of the most ubiquitous replicons, which play a key role in horizontal gene transfer in cold environments.

Background: Halomonas sp. ZM3 was isolated from Zelazny Most post-flotation mineral waste repository (Poland), which is highly contaminated with heavy metals and various organic compounds. Mobile DNA of the strain (i.e. plasmids and transposons) were analyzed in order to identify genetic information enabling adaptation of the bacterium to the harsh environmental conditions., Results: The analysis revealed that ZM3 carries plasmid pZM3H1 (31,370 bp), whose replication system may be considered as an archetype of a novel subgroup of IncU-like replicons. pZM3H1 is a narrow host range, mobilizable plasmid (encodes a relaxase of the MOBV family) containing mercury resistance operon (mer) and czcD genes (mediate resistance to zinc and cobalt), which are part of a large truncated Tn3 family transposon. Further analysis demonstrated that the phenotypes determined by the pZM3H1 resistance cassette are highly dependent on the host strain. In another strand of the study, the trap plasmid pMAT1 was employed to identify functional transposable elements of Halomonas sp. ZM3. Using the sacB positive selection strategy two insertion sequences were identified: ISHsp1 - representing IS5 group of IS5 family and ISHsp2 - a distinct member of the IS630 family., Conclusions: This study provides the first detailed description of mobile DNA in a member of the family Halomonadaceae. The identified IncU plasmid pZM3H1 confers resistance phenotypes enabling adaptation of the host strain to the Zelazny Most environment. The extended comparative analysis has shed light on the distribution of related IncU plasmids among bacteria, which, in many cases, reflects the frequency and direction of horizontal gene transfer events. Our results also identify plasmid-encoded modules, which may form the basis of novel shuttle vectors, specific for this group of halophilic bacteria.

The complete nucleotide sequence of plasmid pP62BP1 (34,467 bp), isolated from Arctic Psychrobacter sp. DAB_AL62B, was determined and annotated. The conserved plasmid backbone is composed of several genetic modules, including a replication system (REP) with similarities to the REP region of the iteron-containing plasmid pPS10 of Pseudomonas syringae. The additional genetic load of pP62BP1 includes two highly related type II restriction-modification systems and a set of genes (slfRCHSL) encoding enzymes engaged in the metabolism of organic sulfates, plus a putative transcriptional regulator (SlfR) of the AraC family. The pP62BP1 slf locus has a compact and unique structure. It is predicted that the enzymes SlfC, SlfH, SlfS and SlfL carry out a chain of reactions leading to the transformation of alkyl sulfates into acyl-CoA, with dodecyl sulfate (SDS) as a possible starting substrate. Comparative analysis of the nucleotide sequences of pP62BP1 and other Psychrobacter spp. plasmids revealed their structural diversity. However, the presence of a few highly conserved DNA segments in pP62BP1, plasmid 1 of P. cryohalolentis K5 and pRWF-101 of Psychrobacter sp. PRwf-1 is indicative of recombinational shuffling of genetic information, and is evidence of lateral gene transfer in the Arctic environment.

Klebsiella pneumoniae 287-w carries three small narrow host range (NHR) plasmids (pIGMS31, pIGMS32, and pIGRK), which could be maintained in several closely related species of Gammaproteobacteria, but not in Alphaproteobacteria. The plasmids contain different mobilization systems (MOB), whose activity in Escherichia coli was demonstrated in the presence of the helper transfer system originating from plasmid RK2. The MOBs of pIGMS31 and pIGMS32 are highly conserved in many bacterial plasmids (members of the MOB family), while the predicted MOB of pIGRK has a unique structure, encoding a protein similar to phage-related integrases. The MOBs of pIGMS31 and pIGMS32 enabled the transfer of heterologous replicons from E. coli into both gammaproteobacterial and alphaproteobacterial hosts, which suggests that these NHR plasmids contain broad host range MOB systems. Such plasmids therefore represent efficient carrier molecules, which may act as natural suicide vectors promoting the spread of diverse genetic information (including other types of mobile elements, e.g. resistance transposons) among evolutionarily distinct bacterial species. Thus, mobilizable NHR plasmids may play a much more important role in horizontal gene transfer than previously thought., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Several trap plasmids (enabling positive selection of transposition events) were used to identify a pool of functional transposable elements (TEs) residing in bacteria of the genus Paracoccus (Alphaproteobacteria). Complex analysis of 25 strains representing 20 species of this genus led to the capture and characterization of (i) 37 insertion sequences (ISs) representing 9 IS families (IS3, IS5, IS6, IS21, IS66, IS256, IS1182, IS1380 and IS1634), (ii) a composite transposon Tn6097 generated by two copies of the ISPfe2 (IS1634 family) containing two predicted genetic modules, involved in the arginine deiminase pathway and daunorubicin/doxorubicin resistance, (iii) 3 non-composite transposons of the Tn3 family, including Tn5393 carrying streptomycin resistance and (iv) a transposable genomic island TnPpa1 (45 kb). Some of the elements (e.g. Tn5393, Tn6097 and ISs of the IS903 group of the IS5 family) were shown to contain strong promoters able to drive transcription of genes placed downstream of the target site of transposition. Through the application of trap plasmid pCM132TC, containing a promoterless tetracycline resistance reporter gene, we identified five ways in which transposition can supply promoters to transcriptionally silent genes. Besides highlighting the diversity and specific features of several TEs, the analyses performed in this study have provided novel and interesting information on (i) the dynamics of the process of transposition (e.g. the unusually high frequency of transposition of TnPpa1) and (ii) structural changes in DNA mediated by transposition (e.g. the generation of large deletions in the recipient molecule upon transposition of ISPve1 of the IS21 family). We also demonstrated the great potential of TEs and transposition in the generation of diverse phenotypes as well as in the natural amplification and dissemination of genetic information (of adaptative value) by horizontal gene transfer, which is considered the driving force of bacterial evolution.

