Small molecule in situ resin capture provides a compound first approach to natural product discovery.

Culture-based microbial natural product discovery strategies fail to realize the extraordinary biosynthetic potential detected across earth's microbiomes. Here we introduce Small Molecule In situ Resin Capture (SMIRC), a culture-independent method to obtain natural products directly from the environments in which they are produced. We use SMIRC to capture numerous compounds including two new carbon skeletons that were characterized using NMR and contain structural features that are, to the best of our knowledge, unprecedented among natural products. Applications across diverse marine habitats reveal biome-specific metabolomic signatures and levels of chemical diversity in concordance with sequence-based predictions. Expanded deployments, in situ cultivation, and metagenomics facilitate compound discovery, enhance yields, and link compounds to candidate producing organisms, although microbial community complexity creates challenges for the later. This compound-first approach to natural product discovery provides access to poorly explored chemical space and has implications for drug discovery and the detection of chemically mediated biotic interactions.

MAR4 Streptomyces: A Unique Resource for Natural Product Discovery.

Marine-derived Streptomyces have long been recognized as a source of novel, pharmaceutically relevant natural products. Among these bacteria, the MAR4 clade within the genus Streptomyces has been identified as metabolically rich, yielding over 93 different compounds to date. MAR4 strains are particularly noteworthy for the production of halogenated hybrid isoprenoid natural products, a relatively rare class of bacterial metabolites that possess a wide range of biological activities. MAR4 genomes are enriched in vanadium haloperoxidase and prenyltransferase genes, thus accounting for the production of these compounds. Functional characterization of the enzymes encoded in MAR4 genomes has advanced our understanding of halogenated, hybrid isoprenoid biosynthesis. Despite the exceptional biosynthetic capabilities of MAR4 bacteria, the large body of research they have stimulated has yet to be compiled. Here we review 35 years of natural product research on MAR4 strains and update the molecular diversity of this unique group of bacteria.

Small Molecule in situ Resin Capture - A Compound First Approach to Natural Product Discovery.

Microbial natural products remain an important resource for drug discovery. Yet, commonly employed discovery techniques are plagued by the rediscovery of known compounds, the relatively few microbes that can be cultured, and laboratory growth conditions that do not elicit biosynthetic gene expression among myriad other challenges. Here we introduce a culture independent approach to natural product discovery that we call the Small Molecule In situ Resin Capture (SMIRC) technique. SMIRC exploits in situ environmental conditions to elicit compound production and represents a new approach to access poorly explored chemical space by capturing natural products directly from the environments in which they are produced. In contrast to traditional methods, this compound-first approach can capture structurally complex small molecules across all domains of life in a single deployment while relying on Nature to provide the complex and poorly understood environmental cues needed to elicit biosynthetic gene expression. We illustrate the effectiveness of SMIRC in marine habitats with the discovery of numerous new compounds and demonstrate that sufficient compound yields can be obtained for NMR-based structure assignment. Two new compound classes are reported including one novel carbon skeleton that possesses a functional group not previously observed among natural products and a second that possesses potent biological activity. We introduce expanded deployments, in situ cultivation, and metagenomics as methods to facilitate compound discovery, enhance yields, and link compounds to producing organisms. This compound first approach can provide unprecedented access to new natural product chemotypes with broad implications for drug discovery.

Metagenomic data reveals type I polyketide synthase distributions across biomes.

