Biosynthetic potential of the sediment microbial subcommunities of an unexplored karst ecosystem and its ecological implications.

Microbial communities from various environments have been studied in the quest for new natural products with a broad range of applications in medicine and biotechnology. We employed an enrichment method and genome mining tools to examine the biosynthetic potential of microbial communities in the sediments of a coastal sinkhole within the karst ecosystem of the Yucatán Peninsula, Mexico. Our investigation led to the detection of 203 biosynthetic gene clusters (BGCs) and 55 secondary metabolites (SMs) within 35 high-quality metagenome-assembled genomes (MAGs) derived from these subcommunities. The most abundant types of BGCs were Terpene, Nonribosomal peptide-synthetase, and Type III polyketide synthase. Some of the in silico identified BGCs and SMs have been previously reported to exhibit biological activities against pathogenic bacteria and fungi. Others could play significant roles in the sinkhole ecosystem, such as iron solubilization and osmotic stress protection. Interestingly, 75% of the BGCs showed no sequence homology with bacterial BGCs previously reported in the MiBIG database. This suggests that the microbial communities in this environment could be an untapped source of genes encoding novel specialized compounds. The majority of the BGCs were identified in pathways found in the genus Virgibacillus, followed by Sporosarcina, Siminovitchia, Rhodococcus, and Halomonas. The latter, along with Paraclostridium and Lysinibacillus, had the highest number of identified BGC types. This study offers fresh insights into the potential ecological role of SMs from sediment microbial communities in an unexplored environment, underscoring their value as a source of novel natural products.

Sediment microbial community structure associated to different ecological types of mangroves in Celestún, a coastal lagoon in the Yucatan Peninsula, Mexico.

Mangroves are unique coastal ecosystems, which have many important ecological functions, as they are a reservoir of many marine species well adapted to saline conditions and are fundamental as sites of carbon storage. Although the microbial contribution to nutrient cycling in these ecosystems has been well recognized, there is a lack of information regarding the microbial composition and structure of different ecological types of mangrove forests. In this study, we characterized the microbial community (Bacteria and Archaea) in sediments associated with five ecological types of mangrove forests in a coastal lagoon dominated by Avicennia germinans and Rhizophora mangle, through 16S rRNA-V4 gene sequencing. Overall, Proteobacteria (51%), Chloroflexi (12%), Gemmatimonadetes (5%) and Planctomycetes (6%) were the most abundant bacterial phyla, while Thaumarchaeota (30%), Bathyarchaeota (21%) and Nanoarchaeaeota (18%) were the dominant archaeal phyla. The microbial composition associated with basin mangroves dominated by Avicennia germinans was significantly different from the other ecological types, which becomes relevant for restoration strategies.

Insights into the Chemical Diversity of Selected Fungi from the Tza Itzá Cenote of the Yucatan Peninsula.

Cenotes are habitats with unique physical, chemical, and biological features. Unexplored microorganisms from these sinkholes represent a potential source of bioactive molecules. Thus, a series of cultivable fungi (Aspergillus spp. NCA257, NCA264, and NCA276, Stachybotrys sp. NCA252, and Cladosporium sp. NCA273) isolated from the cenote Tza Itzá were subjected to chemical, coculture, and metabolomic analyses. Nineteen compounds were obtained and tested for their antimicrobial potential against ESKAPE pathogens, Mycobacterium tuberculosis, and nontuberculous mycobacteria. In particular, phenylspirodrimanes from Stachybotrys sp. NCA252 showed significant activity against MRSA, MSSA, and mycobacterial strains. On the other hand, the absolute configuration of the new compound 17-deoxy-aspergillin PZ (1) isolated from Aspergillus sp. NCA276 was established via single-crystal X-ray crystallography. Also, the chemical analysis of the cocultures between Aspergillus and Cladosporium strains revealed the production of metabolites that were not present or were barely detected in the monocultures. Finally, molecular networking analysis of the LC-MS-MS/MS data for each fungus was used as a tool for the annotation of additional compounds, increasing the chemical knowledge on the corresponding fungal strains. Overall, this is the first systematic chemical study on fungi isolated from a sinkhole in Mexico.

