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Whilst the effects of antibiotics on microorganisms are widely studied, it remains less well-understood how antibiotics affect the physiology of the native producing organisms. Here, using a marine bacterium Photobacterium galatheae S2753 that produces the antibiotic holomycin, we generated a holomycin deficient strain by in-frame deletion of hlmE, the core gene responsible for holomycin production. Mass spectrometry analysis of cell extracts confirmed that ΔhlmE did not produce holomycin and that the mutant was devoid of antibacterial activity. Biofilm formation of ΔhlmE was significantly reduced compared to that of the wild-type S2753 and was restored in an hlmE complementary mutant. Consistently, exogenous holomycin, but not its dimethylated and less antibacterial derivative, S,S'-dimethyl holomycin, restored the biofilm formation of ΔhlmE Furthermore, zinc starvation was found essential for both holomycin production and biofilm formation of S2753, although the molecular mechanism remains elusive. Collectively, these data suggest that holomycin promotes biofilm formation of S2753 via its ene-disulfide group. Lastly, the addition of holomycin in sub-inhibitory concentrations also enhanced the biofilm of four other Vibrionaceae strains. P. galatheae likely gains an ecological advantage from producing holomycin as both an antibiotic and a biofilm stimulator, which facilitates the nutrition acquisition and protects P. galaltheae from environmental stresses. Studying the function of antibiotic compounds in the native producer will shed light on their role in nature and could potentially point to novel bioprospecting strategies. Importance Despite the societal impact of antibiotics, their ecological functions remain elusive and have mostly been studied by exposing non-producing bacteria to sub-inhibitory concentrations. Here, we studied the effects of the antibiotic holomycin on its native producer, Photobacterium galath

Pseudoalteromonas rubra S4059 produces the red pigment prodigiosin, which has pharmaceutical and industrial potential. Here, we targeted a putative prodigiosin-synthesizing transferase PigC, and a pigC in-frame deletion mutant did not produce prodigiosin. However, extractions of the pigC mutant cultures retained antibacterial activity, and bioassay-guided fractionation found antibacterial activity in two fractions of blue color. A precursor of prodigiosin, 4-methoxy-2,2′-bipyrrole-5-carbaldehyde (MBC), was the dominant compound in both the fractions and likely caused the antibacterial activity. Also, a stable blue pigment, di-pyrrolyl-dipyrromethene prodigiosin, was identified from the two fractions. We also discovered antibacterial activity in the sterile filtered (nonextracted) culture supernatant of both wild type and mutant, and both contained a heat-sensitive compound between 30 and 100 kDa. Deletion of prodigiosin production did not affect growth rate or biofilm formation of P. rubra and did not change its fitness, as the mutant and wild type coexisted in equal levels in mixed cultures. In conclusion, a prodigiosin biosynthetic gene cluster (BGC) was identified and verified genetically and chemically in P. rubra S4059 and a stable blue pigment was isolated from the pigC mutant of S4059, suggesting that this strain may produce several prodigiosin-derived compounds of pharmaceutical and/or industrial potential. IMPORTANCE Pigmented Pseudoalteromonas strains are renowned for their production of secondary metabolites, and genome mining has revealed a high number of biosynthetic gene clusters (BGCs) for which the chemistry is unknown. Identification of those BGCs is a prerequisite for linking products to gene clusters and for further exploitation through heterologous expression. In this study, we identified the BGCs for the red, bioactive pigment prodigiosin using genomic, genetic, and metabolomic approaches. We al

New infectious diseases and increase in drug-resistant microbial pathogens emphasize the need for antibiotics with novel mode-of-action. Tetramates represented by fungi-derived tenuazonic acid and bacterial polycyclic tetramate macrolactams (PTMs) are an important family of natural products with a broad spectrum of antimicrobial activities. Despite their potential application as new antibiotics, it remains unknown how PTMs function. In this study, genomic mining revealed that PTM biosynthetic gene clusters (BGCs) are widespread in both Gram-positive and Gram-negative bacteria, and we investigated a sponge endosymbiont Actinoalloteichus hymeniacidonis harboring a potential PTM-BGC. Xanthobaccin A that previously has only been isolated from a Gram-negative bacterium was obtained after a scale-up fermentation, isolation, and structure elucidation through mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Xanthobaccin A as well as two previously reported tetramates, equisetin and ikarugamycin, exhibited antibacterial activities against Bacillus subtilis. In addition, these three tetramates were for the first time to be confirmed as metallophores and the stoichiometry of the complexes were shown to be Fe(III)(equisetin)3/Fe(III)(equisetin)2 and Fe(III)(ikarugamycin)2, respectively. Meanwhile, we found that all three tetramates could reduce ferric into ferrous iron, which triggers the Fenton chemistry reaction. Their antibacterial activity was reduced by adding the radical scavenger, vitamin C. Altogether, our work demonstrates that equisetin and PTMs can act as metallophores and their antimicrobial mechanism is possibly mediated through Fenton chemistry.

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.

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.

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.

Covering: up to 2019Humanity 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.