Replication Protein RepN Encoded by the RC Plasmid of Thermophilic Bacterium Thermoanaerobacterium saccharolyticum: Mutation Analysis and Deletion Mapping of Domains Responsible for Its Lethal Effect

Amino acid sequence analysis of the product encoded by repN of Thermoanaerobacterium saccharolyticum (Clostridium thermosaccharolyticum) pNB2, which is capable of rolling-circle (RC) replication, revealed all known motifs conserved among replication (Rep) proteins that initiate RC replication of plasmids related to pC194/pUB110. Using the T7 expression system in Escherichia coli, RepN was identified as a 35K protein. Its lethal effect on bacterial cells was unusually high for a protein of the kind. Mutation analysis of the potential active centers (Y85F and Y211F) showed that the lethal effect of RepN is not associated with its putative topoisomerase (relaxase) activity. On evidence of deletion mapping, the lethal effect was attributed to the N- and C-terminal domains, each accounting for about 30% of the total protein. The RepN fragments essential for the lethal effect were found to share a motif without appreciable homology to known conserved motifs. The high lethal effect of RepN was assumed to result from duplication of the motif and to play an adaptive role, providing for stable maintenance of the AT-rich plasmid in thermophilic bacterial cells.     

Mutational Analysis of the Conserved Motif of the ArdA Antirestriction Protein Encoded by Self-transmissible IncI Plasmid ColIb-P9

A study was made of the functional role of the ArdA antirestriction motif (130-LLADVPETVALYFD-143) conserved among all known Ard (alleviation of restriction of DNA) proteins, which are encoded by self-transmissible plasmids and specifically inhibit type I restriction–modification systems. Conserved residues of the motif were individually changed, and the resulting mutants tested for in vivo activity. Hydrophobic L130, L131, and V138 were substituted with negatively charged E; negatively charged D133, E136, and D143 substituted with hydrophobic V; and D127, D150, and D154 neighboring the antirestriction motif substituted with V. Four substitutions (L130E, L131E, V138E, and D143V) substantially (25–1000 times) reduced the ArdA activity. The other substitutions within or beyond the motif had no appreciable effect. Substitutions L130A and L131A each reduced the ArdA activity 10- to 20-fold, indicating that high hydrophobicity of L130 and L131 is important for the ArdA function. Thus, the antirestriction role of ArdA is indeed due to its conserved motif.     

Plasmid-encoded antirestriction protein ArdA can discriminate between type I methyltransferase and complete restriction-modification system

Many promiscuous plasmids encode the antirestriction proteins ArdA (alleviation of restriction of DNA) that specifically affect the restriction activity of heterooligomeric type I restriction-modification (R-M) systems in Escherichia coli cells. In addition, a lot of the putative ardA genes encoded by plasmids and bacterial chromosomes are found as a result of sequencing of complete genomic sequences, suggesting that ArdA proteins and type I R-M systems that seem to be widespread among bacteria may be involved in the regulation of gene transfer among bacterial genomes. Here, the mechanism of antirestriction action of ArdA encoded by IncI plasmid ColIb-P9 has been investigated in comparison with that of well-studied T7 phage-encoded antirestriction protein Ocr using the mutational analysis, retardation assay and His-tag affinity chromatography. Like Ocr, ArdA protein was shown to be able to efficiently interact with EcoKI R-M complex and affect its in vivo and in vitro restriction activity by preventing its interaction with specific DNA. However, unlike Ocr, ArdA protein has a low binding affinity to EcoKI Mtase and the additional C-terminal tail region (VF-motif) is needed for ArdA to efficiently interact with the type I R-M enzymes. It seems likely that this ArdA feature is a basis for its ability to discriminate between activities of EcoKI Mtase (modification) and complete R-M system (restriction) which may interact with unmodified DNA in the cells independently. These findings suggest that ArdA may provide a very effective and delicate control for the restriction and modification activities of type I systems and its ability to discriminate against DNA restriction in favour of the specific modification of DNA may give some advantage for efficient transmission of the ardA-encoding promiscuous plasmids among different bacterial populations.     

Nucleotide sequence of the pNB2 plasmid from thermophilic bacteria Clostridium thermosaccharolyticum]

The complete nucleotide sequence of pNB2, a 1.9-kilobases cryptic plasmid from thermophilic Clostridium thermosaccharolyticum has been determined. The plasmid consists of 1882 base pairs and has a G+C composition of 27.2%. The sequence contains three open reading frames capable of coding for polypeptides two of which were identified in maxicell Escherichia coli extracts. Our future studies are directed toward a construction of pNB2-derivatives as vectors for Clostridia.     

Replication protein RepN encoded by the RC plasmid of thermophilic bacterium Thermoanaerobacterium saccharolyticum: mutational analysis and deletion mapping of domains responsible for its lethal effect

Amino acid sequence analysis of the product encoded by repN of Thermoanaerobacterium saccharolyticum (Clostridium thermosaccharolyticum) pNB2, which is capable of rolling-circle (RC) replication, revealed all known motifs conserved among replication (Rep) proteins that initiate RC replication of plasmids related to pC194/pUB110. Using the T7 expression system in Escherichia coli, RepN was identified as a 35K protein. Its lethal effect on bacterial cells was unusually high for a protein of the kind. Mutation analysis of the potential active centers (Y85F and Y211F) showed that the lethal effect of RepN is not associated with its putative topoisomerase (relaxase) activity. On evidence of deletion mapping, the lethal effect was attributed to the N- and C-terminal domains, each accounting for about 30% of the total protein. The RepN fragments essential for the lethal effect were found to share a motif, which showed no appreciable homology to known conserved motifs. The high lethal effect of RepN was assumed to result from duplication of the motif and to play an adaptive role, providing for the stable maintenance of the AT-rich plasmid in thermophilic bacterial cells.     