Changes in the sediment microbial community structure of coastal and inland sinkholes of a karst ecosystem from the Yucatan peninsula.

The karst underground river ecosystem of Yucatan peninsula is composed of cave systems and sinkholes. The microbial diversity of water from this underground river has been studied, but, structure of the microbial community in its cave sediments remained largely unknown. Here we describe how the microbial community structure of these sediments changes due to different environmental conditions found in sediment zones along the caves of a coastal and an inland sinkhole. We found that dominant microbial groups varied according to the type of sinkhole (Coastal: Chloroflexi and Crenarchaeota; inland: Methylomirabilota and Acidobacteriota) and that the community structures differed both among sinkhole types, and within the sediment zones that were studied. These microorganisms are associated with different types of metabolism, and differed from a microbial community dominated by sulfate reducers at the coastal sinkhole, to one dominated by methylotrophs at the inland sinkhole, suggesting there are biogeochemical processes in the coastal and inland sinkholes that lead to changes in the microbial composition of the underground river ecosystem's sediments. Our results suggest sediments from unexplored sinkhole caves are unique environmental niches with distinct microbial assemblages that putatively play an important role in the biogeochemical cycles of these ecosystems.

A Meta-Omics Analysis Unveils the Shift in Microbial Community Structures and Metabolomics Profiles in Mangrove Sediments Treated with a Selective Actinobacterial Isolation Procedure.

Mangrove sediment ecosystems in the coastal areas of the Yucatan peninsula are unique environments, influenced by their karstic origin and connection with the world's largest underground river. The microbial communities residing in these sediments are influenced by the presence of mangrove roots and the trading chemistry for communication between sediment bacteria and plant roots can be targeted for secondary metabolite research. To explore the secondary metabolite production potential of microbial community members in mangrove sediments at the "El Palmar" natural reserve in Sisal, Yucatan, a combined meta-omics approach was applied. The effects of a cultivation medium reported to select for actinomycetes within mangrove sediments' microbial communities was also analyzed. The metabolome of the microbial communities was analyzed by high-resolution liquid chromatography-tandem mass spectrometry, and molecular networking analysis was used to investigate if known natural products and their variants were present. Metagenomic results suggest that the sediments from "El Palmar" harbor a stable bacterial community independently of their distance from mangrove tree roots. An unexpected decrease in the observed abundance of actinomycetes present in the communities occurred when an antibiotic-amended medium considered to be actinomycete-selective was applied for a 30-day period. However, the use of this antibiotic-amended medium also enhanced production of secondary metabolites within the microbial community present relative to the water control, suggesting the treatment selected for antibiotic-resistant bacteria capable of producing a higher number of secondary metabolites. Secondary metabolite mining of "El Palmar" microbial community metagenomes identified polyketide synthase and non-ribosomal peptide synthetases' biosynthetic genes in all analyzed metagenomes. The presence of these genes correlated with the annotation of several secondary metabolites from the Global Natural Product Social Molecular Networking database. These results highlight the biotechnological potential of the microbial communities from "El Palmar", and show the impact selective media had on the composition of communities of actinobacteria.

The Diversity, Metabolomics Profiling, and the Pharmacological Potential of Actinomycetes Isolated from the Estremadura Spur Pockmarks (Portugal).

The Estremadura Spur pockmarks are a unique and unexplored ecosystem located in the North Atlantic, off the coast of Portugal. A total of 85 marine-derived actinomycetes were isolated and cultured from sediments collected from this ecosystem at a depth of 200 to 350 m. Nine genera, Streptomyces, Micromonospora, Saccharopolyspora, Actinomadura, Actinopolymorpha, Nocardiopsis, Saccharomonospora, Stackebrandtia, and Verrucosispora were identified by 16S rRNA gene sequencing analyses, from which the first two were the most predominant. Non-targeted LC-MS/MS, in combination with molecular networking, revealed high metabolite diversity, including several known metabolites, such as surugamide, antimycin, etamycin, physostigmine, desferrioxamine, ikarugamycin, piericidine, and rakicidin derivatives, as well as numerous unidentified metabolites. Taxonomy was the strongest parameter influencing the metabolite production, highlighting the different biosynthetic potentials of phylogenetically related actinomycetes; the majority of the chemical classes can be used as chemotaxonomic markers, as the metabolite distribution was mostly genera-specific. The EtOAc extracts of the actinomycete isolates demonstrated antimicrobial and antioxidant activity. Altogether, this study demonstrates that the Estremadura Spur is a source of actinomycetes with potential applications for biotechnology. It highlights the importance of investigating actinomycetes from unique ecosystems, such as pockmarks, as the metabolite production reflects their adaptation to this habitat.

