A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active ß-d-Galactosidase from Paracoccus sp. 32d.

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomyces macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active ß-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems.

Deep eutectic solvents microbial toxicity: Current state of art and critical evaluation of testing methods.

Deep eutectic solvents (DESs) were described at the beginning of 21st century and they consist of a mixture of two or more solid components, which gives rise to a lower melting point compared to the starting materials. Over the years, DESs have proved to be a promising alternative to traditional organic solvents and ionic liquids (ILs) due to their low volatility, low inflammability, easy preparation, and usually low cost of compounds used in their preparation. All these properties encouraged researchers to use them in diverse fields and applications e.g., as extractants for biomolecules and solvents in pharmaceutical and cosmetic industries. Nevertheless, despite undeniable potential of DESs, there is still controversy about their toxicity. Besides the low number of studies on this topic, there are also some contradicting reports on biocompatibility of these solvents. Such misleading reports could be mainly attributed to the lack of well design standard protocol for DESs toxicity determination or the use of out-off-purpose methodology. Thus, to better apply DESs in green and sustainable chemistry, more studies on their impact on organisms at different trophic levels and the use of proper techniques are required. This review focuses on DESs toxicity towards microorganisms and is divided into three parts: The first part provides a brief general introduction to DESs, the second part discusses the methodologies used for assessment of DESs microbial toxicity and the obtained results, and finally in the third part the critical evaluation of the methods is provided, as well as suggestions and guidelines for future research.

Cloning, expression in Komagataella phaffii, and biochemical characterization of recombinant sequence variants of Pseudomonas sp. S9 GDSL-esterase.

Two recombinant Komagataella phaffii (formerly Pichia pastoris) yeast strains for production of two sequential variants of EstS9 esterase from psychrotolerant bacterium Pseudomonas sp. S9, i.e. αEstS9N (a two-domain enzyme consisting of a catalytic domain and an autotransporter domain) and αEstS9Δ (a single-domain esterase) were constructed. However, only one of recombinant K. phaffii strains, namely Komagataella phaffii X-33/pPICZαestS9Δ, allowed to successfully produce and secrete recombinant αEstS9Δ enzyme outside of the host cell. The purified αEstS9Δ esterase was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the acetate (C2) ester. The single-domain αEstS9Δ esterase exhibits the highest activity at 60oC and pH 9.5. In addition, the enzyme retains 90% of its activity after 3 hour incubation at 70-90oC. What should be also noted is that αEstS9Δ esterase produced in the K. phaffii expression system has a much higher specific activity (0.069 U/mg of protein) than the recombinant EstS9Δ esterase produced in an E. coli expression system (0.0025 U/mg of protein) (Wicka et al., 2016, Acta Biochim Pol 63: 117-125. https://doi.org/10.18388/abp.2015_1074).

Determination of phenol biodegradation pathways in three psychrotolerant yeasts, Candida subhashii A011, Candida oregonensis B021 and Schizoblastosporion starkeyi-henricii L012, isolated from Rucianka peatland.

In this study, three psychrotolerant phenol-degrading yeast strains Candida subhashii (strain A011), Candida oregonenis (strain B021) and Schizoblastosporion starkeyi-henricii (strain L012) isolated from Rucianka peatland were examined to determine which alternative metabolic pathway for phenol biodegradation is used by these microorganisms. All yeast strains were cultivated in minimal salt medium supplemented with phenol at 500, 750 and 1000 mg l-1 concentration with two ways of conducting phenol biodegradation experiments: with and without the starving step of yeast cells. For studied yeast strains, no catechol 2,3-dioxygenase activities were detected by enzymatic assay and no products of catechol meta-cleavage in yeast cultures supernatants (GC-MS analysis), were detected. The detection of catechol 1,2-dioxygenase activity and the presence of cis,cis-muconic acid in the analyzed samples revealed that all studied psychrotolerant yeast strains were able to metabolize phenol via the ortho-cleavage pathway. Therefore, they may be tested in terms of their use to develop biotechnology for the production of cis,cis-muconic acid, a substrate used in the production of plastics (PET) and other valuable goods.

