Figure 1.
Identification of chitin-binding and –degrading proteins in the supernatants of cultured P. larvae.
(A) Chitin-binding proteins isolated via chitin-beads from culture supernatants of ATCC9545 (P. larvae ERIC I) and DSM25430 (P. larvae ERIC II) were subjected to SDS-PAGE analysis. PlCBP49 is marked by an arrowhead. (B) Proteins isolated via chitin-beads from culture supernatants of ATCC9545 (P. larvae ERIC I) and DSM25430 (P. larvae ERIC II) were subjected to zymography using ethylene glycol chitin- (EGC-) impregnated gels to assess their chitinolytic activity. PlCBP49 is marked by an arrow head.
Figure 2.
Identification of PlCBP49 as a novel member of the AA10 (formerly CBM33) family of LPMOs.
(A) Peptide sequences obtained from sequencing PlCBP49I from ATCC9545 and PlCBP49II from DSM25430 are shown in comparison to the corresponding sequences of S. marcescens CBP21 (GenBank acc. no.: BAA31569). (B) In silico-translation of the putative P. larvae PlCBP49 ORF followed by domain analysis revealed the presence of an N-terminal CBM33 (AA10) module, two FN-III-repeats and an additional small, C-terminal chitin-binding domain (CBM 5/12). (C) Amino acid alignment of the AA10 domain of P. larvae PlCBP49 with three other members of the AA10 family of LPMOs (CBP21, GenBank acc. no.: BAA31569; EfCBM33A, GenBank acc. no.: AAO80225; CBD3, Genbank acc. no.: EEM95937) revealed the existence of a signal peptide (framed) and several conserved amino acids (arrows and asterisks) which are described to be involved in chitin-binding and –degradation.
Figure 3.
Inhibition of PlCBP49 chitin degradation through KCN and EDTA but not through caffeine.
Chitin-binding fractions of P. larvae were subjected to zymography and activity of PlCBP49 was probed by adding 20 mM EDTA or 2 mM KCN or 20 mM caffeine as inhibitors. CBP49 activity could be inhibited by EDTA and KCN but not by caffeine. A representative result obtained with the chitin-binding fraction of ATCC9545 (P. larvae ERIC I) is shown.
Figure 4.
Interruption of the cbp-gene in ATCC9545 and DSM25430 leads to lack of both PlCBP49 expression and chitinolytic activity.
(A) SDS-PAGE analysis of the culture supernatant fractions bound to chitin coated beads of wild-type (ATCC9545, DSM25430) and mutant bacteria (ATCC9545 Δcbp, DSM25430 Δcbp) confirmed the absence of PlCBP49 expression in the knockout strains. Bands corresponding to PlCBP49 are marked by an arrow head. (B) Zymography of the culture supernatants of wild-type (ATCC9545, DSM25430) and mutant bacteria (ATCC9545 Δcbp, DSM25430 Δcbp) confirmed the absence of PlCBP49 activity in the knockout strains. Bands corresponding to PlCBP49 chitinolytic activity are marked by an arrow head.
Figure 5.
Degradation of insoluble chitin structures by P. larvae PlCBP49.
Peritrophic matrices were isolated from Spodoptera frugiperda larvae and incubated in an Ussing-chamber (A) with mock-treated chitin beads as negative control and chitin-binding fractions of either wild type bacteria or the corresponding mutant bacteria. Methylene blue efflux was used as a measure for permeability (B). PM permeability was significantly higher than negative control after incubation with ATCC9545 wt and DSM25430 wt chitin binding fractions. After incubation with ATCC9545 Δcbp and DSM25430 Δcbp chitin-binding fractions PM permeability was significantly lower compared to the incubation with chitin-binding fractions of the wild-type strains (ATCC9545, DSM25430). Bars represent mean values + SEM of at least three independent experiments, analyzed by student's t-test; *p value<0.05 and **p value<0.01.
Figure 6.
Knocking out PlCBP49 expression impairs PM degradation in infected larvae and nearly abolishes P. larvae virulence.
(A) PM from 6-day old non-infected control larvae can be isolated as nearly intact structures. A representative macroscopic picture of an unstained PM is shown. (B) After methylene blue-basic fuchsine staining of chitin, the isolated PM of non-infected control larvae could be visualized as intact, pink stained structure. (C) Methylene blue-basic fuchsine staining of PM isolated from 6-day old larvae infected with ATCC9545 wild type bacteria revealed no intact structure but instead only red stained dots most likely remnants of the degraded PM can be observed. (D) Methylene blue-basic fuchsine staining of PM isolated from 6-day old larvae infected with the mutant strain ATCC9545 Δcbp showed an intact, pink stained PM structure. (E) Larvae were infected with wild type bacteria (ATCC9545, DSM25430) or the corresponding mutant strains (ATCC9545 Δcbp, DSM25430 Δcbp) and total mortality was calculated for each group. Data represent mean values + SEM of three independent infection assays with 30 larvae each. Data were analyzed by student's t-test; ***p<0.001.
Table 1.
Primers used for sequence analysis of cbp49, screening of P. larvae isolates for cbp49, and construction of gene knockouts in P. larvae ATCC9545 (ERIC I) and DSM25430 (ERIC II).
Figure 7.
Disruption of the gene coding for PlCBP49 in P. larvae ATCC9545 (ERIC I) and in P. larvae DSM25430 (ERIC II).
(A) Migration properties of amplicons from the wild-type (ATCC9545 wt, DSM25430 wt) and mutant bacteria (ATCC9545 Δcbp, DSM25430 Δcbp) confirmed the successful insertion of the targetron into the gene cbp49. (B) Growth curves of the knockout strain ATCC9545 Δcbp (open circle) in comparison to the parent wild-type strain ATCC9545 (closed circle). (C) Growth curves of the knockout strain DSM25430 Δcbp (open triangle) in comparison to the parent wild-type strain DSM25430 (closed triangle). Both results showed that disrupting PlCBP49 expression did not influence bacterial growth.