Overexpression of the Key Virulence Proteases Bace16 and Bae16 in <i>Bacillus nematocida</i> B16 to Improve Its Nematocidal Activity

Abstract

Proteases Bace16 and Bae16, an alkaline serine protease and a neutral protease, respectively, in the nematocidal bacterium Bacillus nematocida B16, have been identified as two key virulence factors and shown to have remarkable nematotoxic activities against the free-living nematode Panagrellus redivius and the plant parasite nematode Bursaphelenchus xylophilus. To facilitate the successful biological control application of this organism in the field, we genetically altered the strain B. nematocida B16 and optimized its growth condition to overexpress these two pathogenic proteases. The recombinant integration vectors of pAX01-Bace16 and pAX01-Bae16 for overexpressing the two proteases were constructed and successfully transformed into competent cells of the bacterium B. nematocida B16. The optimal induction condition for overexpressing Bace16 is 2% xylose at 37°C for 48 h. Our analyses showed that the proteolytic activity and nematocidal activity of the strain overexpressing Bace16 increased by about 62 and 80%, respectively, over the wild-type strain. However, our tested induction conditions could not significantly improve either the proteolytic activity or the nematocidal activity of the Bae16 overexpression mutant.

ResDE Two-Component Regulatory System Mediates Oxygen Limitation-Induced Biofilm Formation by Bacillus amyloliquefaciens SQR9

“…To delete the targeted genes in B. amyloliquefaciens SQR9, 1-kb fragments located upstream and downstream of the target gene were amplified. The 1.6-kb erythromycin resistance gene (Em r ) was amplified from plasmid pAX01 (51), which partially overlapped the upstream and downstream fragments of each target gene. The upstream and downstream fragments were fused with the erythromycin resistance gene by overlap extension.…”

A novel serine protease, Sep1, from Bacillus firmus DS-1 has nematicidal activity and degrades multiple intestinal-associated nematode proteins

“…To identify the nematicidal virulence factors in B. firmus strain DS-1, we sequenced the genome of B. firmus DS-1 (accession number APVL00000000) with Illumina sequencing technology by BGI Shenzhen 24 . Using the reported nematicidal virulence-associated genes, including Bace16 of Bacillus nematocida 10 , Bmp1 from Bacillus thuringiensis 25 , and all other proteases mentioned in Bmp1 research 25 , as well as the hemolysin superfamily proteins, peptidase_S8, peptidase_M6, peptidase_M7, and peptidase_M48 superfamily proteins, a whole genome InterproScan analysis of the B. firmus DS-1 genome was conducted, and the results showed that the B. firmus DS-1 genome harbors multiple potential extracellular protease genes in the genome ( Table 1 ).…”

“…Because Sep1 has similar conserved domains and enzymatic characteristics as the reported extracellular proteases from nematicidal bacteria 10 32 and nematode-trapping fungi 26 27 28 , there is a possibility that Sep1 is an extracellular protease that can destroy the nematode physical barrier. To confirm this hypothesis, we then investigated whether the Sep1 protein degraded tissues from the nematodes C. elegans and M. incognita .…”

“…The collagenase of B. thuringiensis could damage the intestine and is involved in pathogenesis of C. elegans 34 . B. nematocida can secrete neutral protease Bace16 and Bae16 to target nematode intestinal tissue 10 32 . For nematophagous fungi, infectious proteases participate in several steps of host infection 42 .…”

“…The nematophagous fungi include diverse phylogenetic groups, such as Ascomycota , Basidiomycota , Zygomycota , and Chytridiomycota 1 8 . Bacillus , Pseudomonas , and Pasteuria represent the dominant populations of nematophagous bacteria in soil 1 9 , especially Bacillus nematocida , and their toxicity against PPNs suggests they will be ideal biocontrol agents 10 . Moreover, nematicidal cry genes such as cry5B and cry6A from Bacillus thuringiensis have been identified and applied to GM crops to control Meloidogyne spp 11 12 .…”