Antagonism of volatile organic compounds of the Bacillus sp. against Fusarium kalimantanense.

Fusarium kalimantanense is a genetic lineage of Fusarium oxysporum f. sp. cubense (Foc) and belongs to the Fusarium oxysporum species complex (FOSC). This pathogen is a causative agent of Panama disease, an infection that has caused damage to the banana crop worldwide. Bacillus sp. (LPPC170) showed preliminary antagonist activity against F. kalimantanense (LPPC130) in vitro tests from the cultivation of axenic culture and co-culture with inhibition of mycelial growth of phytopathogen of 41.23%. According to these findings, volatile organic compounds (VOCs) emitted from Bacillus sp. were obtained by solid-phase microextraction and identified by gas chromatography coupled with a mass spectrometer (GC-MS). The multivariate data analysis tool (PLS-DA and Heatmap) identified short-chain organic acids as the main antagonistic VOCs responsible for inhibiting the mycelial growth of LPPC130. Acetic acid, propanoic acid, butanoic acid, valeric acid, and isovaleric acid exhibited a strong inhibitory effect on the mycelial growth of LPPC130, with inhibition of 20.68%, 33.30%, 26.87%, 43.71%, and 53.10%, respectively. Scanning electron microscopy revealed that VOCs caused damage to the vegetative and reproductive structures of the fungus. These results suggest Bacillus LPPC170 as an excellent biocontrol tool against the phytopathogen causative agents of Panama disease.

Expression and efficient secretion of a functional chitinase from Chromobacterium violaceum in Escherichia coli.

BACKGROUND: Chromobacterium violaceum is a free-living ß-proteobacterium found in tropical and subtropical regions. The genomic sequencing of C. violaceum ATCC 12472 has revealed many genes that underpin its adaptability to diverse ecosystems. Moreover, C. violaceum genes with potential applications in industry, medicine and agriculture have also been identified, such as those encoding chitinases. However, none of the chitinase genes of the ATCC 12472 strain have been subjected to experimental validation. Chitinases (EC 3.2.1.14) hydrolyze the ß-(1,4) linkages in chitin, an abundant biopolymer found in arthropods, mollusks and fungi. These enzymes are of great biotechnological interest as potential biocontrol agents against pests and pathogens. This work aimed to experimentally validate one of the chitinases from C. violaceum. RESULTS: The open reading frame (ORF) CV2935 of C. violaceum ATCC 12472 encodes a protein (439 residues) that is composed of a signal peptide, a chitin-binding domain, a linker region, and a C-terminal catalytic domain belonging to family 18 of the glycoside hydrolases. The ORF was amplified by PCR and cloned into the expression vector pET303/CT-His. High levels of chitinolytic activity were detected in the cell-free culture supernatant of E. coli BL21(DE3) cells harboring the recombinant plasmid and induced with IPTG. The secreted recombinant protein was purified by affinity chromatography on a chitin matrix and showed an apparent molecular mass of 43.8 kDa, as estimated by denaturing polyacrylamide gel electrophoresis. N-terminal sequencing confirmed the proper removal of the native signal peptide during the secretion of the recombinant product. The enzyme was able to hydrolyze colloidal chitin and the synthetic substrates p-nitrophenyl-ß-D-N,N'-diacetylchitobiose and p-nitrophenyl-ß-D-N,N',N"-triacetylchitotriose. The optimum pH for its activity was 5.0, and the enzyme retained ~32% of its activity when heated to 60°C for 30 min. CONCLUSIONS: A C. violaceum chitinase was expressed in E. coli and purified by affinity chromatography on a chitin matrix. The secretion of the recombinant protein into the culture medium was directed by its native signal peptide. The mature enzyme was able to hydrolyze colloidal chitin and synthetic substrates. This newly identified signal peptide is a promising secretion factor that should be further investigated in future studies, aiming to demonstrate its usefulness as an alternative tool for the extracellular production of recombinant proteins in E. coli.