Minireplicon from pBtoxis of Bacillus thuringiensis subsp. israelensis.
ABSTRACT: A 2.2-kb fragment containing a replicon from pBtoxis, the large plasmid that encodes the insecticidal endotoxins of Bacillus thuringiensis subsp. israelensis, was identified, cloned, and sequenced. This fragment contains cis elements, including iterons, found in replication origins of other large plasmids and suggests that pBtoxis replicates by a type A theta mechanism. Two genes, pBt156 and pBt157, encoding proteins of 54.4 kDa and 11.8 kDa, respectively, were present in an operon within this minireplicon, and each was shown by deletion analysis to be essential for replication. The deduced amino acid sequences of the 54.4-kDa and 11.8-kDa proteins showed no substantial homology with known replication (Rep) proteins. However, the 54.4-kDa protein contained a conserved FtsZ domain, and the 11.8 kDa protein contained a helix-turn-helix motif. As FtsZ proteins have known functions in bacterial cell division and the helix-turn-helix motif is present in Rep proteins, it is likely that these proteins function in plasmid replication and partitioning. The minireplicon had a copy number of two or three per chromosome equivalent in B. thuringiensis subsp. israelensis but did not replicate in B. cereus, B. megaterium, or B. subtilis. A plasmid constructed to synthesize large quantities of the Cry11A and Cyt1A endotoxins demonstrated that this minireplicon can be used to engineer vectors for cry and cyt gene expression.
Project description:Strains of Bacillus thuringiensis such as B. thuringiensis subsp. israelensis (ONR-60A) and B. thuringiensis subsp. morrisoni (PG-14) pathogenic for mosquito larvae produce a complex parasporal body consisting of several protein endotoxins synthesized during sporulation that form an aggregate of crystalline inclusions bound together by a multilamellar fibrous matrix. Most studies of these strains focus on the molecular biology of the endotoxins, and although it is known that parasporal body structural integrity is important to achieving high toxicity, virtually nothing is known about the matrix that binds the toxin inclusions together. In the present study, we undertook a proteomic analysis of this matrix to identify proteins that potentially mediate assembly and stability of the parasporal body. In addition to fragments of their known major toxins, namely, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, we identified peptides with 100% identity to regions of Bt152, a protein coded for by pBtoxis of B. thuringiensis subsp. israelensis, the plasmid that encodes all endotoxins of this subspecies. As it is known that the Bt152 gene is expressed in B. thuringiensis subsp. israelensis, we disrupted its function and showed that inactivation destabilized the parasporal body matrix and, concomitantly, inclusion aggregation. Using fluorescence microscopy, we further demonstrate that Bt152 localizes to the parasporal body in both strains, is absent in other structural or soluble components of the cell, including the endospore and cytoplasm, and in ligand blots binds to purified multilamellar fibrous matrix. Together, the data show that Bt152 is essential for stability of the parasporal body of these strains.
Project description:Differences in activation between spores from strains of Bacillus thuringiensis subsp. israelensis with and without the toxin-encoding plasmid pBtoxis are demonstrated. Following alkaline activation, the strain bearing pBtoxis shows a significantly greater germination rate. Expression of just three genes constituting a previously identified, putative ger operon from this plasmid is sufficient to produce the same phenotype and characterizes this operon as a genetic determinant of alkaline activation.
Project description:The entire 127,923-bp sequence of the toxin-encoding plasmid pBtoxis from Bacillus thuringiensis subsp. israelensis is presented and analyzed. In addition to the four known Cry and two known Cyt toxins, a third Cyt-type sequence was found with an additional C-terminal domain previously unseen in such proteins. Many plasmid-encoded genes could be involved in several functions other than toxin production. The most striking of these are several genes potentially affecting host sporulation and germination and a set of genes for the production and export of a peptide antibiotic.
