The ability to correlate the production of specialized metabolites to the genetic capacity of the organism that produces such molecules has become an invaluable tool in aiding the discovery of biotechnologically applicable molecules. Here, we accomplish this task by matching molecular families with gene cluster families, making these correlations to 60 microbes at one time instead of connecting one molecule to one organism at a time, such as how it is traditionally done. We can correlate these families through the use of nanospray desorption electrospray ionization MS/MS, an ambient pressure MS technique, in conjunction with MS/MS networking and peptidogenomics. We matched the molecular families of peptide natural products produced by 42 bacilli and 18 pseudomonads through the generation of amino acid sequence tags from MS/MS data of specific clusters found in the MS/MS network. These sequence tags were then linked to biosynthetic gene clusters in publicly accessible genomes, providing us with the ability to link particular molecules with the genes that produced them. As an example of its use, this approach was applied to two unsequenced Pseudoalteromonas species, leading to the discovery of the gene cluster for a molecular family, the bromoalterochromides, in the previously sequenced strain P. piscicida JCM 20779^T. The approach itself is not limited to 60 related strains, because spectral networking can be readily adopted to look at molecular family–gene cluster families of hundreds or more diverse organisms in one single MS/MS network. Significance The paper introduces the concepts of molecular families (MFs) and gene cluster families (GCFs). We define MFs as structurally related molecules based on their mass spectral fragmentation patterns, whereas GCFs are biosynthetic gene clusters that show similar gene cluster organization with a high degree of sequence similarity. We use MS/MS networking as a tool to map the molecular network of more than 60 organisms, most of which are unsequenced, and locate their nonribosomal peptide MFs. These MFs from unsequenced organisms are then connected to GCFs of publicly available genome sequences of closely related organisms.

Coral reefs are intricate ecosystems that harbor diverse organisms, including 25% of all marine fish. Healthy corals exhibit a complex symbiosis between coral polyps, endosymbiotic alga, and an array of microorganisms, called the coral holobiont. Secretion of specialized metabolites by coral microbiota is thought to contribute to the defense of this sessile organism against harmful biotic and abiotic factors. While few causative agents of coral diseases have been unequivocally identified, fungi have been implicated in the massive destruction of some soft corals worldwide. Because corals are nocturnal feeders, they may be more vulnerable to fungal infection at night, and we hypothesized that the coral microbiota would have the capability to enhance their defenses against fungi in the dark. A Pseudoalteromonas sp. isolated from a healthy octocoral displayed light-dependent antifungal properties when grown adjacent to Penicilliumcitrinum (P. citrinum) isolated from a diseased Gorgonian octocoral. Microbial MALDI-imaging mass spectrometry (IMS) coupled with molecular network analyses revealed that Pseudoalteromonas produced higher levels of antifungal polyketide alteramides in the dark than in the light. The alteramides were inactivated by light through a photoinduced intramolecular cyclization. Further NMR studies led to a revision of the stereochemical structure of the alteramides. Alteramide A exhibited antifungal properties and elicited changes in fungal metabolite distributions of mycotoxin citrinin and citrinadins. These data support the hypothesis that coral microbiota use abiotic factors such as light to regulate the production of metabolites with specialized functions to combat opportunistic pathogens at night.

The present research aimed to evaluate the antibacterial activity of volatile organic compounds (VOCs) produced by octocoral-associated bacteria Bacillus sp. BO53 and Pseudoalteromonas sp. GA327. The volatilome bioactivity of both bacteria species was evaluated against human pathogenic antibiotic-resistant bacteria, methicillin-resistant Staphylococcus aureus, Acinetobacter baumanni, and Pseudomonas aeruginosa. In this regard, the in vitro tests showed that Bacillus sp. BO53 VOCs inhibited the growth of P. aeruginosa and reduced the growth of S. aureus and A. baumanni. Furthermore, Pseudoalteromonas sp. GA327 strongly inhibited the growth of A. baumanni, and P. aeruginosa. VOCs were analyzed by headspace solid-phase microextraction (HS-SPME) joined to gas chromatography-mass spectrometry (GC-MS) methodology. Nineteen VOCs were identified, where 5-acetyl-2-methylpyridine, 2-butanone, and 2-nonanone were the major compounds identified on Bacillus sp. BO53 VOCs; while 1-pentanol, 2-butanone, and butyl formate were the primary volatile compounds detected in Pseudoalteromonas sp. GA327. We proposed that the observed bioactivity is mainly due to the efficient inhibitory biochemical mechanisms of alcohols and ketones upon antibiotic-resistant bacteria. This is the first report which describes the antibacterial activity of VOCs emitted by octocoral-associated bacteria.

The marine bacterial genus Pseudoalteromonas is known for their ability to produce antimicrobial compounds. The metabolite-producing capacity of Pseudoalteromonas has been associated with strain pigmentation; however, the genomic basis of their antimicrobial capacity remains to be explained. In this study, we sequenced the whole genome of six Pseudoalteromonas strains (three pigmented and three non-pigmented), with the purpose of identifying biosynthetic gene clusters (BGCs) associated to compounds we detected via microbial interactions along through MS-based molecular networking. The genomes were assembled and annotated using the SPAdes and RAST pipelines and mined for the identification of gene clusters involved in secondary metabolism using the antiSMASH database. Nineteen BGCs were detected for each non-pigmented strain, while more than thirty BGCs were found for two of the pigmented strains. Among these, the groups of genes of nonribosomal peptide synthetases (NRPS) that code for bromoalterochromides stand out the most. Our results show that all strains possess BGCs for the production of secondary metabolites, and a considerable number of distinct polyketide synthases (PKS) and NRPS clusters are present in pigmented strains. Furthermore, the molecular networking analyses revealed two new molecules produced during microbial interactions: the dibromoalterochromides D/D’ (11–12).