Open Access
Baculovirus expression and functional analysis of Vpa2 proteins from Bacillus thuringiensis
(MDPI, 2020) Simón de Goñi, Oihane; Palma Dovis, Leopoldo; Fernández González, Ana Beatriz; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
The mode of action underlying the insecticidal activity of the Bacillus thuringiensis (Bt) binary pesticidal protein Vpa1/Vpa2 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.
Open Access
UV protection and insecticidal activity of microencapsulated Vip3Ag4 protein in Bacillus megaterium
(Elsevier, 2024-06-17) Palma Dovis, Leopoldo; Ruiz de Escudero Fuentemilla, Íñigo; Mañeru Oria, Francisco Javier; Berry, Colin; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
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.
Open Access
Genome sequence analysis of native Xenorhabdus strains isolated from Entomopathogenic nematodes in Argentina
(MDPI, 2024) Palma Dovis, Leopoldo; Frizzo, Laureano; Kaiser, Sebastian; Berry, Colin; Caballero Murillo, Primitivo; Bode, Helge B.; Valle, Eleodoro E. del; Institute for Multidisciplinary Research in Applied Biology - IMAB
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.

Palma Dovis, Leopoldo

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