Long -Term Changes in the Fish Fauna of the Karkinitsky Gulf of the Black Sea

Russian Journal of Marine Biology - The summarized composition of the fish fauna of the Karkinitsky Gulf of the Black Sea was established for the first time on the basis of literature and original...     

Effect of the Razdol’naya River on structure of the free-living nematode community of the Amursky Bay,Sea of Japan

The effect of the Razdol’naya River on the littoral and sublittoral nematode community was studied in the innermost part of the Amursky Bay. Thanks to the seasonal abundance of juveniles of macrobenthic animals, pseudomacrobenthic species prevailed in the meiobenthos littoral community. Juvenile amphipods dominated (39.5%), the percentage of nematodes was 18.4% (45 species). The nematodes dominated in the meiobenthos community (65.7%, 71 species) at the sublittoral zone. The similarity of the species structure of littoral and sublittoral nematodes reached 36.5%. Low population density (2.6±1.7 thousand ind/m²) and poor species diversity of nematodes (7 species), the dominating form being Oncholaimium paraolium, were recorded at the littoral zone, which was subjected to direct riverine influence (1‰salinity). The correlation analysis revealed no relationship between salinity and population density of nematodes at the littoral zone. At the sublittoral zone (at stations of salinity less than 2.85‰) the parameters of population density (73.9±32.3 thousand ind/m²) and species diversity (12 species) of nematodes were the lowest, Parodontophora timmica dominated. A poor expressed dependence between the density of nematode populations and salinity was revealed. The correlation between the average size of ground particles and population density of nematodes was recorded. The greatest similarity of the species structure of littoral and sublittoral nematodes (26.67%) was recorded on gravel-sand bottoms.     

Antirestriction protein Ard (Type C) encoded by IncW plasmid pSa has a high similarity to the "protein transport" domain of TraC1 primase of promiscuous plasmid RP4

The IncW plasmid pSa contains the gene ard encoding an antirestriction function that is specific for type I restriction and modification systems. The nucleotide sequence of ard was determined and an appropriate polypeptide of about 33 kDa was identified in Escherichia coli T7 expression system. Analysis of deduced amino acid sequence of Ard encoded by pSa revealed that this protein has no significant similarities with the known Ard proteins (ArdA and ArdB types) except the "antirestriction" motif (14 amino acid residues in length) conserved for all known Ard proteins. This finding suggests that pSa Ard may be classified as a new type of Ard proteins which we designated ArdC. The remarkable feature of ArdC is that it has a high degree of similarity (about 38 % identity) to the N-terminal region of RP4 TraC1 primase which includes about 300 amino acid residues and seems to be essential for binding to the single-stranded DNA and TraC1 protein transport to the recipient cells during the conjugal transfer of plasmid DNA. ArdC also binds to single-stranded DNA. In addition, this protein is able in vitro to protect the single-stranded but not double-stranded plasmid DNA against the activity of type II restriction endonuclease HhaI that cleaves both single and double-stranded DNA. We suggest that like TraC1, ArdC would be transported as a result of their interaction with the single-stranded DNA of transferred plasmid strand during conjugative passage through the cell envelope to the recipient bacterium. Such properties of ArdC protein might be useful to protect immediately the incoming single-stranded DNA from the host endonucleases.     

Acclimation and introduction of hydrobionts ships’ ballast water organisms in the Port of Vladivostok

Work on the investigation of organisms of ballast water and sediments of ships sailing on Russia-Japan and Russia-China lines was started in the Port of Vladivostok for the first time for the Far East seas of Russia. In total, 145 taxa, 37 microalgal species, 24 holoplankton species, 22 meroplankton taxa, and 10 meiofauna groups were revealed, 24 species of microscopic mycelia fungi were attributed, and 28 morphologically distinct bacterial stains were isolated. Potentially toxic microalgal species were found: the dinoflagellates Dinophysis acuminate and Prorocentrum cordatum and the diatom Pseudo-nitzschia pungens. The tropical-subtropical copepod Pseudocalanus inopinus was recorded in ballast water. Polychaete larvae of the Polydora genus with an unusual morphology, which were not occurring in the Peter the Great Bay previously, were recorded as probable southern migrants from the coastal waters of the southwest coast of Honshu Island. Live larvae of the hermit crab Diogenes nitidimanus were revealed in ballast water, supporting the possibility of introduction of this species in the Peter the Great Bay. Based on an analysis of the total number of colony-forming units of heterotroph microorganisms (within the order of 10³–10⁴ cells/ml), the ballast water of ships sailing on the Russia-Japan line was characterized as moderately polluted. The detection of Escherichia coli strains, exceeding the standards quoted in the International Convention by three times or more, showed the potential danger of a mass bioinvasion. Conditionally pathogenic and toxinogenic mycelial fungi, which are able to induce mycoses and mycotoxicoses in invertebrates and fishes, were isolated from ballast water. It was determined that ballast water of the “rusty” type was poorly suited for the existence of zoo- and phytoplankton forms. In the case of the construction of an oil terminal in the Peter the Great Bay, a sharp increase of the introduction of exotic species via the ballast water and foulings of tankers is inevitable