Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats

Abstract

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that accumulates pesticidal proteins (Cry and Cyt) in parasporal crystals. Proteins from the Cry5, App6 (formerly Cry6), Cry12, Cry13, Cry14, Cry21, and Xpp55 (formerly Cry55) families have been identified as toxic to nematodes. In this study, a total of 846 Bt strains belonging to four collections were analyzed to determine the diversity and distribution of the Bt Cry nematicidal protein genes. We analyzed their presence by PCR, and positives were confirmed by sequencing. As a result, 164 Bt isolates (20%) contained at least one gene coding for nematicidal Cry proteins. The cry5 and cry21 genes were enriched in collection 1 and were often found together in the same strain. Differently, in collection 4, obtained from similar habitats but after 10 years, cry14 was the gene most frequently found. In collection 2, cry5 and app6 were the most abundant genes, and collection 3 had a low incidence of any of these genes. The results point to high variability in the frequencies of the studied genes depending on the timing, geographical origins, and sources. The occurrence of cry1A, cry2, and cry3 genes was also analyzed and showed that the nematicidal Cry protein genes were frequently accompanied by cry1A + cry2. The expression of the genes was assessed by mass spectrometry showing that only 14% of the positive strains produced nematicidal proteins. To our knowledge, this is the first comprehensive screening that examines the presence and expression of genes from the seven known Bt Cry nematicidal families.

Keywords: Bt toxins; Cry genes; Cry proteins; Nematode; PCR screening; Proteomics.

Fig. 1

Agarose gel electrophoresis of PCR products obtained using 13 different primer pairs for the detection of nematicidal Cry protein genes. Lane1: V-C5 with primers Cry5_F/R. Lane2: V-C5 with primers Cry5_F₁/R₁. Lane3: V-AB6 with primers Cry6_F/R. Lane4: V-AB6 with primers Cry6A_F₁/R₁. Lane5: O-V12 with primers Cry12_F/R. Lane6: O-V12 with primers Cry12_F₁/R₁. Lane7: NRRL B-18246 with primers Cry13_F/R. Lane8: NRRL B-18246 with primers Cry13_F₁/R₁. Lane9: A-V14 with primers Cry14_F/R. Lane10: A-V14 with primers Cry14_F₁/R₁. Lane11: N-S21 with primers Cry21_F/R. Lane12: YBT-1518 with primers Cry55_F/R. Lane13: YBT-1518 with primers Cry55_F₁/R₁. Lane M: 100 bp DNA ladder (ThermoScientific, Vilnius, Lithuania)

Fig. 2

Agarose gel electrophoresis of PCR products obtained using primers Cry5_F₁/R₁ for detection of cry5 gene in 11 field-collected Bt strains randomly selected (lanes 1–11). Lane C + : strain BMB171. Lane C − : negative control (no DNA). Lane M: 100 bp DNA ladder (ThermoScientific, Vilnius, Lithuania)

Fig. 3

Scanning electron microscopy of Bt strains showing parasporal crystal inclusions with different morphologies. a Bipyramidal, b round, c irregular, d adhered to the spore, e two-crystals, and f small. In the images, white arrows point to crystals and empty arrows point to spores