Figure 2

Confirmation of the intracellular location of ChiA74∆s p in Escherichia coli and Bacillus thuringiensis 4Q7 using a chimeric construct with the green fluorescent protein (GFP) gene. (A) Schematic illustration that shows the fusion of chiA74∆sp with gfp. Panel 1, two constructs were developed, under regulation of the chiA74 wildtype promoter (wp) or under control of the cytA-p/ STAB-SD system. Lollipop indicates transcriptional terminator. Oligonucleotides used to make chimeric construct with gfp are shown in Table 1. Panel 2, confirmation of chimeric constructs by PCR, primers used for amplification are showed in parenthesis and in Table 1. L, 1 kb (kilobase) DNA Ladder (New England Biolabs); Lane 1, pEHchiA74∆sp-gfp (primers: chiA74-1, chiA74-3); lane 2, pEHchiA74∆sp-gfp (primers: chiA74-1, gfp-3); lane 3, pEHchiA74∆sp-gfp (primers: gfp-1, chiA74-4); lane 4, pEBchi A74∆sp-gfp (primers: cytSTAB-1, chiA74-4); Lane 5, pEBchi A74∆sp-gfp (primers: cytSTAB-1, gfp-3); lane 6, pEBchi A74∆sp-gfp (primers: gfp-1, chiA74-4). (B) Phase contrast (left) and fluorescence micrographs (right) of recombinant strains of E. coli and B. thuringiensis strain 4Q7 expressing the chimeric protein ChiA74∆sp-GFP. Panel 1, E. coli- pEHchiA74∆sp-gfp; panel 2, 4Q7- pEHchiA74∆sp-gfp; panel 3, E. coli- pEBchiA74∆sp-gfp; panel 4, 4Q7- pEBchiA74∆sp-gfp; panel 5, E. coli DH5α; panel 6, 4Q7. Samples of recombinant strains of B. thuringiensis were collected at ~ 72 h.