Project description:In Bacillus thuringiensis subsp. israelensis all of the insecticidal toxins are encoded on a single, large plasmid, pBtoxis. Sequencing of this plasmid revealed 125 potential coding sequences, many of which have predicted functions in gene regulation and physiological processes, such as germination. As a first step in understanding the possible role of pBtoxis in its host bacterium, a survey of the transcription of genes with predicted functions was carried out. Whereas many coding sequences, including those previously identified as probable pseudogenes, were not transcribed, mRNA was detected for 29 of the 40 sequences surveyed. Several of these sequences, including eight with similarities to the sequences of known transcriptional regulators, may influence wider gene regulation and thus may alter the phenotype of the host bacterium.
Project description:Cyt1A protein is a cytolytic protein encoded by the cyt gene of Bacillus thuringiensis subsp. israelensis (Bti) as part of the parasporal crystal proteins produced during the sporulation. Cyt1A protein is unique compared to the other endotoxins present in these parasporal crystals. Unlike ?-endotoxins, Cyt1A protein does not require receptors to bind to the target cell and activate the toxicity. It has the ability to affect a broad range of cell types and organisms, due to this characteristic. Cyt1A has been recognized to not only target the insect cells directly, but also recruit other endotoxins by acting as receptors. Due to these mode of actions, Cyt1A has been studied for its cytolytic activity against human cancer cell lines, although not extensively. In this study, we report a novel Cyt1A protein produced by a Bti strain QBT229 isolated from Qatar. When tested for its cytotoxicity against lung cancer cells, this local strain showed considerably higher activity compared to that of the reference Bti and other strains tested. The possible reasons for such enhanced activity were explored at the gene and protein levels. It was evidenced that five consecutive amino acid replacements in the ?8 sheet of the Cyt1A protein enhanced the cytotoxicity against the lung epithelial cancer cells. Such novel Cyt1A protein with high cytotoxicity against lung cancer cells has been characterized and reported through this study.
Project description:Mosquitocidal Bacillus thuringiensis strains show as a common feature the presence of toxic proteins with cytolytic and hemolytic activities, Cyt1Aa1 being the characteristic cytolytic toxin of Bacillus thuringiensis subsp. israelensis. We have detected the presence of another cyt gene in this subspecies, highly homologous to cyt2An1, coding for the 29-kDa cytolytic toxin from B. thuringiensis subsp. kyushuensis. This gene, designated cyt2Ba1, maps upstream of cry4B coding for the 130-kDa crystal toxin, on the 72-MDa plasmid of strain 4Q2-72. Sequence analysis revealed, as a remarkable feature, a 5' mRNA stabilizing region similar to those described for some cry genes. PCR amplification and Southern analysis confirmed the presence of this gene in other mosquitocidal subspecies. Interestingly, anticoleopteran B. thuringiensis subsp. tenebrionis belonging to the morrisoni serovar also showed this gene. On the other hand, negative results were obtained with the anti-lepidopteran strains B. thuringiensis subsp. kurstaki HD-1 and subsp. aizawai HD-137. Western analysis failed to reveal Cyt2A-related polypeptides in B. thuringiensis subsp. israelensis 4Q2-72. However, B. thuringiensis subsp. israelensis 1884 and B. thuringiensis subsp. tenebrionis did show cross-reactive products, although in very small amounts.
