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Biomedical subjects

P Cegłowski

Publications and source records attributed to P Cegłowski.

12 recordsLinked to original sources
PubMed2007

Mosaic structure of p1658/97, a 125-kilobase plasmid harboring an active amplicon with the extended-spectrum beta-lactamase gene blaSHV-5.

Escherichia coli isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum beta-lactamase (ESBL) of the SHV type. The beta-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64, Providencia rettgeri genomic island R391, Vibrio cholerae STX transposon, Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in E. coli. The presence of an active partition system (sopABC locus) and two postsegregational killing systems (pemIK and hok/sok) indicates that the plasmid should be stably maintained in E. coli populations. The conjugative transfer is ensured by the operons of the tra and trb genes. We also demonstrate that the plasmidic segment undergoing amplification contains the blaSHV-5 gene and is homologous to a 7.9-kb fragment of the K. pneumoniae chromosome. The amplicon displays the structure of a composite transposon of type I.

DNA, Bacterial↗
PubMed2001

Mechanisms of plasmid stable maintenance with special focus on plasmid addiction systems.

The stable inheritance of bacterial plasmids is achieved by a number of different mechanisms. Among them are resolution of plasmid oligomers into monomers, active plasmid partitioning into dividing cells and selective killing of plasmid-free segregants. A special focus is given to the last mechanism. It involves a stable toxin and an unstable antidote. The antidotes neutralize their cognate toxins or prevent their synthesis. The different decay rates of the toxins and the antidotes underlie molecular mechanisms of toxin activation in plasmid-free cells. By eliminating of plasmid-free cells from the population of plasmid-bearing ones the toxin-antidote couples therefore act as plasmid addiction systems.

Bacteriophages↗
PubMed1995

Secretion of streptokinase fusion proteins from Escherichia coli cells through the hemolysin transporter.

The hemolysin (HlyA) secretion system was used to achieve the sec-independent secretion of streptokinase (Skc) originating from Streptococcus equisimilis into the medium by Escherichia coli cells. The in-frame fusions of the skc gene, either possessing or lacking a region encoding the signal peptide (SP) with the 3'-end of the hlyA gene of various lengths were analysed. All hybrids retained Skc activity. Hybrid proteins devoided of the N-terminal SP, regardless of length of the hlyA secretion signal (62 vs. 194 amino acids), were secreted into the medium by the E. coli HlyA transporter at similar levels. Considerable amounts of hybrid proteins were still, however, associated with E. coli cells, mainly in the degraded form.

Bacterial Proteins↗
PubMed1995

Cloning and molecular analysis of the dihydrofolate reductase gene from Lactococcus lactis.

The Lactococcus lactis gene encoding trimethoprim resistance has been cloned and expressed in Escherichia coli and Bacillus subtilis. Several lines of evidence indicate that the cloned gene encodes dihydrofolate reductase (DHFR). (i) It fully complements the fol "null" mutation in E. coli. (ii) Nucleotide sequencing of the cloned fragment revealed the presence of one open reading frame encoding a protein that shares homology with the family of bacterial DHFR enzymes. (iii) Overexpression of this open reading frame in E. coli resulted in the appearance in cell extracts of a protein of the expected size as well as in a dramatic increase of DHFR activity. In cell extracts, the DHFR activity was not inhibited by low trimethoprim concentration. By Northern (RNA) blotting and primer extension analyses, the size and the start point of the dhfr transcript, respectively, have been determined. Results of these experiments indicate that in L. lactis the dhfr gene represents part of a larger transcription unit.

Amino Acid Sequence↗
PubMed1994

Gene organization of the Streptococcus pyogenes plasmid pDB101: sequence analysis of the orf eta-copS region.

