Toxicity of the bacteriophage λ cII gene product to Escherichia coli arises from inhibition of host cell DNA replication

AbstractThe bacteriophage λ cII gene codes for a transcriptional activator protein which is a crucial regulator at the stage of the “lysis-versus-lysogeny” decision during phage development. The CII protein is highly toxic to the host, Escherichia coli, when overproduced. However, the molecular mechanism of this toxicity is not known. Here we demonstrate that DNA synthesis, but not total RNA synthesis, is strongly inhibited in cII-overexpressing E. coli cells. The toxicity was also observed when the transcriptional stimulator activity of CII was abolished either by a point mutation in the cII gene or by a point mutation, rpoA341, in the gene coding for the RNA polymerase α subunit. Moreover, inhibition of cell growth, caused by both wild-type and mutant CII proteins in either rpoA+ or rpoA341 hosts, could be relieved by overexpression of the E. coli dnaB and dnaC genes. In vitro replication of an oriC-based plasmid DNA was somewhat impaired by the presence of the CII, and several CII-resistant E. coli strains contain mutations near dnaC. We conclude that the DNA replication machinery may be a target for the toxic activity of CII

Expression and display of UreA of Helicobacter acinonychis on the surface of Bacillus subtilis spores

<p>Abstract</p> <p>Background</p> <p>The bacterial endospore (spore) has recently been proposed as a new surface display system. Antigens and enzymes have been successfully exposed on the surface layers of the <it>Bacillus subtilis </it>spore, but only in a few cases the efficiency of expression and the effective surface display and have been determined. We used this heterologous expression system to produce the A subunit of the urease of the animal pathogen <it>Helicobater acinonychis</it>. Ureases are multi-subunit enzymes with a central role in the virulence of various bacterial pathogens and necessary for colonization of the gastric mucosa by the human pathogen <it>H. pylori</it>. The urease subunit UreA has been recognized as a major antigen, able to induce high levels of protection against challenge infections.</p> <p>Results</p> <p>We expressed UreA from <it>H. acinonychis </it>on the <it>B. subtilis </it>spore coat by using three different spore coat proteins as carriers and compared the efficiency of surface expression and surface display obtained with the three carriers. A combination of western-, dot-blot and immunofluorescence microscopy allowed us to conclude that, when fused to CotB, UreA is displayed on the spore surface (ca. 1 × 10³recombinant molecules per spore), whereas when fused to CotC, although most efficiently expressed (7-15 × 10³recombinant molecules per spore) and located in the coat layer, it is not displayed on the surface. Experiments with CotG gave results similar to those with CotC, but the CotG-UreA recombinant protein appeared to be partially processed.</p> <p>Conclusion</p> <p>UreA was efficiently expressed on the spore coat of <it>B. subtilis </it>when fused to CotB, CotC or CotG. Of these three coat proteins CotC allows the highest efficiency of expression, whereas CotB is the most appropriate for the display of heterologous proteins on the spore surface.</p

Mapping of a transcription promoter located inside the priA gene of the Bacillus subtilis chromosome

The genome sequence of the Gram-positive soil bacterium Bacillus subtilis was completed in 1997 (Kunst et al., 1998) and the results included the identification of a putative transcription unit encompassing the yloI to yloS genes. Within this region of the B. subtilis chromosome 11 putative open reading frames were found with a wide diversity of probable functions. In this work we have analyzed transcription in the region of the priA-cpgA genes and we have mapped a promoter which is located inside the priA gene and its activity directs transcription of the def-yloM genes. Moreover, this transcript can be extended at low level to the prpC-priK-cpgA genes. Analysis of the sequence in proximity of the transcription start site revealed a sequence suitable for the housekeeping σA subunit of RNA polymerase. Analysis of the β-glactosidase activity of transcription fusions revealed that the identified promoter is active at low level and its activity is increased during late exponential phase of growth

PrpE, a PPP protein phosphatase from Bacillus subtilis with unusual substrate specificity.

Bacillus subtilis is a Gram-positive bacterium with a relatively large number of protein phosphatases. Previous studies have shown that some Ser/Thr phosphatases play an important role in the life cycle of this bacterium [Losick and Stragier (1992) Nature (London) 355, 601-604; Yang, Kang, Brody and Price (1996) Genes Dev. 10, 2265-2275]. In this paper, we report the biochemical properties of a putative, previously uncharacterized phosphatase, PrpE, belonging to the PPP family. This enzyme shares homology with other PPP phosphatases as well as with symmetrical diadenosine tetraphosphatases related to ApaH (symmetrical Ap(4)A hydrolase) from Escherichia coli. A His-tagged recombinant PrpE was purified from E. coli and shown to have Ni(2+)-dependent and okadaic acid-resistant phosphatase activity against a synthetic phosphorylated peptide and hydrolase activity against diadenosine 5',5"'-tetraphosphate. Unexpectedly, PrpE was able to remove phosphate from phosphotyrosine, but not from phosphothreonine or phosphoserine

