Molecular dissection of the replication system of plasmid pIGRK encoding two in-frame Rep proteins with antagonistic functions.

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.

Expression and purification of recombinant human insulin from E. coli 20 strain.

The number of people with diabetes is estimated to be over 370 million, in 2030 it will increase to 552 million. In Poland, the number of people with diabetes is estimated to be 3.5 million (9.1%). According to the estimates of the International Diabetes Federation, the percentage of patients in the adult Polish population will increase to around 11% over the next 20 years. Despite the appearance of insulin analogues on the pharmaceutical market, insulin delivery is still the most effective method of pharmacotherapy in cases of extremely high hyperglycemia. A new bacterial host strain (Escherichia coli 20) was obtained at the Institute of Biotechnology and Antibiotics and a new pIBAINS expression vector was constructed that provides greater efficiency in the production of recombinant human insulin. In the IBA Bioengineering Department, successful attempts were made to produce recombinant human insulin on a laboratory and quarter-technical scale, and several batches were performed on a semi-technical scale. The production process has been divided into several stages: 1. biosynthesis of insulin in the fermenter, 2. isolation, purification and dissolution of inclusion bodies, 3. protein renaturation, 4. enzymatic reaction with trypsin, 5. multi-stage purification of insulin using low-pressure and HPLC techniques. At each stage of insulin production, qualitative and quantitative analyses were performed to confirm identity and purity. In particular, the molecular weight of insulin, the amount of insulin and the content of protein impurities were studied. The results of these experiments are presented in this work.

The Redox Balance in Erythrocytes, Plasma, and Periosteum of Patients with Titanium Fixation of the Jaw.

Titanium miniplates and screws are commonly used for fixation of jaw fractured or osteotomies. Despite the opinion of their biocompatibility, in clinical practice symptoms of chronic inflammation around the fixation develop in some patients, even many years after the application of miniplates and screws. The cause of these complications is still an unanswered question. Taking into account that oxidative stress is one of the toxic action of titanium, we have evaluated the antioxidant barrier as well as oxidative stress in the erythrocytes, plasma and periosteum covering the titanium fixation of the jaw. The study group was composed of 32 patients aged 20-30 with inserted miniplates and screws. The antioxidant defense: catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase-1 (SOD1), uric acid (UA), total antioxidant capacity (TAC), as well as oxidative damage products: advanced oxidation protein products (AOPP), advanced glycation end products (AGE), dityrosine, kynurenine, N-formylkynurenine, tryptophan, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), total oxidant status (TOS), and oxidative status index (OSI) were evaluated. SOD1 activity (↓37%), and tryptophan levels (↓34%) showed a significant decrease while AOPP (↑25%), TOS (↑80%) and OSI (↑101%) were significantly elevated in maxillary periosteum of patients who underwent bimaxillary osteotomies as compared to the control group. SOD-1 (↓55%), TAC (↓58.6%), AGE (↓60%) and N-formylkynurenine (↓34%) was statistically reduced while AOPP (↑38%), MDA (↑29%), 4-HNE (↑114%), TOS (↑99%), and OSI (↑381%) were significantly higher in the mandibular periosteum covering miniplates/screw compared with the control tissues. There were no correlations between antioxidants and oxidative stress markers in the periosteum of all patients and the blood. As exposure to the Ti6Al4V titanium alloy leads to disturbances of redox balance in the periosteum surrounding titanium implants of the maxilla and the mandible so antioxidant supplementation should be recommended to the patients undergoing treatment of dentofacial deformities with the use of titanium implants. The results we obtained may also indicate a need to improve the quality of titanium jaw fixations through increase of TiO2 passivation layer thickness or to develop new, the most highly biodegradable materials for their production.

Diversity and Global Distribution of IncL/M Plasmids Enabling Horizontal Dissemination of ß-Lactam Resistance Genes among the Enterobacteriaceae.

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 ß-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 ß-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.

pIGWZ12--A cryptic plasmid with a modular structure.

We studied the detailed structure of the cryptic plasmid pIGWZ12, which was isolated from an Escherichia coli strain. pIGWZ12 is composed of two structural modules of distinct evolutionary origin. The REP module, which contains all the features necessary for replication and stable maintenance in the bacterial cell, was assigned by genotyping to the IncF family. The MOB module, which is responsible for plasmid mobilization, shows significant homology to MOBQ modules from broad-host-range plasmids belonging to the RSF1010/R1162 family. We showed that iterons located in the origin of replication are the target for specific binding by the replication initiator protein RepApIGWZ12. Furthermore, we proved that the promoter for the repA gene overlaps with the iterons, and that the latter are the sole determinant of incompatibility. We performed a mutagenesis analysis of the MOBpIGWZ12 module and characterized the roles played by all identified genes (mobA and mobC), as well as the role played by oriT in mobilization. Finally, we showed that it was possible to remove the MOB module from pIGWZ12 without any loss in plasmid replication and stability. Furthermore, the MOBpIGWZ12 module was fully functional after subcloning into another plasmid. Therefore, pIGWZ12 is yet another example of modular structure in small cryptic plasmids.