The gene organization of the broad-host-range low-copy-number pSM19035-derived plasmid pDB101 is presented. Analysis of the 19,202-bp sequence revealed thirteen different open reading frames (orfs). Nine of these orfs (repS-orf-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1 have been previously identified [Cegłowski et al., Gene 136 (1993) 1-12]. The extraordinarily long inverted repeated sequence, which includes orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta, comprises 76% of the pDB101 molecule. The gene order in pDB101 is repS-orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1-orf zeta-orf epsilon-orf delta-orf gamma-orf beta-orf alpha-orf eta-orf theta-orf1-copS. The organization of genes of the orf eta-orf gamma region resembles the organization of genes in the orfA-orfI region of pAM beta 1. Except for Orf1, bands of radioactive proteins corresponding to the molecular mass of the deduced reading frames (26.7, 14.3 and 10.3 kDa) were detected using the T7 promoter-expression system. The orf1 encoded a product (deduced molecular mass 28.3 kDa) which shows anomalous electrophoretical mobility corresponding to 60 kDa. The copS- and orf1-encoded proteins share homology to plasmid copy number control systems and Gram+ cocci surface proteins, respectively. The orf eta and orf theta encode proteins with unknown activity.

Amino Acid Sequence↗
PubMed1993

Analysis of the stabilization system of pSM19035-derived plasmid pBT233 in Bacillus subtilis.

The low-copy-number, 9.0-kb pSM19035-derived plasmid pBT233, is stably inherited in Bacillus subtilis. The complete nucleotide (nt) sequence of pBT233 has been determined. Analysis of the nt sequence revealed nine major open reading frames (orfs). The repS, erm1 and erm2 genes have been assigned to three of these orfs, and given the gene order, repS-orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1. The organization of genes of the repS-orf gamma region resembles the organization of genes in the repE-orfI region of pAM beta 1. Messenger RNA species of molecular weights corresponding to repS, orf alpha + orf beta, orf gamma, orf delta and orf epsilon + orf zeta were detected by Northern blotting. Proteins of 23.8, 81.3, 34.4, 10.7 and 32.4 kDa correspond to Orfs beta, gamma, delta, epsilon and zeta, respectively. Bands of radioactive proteins of 25, 81, 34, 10 and 32 kDa were detected using the T7 promoter-expression system. The orf beta and orf gamma encode proteins that share homology to site-specific recombinases and type-I topoisomerases, respectively. The orfs, delta, epsilon and zeta, encode proteins with unknown activity. Deletion of a 1.5-kb segment (nt 2999-4552) with coding capacity for orf beta, orf gamma and orf delta does not seem to affect plasmid maintenance. Removal of a 3.0-kb fragment (nt 4598-7689) with coding capacity for orf epsilon and orf zeta reduced plasmid segregational stability, but deletion of a 5.2-kb DNA segment (nt 2546-7826) abolished it.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Isolation, sequence and expression in Escherichia coli, Bacillus subtilis and Lactococcus lactis of the DNase (streptodornase)-encoding gene from Streptococcus equisimilis H46A.

A partial library of BclI-generated chromosomal DNA fragments from Streptococcus equisimilis H64A (Lancefield Group C) was constructed in Escherichia coli. Clones displaying either streptokinase or deoxyribonuclease (streptodornase; SDC) activities were isolated. The gene (sdc) expressing the SDC activity was allocated on the 1.1-kb AccI DNA subfragment. Sequence analysis of this DNA fragment revealed the presence of one open reading frame, which could encode a protein of 36.8 kDa. The N-terminal portion of the deduced protein exhibited features characteristic of prokaryotic signal peptides. The sdc gene was expressed in E. coli, Bacillus subtilis and Lactococcus lactis. As observed for S. equisimilis, in the heterologous Gram + hosts, at least part of the SDC protein was secreted into the medium.

Amino Acid Sequence↗
PubMed1982

Construction of plasmid vectors for gene cloning in Escherichia coli and Bacillus subtilis.

The construction and some properties of new hybrid plasmids which are able to replicate in both Escherichia coli and Bacillus subtilis are presented. A 5.5 Md hybrid plasmid pJP9 was constructed from pBR322 (Tc, Ap) and pUB110 (Nm) plasmids. pIM1 (7.0 Md) and pIM3 (7.7 Md) plasmids are its different erythromycin resistant derivatives. Tetracycline, ampicillin, neomycin and possibly erythromycin resistance genes are expressed in E. coli while neomycin and erythromycin resistance genes are expressed in B. subtilis. Insertional inactivation of only one gene is possible using the pJP9 plasmid as a vector in B. subtilis. However, insertional inactivation of at least two different genes can be achieved and monitored in E. coli and B. subtilis transformants in cloning experiments with PIM1 and pIM3 plasmids. Insertional inactivation of antibiotic resistance genes present in pJP9 plasmid was achieved by cloning of Streptococcus sanguis DNA fragments generated by appropriate restriction endonucleases. The pJP9 plasmid and its derivatives were found to be stable in both hosts cells.