Active Site Architecture and Reaction Mechanism Determination of Cold Adapted ß-d-galactosidase from Arthrobacter sp. 32cB.

ArthßDG is a dimeric, cold-adapted ß-d-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its ArthßDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The ArthßDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The resulting three-dimensional structures of complexes: ArthßDG_E441Q/LACs and ArthßDG/IPTG (ligand bound in shallow mode) and structures of complexes ArthßDG_E441Q/LACd, ArthßDG/ONPG (ligands bound in deep mode), and galactose ArthßDG/GAL and their analysis enabled structural characterization of the hydrolysis reaction mechanism. Furthermore, comparative analysis with mesophilic analogs revealed the most striking differences in catalysis mechanisms. The key role in substrate transfer from shallow to deep binding mode involves rotation of the F581 side chain. It is worth noting that the 10-aa loop restricting access to the active site in mesophilic GH2 ßDGs, in ArthßDG is moved outward. This facilitates access of substrate to active site. Such a permanent exposure of the entrance to the active site may be a key factor for improved turnover rate of the cold adapted enzyme and thus a structural feature related to its cold adaptation.

N-phosphonomethylglycine utilization by the psychrotolerant yeast Solicoccozyma terricola M 3.1.4.

Solicoccozyma terricola M 3.1.4., the yeast strain isolated from soil sample from blueberry cultivation in Miedzyrzec Podlaski in Poland, is capable to split of phosphorus to nitrogen and nitrogen to carbon bonds in N-phosphonomethylglycine (PMG, glyphosate). The biodegradation process proceeds in the phosphate-independent manner. It is the first example of a psychrotolerant yeast strain able to degrade PMG via CN bond cleavage accompanied by AMPA formation and not like in most microorganisms via CP bond disruption followed by the sarcosine pathway. Glyphosate oxidoreductase (GOX) type activity was detected in cell-free extracts prepared from S. terricola M 3.1.4. pregrown on 4 mM PMG as a sole phosphorus and nitrogen source in cultivation medium.

Hydrogen Production from Energy Poplar Preceded by MEA Pre-Treatment and Enzymatic Hydrolysis.

The need to pre-treat lignocellulosic biomass prior to dark fermentation results primarily from the composition of lignocellulose because lignin hinders the processing of hard wood towards useful products. Hence, in this work a two-step approach for the pre-treatment of energy poplar, including alkaline pre-treatment and enzymatic saccharification followed by fermentation has been studied. Monoethanolamine (MEA) was used as the alkaline catalyst and diatomite immobilized bed enzymes were used during saccharification. The response surface methodology (RSM) method was used to determine the optimal alkaline pre-treatment conditions resulting in the highest values of both total released sugars (TRS) yield and degree of lignin removal. Three variable parameters (temperature, MEA concentration, time) were selected to optimize the alkaline pre-treatment conditions. The research was carried out using the Box-Behnken design. Additionally, the possibility of the re-use of both alkaline as well as enzymatic reagents was investigated. Obtained hydrolysates were subjected to dark fermentation in batch reactors performed by Enterobacter aerogenes ATCC 13048 with a final result of 22.99 mL H2/g energy poplar (0.6 mol H2/mol TRS).

Isolation and Characterization of Phenol-Degrading Psychrotolerant Yeasts.

In this study, the potential of selected psychrotolerant yeast strains for phenol biodegradation was studied. From 39 strains isolated from soil and water samples from Rucianka peat bog, three psychrotolerant yeast strains, A011, B021, and L012, showed the ability to degrade phenol. The result shows that all three yeast strains could degrade phenol at 500 and 750 mg l-1 concentration, whereas strains A011 and L012 could degrade phenol at 1000 mg l-1 concentration. The time needed for degradation of each phenol concentration was no longer than 2 days. Strains A011, B021, and L012 were identified based on 26S rDNA and ITS sequence analysis as belonging to species Candida subhashii, Candida oregonensis, and Schizoblastosporion starkeyi-henricii, respectively.

Structural studies of a cold-adapted dimeric ß-D-galactosidase from Paracoccus sp. 32d.