The combination of recombinant and non-recombinant Bacillus subtilis spore display technology for presentation of antigen and adjuvant on single spore

Abstract Background Bacillus subtilis spores can be used for presentation of heterologous proteins. Two main approaches have been developed, the recombinant one, requiring modification of bacterial genome to express a protein of interest as a fusion with spore-coat protein, and non-recombinant, based on the adsorption of a heterologous protein onto the spore. So far only single proteins have been displayed on the spore surface. Results We have used a combined approach to adsorb and display FliD protein of Clostridium difficile on the surface of recombinant IL-2-presenting spores. Such spores presented FliD protein with efficiency comparable to FliD-adsorbed spores produced by wild-type 168 strain and elicited FliD-specific immune response in intranasally immunized mice. Conclusions Our results indicate that such dual display technology may be useful in creation of spores simultaneously presenting adjuvant and antigen molecules. Regarding the characteristics of elicited immune response it seems plausible that such recombinant IL-2-presenting spores with adsorbed FliD protein might be an interesting candidate for vaccine against infections with Clostridium difficile

The choice of the anchoring protein influences the interaction of recombinant Bacillus spores with the immune system

The technology of display of heterologous proteins on the surface of Bacillus subtilis spores enables use of these structures as carriers of antigens for mucosal vaccination. Currently, there are no technical possibilities to predict whether a designed fusion will be efficiently displayed on the spore surface and how such recombinant spores will interact with cells of the immune system. In this study, we compared four variants of B. subtilis spores presenting a fragment of a FliD protein from Clostridium difficile in fusion with CotB, CotC, CotG or CotZ spore coat proteins. We show that these spores promote their own phagocytosis and activate both, the J774 macrophages and JAWSII dendritic cells of murine cell lines. Moreover, we used these spores for mucosal immunization of mice. We conclude that the observed effects vary with the type of displayed FliD-spore coat protein fusion and seem to be mostly independent of its abundance and localization in the spore coat structure

Mucosal adjuvant activity of IL-2 presenting spores of bacillus subtilis in a murine model of Helicobacter pylori vaccination.

The endospores of Bacillus subtilis are now widely used as a platform for presentation of heterologous proteins and due to their safety record and high resistance to harsh environmental conditions can be considered as potential vehicles for oral vaccination. In this research we show that recombinant B. subtilis spores presenting a fragment of the Helicobacter acinonychis UreB protein and expressing the ureB gene under vegetative promoter elicit a strong cellular immune response in orally immunized mice when co-administered with spores presenting IL-2. We show for the first time the successful application of two types of recombinant spores, one carrying an antigen and the other an adjuvant, in a single oral immunization

Protective effects of voltage-gated calcium channel antagonists against zinc toxicity in SN56 neuroblastoma cholinergic cells

One of the pathological site effects in excitotoxic activation is Zn2+ overload to postsynaptic neurons. Such an effect is considered to be equivalent to the glutamate component of excitotoxicity. Excessive uptake of Zn2+ by active voltage-dependent transport systems in these neurons may lead to significant neurotoxicity. The aim of this study was to investigate whether and which antagonists of the voltage gated calcium channels (VGCC) might modify this Zn2+-induced neurotoxicity in neuronal cells. Our data demonstrates that depolarized SN56 neuronal cells may take up large amounts of Zn2+ and store these in cytoplasmic and mitochondrial sub-fractions. The mitochondrial Zn2+ excess suppressed pyruvate uptake and oxidation. Such suppression was caused by inhibition of pyruvate dehydrogenase complex, aconitase and NADP-isocitrate dehydrogenase activities, resulting in the yielding of acetyl-CoA and ATP shortages. Moreover, incoming Zn2+ increased both oxidized glutathione and malondialdehyde levels, known parameters of oxidative stress. In depolarized SN56 cells, nifedipine treatment (L-type VGCC antagonist) reduced Zn2+ uptake and oxidative stress. The treatment applied prevented the activities of PDHC, aconitase and NADP-IDH enzymes, and also yielded the maintenance of acetyl-CoA and ATP levels. Apart from suppression of oxidative stress, N- and P/Q-type VGCCs presented a similar, but weaker protective influence. In conclusion, our data shows that in the course of excitotoxity, impairment to calcium homeostasis is tightly linked with an excessive neuronal Zn2+ uptake. Hence, the VGCCs types L, N and P/Q share responsibility for neuronal Zn2+ overload followed by significant energy-dependent neurotoxicity. Moreover, Zn2+ affects the target tricarboxylic acid cycle enzymes, yields acetyl-CoA and energy deficits as well