Bacillus thuringiensis strains isolated from Qatari soil, synthesizing δ-endotoxins highly active against the disease vector insect Aedes aegypti Bora Bora

© 2020 Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that has been recognized as an effective bioinsecticide active against plant, animal and human pathogenic and disease vector insects. During its sporulation phase, Bt produces crystals consisting of δ-endotoxins, which upon ingestion kill specifically insect larvae. Bt subsp. israelensis (Bti) is very active against dipteran insects. Bti based bioinsecticides are considered as a sustainable solution to control the Dipteran insects responsible of plant, animal and human diseases. In this study, Bti strains isolated from Qatar soil were analyzed for their insecticidal activities against the dipteran insect Aedes aegypti Bora Bora (Culicidae, Diptera) and for their δ-endotoxins yields per cell. Among the local Bti strains, four exceptional strains producing spherical crystals, were found to be more insecticidal than the reference strain Bti H14. When tested for their δ-endotoxin yield, the Bti QBT217 strain, producing typical spherical crystals and having the best insecticidal activity, was recognized as the best candidate strain for potential bioinsecticide production and biological control of dipteran insects, particularly the disease vector insect A. aegypti.This work was supported by the Qatar National Research Fund [GSRA2-1-0604-14015] and the publication of this article was supported by the Qatar National Library, both of which are members of the Qatar foundation

Identification of Novel Pathogenicity Loci in Clostridium perfringens Strains That Cause Avian Necrotic Enteritis

Type A Clostridium perfringens causes poultry necrotic enteritis (NE), an enteric disease of considerable economic importance, yet can also exist as a member of the normal intestinal microbiota. A recently discovered pore-forming toxin, NetB, is associated with pathogenesis in most, but not all, NE isolates. This finding suggested that NE-causing strains may possess other virulence gene(s) not present in commensal type A isolates. We used high-throughput sequencing (HTS) technologies to generate draft genome sequences of seven unrelated C. perfringens poultry NE isolates and one isolate from a healthy bird, and identified additional novel NE-associated genes by comparison with nine publicly available reference genomes. Thirty-one open reading frames (ORFs) were unique to all NE strains and formed the basis for three highly conserved NE-associated loci that we designated NELoc-1 (42 kb), NELoc-2 (11.2 kb) and NELoc-3 (5.6 kb). The largest locus, NELoc-1, consisted of netB and 36 additional genes, including those predicted to encode two leukocidins, an internalin-like protein and a ricin-domain protein. Pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed that the NE strains each carried 2 to 5 large plasmids, and that NELoc-1 and -3 were localized on distinct plasmids of sizes ∼85 and ∼70 kb, respectively. Sequencing of the regions flanking these loci revealed similarity to previously characterized conjugative plasmids of C. perfringens. These results provide significant insight into the pathogenetic basis of poultry NE and are the first to demonstrate that netB resides in a large, plasmid-encoded locus. Our findings strongly suggest that poultry NE is caused by several novel virulence factors, whose genes are clustered on discrete pathogenicity loci, some of which are plasmid-borne

Partial Restoration of Antibacterial Activity of the Protein Encoded by a Cryptic Open Reading Frame (cyt1Ca) from Bacillus thuringiensis subsp. israelensis by Site-Directed Mutagenesis

Insecticidal crystal proteins of Bacillus thuringiensis belong to two unrelated toxin families: receptor-specific Cry toxins against insects and Cyt toxins that lyse a broad range of cells, including bacteria, via direct binding to phospholipids. A new cyt-like open reading frame (cyt1Ca) encoding a 60-kDa protein, has recently been discovered (C. Berry et al., Appl. Environ. Microbiol. 68:5082-5095, 2002). Cyt1Ca displays the structure of a two-domain fusion protein: the N-terminal moiety resembles the full-length Cyt toxins, and the C-terminal moiety is similar to the receptor-binding domains of several ricin-like toxins, such as Mtx1. Neither the larvicidal activity of cyt1Ca expressed in Escherichia coli nor the hemolytic effect of His-tagged purified Cyt1Ca has been observed (R. Manasherob et al., unpublished). This was attributed to five amino acid differences between the sequences of its N-terminal moiety and Cyt1Aa. The 3′ end of cyt1Ca was truncated (removing the ricin-binding domain of Cyt1Ca), and six single bases were appropriately changed by site-directed mutagenesis, sequentially replacing the noncharged amino acids by charged ones, according to Cyt1Aa, to form several versions. Expression of these mutated cyt1Ca versions caused loss of the colony-forming ability of the corresponding E. coli cells to different extents compared with the original gene. In some mutants this antibacterial effect was associated by significant distortion of cell morphology and in others by generation of multiple inclusion bodies spread along the cell envelope. The described deleterious effects of mutated cyt1Ca versions against E. coli may reflect an evolutionary relationship between Cyt1Aa and Cyt1Ca

YmdB: a stress-responsive ribonuclease-binding regulator of E. coli RNase III activity

