Positive selection analysis of Cyt proteins from Bacillus thuringiensis: a conservative trend driven by negative (purifying) selection.

Bacillus thuringiensis is a Gram-positive entomopathogenic bacterium that produces different pesticidal proteins: vegetative insecticidal proteins (Vpb1/Vpa2, Vip3, and Vpb4) during vegetative growth and δ-endotoxins (Cry and Cyt) during sporulation, which accumulate into parasporal crystals. Cyt proteins are the smaller subset of δ-endotoxins targeting Diptera species. While Cry and Vip3 proteins undergo positive selection, our analysis suggests that Cyt proteins evolve following a conservative trend driven negative (purifying) selection.

Draft genome sequence of Bacillus thuringiensis strain V-AB8.18, a novel isolate with potential nematicidal activity.

We report the draft genome of Bacillus thuringiensis strain V-AB8.18, comprising 308 contigs totaling 6,182,614 bp, with 35% G + C content. It contains 6,151 putative protein-coding genes, including App6 and Cry5-like crystal proteins, exhibiting 99% pairwise identity to nematicidal proteins App6Aa2 and Cry5Ba2, active against Meloidogyne incognita and Meloidogyne hapla.

UV protection and insecticidal activity of microencapsulated Vip3Ag4 protein in Bacillus megaterium.

In this study, secretable Vip3Ag4 protein was encapsulated in Bacillus megaterium and used for quantitative bioassays, in order to determine the UV photoprotective capacity of the cell, for preventing inactivation of the insecticidal activity of the protein. The non-encapsulated and purified protein was exposed to the UV light showing a LC50 of 518 ng/cm2 against Spodoptera littoralis larvae, whereas the exposed encapsulated protein exhibited 479 ng/cm2. In addition to the capability to accumulate Vip3 proteins for the development of novel insecticidal formulates, the B. megaterium cell has demonstrated to provide moderate protection against the deleterious action of UV light.

First report of Pectobacterium brasiliense causing blackleg and soft rot of potato in Argentina.

Blackleg and soft rot diseases represent a major threat to the health of potato (Solanum tuberosum) and other vegetable, ornamental and fruit crops worldwide; their main causal agents are species of Pectobacterium and Dickeya. In May 2022, 60% of potato plants (cv. Spunta) in a production field in Córdoba, Argentina (31°32'36''S 64°09'46''W) showed soft rot, blackleg and wilt. To isolate the causal agent, decayed plant tissues were disinfected in 2% NaClO, macerated in sterile water and streaked on crystal violet pectate (CVP) medium. Plates were incubated at 28°C for 48 h. Colonies that produced a pit on CVP medium were purified on nutrient agar. Two of the isolates, called 1Aia and 1B, were characterized by tests commonly employed for the identification of pectinolytic bacteria (Schaad et al. 2001). Both produced Gram-negative rods that were facultatively anaerobic, oxidase negative, nonfluorescent on King´s B, resistant to erythromycin and caused soft rot of potato slices. In addition, these isolates did not produce the blue pigment indigoidine and grew on nutrient glucose agar containing 5% NaCl. Phenotypic characteristics of the isolates 1Aia and 1B were compatible with Pectobacterium spp. Genomic DNA was extracted using the commercially available Wizard® Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions for the purification of DNA from Gram-negative bacteria. The isolates were positive in a PCR assay for Pectobacterium brasiliense (Duarte et al. 2004). The purified DNA of isolate 1Aia was used to construct a pooled Illumina library, which was sequenced at the Genomics Unit from the National Institute of Agricultural Technology (INTA, Argentina), by using high-throughput Illumina sequencing technology. Average nucleotide identity (ANI) calculation performed by FastANI v0.1.3 (Jain et al. 2018) showed 96.11% identity between the genome of the type strain LMG 21371 of P. brasiliense (Acc. no. JQOE00000000) and our strain 1Aia (Acc. no. JAYGXQ000000000). For pathogenicity test, 3-weeks-old potato plants (cv. Spunta) planted in pots were infiltrated with 10 µl of a bacterial suspension (1x107 CFU/ml) 5 cm above the base of the stem using a sterile syringe. Negative controls were infiltrated with sterile water. Plants were kept under greenhouse conditions and regularly watered. The experiment was performed twice with six plants per treatment. Two days after inoculation, plants treated with P. brasiliense strain 1Aia or 1B showed necrotic lesions on the stems and tubers soft rot symptoms while control plants remained asymptomatic. To fulfill Koch´s postulates, bacteria were re-isolated from symptomatic plants. Re-isolated bacteria, called 1Aia d and 1B d, were confirmed as P. brasiliense according to biochemical and PCR results, as outlined above. Also, the % ANI value between P. brasiliense isolates 1Aia and 1Aia d was 99.99% (Acc. no. JAYGXR000000000). To our knowledge, this is the first report of the occurrence of P. brasiliense in Argentina. This pathogen has been observed causing blackleg and tuber soft rot on potato in Brazil (Duarte et al. 2004), Netherlands (Nunes Leite et al. 2014), Switzerland (de Werra et al. 2015), Russia (Voronina et al. 2019), Serbia (Loc et al. 2022) and USA (Zhang et al. 2023), among other countries worldwide. Due to the important economic and nutritional value of the crop, the distribution of P. brasiliense needs to be investigated and monitored in order to develop effective control strategies.