The metabolic core of the prokaryotic community from deep-sea sediments of the southern Gulf of Mexico shows different functional signatures between the continental slope and abyssal plain.

Marine sediments harbor an outstanding level of microbial diversity supporting diverse metabolic activities. Sediments in the Gulf of Mexico (GoM) are subjected to anthropic stressors including oil pollution with potential effects on microbial community structure and function that impact biogeochemical cycling. We used metagenomic analyses to provide significant insight into the potential metabolic capacity of the microbial community in Southern GoM deep sediments. We identified genes for hydrocarbon, nitrogen and sulfur metabolism mostly affiliated with Alpha and Betaproteobacteria, Acidobacteria, Chloroflexi and Firmicutes, in relation to the use of alternative carbon and energy sources to thrive under limiting growth conditions, and metabolic strategies to cope with environmental stressors. In addition, results show amino acids metabolism could be associated with sulfur metabolism carried out by Acidobacteria, Chloroflexi and Firmicutes, and may play a crucial role as a central carbon source to favor bacterial growth. We identified the tricarboxylic acid cycle (TCA) and aspartate, glutamate, glyoxylate and leucine degradation pathways, as part of the core carbon metabolism across samples. Further, microbial communities from the continental slope and abyssal plain show differential metabolic capacities to cope with environmental stressors such as oxidative stress and carbon limiting growth conditions, respectively. This research combined taxonomic and functional information of the microbial community from Southern GoM sediments to provide fundamental knowledge that links the prokaryotic structure to its potential function and which can be used as a baseline for future studies to model microbial community responses to environmental perturbations, as well as to develop more accurate mitigation and conservation strategies.

A vibriosis outbreak in the Pacific white shrimp, Litopenaeus vannamei reared in biofloc and clear seawater.

In May and June 2015, moderate and severe lesions were observed in Litopenaeus vannamei reared in clear seawater while, at the same time, lesions in shrimp reared in biofloc were considerably fewer. The signs of disease included anorexia, lethargy, melanization, expanded chromatophores, luminescence and necrotic areas in the uropods, suggesting a possible vibriosis. However, lesions observed in shrimp reared in biofloc disappeared after a certain time and without mortality in tanks, whereas mortality and severe signs continued to be observed in shrimp reared in clear seawater. To treat the possible vibriosis, oxytetracycline was administered only in clear seawater tanks, but the results were not successful. Bacterial cultures from hepatopancreas tissues of shrimp from both rearing systems confirmed a vibriosis outbreak only in the clear seawater system. Subsequently, Vibrio harveyi, Vibrio rotiferianus, Photobacterium sp. and Photobacterium damselae were identified from bacterial culture previously isolated for both rearing systems by molecular methods. Shewanella sp. was isolated and identified only in biofloc. To understand the possible pathogenicity and resistance mechanisms of the Vibiro strains for both rearing systems, pathogenicity (toxR) and oxytetracycline resistance-related genes (tet(B), tet(D), tet(G)) were determined. Although these genes were expressed for both rearing systems, biofloc proved to have the ability to control the development of the disease, in comparison to clear water, where the vibriosis was evident regardless of the administration of oxytetracycline as a treatment.

Ecological Strategies Behind the Selection of Cultivable Actinomycete Strains from the Yucatan Peninsula for the Discovery of Secondary Metabolites with Antibiotic Activity.

