Genome Mining, Microbial Interactions, and Molecular Networking Reveals New Dibromoalterochromides from Strains of Pseudoalteromonas of Coiba National Park-Panama.

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).

Antimicrobial-producing Pseudoalteromonas from the marine environment of Panama shows a high phylogenetic diversity and clonal structure.

Pseudoalteromonas is a genus of marine bacteria often found in association with other organisms. Although several studies have examined Pseudoalteromonas diversity and their antimicrobial activity, its diversity in tropical environments is largely unexplored. We investigated the diversity of Pseudoalteromonas in marine environments of Panama using a multilocus phylogenetic approach. Furthermore we tested their antimicrobial capacity and evaluated the effect of recombination and mutation in shaping their phylogenetic relationships. The reconstruction of clonal relationships among 78 strains including 15 reference Pseudoalteromonas species revealed 43 clonal lineages, divided in pigmented and non-pigmented strains. In total, 39 strains displayed moderate to high activity against Gram-positive and Gram-negative bacteria and fungi. Linkage disequilibrium analyses showed that the Pseudoalteromonas strains of Panama have a highly clonal structure and that, although present, recombination is not frequent enough to break the association among alleles. This clonal structure is in contrast to the high rates of recombination generally reported for aquatic and marine bacteria. We propose that this structure is likely due to the symbiotic association with marine invertebrates of most strains analyzed. Our results also show that there are several putative new species of Pseudoalteromonas in Panama to be described.

MS/MS networking guided analysis of molecule and gene cluster families.

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.

Imaging mass spectrometry of a coral microbe interaction with fungi.

Fungal infections are increasing worldwide, including in the aquatic environment. Microbiota that coexist with marine life can provide protection against fungal infections by secretion of metabolites with antifungal properties. Our laboratory has developed mass spectrometric methodologies with the goal of improving our functional understanding of microbial metabolites and guiding the discovery process of anti-infective agents from natural sources. GA40, a Bacillus amyloliquefaciens strain isolated from an octocoral in Panama, displayed antifungal activity against various terrestrial and marine fungal strains. Using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), the molecular species produced by this microbe were visualized in a side-by-side interaction with two representative fungal strains, Aspergillus fumigatus and Aspergillus niger. The visualization was performed directly on the agar without the need for extraction. By evaluating the spatial distributions, relative intensities and m/z values of GA40 secreted metabolites in the fungal interactions and singly grown control colonies, we obtained insight into the antifungal activity of secreted metabolites. Annotation of GA40 metabolites observed in MALDI-IMS was facilitated by MS/MS networking analysis, a mass spectrometric technique that clusters metabolites with similar MS/MS fragmentation patterns. This analysis established that the predominant GA40 metabolites belong to the iturin family. In a fungal inhibition assay of A. fumigatus, the GA40 iturin metabolites were found to be responsible for the antifungal properties of this Bacillus strain.

Antibacterial Activity of Volatile Organic Compounds Produced by the Octocoral-Associated Bacteria Bacillus sp. BO53 and Pseudoalteromonas sp. GA327.

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.

Microbiota of healthy corals are active against fungi in a light-dependent manner.

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 Penicillium citrinum (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.