The crystal structure of a novel dimeric ß-D-galactosidase from Paracoccus sp. 32d (ParßDG) was solved in space group P212121 at a resolution of 2.4 Šby molecular replacement with multiple models using the BALBES software. This enzyme belongs to glycoside hydrolase family 2 (GH2), similar to the tetrameric and hexameric ß-D-galactosidases from Escherichia coli and Arthrobacter sp. C2-2, respectively. It is the second known structure of a cold-active GH2 ß-galactosidase, and the first in the form of a functional dimer, which is also present in the asymmetric unit. Cold-adapted ß-D-galactosidases have been the focus of extensive research owing to their utility in a variety of industrial technologies. One of their most appealing applications is in the hydrolysis of lactose, which not only results in the production of lactose-free dairy, but also eliminates the `sandy effect' and increases the sweetness of the product, thus enhancing its quality. The determined crystal structure represents the five-domain architecture of the enzyme, with its active site located in close vicinity to the dimer interface. To identify the amino-acid residues involved in the catalytic reaction and to obtain a better understanding of the mechanism of action of this atypical ß-D-galactosidase, the crystal structure in complex with galactose (ParßDG-Gal) was also determined. The catalytic site of the enzyme is created by amino-acid residues from the central domain 3 and from domain 4 of an adjacent monomer. The crystal structure of this dimeric ß-D-galactosidase reveals significant differences in comparison to other ß-galactosidases. The largest difference is in the fifth domain, named Bgal_windup domain 5 in ParßDG, which contributes to stabilization of the functional dimer. The location of this domain 5, which is unique in size and structure, may be one of the factors responsible for the creation of a functional dimer and cold-adaptation of this enzyme.

Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp. S9 isolated from Spitsbergen island soil.

An estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa. Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli strains for production of EstS9N (a two domain enzyme) and EstS9Δ (a one domain enzyme) proteins were constructed, respectively. Both recombinant proteins were successfully produced as inclusion bodies and then purified under denaturing conditions. However, because of the low enzymatic activity of the refolded EstS9Δ protein, only the EstS9N protein was further characterized. The purified and refolded EstS9N protein was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the butyrate (C4) ester. With p-nitrophenyl butyrate as the substrate, the enzyme displayed optimal activity at 35°C and pH 9.0. Additionally, the EstS9N esterase retained ~90% of its activity from 25-40°C and ~40% of its activity at 10°C. Moreover, analysis of its kinetic parameters (Km, kcat, kcat/Km) toward p-nitrophenyl butyrate determined at 15°C and 25°C confirmed that the EstS9 enzyme is cold-adapted. To the best of our knowledge, EstS9 is the third characterized cold-active GDSL-esterase and the first one confirmed to contain an autotransporter domain characteristic for enzymes secreted by the type V secretion system.

Cloning and characterization of a novel cold-active glycoside hydrolase family 1 enzyme with ß-glucosidase, ß-fucosidase and ß-galactosidase activities.

BACKGROUND: Cold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products of the enzymatic reaction, (iv) prevents undesired chemical transformations, and (v) prevents the loss of volatile compounds. RESULTS: A bglMKg gene that encodes a monomeric cold-active glycoside hydrolase family 1 enzyme with an apparent molecular mass of 50 kDa was isolated by the functional screening of a marine metagenomic library. The BglMKg enzyme was expressed in E. coli, purified by FPLC and characterized. The recombinant BglMKg could effectively hydrolyze various chromogenic substrates and ß-linked oligosaccharides, and had remarkably high ß-galactosidase, ß-glucosidase and ß-fucosidase activities. Because of the lack of information about the usefulness of ß-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for ß-glucosidase or ß-galactosidase. The BglMKg had maximal ß-galactosidase and ß-glucosidase activities at approximately 40°C and 45°C, respectively. The optimum pH for ß-galactosidase activity was 6.5, whereas the optimum pH for ß-glucosidase activity was 7.5. In general, the enzyme was stable below 30°C and from pHs 6.0 to 8.0. The results of the kinetic studies revealed that BglMKg more efficiently hydrolyzed ß-glucosidase substrates than ß-galactosidase ones. CONCLUSIONS: BglMKg is a small, monomeric, cold-active ß-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.

