Both Gram-positive and Gram-negative bacteria can take up exogenous DNA when they are in a competent state either naturally or artificially. However, the thick peptidoglycan layer in Gram-positive bacteria's cell wall is considered as a possible barrier to DNA uptake. In the present work, two transformation techniques have been evaluated in assessing the protocol's ability to introduce foreign DNA, pBBRGFP-45 plasmid which harbors kanamycin resistance and green fluorescent protein (GFP) genes into a Gram-positive bacterium, Bacillus cereus EB2. B. cereus EB2 is an endophytic bacterium, isolated from oil palm roots. A Gram-negative bacterium, Pseudomonas aeruginosa EB35 was used as a control sample for both transformation protocols. The cells were made competent using respective chemical treatment to Gram-positive and Gram-negative bacteria, and kanamycin concentration in the selective medium was also optimized. Preliminary findings using qualitative analysis of colony polymerase chain reaction (PCR)-GFP indicated that the putative positive transformants for B. cereus EB2 were acquired using the second transformation protocol. The positive transformants were then verified using molecular techniques such as observation of putative colonies on specific media under UV light, plasmid extraction, and validation analyses, followed by fluorescence microscopy. Conversely, both transformation protocols were relatively effective for introduction of plasmid DNA into P. aeruginosa EB35. Therefore, this finding demonstrated the potential of chemically prepared competent cells and the crucial step of heat-shock in foreign DNA transformation process of Gram-positive bacterium namely B. cereus was required for successful transformation.
This study aimed to validate the colonisation capability of endophytic bacteria (EB) isolates, Bacillus cereus EB2 and Pseudomonas aeruginosa EB35, that previously exhibited their potentials as biological control agents (BCAs) against the Ganoderma spp., a pathogen for Ganoderma disease in oil palm. Here, we demonstrated a rapid method to determine the colonisation capacity of the selected EB using oil palm tissue culture plantlets and a green fluorescent protein (GFP) visual marker. Wounded plantlet roots were inoculated with GFP-tagged B. cereus EB2 and P. aeruginosa EB35 while the plantlets without EB inoculation served as controls. The GFP signals appeared as bright green spots or lines in the inoculated GFP-tagged EB cells in root and leaf plantlet tissues, respectively, under the confocal laser scanning microscopy (CLSM) 5 days post-inoculation. In contrast, there was no intense GFP spots in neither the control root nor leaf tissues. The cracks in the roots by wounding facilitated the entry of the GFP-tagged EB cells into root tissues, allowing for endophytically colonisation of the root and above-ground tissues. Subsequent result of polymerase chain reaction (PCR)-GFP analysis further displayed the endophytic nature and early chronological colonisation of the tested EB. This is a preliminary report on root colonisation by a Gram-positive endophyte, B. cereus EB2 and leaf tissues colonisation by both EB isolates as internal colonisers, demonstrating their potential as BCAs to protect oil palm against Ganoderma spp. for a sustainable disease management.
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