Bacillus thuringiensis: A Broader View of Its Biocidal Activity.

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that forms spores and produces parasporal crystalline inclusions containing Cry and Cyt proteins [...].

Genome Sequence Analysis of Native Xenorhabdus Strains Isolated from Entomopathogenic Nematodes in Argentina.

Entomopathogenic nematodes from the genus Steinernema (Nematoda: Steinernematidae) are capable of causing the rapid killing of insect hosts, facilitated by their association with symbiotic Gram-negative bacteria in the genus Xenorhabdus (Enterobacterales: Morganellaceae), positioning them as interesting candidate tools for the control of insect pests. In spite of this, only a limited number of species from this bacterial genus have been identified from their nematode hosts and their insecticidal properties documented. This study aimed to perform the genome sequence analysis of fourteen Xenorhabdus strains that were isolated from Steinernema nematodes in Argentina. All of the strains were found to be able of killing 7th instar larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae). Their sequenced genomes harbour 110 putative insecticidal proteins including Tc, Txp, Mcf, Pra/Prb and App homologs, plus other virulence factors such as putative nematocidal proteins, chitinases and secondary metabolite gene clusters for the synthesis of different bioactive compounds. Maximum-likelihood phylogenetic analysis plus average nucleotide identity calculations strongly suggested that three strains should be considered novel species. The species name for strains PSL and Reich (same species according to % ANI) is proposed as Xenorhabdus littoralis sp. nov., whereas strain 12 is proposed as Xenorhabdus santafensis sp. nov. In this work, we present a dual insight into the biocidal potential and diversity of the Xenorhabdus genus, demonstrated by different numbers of putative insecticidal genes and biosynthetic gene clusters, along with a fresh exploration of the species within this genus.

Bacillus thuringiensis Bt_UNVM-84, a Novel Strain Showing Insecticidal Activity against Anthonomus grandis Boheman (Coleoptera: Curculionidae).

Bacillus thuringiensis is a Gram-positive bacterium known for its insecticidal proteins effective against various insect pests. However, limited strains and proteins target coleopteran pests like Anthonomous grandis Boheman, causing substantial economic losses in the cotton industry. This study focuses on characterizing a Bacillus sp. strain, isolated from Oncativo (Argentina), which exhibits ovoid to amorphous parasporal crystals and was designated Bt_UNVM-84. Its genome encodes genes for the production of two pairs of binary Vpb1/Vpa2 proteins and three Cry-like proteins showing similarity with different Cry8 proteins. Interestingly, this gene content was found to be conserved in a previously characterized Argentine isolate of B. thuringiensis designated INTA Fr7-4. SDS-PAGE analysis revealed a major band of 130 kDa that is proteolytically processed to an approximately 66-kDa protein fragment by trypsin. Bioassays performed with spore-crystal mixtures demonstrated an interesting insecticidal activity against the cotton boll weevil A. grandis neonate larvae, resulting in 91% mortality. Strain Bt_UNVM-84 is, therefore, an interesting candidate for the efficient biological control of this species, causing significant economic losses in the cotton industry in the Americas.