Plasmid pAMI7 of the methylotrophic bacterium Paracoccus aminophilus JCM 7686 (Alphaproteobacteria) encodes a functional type II restriction-modification (R-M) system designated PamI. Homologous systems were identified in the genomes of distinct taxonomic groups of Bacteria and Archaea, which provides evidence that horizontal gene transfer has contributed to the wide dissemination of R-M modules - even between domains. Analysis of the cleavage specificity of the R.PamI endonuclease revealed that this protein is an isoschizomer of restriction enzyme NcoI. Interestingly, bioinformatic analyses suggest that R.PamI and NcoI are accompanied by methyltransferases of different methylation specificities (C5-methylcytosine and N4-methylcytosine methyltransferases, respectively), which possibly exemplifies recombinational shuffling of genes coding for individual components of R-M systems. The PamI system can stabilize plasmid pAMI7 in a bacterial population, most probably at the postsegregational level. Therefore, it functions in an analogous manner to plasmid-encoded toxin-antitoxin (TA) systems. Since the TA system of pAMI7 is nonfunctional, it is highly probable that this lack is compensated by the stabilizing activity of PamI. This indicates the crucial role of the analyzed R-M system in the stable maintenance of pAMI7, which is, to our knowledge, the first report of 'symbiosis' between a R-M system and a plasmid in the Alphaproteobacteria., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

We have developed a DIY (Do It Yourself) series of genetic cassettes, which facilitate construction of novel versatile vectors for Alphaproteobacteria. All the cassettes are based on defined genetic modules derived from three natural plasmids of Paracoccus aminophilus JCM 7686. We have constructed over 50 DIY cassettes, which differ in structure and specific features. All of them are functional in eight strains representing three orders of Alphaproteobacteria: Rhodobacterales, Rhizobiales and Caulobacterales. Besides various replication and stabilization systems, many of the cassettes also contain selective markers appropriate for Alphaproteobacteria (40 cassettes) and genetic modules responsible for mobilization for conjugal transfer (24 cassettes). All the DIY cassettes are bordered by different types of polylinkers, which facilitate vector construction. Using these DIY cassettes, we have created a set of compatible Escherichia coli-Alphaproteobacteria mobilizable shuttle vectors (high or low copy number in E. coli), which will greatly assist the genetic manipulation of Alphaproteobacteria., (Copyright © 2011 Elsevier B.V. All rights reserved.)

N,N-Dimethylformamide (DMF), a toxic solvent used in the chemical industry, is frequently present in industrial wastes. Plasmid pAMI2 (18.6 kb) of Paracoccus aminophilus JCM 7686 carries genetic information which is crucial for methylotrophic growth of this bacterium, using DMF as the sole source of carbon and energy. Besides a conserved backbone related to pAgK84 of Agrobacterium radiobacter K84, pAMI2 carries a three-gene cluster coding for the protein DmfR, which has sequence similarities to members of the LuxR family of transcription regulators, and two subunits (DmfA1 and DmfA2) of N,N-dimethylformamidase, an enzyme of high substrate specificity that catalyzes the first step in the degradation of DMF. Genetic analysis revealed that these genes, which are all placed in the same orientation, constitute an inducible operon whose expression is activated in the presence of DMF by the positive transcription regulator DmfR. This operon was used to construct a strain able to degrade DMF at high concentrations that might be used in the biotreatment of DMF-containing industrial wastewaters. To our knowledge, this is the first study to provide insights into the genetic organization and regulation as well as the dissemination in bacteria of genes involved in the enzymatic breakdown of DMF.