SummaryThe entomopathogen Bacillus thuringiensis species harbours numerous plasmids essentially studied for their involvement in pathogenicity, as Cry-plasmids. The life cycle of B. thuringiensis in the insect host is regulated by the sequential activation of quorum sensing systems to kill, survive and sporulate. In this study, we characterize a new quorum sensing system belonging to the Rap-Phr family. The Rap8-Phr8 system is borne by the pHT8_1 plasmid, a small cryptic plasmid from the B. thuringiensis var. kurstaki HD73 strain. Our results demonstrate that the Rap8 protein inhibits sporulation and biofilm formation through the Spo0A pathway. The Rap8 activity is inhibited by the mature Phr8 heptapeptide YAHGKDI. The key residues specific for the Rap phosphatase activity are conserved in Rap8 suggesting a common mode of action. Interestingly, we show that the Rap8-Phr8 system is specifically required for regulating sporulation of B. thuringiensis in insect larvae. This system may allow the bacteria to exert a tight control of the sporulation process in the host cadaver for optimizing the multiplication, the survival and the dissemination of the bacteria. Thus, our results suggest that pHT8_1 provides advantages for adaptation and evolution of B. thuringiensis in its ecological niche.

ABSTRACT. Elasmopalpus lignosellus (Zeller, 1848) (Lepidoptera, Pyralidae) is an insect pest of 60 economically important crops, including sugarcane, wheat, soybean, rice, beans, sorghum, peanuts, and cotton. The aim of this work was to select and characterize Bacillus thuringiensis isolates with insecticidal activity against E. Lignosellus that could be used as an alternative method of control. Selective bioassays were done to evaluate the toxicity of 47 isolates against first instar larvae of E. lignosellus. For the most toxic bacterial strains, the lethal concentration (LC 50 ) was estimated and morphological, biochemical and molecular methods were used to characterize the isolates. Among the 47 isolates tested, 12 caused mortality above 85% and showed LC 50 values from 0.038E+8 to 0.855E+8 spores mL -1. Isolates BR83, BR145, BR09, BR78, S1534, and S1302 had the lowest LC 50 values and did not differ from the standard HD-1 strain; the exception was BR83.The protein profiles produced bands with molecular masses of 60-130 kDa. The genes cry1, cry2, cry3, and cry11 were identified in the molecular characterization. The morphological analysis identified three different crystal inclusions: bipyramidal, spherical and cuboidal. Among the tested isolates, 12 isolates have potential for biotechnological control of E. Lignosellus by development of new biopesticides or genetically modified plants.Keywords: biological control, cry genes, entomopathogenic bacteria, lesser cornstalk borer. Seleção e caracterização de linhagens de Bacillus thuringiensis ativas contra Elasmopalpus lignosellus (Zeller, 1848) (Lepidoptera, Pyralidae)RESUMO. Elasmopalpus lignosellus (Zeller, 1848) (Lepidoptera, Pyralidae) é considerada praga para mais de 60 espécies de plantas cultivadas como o milho, cana-de-açúcar, trigo, soja, arroz, feijão, sorgo, amendoim e algodão. O objetivo deste trabalho foi selecionar e caracterizar isolados de Bacillus thuringiensis virulentos a E. lignosellus atuando como método alternativo de controle. Assim, 47 isolados foram avaliados em bioensaios seletivos contra lagartas de 1º ínstar de E. lignosellus. Para os isolados mais tóxicos, a concentração letal (CL 50 ) foi estimada e a sua caracterização foi realizada por métodos morfológicos, bioquímicos e moleculares. Dos 47 isolados testados, 12 causaram mortalidade acima 85% e apresentaram CL 50 entre 0.038E+8 a 0.855E+8 esporos mL -1 . Os menores valores de CL 50 foram obtidos pelos isolados BR83, BR145, BR09, BR78, S1534 e S1302, os quais não diferiram da linhagem padrão HD-1, com exceção do BR83. O perfil proteico revelou proteínas Cry entre 60 e 130 kDa, e a caracterização molecular mostrou a presença dos genes cry1, cry2, cry3 e cry11. A análise morfológica identificou três diferentes inclusões cristalinas: bipiramidais, esféricas e cuboides. Entre os isolados avaliados, 12 apresentam potencial biotecnológico para controle de E. lignosellus via formulação de novos bioinseticida ou produção de plantas transgênicas.Palavras-chave: controle biológico, genes cry...