A study on the interaction of rhodamine B with methylthioadenosine phosphorylase protein sourced from an Antarctic soil metagenomic library.

The presented study examines the phenomenon of the fluorescence under UV light excitation (312 nm) of E. coli cells expressing a novel metagenomic-derived putative methylthioadenosine phosphorylase gene, called rsfp, grown on LB agar supplemented with a fluorescent dye rhodamine B. For this purpose, an rsfp gene was cloned and expressed in an LMG194 E. coli strain using an arabinose promoter. The resulting RSFP protein was purified and its UV-VIS absorbance spectrum and emission spectrum were assayed. Simultaneously, the same spectroscopic studies were carried out for rhodamine B in the absence or presence of RSFP protein or native E. coli proteins, respectively. The results of the spectroscopic studies suggested that the fluorescence of E. coli cells expressing rsfp gene under UV illumination is due to the interaction of rhodamine B molecules with the RSFP protein. Finally, this interaction was proved by a crystallographic study and then by site-directed mutagenesis of rsfp gene sequence. The crystal structures of RSFP apo form (1.98 Å) and complex RSFP/RB (1.90 Å) show a trimer of RSFP molecules located on the crystallographic six fold screw axis. The RSFP complex with rhodamine B revealed the binding site for RB, in the pocket located on the interface between symmetry related monomers.

A novel cold-active ß-D-galactosidase from the Paracoccus sp. 32d--gene cloning, purification and characterization.

BACKGROUND: ß-D-Galactosidases (EC 3.2.1.23) catalyze the hydrolysis of terminal non-reducing ß-D-galactose residues in ß-D-galactosides. Cold-active ß-D-galactosidases have recently become a focus of attention of researchers and dairy product manufactures owing to theirs ability to: (i) eliminate of lactose from refrigerated milk for people afflicted with lactose intolerance, (ii) convert lactose to glucose and galactose which increase the sweetness of milk and decreases its hydroscopicity, and (iii) eliminate lactose from dairy industry pollutants associated with environmental problems. Moreover, in contrast to commercially available mesophilic ß-D-galactosidase from Kluyveromyces lactis the cold-active counterparts could make it possible both to reduce the risk of mesophiles contamination and save energy during the industrial process connected with lactose hydrolysis. RESULTS: A genomic DNA library was constructed from soil bacterium Paracoccus sp. 32d. Through screening of the genomic DNA library on LB agar plates supplemented with X-Gal, a novel gene encoding a cold-active ß-D-galactosidase was isolated. The in silico analysis of the enzyme amino acid sequence revealed that the ß-D-galactosidase Paracoccus sp. 32d is a novel member of Glycoside Hydrolase Family 2. However, owing to the lack of a BGal_small_N domain, the domain characteristic for the LacZ enzymes of the GH2 family, it was decided to call the enzyme under study 'BgaL'. The bgaL gene was cloned and expressed in Escherichia coli using the pBAD Expression System. The purified recombinant BgaL consists of two identical subunits with a combined molecular weight of about 160 kDa. The BgaL was optimally active at 40°C and pH 7.5. Moreover, BgaL was able to hydrolyze both lactose and o-nitrophenyl-ß-D-galactopyranoside at 10°C with Km values of 2.94 and 1.17 mM and kcat values 43.23 and 71.81 s-1, respectively. One U of the recombinant BgaL would thus be capable hydrolyzing about 97% of the lactose in 1 ml of milk in 24 h at 10°C. CONCLUSIONS: A novel bgaL gene was isolated from Paracoccus sp. 32d encoded a novel cold-active ß-D-galactosidase. An E. coli expression system has enabled efficient production of soluble form of BgaL Paracoccus sp. 32d. The amino acid sequence analysis of the BgaL enzyme revealed notable differences in comparison to the result of the amino acid sequences analysis of well-characterized cold-active ß-D-galactosidases belonging to Glycoside Hydrolase Family 2. Finally, the enzymatic properties of Paracoccus sp. 32d ß-D-galactosidase shows its potential for being applied to development of a new industrial biocatalyst for efficient lactose hydrolysis in milk.