Baculovirus Expression and Functional Analysis of Vpa2 Proteins from Bacillus thuringiensis.

The mode of action underlying the insecticidal activity of the Bacillus thuringiensis (Bt) binary pesticidal protein Vpb/Vpa (formerly Vip1/Vip2) is uncertain. In this study, three recombinant baculoviruses were constructed using Bac-to-Bac technology to express Vpa2Ac1 and two novel Vpa2-like genes, Vpa2-like1 and Vpa2-like2, under the baculovirus p10 promoter in transfected Sf9 cells. Pairwise amino acid analyses revealed a higher percentage of identity and a lower number of gaps between Vpa2Ac1 and Vpa2-like2 than to Vpa2-like1. Moreover, Vpa2-like1 lacked the conserved Ser-Thr-Ser motif, involved in NAD binding, and the (F/Y)xx(Q/E)xE consensus sequence, characteristic of the ARTT toxin family involved in actin polymerization. Vpa2Ac1, Vpa2-like1 and Vpa2-like2 transcripts and proteins were detected in Sf9 culture cells, but the signals of Vpa2Ac1 and Vpa2-like2 were weak and decreased over time. Sf9 cells infected by a recombinant bacmid expressing Vpa2-like1 showed typical circular morphology and produced viral occlusion bodies (OBs) at the same level as the control virus. However, expression of Vpa2Ac1 and Vpa2-like2 induced cell polarization, similar to that produced by the microfilament-destabilizing agent cytochalasin D and OBs were not produced. The presence of filament disrupting agents, such as nicotinamide and nocodazole, during transfection prevented cell polarization and OB production was observed. We conclude that Vpa2Ac1 and Vpa2-like2 proteins likely possess ADP-ribosyltransferase activity that modulated actin polarization, whereas Vpa2-like1 is not a typical Vpa2 protein. Vpa2-like2 has now been designated Vpa2Ca1 (accession number AAO86513) by the Bacillus thuringiensis delta-endotoxin nomenclature committee.

Phylogenetic determinants of toxin gene distribution in genomes of Brevibacillus laterosporus.

Brevibacillus laterosporus is a globally ubiquitous, spore forming bacterium, strains of which have shown toxic activity against invertebrates and microbes and several have been patented due to their commercial potential. Relatively little is known about this bacterium. Here, we examined the genomes of six published and five newly determined genomes of B. laterosporus, with an emphasis on the relationships between known and putative toxin encoding genes, as well as the phylogenetic relationships between strains. Phylogenetically, strain relationships are similar using average nucleotide identity (ANI) values and multi-gene approaches, although PacBio sequencing revealed multiple copies of the 16S rDNA gene which lessened utility at the strain level. Based on ANI values, the New Zealand isolates were distant from other isolates and may represent a new species. While all of the genomes examined shared some putative toxicity or virulence related proteins, many specific genes were only present in a subset of strains.

Draft Genome Sequence of Bacillus cereus CITVM-11.1, a Strain Exhibiting Interesting Antifungal Activities.

Bacillus cereus is a gram-positive, spore-forming bacterium possessing an important and historical record as a human-pathogenic bacterium. However, several strains of this species exhibit interesting potential to be used as plant growth-promoting rhizobacteria. Here, we report the draft genome sequence of B. cereus strain CITVM-11.1, which consists of 37 contig sequences, accounting for 5,746,486 bp (with a GC content of 34.8%) and 5,752 predicted protein-coding sequences. Several of them could potentially be involved in plant-bacterium interactions and may contribute to the strong antagonistic activity shown by this strain against the charcoal root rot fungus, Macrophomina phaseolina. This genomic sequence also showed a number of genes that may confer this strain resistance against several polluting heavy metals and for the bioconversion of mycotoxins.