The broad cellular actions of RNase III family enzymes include ribosomal RNA (rRNA) processing, mRNA decay, and the generation of noncoding microRNAs in both prokaryotes and eukaryotes. Here we report that YmdB, an evolutionarily conserved 18.8-kDa protein of Escherichia coli of previously unknown function, is a regulator of RNase III cleavages. We show that YmdB functions by interacting with a site in the RNase III catalytic region, that expression of YmdB is transcriptionally activated by both cold-shock stress and the entry of cells into stationary phase, and that this activation requires the σ-factor-encoding gene, rpoS. We discovered that down-regulation of RNase III activity occurs during both stresses and is dependent on YmdB production during cold shock; in contrast, stationary-phase regulation was unperturbed in ymdB-null mutant bacteria, indicating the existence of additional, YmdB-independent, factors that dynamically regulate RNase III actions during normal cell growth. Our results reveal the previously unsuspected role of ribonuclease-binding proteins in the regulation of RNase III activity

Effect of trabecular metal on the elution of gentamicin from Palacos cement

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Germination, Growth, and Sporulation of Bacillus thuringiensis subsp. israelensis in Excreted Food Vacuoles of the Protozoan Tetrahymena pyriformis

Spores of Bacillus thuringiensis subsp. israelensis and their toxic crystals are bioencapsulated in the protozoan Tetrahymena pyriformis, in which the toxin remains stable. Each T. pyriformis cell concentrates the spores and crystals in its food vacuoles, thus delivering them to mosquito larvae, which rapidly die. Vacuoles containing undigested material are later excreted from the cells. The fate of spores and toxin inside the food vacuoles was determined at various times after excretion by phase-contrast and electron microscopy as well as by viable-cell counting. Excreted food vacuoles gradually aggregated, and vegetative growth of B. thuringiensis subsp. israelensis was observed after 7 h as filaments that stemmed from the aggregates. The outgrown cells sporulated between 27 and 42 h. The spore multiplication values in this system are low compared to those obtained in carcasses of B. thuringiensis subsp. israelensis-killed larvae and pupae, but this bioencapsulation represents a new possible mode of B. thuringiensis subsp. israelensis recycling in nontarget organisms

Effect of RNase E and/or G on SOS Response.

<p>(A) Effect of <i>rne</i> and <i>rne rng</i> double null mutations on SOS. β-galactosidase activity encoded by a chromosomally inserted <i>sulA-lacZ</i> fusion was measured in <i>E. coli</i> cells SC5074 (<i>rne,</i> plac03-<i>rne</i>, circles) and SC5070 (<i>rne rng,</i> plac12-<i>rng</i>, rectangles). Cells expressing RNase E or G from an IPTG regulated promoter were cultured, collected, and washed at OD₆₀₀ = 0.1 and then re-cultured for 2 h with or without IPTG to complement or deplete RNase E or G; at time 0 the O.D of the various cultures was adjusted to the same level and SOS was induced by addition of 0.1 µg/ml of MTC. β-galactosidase activity was measured in SC5083 <i>E. coli</i> cells grown in the presence of 0.042 mM IPTG to induce RNase E (•) or in cultures lacking IPTG (○). Both RNase E and G were depleted in <i>E. coli</i> cells containing an <i>rne</i> null mutation complemented by an IPTG-inducible RNase G gene (SC5070; <i>rne::cm, rng::km</i>, plac12-<i>rng</i>) and then tested for later growth in the presence (▪) or absence (□) of IPTG (0.42 mM). The values are averages of at least three independent experiments calculated as the percent of the highest level of β-galactosidase activity (in Miller Units) accumulated in each experiment and standard deviation is indicated by error bars (bars = s.d.). After completion of the experiments we found that <i>rng</i> on the plasmid plac12-<i>rng</i> that was overexpressed is a variant containing a point mutation that not interfere with its ability to compliment the <i>rne</i>. (B) β-galactosidase activity encoded by a chromosomally inserted <i>sulA-lacZ</i> fusion was measured in syngenic strains after addition of MTC (0.1 µg/ml): WT parental cells (SC5080), (□); <i>rng</i> deletion, SC5077 (▴); <i>rne^ts</i>, SC5079 (○), double mutant <i>rng, rne^ts</i>, SC5078 (•) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038426#pone.0038426.s003" target="_blank">Table S1</a>). All strains were shifted from 30°C to 42°C at OD₆₀₀ = 0.1, 2 h prior to SOS induction to inactivate the RNase E in <i>ts</i> mutants. (C) <i>De novo</i> production of β-galactosidase protein from pCM400, Tc^R plasmid by <i>rne</i> mutant (SC5083, <i>rne::cm</i>, plac03-<i>rne</i>, pCM400, Tc^R) cells depleted of RNase E by removal of IPTG, as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038426#pone-0038426-g001" target="_blank">Figure 1A</a>. Arabinose-induced β<i>-</i>galactosidase activity was measured after depletion (○), or in the presence of IPTG-induced RNase E (•). (D) Recovery of SOS response after return of SC5078 strain (double mutant <i>rng, rne^ts</i>) to permissive temperature. MTC (0.1 µg/ml) was added at time 0 to cells grown at 30°C for 90 min; β-galactosidase activity was measured in cultures maintained at 30°C (○), shifted to and maintained at 42°C (▴), or returned to 30°C after 90 min incubation at 42°C (•), (bars = s.d.).</p