Identification and molecular modeling of a novel lipase from an Antarctic soil metagenomic library.

In this work, we present the construction of a metagenomic library in Escherichia coli using pUC19 vector and environmental DNA directly isolated from Antarctic topsoil and screened for lipolytic enzymes. Screening on agar supplemented with olive oil and rhodamine B revealed one clone with lipolytic activity (Lip1) out of 1000 E. coli clones. This clone harbored a plasmid, pLip1, which has an insert of 4722 bp that was completely sequenced from both directions. Further analysis of the insert showed three open reading frames (ORFs). ORF2 encoded a protein (Lip1 ) of 469 amino acids with 93% identity to the uncultured Pseudomonas sp. lipase LipJ03. Amino acid sequence comparison and phylogenetic analysis indicated that Lip1 lipase was closely related to family I subfamily 3. Furthermore, we present a three-dimensional model of lipase Lip1 which was generated based on the two known structures of mesophilic lipases from Pseudomonas sp. MIS 38 (PML lipase, PDB; 2Z8X) and Serratia marcescens (SML lipase, PDB: 2QUB). Finally, we report the results of comparisons between lipase Lip1 and mesophilic lipases and point out similarities and differences in the catalytic site and in other parts of the analyzed structures.

Purification and biochemical characteristic of a cold-active recombinant esterase from Pseudoalteromonas sp. 643A under denaturing conditions.

In this paper production of a cold-active esterase EstA from the Antarctic bacterium Pseudoalteromonas sp. 643A in E. coli expression system was described. The purification and biochemical characteristic of EstA were performed in the presence of urea and then compared with results obtained for the esterase with no addition of urea and isolated from the native source. In both cases the cold-active enzyme displayed similar properties. However, the differences concerning thermal activity were observed. The optimal temperature for recombinant esterase in the presence of urea (1 M) was about 15 degrees C lower in comparison with enzyme isolated from the native source. Furthermore, the EstA was found to be more thermolabile in denaturant conditions. The differences were presumably caused by slightly changed protein structure in the presence of urea. The preservation of activity of EstA dissolved in buffer containing 8 M urea suggests that the protein structure is retained and it does not undergo dramatic changes due to high urea concentration. This thesis was confirmed with FT-IR data.

An MTA phosphorylase gene discovered in the metagenomic library derived from Antarctic top soil during screening for lipolytic active clones confers strong pink fluorescence in the presence of rhodamine B.

In this work, we present the construction of a metagenomic library in Escherichia coli using the pUC19 vector and environmental DNA directly isolated from Antarctic topsoil and screened for lipolytic enzymes. Unexpectedly, the screening on agar supplemented with olive oil and rhodamine B revealed one unusual pink fluorescent clone (PINKuv) out of 85 000 clones. This clone harbored a plasmid, pPINKuv, which has an insert of 8317 bp that has been completely sequenced. Further analysis of the insert showed eight ORFs. Three ORFs among these exhibited similarities to Psychrobacter arcticus genes. A nucleotide sequence designated as ORF4 encoded a protein with 93% identity to the methylthioadenosine phosphorylase of P. arcticus. This protein was responsible for the observed pink fluorescence of the PINKuv clone in the presence of rhodamine B. We found that colonies of recombinant E. coli TOP10F'/pUC19-ORF4 strain showed pink fluorescence under UV illumination on the Luria-Bertani agar supplemented with rhodamine B after culturing at 25, 30 and 37 degrees C. The same effect was achieved using other E. coli strains such as DH5alpha, LMG194, JM101 and BL21(DE3) pLysS. The results presented here will provide the basis for further studies on the use of the discovered gene as a new reporter gene for molecular biology applications.

Structure of EstA esterase from psychrotrophic Pseudoalteromonas sp. 643A covalently inhibited by monoethylphosphonate.