Project description:The segregation of plasmid DNA typically requires three elements: a DNA centromere site, an NTPase, and a centromere-binding protein. Because of their simplicity, plasmid partition systems represent tractable models to study the molecular basis of DNA segregation. Unlike eukaryotes, which utilize the GTPase tubulin to segregate DNA, the most common plasmid-encoded NTPases contain Walker-box and actin-like folds. Recently, a plasmid stability cassette on Bacillus thuringiensis pBtoxis encoding a putative FtsZ/tubulin-like NTPase called TubZ and DNA-binding protein called TubR has been described. How these proteins collaborate to impart plasmid stability, however, is unknown. Here we show that the TubR structure consists of an intertwined dimer with a winged helix-turn-helix (HTH) motif. Strikingly, however, the TubR recognition helices mediate dimerization, making canonical HTH-DNA interactions impossible. Mutagenesis data indicate that a basic patch, encompassing the two wing regions and the N termini of the recognition helices, mediates DNA binding, which indicates an unusual HTH-DNA interaction mode in which the N termini of the recognition helices insert into a single DNA groove and the wings into adjacent DNA grooves. The TubZ structure shows that it is as similar structurally to eukaryotic tubulin as it is to bacterial FtsZ. TubZ forms polymers with guanine nucleotide-binding characteristics and polymer dynamics similar to tubulin. Finally, we show that the exposed TubZ C-terminal region interacts with TubR-DNA, linking the TubR-bound pBtoxis to TubZ polymerization. The combined data suggest a mechanism for TubZ-polymer powered plasmid movement.
Project description:Insecticidal Bacillus thuringiensis (Bt) delta-endotoxins are cytolytic to a range of insect cell lines in vitro. Addition of Bt var. aizawai or var. israelensis toxins to Mamestra brassicae (cabbage moth) cells in vitro increased intracellular cyclic AMP, which was paralleled by activation of adenylate cyclase in isolated membranes. Var. kurstaki toxin, which does not lyse M. brassicae cells, had no effect on cyclic AMP concentrations in intact cells, but was able to stimulate adenylate cyclase in membrane preparations. In contrast, the bee-venom toxin melittin, which is also cytolytic, increased intracellular cyclic AMP in whole cells, but inhibited adenylate cyclase in isolated membranes. Octopamine and forskolin also increased cyclic AMP in cells, but were not cytolytic. When added to cells at concentrations exceeding their LC90 (concentration causing 90% cell death), melittin and var. israelensis toxins caused cell lysis without a concomitant increase in intracellular cyclic AMP. Taken together, these results suggest that activation of adenylate cyclase by cytolytic toxins is a secondary effect (related perhaps to interactions of these toxins with membrane lipids) and is neither necessary nor sufficient for cytolysis.
Project description:Lactobacillus plantarum A112 has four different plasmids. Plus-origin-specific probes were used to determine that the smallest, cryptic plasmid, pA1 (2,820 bp), showed homology to the pE194 plasmid family. This subclass of plasmids uses the rolling-circle mode of replication. Subsequent analysis of plasmid pA1 demonstrated that it generates single-stranded DNA intermediates, and sequence analysis revealed that it contains three putative open reading frames (ORFs): ORF1, ORF2, and ORF3, which could encode proteins designated RepA (47 amino acids [aa]) and RepB (196 aa) and a protein of 103 aa, respectively. Two of these proteins, RepA (5.6 kDa) and RepB (26 kDa), were identified in in vitro transcription translation assays. The RepA protein contains a characteristic alpha-helix-turn-alpha-helix motif typical of DNA-binding proteins that act as DNA-binding repressors. The RepB protein shows a significant similarity with replication initiation proteins of the pE194 family of plasmids that use the rolling-circle mode of replication. Plasmid pA1 is able to replicate in Escherichia coli and Lactobacillus lactis subsp. lactis as well as in other L. plantarum strains.
Project description:During replication of human parainfluenza virus type 3 (HPIV3), the 96-nucleotide antigenomic promoter (AGP) of HPIV3 directs the synthesis of genomic RNA. Previous work showed that nucleotides 1-12 were critical in promoting replication of an HPIV3 minireplicon, but nucleotides 13-96 were not investigated. In this study, the role of nucleotides 13-96 in AGP function was analyzed by creating and assaying mutations in an HPIV3 minireplicon. A replication promoting element known as promoter element II (nt 79-96) was confirmed in the HPIV3 AGP. Additionally, nucleotides 13-39 were found to constitute an additional positive-acting cis-element. However, detailed analysis of the 13-39 element revealed a complicated control element with both stimulatory and repressing elements. Specifically, nucleotides 21-28 were shown to repress RNA replication, while flanking sequences had a stimulatory effect.