The Vip3Ag4 Insecticidal Protoxin from Bacillus thuringiensis Adopts A Tetrameric Configuration That Is Maintained on Proteolysis.

The Vip3 proteins produced during vegetative growth by strains of the bacterium Bacillus thuringiensis show insecticidal activity against lepidopteran insects with a mechanism of action that may involve pore formation and apoptosis. These proteins are promising supplements to our arsenal of insecticidal proteins, but the molecular details of their activity are not understood. As a first step in the structural characterisation of these proteins, we have analysed their secondary structure and resolved the surface topology of a tetrameric complex of the Vip3Ag4 protein by transmission electron microscopy. Sites sensitive to proteolysis by trypsin are identified and the trypsin-cleaved protein appears to retain a similar structure as an octomeric complex comprising four copies each of the ~65 kDa and ~21 kDa products of proteolysis. This processed form of the toxin may represent the active toxin. The quality and monodispersity of the protein produced in this study make Vip3Ag4 a candidate for more detailed structural analysis using cryo-electron microscopy.

Is the Insect World Overcoming the Efficacy of Bacillus thuringiensis?

The use of chemical pesticides revolutionized agriculture with the introduction of DDT (Dichlorodiphenyltrichloroethane) as the first modern chemical insecticide. However, the effectiveness of DDT and other synthetic pesticides, together with their low cost and ease of use, have led to the generation of undesirable side effects, such as pollution of water and food sources, harm to non-target organisms and the generation of insect resistance. The alternative comes from biological control agents, which have taken an expanding share in the pesticide market over the last decades mainly promoted by the necessity to move towards more sustainable agriculture. Among such biological control agents, the bacterium Bacillus thuringiensis (Bt) and its insecticidal toxins have been the most studied and commercially used biological control agents over the last 40 years. However, some insect pests have acquired field-evolved resistance to the most commonly used Bt-based pesticides, threatening their efficacy, which necessitates the immediate search for novel strains and toxins exhibiting different modes of action and specificities in order to perpetuate the insecticidal potential of this bacterium.

Insecticidal spectrum and mode of action of the Bacillus thuringiensis Vip3Ca insecticidal protein.

The Vip3Ca protein, discovered in a screening of Spanish collections of Bacillus thuringiensis, was known to be toxic to Chrysodeixis chalcites, Mamestra brassicae and Trichoplusia ni. In the present study, its activity has been tested with additional insect species and we found that Cydia pomonella is moderately susceptible to this protein. Vip3Ca (of approximately 90kDa) was processed to an approximately 70kDa protein when incubated with midgut juice in all tested species. The kinetics of proteolysis correlated with the susceptibility of the insect species to Vip3Ca. The activation was faster to slower in the following order: M. brassicae (susceptible), Spodoptera littoralis (moderately susceptible), Agrotis ipsilon and Ostrinia nubilalis (slightly susceptible). Processing Vip3Ca by O. nubilalis or M. brassicae midgut juice did not significantly changed its toxicity to either insect species, indicating that the low susceptibility of O. nubilalis is not due to a problem in the midgut processing of the toxin. M. brassicae larvae fed with Vip3Ca showed binding of this toxin to the apical membrane of the midgut epithelial cells. Histopathological inspection showed sloughing of the epithelial cells with further disruption, which suggests that the mode of action of Vip3Ca is similar to that described for Vip3Aa. Biotin-labeled Vip3Ca and Vip3Aa bound specifically to M. brassicae brush border membrane vesicles and both toxins competed for binding sites. This result suggests that insects resistant to Vip3A may also be cross-resistant to Vip3C, which has implications for Insect Resistance Management (IRM).

Draft Genome Sequence of Photorhabdus luminescens Strain DSPV002N Isolated from Santa Fe, Argentina.

Here, we report the draft genome sequence of Photorhabdus luminescens strain DSPV002N, which consists of 177 contig sequences accounting for 5,518,143 bp, with a G+C content of 42.3% and 4,701 predicted protein-coding genes (CDSs). From these, 27 CDSs exhibited significant similarity with insecticidal toxin proteins from Photorhabdus luminescens subsp. laumondii TT01.