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Our understanding of the role of secondary metabolites in microbial communities is challenged by intrinsic limitations of culturing bacteria under laboratory conditions and hence cultivation independent approaches are needed. Here, we present a protocol termed Secondary Metabolite FISH (SecMet-FISH), combining advantages of gene-targeted fluorescence in situ hybridization (geneFISH) with in-solution methods (in-solution FISH) to detect and quantify cells based on their genetic capacity to produce secondary metabolites. The approach capitalizes on the conserved nature of biosynthetic gene clusters (BGCs) encoding adenylation (AD) and ketosynthase (KS) domains, and thus selectively targets the genetic basis of non-ribosomal peptide and polyketide biosynthesis. The concept relies on the generation of amplicon pools using degenerate primers broadly targeting AD and KS domains followed by fluorescent labeling, detection, and quantification. Initially, we obtained AD and KS amplicons from Pseuodoalteromonas rubra , which allowed us to successfully label and visualize BGCs within P. rubra cells, demonstrating the feasibility of SecMet-FISH. Next, we adapted the protocol and optimized it for hybridization in both Gram-negative and Gram-positive bacterial cell suspensions, enabling high-throughput single cell analysis by flow cytometry. Ultimately, we used SecMet-FISH to successfully distinguish secondary metabolite producers from non-producers in a five-member synthetic community. [ABSTRACT FROM AUTHOR]

Deciphering the cues that stimulate microorganisms to produce their full secondary metabolic potential promises to speed up the discovery of novel drugs. Ecology-relevant conditions, including carbon-source(s) and microbial interactions, are important effectors of secondary metabolite production. Vice versa secondary metabolites are important mediators in microbial interactions, although their exact natural functions are not always completely understood. In this study, we investigated the effects of microbial interactions and in-culture produced antibiotics on the production of secondary metabolites by Vibrio coralliilyticus and Photobacterium galatheae , two co-occurring marine Vibrionaceae. In co-culture, production of andrimid by V. coralliilyticus and holomycin by P. galatheae , were, compared to monocultures, increased 4.3 and 2.7 fold, respectively. Co-cultures with the antibiotic deficient mutant strains (andrimid− and holomycin−) did not reveal a significant role for the competitor's antibiotic as stimulator of own secondary metabolite production. Furthermore, we observed that V. coralliilyticus detoxifies holomycin by sulphur-methylation. Results presented here indicate that ecological competition in Vibrionaceae is mediated by, and a cue for, antibiotic secondary metabolite production. [ABSTRACT FROM AUTHOR]

The development and spread of multidrug resistant pathogens have reinforced the urgency to find novel natural products with antibiotic activity. In bacteria, orphan biosynthetic gene clusters (BGCs) far outnumber the BGCs for which chemistry is known, possibly because they are transcriptionally silent under laboratory conditions. A strategy to trigger the production of this biosynthetic potential is to challenge the microorganism with low concentrations of antibiotics, and by using a Burkholderia genetic reporter strain (Seyedsayamdost, Proc Natl Acad Sci 111:7266–7271), we found BGC unsilencing activity for the antimicrobial andrimid, produced by the marine bacterium Vibrio coralliilyticus. Next, we challenged another marine Vibrionaceae, Photobacterium galatheae , carrier of seven orphan BGCs with sub-inhibitory concentrations of andrimid. A combined approach of transcriptional and chemical measurements of andrimid-treated P. galatheae cultures revealed a 10-fold upregulation of an orphan BGC and, amongst others, a 1.6–2.2-fold upregulation of the gene encoding the core enzyme for biosynthesis of holomycin. Also, addition of andrimid caused an increase, based on UV-Vis peak area, of 4-fold in production of the antibiotic holomycin. Transcriptional measurements of stress response related genes in P. galatheae showed a co-occurrence of increased transcript levels of rpoS (general stress response) and andrimid induced holomycin overproduction, while in trimethoprim treated cultures attenuation of holomycin production coincided with a transcriptional increase of recA (SOS stress response). This study shows that using antimicrobial compounds as activators of secondary metabolism can be a useful strategy in eliciting biosynthetic gene clusters and facilitate natural product discovery. Potentially, such interactions could also have ecological relevant implications. [ABSTRACT FROM AUTHOR]

Covering: up to 2019 Humanity is in dire need for novel medicinal compounds with biological activities ranging from antibiotic to anticancer and anti-dementia effects. Recent developments in genome sequencing and mining have revealed an unappreciated potential for bioactive molecule production in marine Proteobacteria. Also, novel bioactive compounds have been discovered through molecular manipulations of either the original marine host bacteria or in heterologous hosts. Nevertheless, in contrast to the large repertoire of such molecules as predicted by in silico analysis, few marine bioactive compounds have been reported. This review summarizes the recent advances in the study of natural products from marine Proteobacteria. Here we present successful examples on genetic engineering of biosynthetic gene clusters of natural products from marine Proteobacteria. We also discuss the future prospects of discovering novel bioactive molecules via both heterologous production methodology and the development of marine Proteobacteria as new cell factories. [ABSTRACT FROM AUTHOR]

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential. [ABSTRACT FROM AUTHOR]