The quest for novel natural products has recently focused on the marine environment as a source for novel microorganisms. Although isolation of marine-derived actinomycete strains is now common, understanding their distribution in the oceans and their adaptation to this environment can be helpful in the selection of isolates for further novel secondary metabolite discovery. This study explores the taxonomic diversity of marine-derived actinomycetes from distinct environments in the coastal areas of the Yucatan Peninsula and their adaptation to the marine environment as a first step towards novel natural product discovery. The use of simple ecological principles, for example, phylogenetic relatedness to previously characterized actinomycetes or seawater requirements for growth, to recognize isolates with adaptations to the ocean in an effort to select for marine-derived actinomycete to be used for further chemical studies. Marine microbial environments are an important source of novel bioactive natural products and, together with methods such as genome mining for detection of strains with biotechnological potential, ecological strategies can bring useful insights in the selection and identification of marine-derived actinomycetes for novel natural product discovery.

Intra-clade metabolomic profiling of MAR4 Streptomyces from the Macaronesia Atlantic region reveals a source of anti-biofilm metabolites.

The search for new and effective strategies to reduce bacterial biofilm formation is of utmost importance as bacterial resistance to antibiotics continues to emerge. The use of anti-biofilm agents that can disrupt recalcitrant bacterial communities can be an advantageous alternative to antimicrobials, as their use does not lead to the development of resistance mechanisms. Six MAR4 Streptomyces strains isolated from the Madeira Archipelago, at the unexplored Macaronesia Atlantic ecoregion, were used to study the chemical diversity of produced hybrid isoprenoids. These marine actinomycetes were investigated by analysing their crude extracts using LC-MS/MS and their metabolomic profiles were compared using multivariate statistical analysis (principal component analysis), showing a separation trend closely related to their phylogeny. Molecular networking unveiled the presence of a class of metabolites not previously described from MAR4 strains and new chemical derivatives belonging to the napyradiomycin and marinone classes. Furthermore, these MAR4 strains produce metabolites that inhibit biofilm formation of Staphylococcus aureus and Marinobacter hydrocarbonoclasticus. The anti-biofilm activity of napyradiomycin SF2415B3 (1) against S. aureus was confirmed.

Anaerobic Methane Oxidation Driven by Microbial Reduction of Natural Organic Matter in a Tropical Wetland.

Wetlands constitute the main natural source of methane on Earth due to their high content of natural organic matter (NOM), but key drivers, such as electron acceptors, supporting methanotrophic activities in these habitats are poorly understood. We performed anoxic incubations using freshly collected sediment, along with water samples harvested from a tropical wetland, amended with 13C-methane (0.67 atm) to test the capacity of its microbial community to perform anaerobic oxidation of methane (AOM) linked to the reduction of the humic fraction of its NOM. Collected evidence demonstrates that electron-accepting functional groups (e.g., quinones) present in NOM fueled AOM by serving as a terminal electron acceptor. Indeed, while sulfate reduction was the predominant process, accounting for up to 42.5% of the AOM activities, the microbial reduction of NOM concomitantly occurred. Furthermore, enrichment of wetland sediment with external NOM provided a complementary electron-accepting capacity, of which reduction accounted for ∼100 nmol 13CH4 oxidized · cm-3 · day-1 Spectroscopic evidence showed that quinone moieties were heterogeneously distributed in the wetland sediment, and their reduction occurred during the course of AOM. Moreover, an enrichment derived from wetland sediments performing AOM linked to NOM reduction stoichiometrically oxidized methane coupled to the reduction of the humic analogue anthraquinone-2,6-disulfonate. Microbial populations potentially involved in AOM coupled to microbial reduction of NOM were dominated by divergent biota from putative AOM-associated archaea. We estimate that this microbial process potentially contributes to the suppression of up to 114 teragrams (Tg) of CH4 · year-1 in coastal wetlands and more than 1,300 Tg · year-1, considering the global wetland area.IMPORTANCE The identification of key processes governing methane emissions from natural systems is of major importance considering the global warming effects triggered by this greenhouse gas. Anaerobic oxidation of methane (AOM) coupled to the microbial reduction of distinct electron acceptors plays a pivotal role in mitigating methane emissions from ecosystems. Given their high organic content, wetlands constitute the largest natural source of atmospheric methane. Nevertheless, processes controlling methane emissions in these environments are poorly understood. Here, we provide tracer analysis with 13CH4 and spectroscopic evidence revealing that AOM linked to the microbial reduction of redox functional groups in natural organic matter (NOM) prevails in a tropical wetland. We suggest that microbial reduction of NOM may largely contribute to the suppression of methane emissions from tropical wetlands. This is a novel avenue within the carbon cycle in which slowly decaying NOM (e.g., humic fraction) in organotrophic environments fuels AOM by serving as a terminal electron acceptor.

