Variations of Indole Metabolites and NRPS-PKS Loci in Two Different Virulent Strains of Xenorhabdus hominickii

Abstract

Xenorhabdus hominickii ANU1 is known to be an entomopathogenic bacterium symbiotic to nematode Steinernema monticolum. Another bacterial strain X. hominickii DY1 was isolated from a local population of S. monticolum. This bacterial strain X. hominickii DY1 was found to exhibit high insecticidal activities against lepidopteran and coleopteran species after hemocoelic injection. However, these two X. hominickii strains exhibited significant variations in insecticidal activities, with ANU1 strain being more potent than DY1 strain. To clarify their virulence difference, bacterial culture broths of these two strains were compared for secondary metabolite compositions. GC-MS analysis revealed that these two strains had different compositions, including pyrrolopyrazines, piperazines, cyclopeptides, and indoles. Some of these compounds exhibited inhibitory activities against phospholipase A2 to block eicosanoid biosynthesis and induce significant immunosuppression. They also exhibited significant insecticidal activities after oral feeding, with indole derivatives being the most potent. More kinds of indole derivatives were detected in the culture broth of ANU1 strain. To investigate variations in regulation of secondary metabolite production, expression level of leucine-responsive regulatory protein (Lrp), a global transcription factor, was compared. ANU1 strain exhibited significantly lower Lrp expression level than DY1 strain. To assess genetic variations associated with secondary metabolite synthesis, bacterial loci encoding non-ribosomal protein synthase and polyketide synthase (NRPS-PKS) were compared. Three NRPS and four PKS loci were predicted from the genome of X. hominickii. The two bacterial strains exhibited genetic variations (0.12∼0.67%) in amino acid sequences of these NRPS-PKS. Most NRPS-PKS genes exhibited high expression peaks at stationary phase of bacterial growth. However, their expression levels were significantly different between the two strains. These results suggest that differential virulence of the two bacterial strains is caused by the difference in Lrp expression level, leading to difference in the production of indole compounds and other NRPS-PKS-associated secondary metabolites.

Immune mediation of HMG-like DSP1 via Toll-Spätzle pathway and its specific inhibition by salicylic acid analogs

“…Total RNA extraction, cDNA preparation, and RT-qPCR followed the procedure described by Mollah et al [ 48 ]. Briefly, the synthesized single-stranded cDNA was used as template for RT-PCR with 35 rounds of a temperature cycle of 94°C for 30 sec, different annealing temperatures for 30 sec, and 72°C for 30 sec after an initial heat treatment at 94°C for 2 min with gene-specific primers ( S1 Table ).…”

“…X . hominickii was cultured in 1 L of TSB at 28°C for 48 h. After centrifuging cultured broth at 10,000 × g for 20 min at 4°C, the resulting supernatant was used for organic extraction as described by Mollah et al [ 48 ]. Briefly, the supernatant was mixed with the same volume of hexane.…”

“…Pathogenic bacteria including X. hominickii possess a gene cluster (polyketide ketone synthase: PKS) associated with the biosynthesis of virulent factors [47]. EMP is likely to be a product of PKS [48].…”

Salicylic Acid, a Plant Hormone, Suppresses Phytophagous Insect Immune Response by Interrupting HMG-Like DSP1

“…Total RNA extraction, complementary DNA (cDNA) preparation, and quantitative reverse transcription (RT-q)PCR were performed following the methods described previously (Mollah et al, 2020a , b ). The synthesized first-stranded cDNA was used as a template for PCR amplification with 35 cycles of 94°C for 30 s, 58.8°C for 30 s, and 72°C for 30 s after an initial heat treatment at 94°C for 2 min with the gene-specific primers listed in Supplementary Table S2 .…”

Microbial Toxins in Insect and Nematode Pest Biocontrol

“…An isocyanide-containing compound rhabducin produced by Xenorhabdus can also inhibit phenoloxidase and thereby melanization [ 42 ]. In addition, phurealipids (urea compounds) made by these bacteria can prevent the expression of antimicrobial peptide genes in the insect hosts, a part of the humoral component of immunity [ 43 ]. It is no surprise that these bacterial products induce fatal septicemia and toxemia culminating in the death of target insects in 24 to 48 h [ 44 ].…”