Disassembly of the Coliphage λ Replication Complex Due to Heat Shock Induction of thegroEOperon

AbstractWe have found previously that, in contrast to the free O initiator protein of λ phage or plasmid rapidly degraded by theEscherichia coliClpP/ClpX protease, the λO present in the replication complex (RC) is protected from proteolysis. In amino acid-starvedE. coli relAcells, a temperature shift from 30 to 43° did not affect RC integrity, as judged from the unchanged level of stable λO observed; however, the same temperature shift in a complete medium resulted in the decay of this λO fraction, which suggested disassembly of the RC. Examination of this phenomenon revealed that for λ RC disassembly, heat shock induction of thegroEoperon, coding for molecular chaperones of the Hsp60 class, is indispensable. Heat shock induction of thegroEoperon present on a multicopy plasmid inhibited the growth of infecting phage

Replication of plasmids derived from Shiga toxinconverting bacteriophages in starved Escherichia coli

The pathogenicity of Shiga toxin-producing Escherichia coli (STEC) depends on the expression of stx genes that are located on lambdoid prophages. Effective toxin production occurs only after prophage induction, and one may presume that replication of the phage genome is important for an increase in the dosage of stx genes, positively influencing their expression. We investigated the replication of plasmids derived from Shiga toxin (Stx)-converting bacteriophages in starved E. coli cells, as starvation conditions may be common in the intestine of infected humans. We found that, unlike plasmids derived from bacteriophage lambda, the Shiga toxin phage-derived replicons did not replicate in amino acid-starved relA+ and relA” cells (showing the stringent and relaxed responses to starvation, respectively). The presence of the stable fraction of the replication initiator O protein was detected in all tested replicons. However, while ppGpp, the stringent response effector, inhibited the activities of the l PR promoter and its homologues from Shiga toxin-converting bacteriophages, these promoters, except for lambda PR, were only weakly stimulated by the DksA protein. We suggest that this less efficient (relative to lambda) positive regulation of transcription responsible for transcriptional activation of the origin contributes to the inhibition of DNA replication initiation of Shiga toxin-converting bacteriophages in starved host cells, even in the absence of ppGpp (as in starved relA” hosts). Possible clinical implications of these results are discussed

A novel method for screening the glutathione transferase inhibitors

<p>Abstract</p> <p>Background</p> <p>Glutathione transferases (GSTs) belong to the family of Phase II detoxification enzymes. GSTs catalyze the conjugation of glutathione to different endogenous and exogenous electrophilic compounds. Over-expression of GSTs was demonstrated in a number of different human cancer cells. It has been found that the resistance to many anticancer chemotherapeutics is directly correlated with the over-expression of GSTs. Therefore, it appears to be important to find new GST inhibitors to prevent the resistance of cells to anticancer drugs. In order to search for glutathione transferase (GST) inhibitors, a novel method was designed.</p> <p>Results</p> <p>Our results showed that two fragments of GST, named F1 peptide (G<b>YW</b>KIKG<b>L</b>V) and F2 peptide (KW<b>R</b>NK<b>K</b>FELGLEFP<b>N</b>L), can significantly inhibit the GST activity. When these two fragments were compared with several known potent GST inhibitors, the order of inhibition efficiency (measured in reactions with 2,4-dinitrochlorobenzene (CDNB) and glutathione as substrates) was determined as follows: tannic acid > cibacron blue > F2 peptide > hematin > F1 peptide > ethacrynic acid. Moreover, the F1 peptide appeared to be a noncompetitive inhibitor of the GST-catalyzed reaction, while the F2 peptide was determined as a competitive inhibitor of this reaction.</p> <p>Conclusion</p> <p>It appears that the F2 peptide can be used as a new potent specific GST inhibitor. It is proposed that the novel method, described in this report, might be useful for screening the inhibitors of not only GST but also other enzymes.</p

Small and Smaller—sRNAs and MicroRNAs in the Regulation of Toxin Gene Expression in Prokaryotic Cells: A Mini-Review

Non-coding small RNAs (sRNAs) have been identified in the wide range of bacteria (also pathogenic species) and found to play an important role in the regulation of many processes, including toxin gene expression. The best characterized prokaryotic sRNAs regulate gene expression by base pairing with mRNA targets and fall into two broad classes: cis-encoded sRNAs (also called antisense RNA) and trans-acting sRNAs. Molecules from the second class are frequently considered as the most related to eukaryotic microRNAs. Interestingly, typical microRNA-size RNA molecules have also been reported in prokaryotic cells, although they have received little attention up to now. In this work we have collected information about all three types of small prokaryotic RNAs in the context of the regulation of toxin gene expression

Effects of the presence of ColE1 plasmid DNA in Escherichia coli on the host cell metabolism

BACKGROUND: Although understanding of physiological interactions between plasmid DNA and its host is important for vector design and host optimization in many biotechnological applications, to our knowledge, global studies on plasmid-host interactions have not been performed to date even for well-characterized plasmids. RESULTS: Escherichia coli cells, either devoid of plasmid DNA or bearing plasmid pOri1 (with a single ColE1 replication origin) or plasmid pOri2 (with double ColE1 replication origins), were cultured in a chemostat. We used a combination of metabolic flux analysis, DNA microarray and enzyme activity analysis methods to explore differences in the metabolism between these strains. We found that the presence of plasmids significantly influenced various metabolic pathways in the host cells, e.g. glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate (PP) pathway. Expression of rpiA, a gene coding for ribose-5-phosphate isomerase A, was considerably decreased in E. coli carrying a high copy number plasmid relative to E. coli carrying a low copy number plasmid and plasmid-free E. coli. The rpiA gene was cloned into an expression vector to construct plasmid pETrpiA. Following induction of pETrpiA-bearing E. coli, which harbored either pOri1 or pOri2, with isopropyl-β-D-thiogalactopyranoside (IPTG), the copy number of pOri1 and pOri2 was sigificantly higher than that measured in a host devoid of pETrpiA. CONCLUSION: The presence of plasmids can significantly influence some metabolic pathways in the host cell. We believe that the results of detailed metabolic analysis may be useful in optimizing host strains, vectors and cultivation conditions for various biotechnological purposes

A lack of Wolbachia-specific DNA in samples from apollo butterfly (Parnassius apollo, Lepidoptera : papilionidae) individuals with deformed or reduced wings

Various insects contain maternally inherited endosymbiotic bacteria which can cause reproductive alterations, modulation of some physiological responses (like immunity, heat shock response, and oxidative stress response), and resistance to viral infections. In butterflies, Wolbachia sp. is the most frequent endosymbiont from this group, occurring in about 30 % of species tested to date. In this report, the presence of Wolbachia-specific DNA has been detected in apollo butterfly (Parnassius apollo). In the isolated population of this insect occurring in Pieniny National Park (Poland), malformed individuals with deformed or reduced wings appear with an exceptionally high frequency. Interestingly, while total DNA isolated from most (about 85 %) normal insects contained Wolbachia-specific sequences detected by PCR, such sequences were absent in a large fraction (70 %) of individuals with deformed wings and in all tested individuals with reduced wings. These results indicate for the first time the correlation between malformation of wings and the absence of Wolbachia sp. in insects. Although the lack of the endosymbiotic bacteria cannot be considered as the sole cause of the deformation or reduction of wings, one might suggest that Wolbachia sp. could play a protective role in the ontogenetic development of apollo butterfly

Differential inhibition of transcription from σ70- and σ32-dependent promoters by rifampicin