A computationally simplistic poly-phasic approach to explore microbial communities from the Yucatan aquifer as a potential sources of novel natural products.

The need for new antibiotics has sparked a search for the microbes that might potentially produce them. Current sequencing technologies allow us to explore the biotechnological potential of microbial communities in diverse environments without the need for cultivation, benefitting natural product discovery in diverse ways. A relatively recent method to search for the possible production of novel compounds includes studying the diverse genes belonging to polyketide synthase pathways (PKS), as these complex enzymes are an important source of novel therapeutics. In order to explore the biotechnological potential of the microbial community from the largest underground aquifer in the world located in the Yucatan, we used a polyphasic approach in which a simple, non-computationally intensive method was coupled with direct amplification of environmental DNA to assess the diversity and novelty of PKS type I ketosynthase (KS) domains. Our results suggest that the bioinformatic method proposed can indeed be used to assess the novelty of KS enzymes; nevertheless, this in silico study did not identify some of the KS diversity due to primer bias and stringency criteria outlined by the metagenomics pipeline. Therefore, additionally implementing a method involving the direct cloning of KS domains enhanced our results. Compared to other freshwater environments, the aquifer was characterized by considerably less diversity in relation to known ketosynthase domains; however, the metagenome included a family of KS type I domains phylogenetically related, but not identical, to those found in the curamycin pathway, as well as an outstanding number of thiolases. Over all, this first look into the microbial community found in this large Yucatan aquifer and other fresh water free living microbial communities highlights the potential of these previously overlooked environments as a source of novel natural products.

The Madeira Archipelago As a Significant Source of Marine-Derived Actinomycete Diversity with Anticancer and Antimicrobial Potential.

Marine-derived actinomycetes have demonstrated an ability to produce novel compounds with medically relevant biological activity. Studying the diversity and biogeographical patterns of marine actinomycetes offers an opportunity to identify genera that are under environmental pressures, which may drive adaptations that yield specific biosynthetic capabilities. The present study describes research efforts to explore regions of the Atlantic Ocean, specifically around the Madeira Archipelago, where knowledge of the indigenous actinomycete diversity is scarce. A total of 400 actinomycetes were isolated, sequenced, and screened for antimicrobial and anticancer activities. The three most abundant genera identified were Streptomyces, Actinomadura, and Micromonospora. Phylogenetic analyses of the marine OTUs isolated indicated that the Madeira Archipelago is a new source of actinomycetes adapted to life in the ocean. Phylogenetic differences between offshore (>100 m from shore) and nearshore (< 100 m from shore) populations illustrates the importance of sampling offshore in order to isolate new and diverse bacterial strains. Novel phylotypes from chemically rich marine actinomycete groups like MAR4 and the genus Salinispora were isolated. Anticancer and antimicrobial assays identified Streptomyces, Micromonospora, and Salinispora as the most biologically active genera. This study illustrates the importance of bioprospecting efforts at unexplored regions of the ocean to recover bacterial strains with the potential to produce novel and interesting chemistry.

Acidobacteria appear to dominate the microbiome of two sympatric Caribbean Sponges and one Zoanthid.

BACKGROUND: Marine invertebrate-associated microbial communities are interesting examples of complex symbiotic systems and are a potential source of biotechnological products. RESULTS: In this work, pyrosequencing-based assessment from bacterial community structures of sediments, two sponges, and one zoanthid collected in the Mexican Caribbean was performed. The results suggest that the bacterial diversity at the species level is higher in the sediments than in the animal samples. Analysis of bacterial communities' structure showed that about two thirds of the bacterial diversity in all the samples belongs to the phyla Acidobacteria and Proteobacteria. The genus Acidobacterium appears to dominate the bacterial community in all the samples, reaching almost 80% in the sponge Hyrtios. CONCLUSIONS: Our evidence suggests that the sympatric location of these benthonic species may lead to common bacterial structure features among their bacterial communities. The results may serve as a first insight to formulate hypotheses that lead to more extensive studies of sessile marine organisms' microbiomes from the Mexican Caribbean.