New cloning and expression vector derived from Escherichia coli plasmid pIGWZ12; a potential vector for a two-plasmid expression system.

We constructed pIGPZ, a new cloning and expression vector derived from Escherichia coli plasmid pIGWZ12::Kan. pIGPZ contains a kanamycin resistance marker, a multiple-cloning-site (MCS) region, and a promoter for constitutive expression of cloned genes. pIGPZ has the same high level of stability as the original plasmid, even in the absence of antibiotic selection. Furthermore, we show that pIGPZ is compatible with ColE1-based plasmids and a pSC101-like plasmid. All the characteristic elements of theta-replicating plasmids were found in the pIGPZ putative origin of replication. Finally, we demonstrate that pIGPZ can be used in a double-plasmid expression system by co-expressing UBP1 protease from pIGPZ with ubi-interferon alpha (IFNA13; GenBank Accession No. NM_006900.3) or ubi-human growth hormone (ubi-hGH; patent No. WO 2005/066208 A2) cloned in another plasmid. In this system, both ubi-interferon alpha and ubi-human growth hormone were deubiquitinated efficiently in E. coli cells.

Mobilizable narrow host range plasmids as natural suicide vectors enabling horizontal gene transfer among distantly related bacterial species.

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.

X-ray absorption near edge structure and extended X-ray absorption fine structure analysis of Fe(II) aqueous and acetone solutions.

X-ray absorption spectroscopy measurements were used to determine the structure of the first coordination shell of Fe(II) ions in aqueous and acetone based solutions. Extended X-ray absorption fine structure analysis coupled with ab initio X-ray absorption near edge structure calculations confirms the octahedral coordination of the iron ion in water based solution. Data collected for acetone rich solutions can be reproduced assuming coexistence of the octahedral Fe(H(2)O)(6)(2+) and tetrahedral [FeCl(4)](2-) complexes. Distortion of the tetrahedral coordination of ion was detected in some of the acetone based solutions.

The complete sequence and segregational stability analysis of a new cryptic plasmid pIGWZ12 from a clinical strain of Escherichia coli.

A new cryptic plasmid from a multi-resistant, multi-plasmid clinical strain of Escherichia coli has been isolated. The sequence of the 4072-base-pair pIGWZ12 (GenBank Accession No. DQ311641) was determined and analyzed. Two open-reading frames that code for proteins involved in plasmid mobilization and initiation of replication were identified. The putative origin of replication possesses all characteristic features of the theta mechanism for replicating plasmids. pIGWZ12 is stably maintained without selective pressure in bacterial cultures (for up to 80 generations), making it a good candidate for engineering a new cloning vector.

The role of Dam methylation in phase variation of Haemophilus influenzae genes involved in defence against phage infection.

Haemophilus influenzae uses phase variation (PV) to modulate the activity of its defence systems against phage infection. The PV of the restriction-modification (R-M) system HindI, the main defence system against phage infection and incoming chromosomal and phage DNA in H. influenzae Rd, is driven by changes of the pentanucleotide repeat tract within the coding sequence of the hsdM gene and is influenced by lack of Dam methylation. Phase-variable resistance/sensitivity to phage infection correlates with changes in lipooligosaccharide (LOS) structure and occurs by slippage of tetranucleotide repeats within the gene lic2A, coding for a step in the biosynthesis of LOS. The lack of Dam activity destabilizes the tetranuclotide (5'-CAAT) repeat tract and increases the frequency of switching from sensitivity to resistance to phage infection more than in the opposite direction. The PV of the lgtC gene does not influence resistance or sensitivity to phage infection. Insertional inactivation of lic2A, but not lgtC or lgtF, leads to resistance to phage infection and to the same structure of the LOS as observed among phase-variable phage-resistant variants. This indicates that in the H. influenzae Rd LOS only the first two sugars (Glc-Gal) extending from the third heptose are part of bacterial phage receptors.

Characterization of a dam mutant of Haemophilus influenzae Rd.

The gene encoding Dam methyltransferase of Haemophilus influenzae was mutagenized by the insertion of a chloramphenicol-resistance cassette into the middle of the Dam coding sequence. This mutant construct was introduced into the H. influenzae chromosome by transformation and selection for Cam(R) transformants. The authors have shown that several phenotypic properties, resistance to antibiotics, dyes and detergent as well as efficiency of transformation, depend on the Dam methylation state of the DNA. Although the major role of the methyl-directed mismatch repair (MMR) system is to repair postreplicative errors, it seems that in H. influenzae its effect is more apparent in repairing DNA damage caused by oxidative compounds. In the dam mutant treated with hydrogen peroxide, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted into single- and double-strand DNA breaks. This is shown by the increased peroxide sensitivity of the dam mutant and the finding that the sensitivity can be suppressed by a mutH mutation inactivating MMR. In the dam mutant treated with nitrofurazone the resulting damage is not converted into DNA breaks but the high sensitivity is also suppressed by a mutH mutation.