AbstractRifampicin is an antibiotic which binds to the β subunit of prokaryotic RNA polymerases and prevents initiation of transcription. It was found previously that production of heat shock proteins in Escherichia coli cells after a shift from 30°C to 43°C is not completely inhibited by this antibiotic. Here we demonstrate that while activity of a pL-lacZ fusion (pL is a σ70-dependent promoter) in E. coli cells is strongly inhibited by rifampicin, a pgroE-lacZ fusion, whose activity is dependent on the σ32 factor, retains significant residual activity even at relatively high rifampicin concentrations. Differential sensitivity to this antibiotic of RNA polymerase holoenzymes containing either the σ70 or the σ32 subunit was confirmed in vitro. Since the effects of an antibiotic that binds to the β subunit can be modulated by the presence of either the σ70 or the σ32 subunit in the holoenzyme, it is tempting to speculate that binding of various σ factors to the core of RNA polymerase results in different conformations of particular holoenzymes, including changes in the core enzyme

Detection of Yersinia pseudotuberculosis in Apollo Butterfly (Parnassius apollo, Lepidoptera: Papilionidae) Individuals from a Small, Isolated, Mountain Population

Yersinia pseudotuberculosis is a bacterium pathogenic to humans and other mammals; however, its insecticidal activity has also been documented in laboratory studies. A small population of Apollo butterfly (Parnassius apollo), reconstituted from less than 30 individuals in 1990s, occurs in Pieniny National Park (Poland). In this report, we demonstrate that a DNA fragment specific to Y. pseudotuberculosis could be detected in 40% of biological samples isolated from insects belonging to the Apollo butterfly population. Although Y. pseudotuberculosis DNA occurred in both normal and malformed insects, the difference between the fractions of infected individuals was statistically significant (p = 0.044 in the Fisher\u27s exact test). No such DNA could be detected in analogous samples from other butterflies (Pieris napi, Pieris rapae, and Zerynthia polyxena) occurring in separate habitats (either a meadow near the city of Cracow, Poland, or in a mountain region of Greece). It is suggested that infection with Y. pseudotuberculosis might weaken the general condition of the P. apollo population from Pieniny and contribute to the appearance of developmental abnormalities of the butterflies. Thus, it appears that Y. pseudotuberculosis infections of insects may be of biological significance in natural environment

Phage display-selected peptides for binding and synthesis of nanoparticles: ZnO as an example

Nanoparticles of metal oxides are widely used in bionanotechnology, particularly in bio-medical applications; e.g., construction of biosensors, separation of biological materials, molecular imaging, and anticancer and antimicrobial therapies. However, synthesis of these nanoparticles using physico-chemical methods is problematic, because such procedures require high-temperature processes and harsh chemical treatments. The use of peptides specifically binding particular nanoparticles or nanostructures and facilitating their synthesis appears to be an encouraging alternative. Specific peptides capable of such reactions may be identified with the use of the phage display method. In this mini-review, zinc oxide is discussed as an exemple material whose nanoparticles can be bound and synthesized by such peptides exposed on the surface of bacteriophage capsids. An analysis of reports on studies into methods of peptide-aided synthesis of ZnO nanoparticles has indicated that, despite the encouraging results obtained so far, further studies are necessary to optimize such procedures. This may also be true for nanoparticles of other materials, particularly metal oxides

Coupling of transcription and replication machineries in the λ DNA replication initiation: evidence for direct interaction of Escherichia coli RNA polymerase and the lambda O protein

Transcription proceeding downstream of the lambda phage replication origin was previously shown to support initial steps of the lambda primosome assembly in vitro and to regulate frequency and directionality of lambda DNA replication in vivo. In this report, the data are presented indicating that the RNA polymerase beta subunit makes a direct contact with the lambdaO protein, a replication initiator of lambda phage. These results suggest that the role of RNA polymerase during the initiation of lambda phage DNA replication may be more complex than solely influencing DNA topology. Results demonstrated in this study also show that gyrase supercoiling activity stimulates the formation of a complex between lambdaO and RNA polymerase, suggesting that the introduction of negative supercoils by DNA gyrase, besides lowering the energy required for DNA strand separation, may play an additional role in modeling protein–protein interactions at early steps of DNA replication initiation