Acidobacteria appear to dominate the microbiome of two sympatric Caribbean Sponges and one Zoanthid

BACKGROUND: Marine invertebrate-associated microbial communities are interesting examples of complex symbiotic systems and are a potential source of biotechnological products. RESULTS: In this work, pyrosequencing-based assessment from bacterial community structures of sediments, two sponges, and one zoanthid collected in the Mexican Caribbean was performed. The results suggest that the bacterial diversity at the species level is higher in the sediments than in the animal samples. Analysis of bacterial communities' structure showed that about two thirds of the bacterial diversity in all the samples belongs to the phyla Acidobacteria and Proteobacteria. The genus Acidobacteriumappears to dominate the bacterial community in all the samples, reaching almost 80% in the sponge Hyrtios. CONCLUSIONS: Our evidence suggests that the sympatric location of these benthonic species may lead to common bacterial structure features among their bacterial communities. The results may serve as a first insight to formulate hypotheses that lead to more extensive studies of sessile marine organisms' microbiomes from the Mexican Caribbean.

Targeted search for actinomycetes from nearshore and deep-sea marine sediments.

Sediment samples collected off the coast of San Diego were analyzed for actinomycete diversity using culture-independent techniques. Eight new operational taxonomic units (OTUs) in the Streptomycetaceae were identified as well as new diversity within previously cultured marine OTUs. Sequences belonging to the marine actinomycete genus Salinispora were also detected, despite the fact that this genus has only been reported from more tropical environments. Independent analyses of marine sediments from the Canary Basin (3814 m) and the South Pacific Gyre (5126 and 5699 m) also revealed Salinispora sequences providing further support for the occurrence of this genus in deep-sea sediments. Efforts to culture Salinispora spp. from these samples have yet to be successful. This is the first report of Salinispora spp. from marine sediments > 1100 m and suggests that the distribution of this genus is broader than previously believed.

Oxygen minimum zones harbour novel viral communities with low diversity.

Oxygen minimum zones (OMZs) are oceanographic features that affect ocean productivity and biodiversity, and contribute to ocean nitrogen loss and greenhouse gas emissions. Here we describe the viral communities associated with the Eastern Tropical South Pacific (ETSP) OMZ off Iquique, Chile for the first time through abundance estimates and viral metagenomic analysis. The viral-to-microbial ratio (VMR) in the ETSP OMZ fluctuated in the oxycline and declined in the anoxic core to below one on several occasions. The number of viral genotypes (unique genomes as defined by sequence assembly) ranged from 2040 at the surface to 98 in the oxycline, which is the lowest viral diversity recorded to date in the ocean. Within the ETSP OMZ viromes, only 4.95% of genotypes were shared between surface and anoxic core viromes using reciprocal BLASTn sequence comparison. ETSP virome comparison with surface marine viromes (Sargasso Sea, Gulf of Mexico, Kingman Reef, Chesapeake Bay) revealed a dissimilarity of ETSP OMZ viruses to those from other oceanic regions. From the 1.4 million non-redundant DNA sequences sampled within the altered oxygen conditions of the ETSP OMZ, more than 97.8% were novel. Of the average 3.2% of sequences that showed similarity to the SEED non-redundant database, phage sequences dominated the surface viromes, eukaryotic virus sequences dominated the oxycline viromes, and phage sequences dominated the anoxic core viromes. The viral community of the ETSP OMZ was characterized by fluctuations in abundance, taxa and diversity across the oxygen gradient. The ecological significance of these changes was difficult to predict; however, it appears that the reduction in oxygen coincides with an increased shedding of eukaryotic viruses in the oxycline, and a shift to unique viral genotypes in the anoxic core.

Significant natural product biosynthetic potential of actinorhizal symbionts of the genus frankia, as revealed by comparative genomic and proteomic analyses.

Bacteria of the genus Frankia are mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genus Frankia has largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences of Frankia strains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from other Frankia strains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis of Frankia sp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed in Frankia sp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in the Frankia genus.