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Background: Bacteria within the genus Photorhabdus maintain mutualistic symbioses with nematodes in complicated lifecycles that also involves insect pathogenic phases. Intriguingly, these bacteria are rich in biosynthetic gene clusters that produce compounds with diverse biological activities. As a basis to better understand the life cycles of Photorhabdus we sequenced the genomes of two recently discovered representative species and performed detailed genomic comparisons with five publically available genomes.
Results: Here we report the genomic details of two new reference Photorhabdus species. By then conducting genomic comparisons across the genus, we show that there are several highly conserved biosynthetic gene clusters. These clusters produce a range of bioactive small molecules that support the pathogenic phase of the integral relationship that Photorhabdus maintain with nematodes.
Conclusions: Photorhabdus contain several genetic loci that allow them to become specialist insect pathogens by efficiently evading insect immune responses and killing the insect host.
Identification of new natural products from nematode-associated bacteria using mass spectrometry
(2023)
This work aims to find unknown natural products produced by bacteria, that live in close association with nematodes and to elucidate their structure by using mass spectrometry.
The first chapter of this work is dedicated to the detection of hitherto unknown natural products by using a metabolomics approach and subsequent structure elucidation of said compounds. This chapter includes metabolomics analysis of Xenorhabdus szentirmaii wild type and knockout mutants, overproduction of the target compound, identification of derivatives from other strains and MS based structure elucidation.
The second and third chapters are about natural products that protect C. elegans from B. thuringiensis infections.
The second chapter deals with natural products that protect the nematode host without killing the pathogen. I deployed molecular biology methods to generate deletion and overproduction strains of a target compound, identified it via LC-MS/MS analysis and used LC-MS/MS and lipidomics to analyse the chemical properties of the active compound.
The third chapter aims at finding natural products, which are produced by Pseudomonas strains MYb11 and MYb12, respectively. These natural products display the ability to protect C. elegans by killing B. thuringiensis. I identified said compounds via fractionation and subsequent bioactivity testing. After identification, I generated production strains of the target compounds and elucidated the structure of the bioactive derivative.
The last chapter deals with the structure elucidation of peptides produced by an unusual GameXPeptide synthetase in Xenorhabdus miraniensis. I analysed producer strains of GameXPeptides using LC-MS and elucidated the structural differences between the known GameXPeptides, produced by P. luminescens TT01, and the unusual ones produced by X. miraniensis.
The Gram-negative bacteria Photorhabdus and Xenorhabdus are known to produce a variety of different natural products (NP). These compounds play different roles since the bacteria live in symbiosis with nematodes and are pathogenic to insect larvae in the soil. Thus, a fine tuned regulatory system controlling NP biosynthesis is indispensable. Global regulators such as Hfq, Lrp, LeuO and HexA have been shown to influence NP production of Photorhabdus and Xenorhabdus. Additionally, photopyrones as quorum sensing (QS) signals were demonstrated to be involved in the regulation of NP production in Photorhabdus. In this study, we investigated the role of another possible QS signal, autoinducer-2 (AI-2), in regulation of NP production. The AI-2 synthase (LuxS) is widely distributed within the bacterial kingdom and has a dual role as a part of the activated methyl cycle pathway, as well as being responsible for AI-2 precursor production. We deleted luxS in three different entomopathogenic bacteria and compared NP levels in the mutant strains to the wild type (WT) but observed no difference to the WT strains. Furthermore, the absence of the small regulatory RNA micA, which is encoded directly upstream of luxS, did not influence NP levels. Phenotypic differences between the P. luminescens luxS deletion mutant and an earlier described luxS deficient strain of P. luminescens suggested that two phenotypically different strains have evolved in different laboratories.
A new cyclic lipopeptide, phototemtide A (1), was isolated from Escherichia coli expressing the biosynthetic gene cluster pttABC from Photorhabdus temperata Meg1. The structure of 1 was elucidated by HR‐ESI‐MS and NMR experiments. The absolute configurations of amino acids and 3‐hydroxyoctanoic acid in 1 were determined by using the advanced Marfey's method and comparison after total synthesis of 1, respectively. Additionally, three new minor derivatives, phototemtides B–D (2–4), were identified by detailed HPLC–MS analysis. Phototemtide A (1) showed weak antiprotozoal activity against Plasmodium falciparum, with an IC50 value of 9.8 μm. The biosynthesis of phototemtides A–D (1–4) was also proposed.
In search for new natural products, which may lead to the development of new drugs for all kind of applications, novel methods are needed. Here we describe the identification of electrophilic natural products in crude extracts via their reactivity against azide as a nucleophile followed by their subsequent enrichment using a cleavable azide-reactive resin (CARR). Using this approach, natural products carrying epoxides and α,β-unsaturated enones as well as several unknown compounds were identified in crude extracts from entomopathogenic Photorhabdus bacteria.