Pathogen-induced activation of disease-suppressive functions in the endophytic root microbiome

Microorganisms living inside plants can promote plant growth and health, but their genomic and functional diversity remain largely elusive. Here, metagenomics and network inference show that fungal infection of plant roots enriched for Chitinophagaceae and Flavobacteriaceae in the root endosphere and for chitinase genes and various unknown biosynthetic gene clusters encoding the production of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). After strain-level genome reconstruction, a consortium of Chitinophaga and Flavobacterium was designed that consistently suppressed fungal root disease. Site-directed mutagenesis then revealed that a previously unidentified NRPS-PKS gene cluster from Flavobacterium was essential for disease suppression by the endophytic consortium. Our results highlight that endophytic root microbiomes harbor a wealth of as yet unknown functional traits that, in concert, can protect the plant inside out.</p

PCR amplification and cloning of six target genes from Burkholderia pseudomallei

Burkholderia pseudomallei (B. pseudomallei) is an aerobic Gram negative bacteria and a causative agent of melioidosis, a disease endemic in several regions worldwide including Southeast Asia and Northern Australia. Melioidosis can be fatal in human, where it causes fever and is commonly present with pneumonia, with or without septicaemia. Treating melioidosis is a challenge, due to its intrinsic resistance to many antibiotics, and occurrence of latent infection. This study is part of a larger project to purify and characterise target proteins from B. pseudomallei to gain fundamental knowledge on how this bacterium behaves, and thus providing us strategies to combat them. In this report, six target genes predicted to be essential by Transposon-Directed Insertion site Sequencing (TraDIS) technique were selected for PCR amplification and cloning trials. Out of the six target genes, three genes encode for hypothetical proteins (BPSL0084, BPSL0086, and BPSL1060), two genes encode for putative annotated protein (BPSL0398 and BPSL1192) and one gene (BPSL1549) encodes for B. pseudomallei lethal factor 1. Genomic DNA of B. pseudomallei strain D286 and K96243 is obtained from School of Biosciences & Biotechnology, UKM. All six target genes were successfully amplified. Sufficient concentration of purified attBPCR products for all target genes was obtained after nested PCR. This is then utilized in the BP recombination reactions to create the entry clones. The entry clones were transformed into Escherichia coli (E. coli) DH5α™ competent cells and plated onto LB agar plate supplemented with 50 µg/mL kanamycin. The positive clones were screened using colony PCR and BsrGI restriction enzyme digestion. At the time of writing, three target genes were successfully cloned and validated by sequencing. In future projects, the entry clones can then be easily transferred into various expression vectors to be expressed and purified for diverse functional protein studies

Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas

CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for tight and inducible control over CRISPR-Cas counter-selection. This control delays CRISPR-Cas counter-selection, granting more time for the editing event (e.g. by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three wild-type bacteria species (Escherichia coli MG1655, Pseudomonas putida KT2440 and Flavobacterium IR1) with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a simple gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria