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.

Bacillus nematocida B16 has been shown to use “Trojan horse” mechanism in pathogenesis that has characteristics of “social” behavior. The ComP-ComA system, a conserved quorum sensing system in the genus Bacillus, functions in many physiological processes including competence development, lipopeptide antibiotic surfactin production, degradative enzyme production and even some unknown functions. Here we investigated the requirement of ComP-ComA system in B. nematocida B16 for its pathogenicity against nematodes. The ΔcomP mutant displayed deficiencies in attracting and killing nematodes, due to the absence of attractive signal molecules and the decreased expressions of virulence factors, respectively. Contrarily, a complemented comP mutant at least partially resumed its pathogenicity. Our data from transcriptional analysis further confirmed that this signaling system directly or indirectly regulated the expressions of two major virulence proteases in the infection of B. nematocida B16. Bioinformatics analyses from comparative genomics also suggested that the potential target genes of transcription factor ComA were involved in the processes such as the synthesis of attractants, production of extracellular degradative enzymes and sortase, secondary metabolites biosynthesis, regulation of transcription factors, mobility, as well as transporters, most of which were different from a saprophytic relative B. subtilis 168. Therefore, our investigation firstly revealed that the participation and necessity of ComP-ComA signaling system in bacterial pathogenesis.

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