The crystal structure of the esterase EstA from the cold-adapted bacterium Pseudoalteromonas sp. 643A was determined in a covalently inhibited form at a resolution of 1.35 A. The enzyme has a typical SGNH hydrolase structure consisting of a single domain containing a five-stranded beta-sheet, with three helices at the convex side and two helices at the concave side of the sheet, and is ornamented with a couple of very short helices at the domain edges. The active site is located in a groove and contains the classic catalytic triad of Ser, His and Asp. In the structure of the crystal soaked in diethyl p-nitrophenyl phosphate (DNP), the catalytic serine is covalently connected to a phosphonate moiety that clearly has only one ethyl group. This is the only example in the Protein Data Bank of a DNP-inhibited enzyme with covalently bound monoethylphosphate.

A new beta-galactosidase with a low temperature optimum isolated from the Antarctic Arthrobacter sp. 20B: gene cloning, purification and characterization.

A psychrotrophic bacterium producing a cold-adapted beta-galactosidase upon growth at low temperatures was classified as Arthrobacter sp. 20B. A genomic DNA library of strain 20B introduced into Escherichia coli TOP10F' and screening on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside)-containing agar plates led to the isolation of beta-galactosidase gene. The beta-galactosidase gene (bgaS) encoding a protein of 1,053 amino acids, with a calculated molecular mass of 113,695 kDa. Analysis of the amino acid sequence of BgaS protein, deduced from the bgaS ORF, suggested that it is a member of the glycosyl hydrolase family 2. A native cold-adapted beta-galactosidase was purified to homogeneity and characterized. It is a homotetrameric enzyme, each subunit being approximately 116 kDa polypeptide as deduced from native and SDS-PAGE, respectively. The beta-galactosidase was optimally active at pH 6.0-8.0 and 25 degrees Celsius. P-nitrophenyl-beta-D-galactopyranoside (PNPG) is its preferred substrate (three times higher activity than for ONPG-o-nitrophenyl-beta-D-galactopyranoside). The Arthrobacter sp. 20B beta-galactosidase is activated by thiol compounds (53% rise in activity in the presence of 10 mM 2-mercaptoethanol), some metal ions (activity increased by 50% for Na(+), K(+) and by 11% for Mn(2+)) and inactivated by pCMB (4-chloro-mercuribenzoic acid) and heavy metal ions (Pb(2+), Zn(2+), Cu(2+)).

Characterization of a cryptic plasmid pSFKW33 from Shewanella sp. 33B.

A cryptic plasmid pSFKW33 from psychrotrophic bacterium Shewanella sp. 33B, an isolate from the Gulf of Gdansk (the Baltic Sea), was sequenced and characterized. It is an 8021bp circular molecule with 38% GC content, which shows a distinctive nucleotide sequence without homology to other known plasmids. The nucleotide sequence analysis predicts eight open reading frames. The deduced amino acid sequence of ORF-1 shared significant similarity with the phage integrase protein from Pseudomonas putida GB-1. The ORF2 product showed some similarity with the hypothetical protein Shal_1786 from Shewanella halifaxensis HAW-EB4. The ORF-3 product revealed high amino acid sequence homology with the Escherichia coli replication initiation protein similar to pi protein of R6K plasmid. Five repeat regions (three perfect 22-bp repeats and two imperfect motifs), a putative ribosome binding site, and -10 and -35 promotor sequences were identified upstream of the ORF-3 (rep). The rep module is very similar to several theta replicating iteron family plasmids, suggesting that pSFKW33 replication follows the same course. The ORF-5 product revealed significant identity with the entry exclusion protein 1 of plasmid pIS2 from enteropathogenic strain E. coli 0111:H. The ORF-6 product showed significant identity with the putative transcriptional regulator protein from Shewanella benthica KT99 belonging to HTH-XRE-family protein. The ORF-8 encoded protein that showed some similarity with the hypothetical protein KT99_00146 from Shewanella benthica KT99. The ORF-4 and ORF-7 encoded putative proteins of 189 and 118 amino acid residues, respectively, with no homologies to any known proteins. The absolute copy number of pSFKW33 was estimated to be five copies per chromosome by real-time PCR.