ArdA genes from pKM101 and from B. bifidum chromosome have a different range of regulated genes.

The ardA genes are present in a wide variety of conjugative plasmids and play an important role in overcoming the restriction barrier. To date, there is no information on the chromosomal ardA genes. It is still unclear whether they keep their antirestriction activity and why bacterial chromosomes contain these genes. In the present study, we confirmed the antirestriction function of the ardA gene from the Bifidobacterium bifidum chromosome. Transcriptome analysis in Escherichia coli showed that the range of regulated genes varies significantly for ardA from conjugative plasmid pKM101 and from the B. bifidum chromosome. Moreover, if the targets for both ardA genes match, they often show an opposite effect on regulated gene expression. The results obtained indicate two seemingly mutually exclusive conclusions. On the one hand, the pleiotropic effect of ardA genes was shown not only on restriction-modification system, but also on expression of a number of other genes. On the other hand, the range of affected genes varies significally for ardA genes from different sources, which indicates the specificity of ardA to inhibited targets. Author Summary. Conjugative plasmids, bacteriophages, as well as transposons, are capable to transfer various genes, including antibiotic resistance genes, among bacterial cells. However, many of those genes pose a threat to the bacterial cells, therefore bacterial cells have special restriction systems that limit such transfer. Antirestriction genes have previously been described as a part of conjugative plasmids, and bacteriophages and transposons. Those plasmids are able to overcome bacterial cell protection in the presence of antirestriction genes, which inhibit bacterial restriction systems. This work unveils the antirestriction mechanisms, which play an important role in the bacterial life cycle. Here, we clearly show that antirestriction genes, which are able to inhibit cell protection, exist not only in plasmids but also in the bacterial chromosomes themselves. Moreover, antirestrictases have not only an inhibitory function but also participate in the regulation of other bacterial genes. The regulatory function of plasmid antirestriction genes also helps them to overcome the bacterial cell protection against gene transfer, whereas the regulatory function of genomic antirestrictases has no such effect.

Disjunct habitat of cryptic Terebellides (Annelida, Trichobranchidae) species shows a phylogenetic link between polychaetes from the White and the North Seas.

Understanding the distribution and biodiversity of marine species is crucial for developing effective conservation strategies and maintaining the health of global ecosystems. Advancements in molecular data utilization have significantly improved our understanding of biodiversity within the genus Terebellides. In this study, we conducted a phylogenetic analysis on polychaete samples from the Kandalaksha Bay, White Sea, revealing their affiliation with a putative undescribed species of the genus Terebellides found in two locations of the North Sea. Interestingly, this species was not detected in the Norwegian and Barents Seas, leading us to propose a disjunct distribution scenario for this Terebellides species. This unique distribution pattern might be attributed to the succession of polychaetes by new species, facilitated by the Gulf Stream and a climate change role in driving shifts in species' ranges and altering marine ecosystem dynamics.

[Anti-Restriction Activity of ArdB Protein against EcoAI Endonuclease].

ArdB proteins are known to inhibit the activity of the type I restriction-modification (RM-I) system, in particular EcoKI (IA family). The mechanism of ArdB's activity still remains unknown; the spectrum of targets inhibited has been poorly studied. In this work, it was shown that the presence of the ardB gene from the R64 plasmid could suppress the activity of EcoAI endonuclease (IB family) in Escherichia coli TG1 cells. Due to the absence of specificity of ArdB to a certain RM-I system (it inhibits both the IA- and IB-family), it can be assumed that the mechanism of the anti-restriction activity of this protein does not depend on the sequence DNA at the recognition site nor the structure of the restriction enzyme of the RM-I systems.

Antirestriction Protein ArdB (R64) Interacts with DNA.

The antirestriction ArdB protein inhibits the endonuclease activity of type I restriction/modification (RM) systems in vivo; however, the mechanism of inhibition remains unknown. In this study, we showed that recombinant ArdB from Escherichia coli cells co-purified with DNA. When overexpressed in E. coli cells, a portion of ArdB protein formed insoluble DNA-free aggregates. Only native ArdB, but not the ArdBΔD141 mutant lacking the antirestriction activity, co-purified with DNA upon anion-exchange and affinity chromatography or total DNA isolation from formaldehyde-treated cells. These observations confirm the hypothesis that ArdB blocks DNA translocation via the R subunits of the R2M2S complex of type I RM enzymes.

[The importance of C-terminal aspartic acid residue (D141) to the antirestriction activity of the ArdB (R64) protein].

Antirestriction proteins of the ArdB/KlcA family are specific inhibitors of restriction (endonuclease) activity of type-I restriction/modification enzymes. The effect of conserved amino acid residues on the antirestriction activity of the ArdB protein encoded by the transmissible R64 (IncI1) plasmid has been investigated. An analysis of the amino acid sequences of ArdB homologues demonstrated the presence of four groups of conserved residues ((1) R16, E32, and W51; (2) Y46 and G48; (3) S81, D83 and E132, and (4) N77, L(I)140, and D141) on the surface of the protein globule. Amino acid residues of the fourth group showed a unique localization pattern with the terminal residue protruding beyond the globule surface. The replacement of two conserved amino acids (D141 and N77) located in the close vicinity of each other on the globule surface showed that the C-terminal D141 is essential for the antirestriction activity of ArdB. The deletion of this residue, as well as replacement by a hydrophobic threonine residue (D141T), completely abolished the antirestriction activity of ArdB. The synonymous replacement of D141 by a glutamic acid residue (D141E) caused an approximately 30-fold decrease of the antirestriction activity of ArdB, and the point mutation N77A caused an approximately 20-fold decrease in activity. The residues D141 and N77 located on the surface of the protein globule are presumably essential for the formation of a contact between ArdB and a currently unknown factor that modulates the activity of type-I restriction/modification enzymes.