Anti-Bacterial Agents↗
PubMed1981

Role of Streptococcus sanguis (strain Wicky) cell surface-located deoxyribonucleic acid-binding factor in transformation of a homologous strain.

In a previous report we demonstrated the presence of a factor binding deoxyribonucleic acid (DNA) in vitro (BF) in cell leakage fluids from transformable Streptococcus sanguis strains Wicky, Challis, and Blackburn. BF originating from strain Wicky was purified to homogeneity, and its properties are described. In this work, it was found that BF occurs at the surface of Wicky cells in two forms, loosely bound (LB-BF) and strongly bound to the cell envelope. It was demonstrated that LB-BF formed fast-sedimenting complexes with exogenous DNA at the surface of Wicky cells. About 10-fold-more DNA became associated as a fast-sedimenting complex in competent than in incompetent cells. Thus, LB-BF is a cell receptor for exogenous DNA. However, the comparison of the effects of some agents on the transformation yield and the formation of LB-BF-DNA complexes, showed that the influence of these agents on both observed phenomena is not parallel and may be even opposite. These results are interpreted to mean that the LB-BF-DNA complexes do not take part in transformation. The problem of participation of BF strongly bound with the cell membrane fraction remains to be elucidated.

Carrier Proteins↗
PubMed1980

Purification and properties of deoxyribonucleic acid binding factor isolated from the surface of Streptococcus sanguis cells.

Deoxyribonucleic acid (DNA) binding factor (BF) was found in surface fluids from competent and noncompetent cells of Streptococcus sanguis strains Challis, Wicky, and Blackburn. Fluids from noncompetent cells exhibited about 10% BF activity compared with extracts from competent cells. BF from competent Wicky cells was purified to homogeneity by electrophoresis and immunodiffusion. Purified BF preparations exhibited slight endonucleolytic activity, directed mainly against single-stranded DNA. Nucleolytic and DNA binding activities present in purified BF could be separated by polyacrylamide gel electrophoresis. Purified BF was sensitive to proteolytic enzymes and to phospholipase D, and its activity was stimulated in the presence of low Triton X-100 concentrations. The protein component of BF is a single, monomeric polypeptide with a molecular weight of 56,000 and an isoelectric point of pH 5.8. Binding of purified BF to DNA was a very rapid process at the optimum temperature, pH, and ionic strength and led to the formation of fast-sedimenting complexes. Purified BF was tested for several properties. It exhibited higher affinity to single- than to double-stranded DNA. It bound poorly to glucosylated phage T4 and single-stranded, synthetic polydeoxyribonucleotides and did not bind to RNA. It protected single-stranded DNA against nuclease S(1) action but did not protect native DNA against deoxyribonuclease I action. No evidence was found for unwinding activity, using double-stranded DNA as a substrate.

Bacterial Proteins↗
PubMed1978

Competence-related increased enzyme release from Streptococcus sanguis (Wicky) cells.

The ablity of competent and noncompetent Streptococcus sanguis (strain Wicky) cells to release enzymes to the environment was studied. Both competent and noncompetent cells leaked the enzymes tested (aldolase, phosphatase and deoxyribonuclease), but the activities liberated from the competent cells were always roughly 2-fold higher than those released from noncompetent cells. This increased enzyme leakage from competent cells occured in all kinds of media and procedures employed. The leakage of enzymes followed a time-dependent kinetics (different for aldolase and phosphatase), was temperature sensitive and had a pH optimum. The increased enzyme release was most likely not due to cell disruption, but seemed to be rather a consequence of alteration in cell barrier permeability. These results strongly support the "unmasking" model proposed for explanation of competence development in bacteria.

Deoxyribonucleases↗