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

When genome-based approach meets the “Old but Good”: revealing genes involved in the antibacterial activity of Pseudomonas sp. P482 against soft rot pathogens

Dickeya solani and Pectobacterium carotovorum subsp. brasiliense are recently established species of bacterial plant pathogens causing black leg and soft rot of many vegetables and ornamental plants. Pseudomonas sp. strain P482 inhibits the growth of these pathogens, a desired trait considering the limited measures to combat these diseases. In this study, we determined the genetic background of the antibacterial activity of P482, and established the phylogenetic position of this strain. Pseudomonas sp. P482 was classified as Pseudomonas donghuensis. Genome mining revealed that the P482 genome does not contain genes determining the synthesis of known antimicrobials. However, the ClusterFinder algorithm, designed to detect atypical or novel classes of secondary metabolite gene clusters, predicted 18 such clusters in the genome. Screening of a Tn5 mutant library yielded an antimicrobial negative transposon mutant. The transposon insertion was located in a gene encoding an HpcH/HpaI aldolase/citrate lyase family protein. This gene is located in a hypothetical cluster predicted by the ClusterFinder, together with the downstream homologs of four nfs genes, that confer production of a non-fluorescent siderophore by P. donghuensis HYS(T). Site-directed inactivation of the HpcH/HpaI aldolase gene, the adjacent short chain dehydrogenase gene, as well as a homolog of an essential nfs cluster gene, all abolished the antimicrobial activity of the P482, suggesting their involvement in a common biosynthesis pathway. However, none of the mutants showed a decreased siderophore yield, neither was the antimicrobial activity of the wild type P482 compromised by high iron bioavailability. A genomic region comprising the nfs cluster and three upstream genes is involved in the antibacterial activity of P. donghuensis P482 against D. solani and P. carotovorum subsp. brasiliense. The genes studied are unique to the two known P. donghuensis strains. This study illustrates that mining of microbial genomes is a powerful approach for predictingthe presence of novel secondary-metabolite encoding genes especially when coupled with transposon mutagenesis

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

Long-term allogeneic hematopoietic cells transplantation survivors proinflammatory cytokine profile compared to their respective donors and immunophenotype differences depending on GvHD history and infection status

Background In the course of allogeneic hematopoietic cell transplantation (allo-HCT) the donor’s hematopoietic progenitor cells are exposed to immense proliferative stress to reconstitute in the recipient the functional hematopoiesis. Moreover, recipients who develop infections or chronic GvHD are subjected to further proliferative stress, especially in the lymphocyte subset. We hypothesized that allo-HCT may induce changes in proinflammatory cytokines profile and immunophenotype in the allo-HCT recipients, especially in patients with cGVHD history. We compared the cytokine profile (Il-6, Il-10, and TNF-) between long-term allo-HCT recipients and their respective donors and we analyzed cytokines profile and the immunophenotype of lymphocytes in long-term recipients grouped according to the infection and GvHD history. Results We have found no differences in the proinflammatory cytokines between allo-HCT recipients and their respective donors, as well as between recipients grouped according to infectious risk status. Immunophenotyping of recipients grouped according to GvHD status revealed an increased percentage of B-cell presenting PD-1 in recipients without a history of GvHD. Conclusions Lack of differences in proinflammatory cytokines concentrations between recipients and donors of allo-HCT would suggest that allo-HCT does not induce acceleration of the inflammageing-resembling phenomenon. No differences in the cytokine profile and immunophenotype between recipients grouped according to infectious risk status suggest that infectious risk is not reflected by the immunophenotype and cytokine profile. Furthermore, the lack of significant differences in immunophenotype of the recipients grouped according to the history of GvHD may suggest that in long-term survivors the immune system tends to stabilize with time

ATP and its N6-substituted analogues: parameterization, molecular dynamics simulation and conformational analysis

In this work we used a combination of classical molecular dynamics and simulated annealing techniques to shed more light on the conformational flexibility of 12 adenosine triphosphate (ATP) analogues in a water environment. We present simulations in AMBER force field for ATP and 12 published analogues [Shah et al. (1997) Proc Natl Acad Sci USA 94: 3565–3570]. The calculations were carried out using the generalized Born (GB) solvation model in the presence of the cation Mg2+. The ion was placed at a close distance (2 Å) from the charged oxygen atoms of the beta and gamma phosphate groups of the −3 negatively charged ATP analogue molecules. Analysis of the results revealed the distribution of inter-proton distances H8–H1′ and H8–H2′ versus the torsion angle ψ (C4–N9-C1′–O4′) for all conformations of ATP analogues. There are two gaps in the distribution of torsion angle ψ values: the first is between −30 and 30 degrees and is described by cis-conformation; and the second is between 90 and 175 degrees, which mostly covers a region of anti conformation. Our results compare favorably with results obtained in experimental assays [Jiang and Mao (2002) Polyhedron 21:435–438]

Transformer-Capsule Model for Intent Detection (Student Abstract)

Intent recognition is one of the most crucial tasks in NLU systems, which are nowadays especially important for designing intelligent conversation. We propose a novel approach to intent recognition which involves combining transformer architecture with capsule networks. Our results show that such architecture performs better than original capsule-NLU network implementations and achieves state-of-the-art results on datasets such as ATIS, AskUbuntu ,and WebApp