Microbial polyketide synthase (PKS) genes encode the biosynthesis of many biomedically or otherwise commercially important natural products. Despite extensive discovery efforts, metagenomic analyses suggest that only a small fraction of nature's polyketide biosynthetic potential has been realized. Much of this potential originates from type I PKSs (T1PKSs), which can be further delineated based on their domain organization and the structural features of the compounds they encode. Notably, phylogenetic relationships among ketosynthase (KS) domains provide an effective method to classify the larger and more complex T1PKS genes in which they occur. Increased access to large metagenomic data sets from diverse habitats provides opportunities to assess T1PKS biosynthetic diversity and distributions through their smaller and more tractable KS domain sequences. Here, we used the web tool NaPDoS2 to detect and classify over 35,000 type I KS domains from 137 metagenomic data sets reported from eight diverse, globally distributed biomes. We found biome-specific separation with soils enriched in KSs from modular cis-acetyltransferase (AT) and hybrid cis-AT KSs relative to other biomes and marine sediments enriched in KSs associated with polyunsaturated fatty acid and enediyne biosynthesis. We linked the phylum Actinobacteria to soil-derived enediyne and cis-AT KSs while marine-derived KSs associated with enediyne and monomodular PKSs were linked to phyla from which the compounds produced by these biosynthetic enzymes have not been reported. These KSs were phylogenetically distinct from those associated with experimentally characterized PKSs suggesting they may be associated with novel structures or enzyme functions. Finally, we employed our metagenome-extracted KS domains to evaluate the PCR primers commonly used to amplify type I KSs and identified modifications that could increase the KS sequence diversity recovered from amplicon libraries. IMPORTANCE Polyketides are a crucial source of medicines, agrichemicals, and other commercial products. Advances in our understanding of polyketide biosynthesis, coupled with the increased availability of metagenomic sequence data, provide new opportunities to assess polyketide biosynthetic potential across biomes. Here, we used the web tool NaPDoS2 to assess type I polyketide synthase (PKS) diversity and distributions by detecting and classifying ketosynthase (KS) domains across 137 metagenomes. We show that biomes are differentially enriched in type I KS domains, providing a roadmap for future biodiscovery strategies. Furthermore, KS phylogenies reveal biome-specific clades that do not include biochemically characterized PKSs, highlighting the biosynthetic potential of poorly explored environments. The large metagenome-derived KS data set allowed us to identify regions of commonly used type I KS PCR primers that could be modified to capture a larger extent of environmental KS diversity. These results facilitate both the search for novel polyketides and our understanding of the biogeographical distribution of PKSs across Earth's major biomes.

Biogeographic patterns of biosynthetic potential and specialized metabolites in marine sediments.

While the field of microbial biogeography has largely focused on the contributions of abiotic factors to community patterns, the potential influence of biotic interactions in structuring microbial communities, such as those mediated by the production of specialized metabolites, remains largely unknown. Here, we examined the relationship between microbial community structure and specialized metabolism at local spatial scales in marine sediment samples collected from the Long-Term Ecological Research (LTER) site in Moorea, French Polynesia. By employing a multi-omic approach to characterize the taxonomic, functional, and specialized metabolite composition within sediment communities, we find that biogeographic patterns were driven by local scale processes (e.g., biotic interactions) and largely independent of dispersal limitation. Specifically, we observed high variation in biosynthetic potential (based on Bray-Curtis dissimilarity) between samples, even within 1 m2 plots, that reflected uncharacterized chemical space associated with site-specific metabolomes. Ultimately, connecting biosynthetic potential to community metabolomes facilitated the in situ detection of natural products and revealed new insights into the complex metabolic dynamics associated with sediment microbial communities. Our study demonstrates the potential to integrate biosynthetic genes and metabolite production into assessments of microbial community dynamics.

Metagenomic Data Reveal Type I Polyketide Synthase Distributions Across Biomes.

Microbial polyketide synthase (PKS) genes encode the biosynthesis of many biomedically important natural products, yet only a small fraction of nature's polyketide biosynthetic potential has been realized. Much of this potential originates from type I PKSs (T1PKSs), which can be delineated into different classes and subclasses based on domain organization and structural features of the compounds encoded. Notably, phylogenetic relationships among PKS ketosynthase (KS) domains provide a method to classify the larger and more complex genes in which they occur. Increased access to large metagenomic datasets from diverse habitats provides opportunities to assess T1PKS biosynthetic diversity and distributions through the analysis of KS domain sequences. Here, we used the webtool NaPDoS2 to detect and classify over 35,000 type I KS domains from 137 metagenomic data sets reported from eight diverse biomes. We found biome-specific separation with soils enriched in modular cis -AT and hybrid cis -AT KSs relative to other biomes and marine sediments enriched in KSs associated with PUFA and enediyne biosynthesis. By extracting full-length KS domains, we linked the phylum Actinobacteria to soil-specific enediyne and cis -AT clades and identified enediyne and monomodular KSs in phyla from which the associated compound classes have not been reported. These sequences were phylogenetically distinct from those associated with experimentally characterized PKSs suggesting novel structures or enzyme functions remain to be discovered. Lastly, we employed our metagenome-extracted KS domains to evaluate commonly used type I KS PCR primers and identified modifications that could increase the KS sequence diversity recovered from amplicon libraries. Importance: Polyketides are a crucial source of medicines, agrichemicals, and other commercial products. Advances in our understanding of polyketide biosynthesis coupled with the accumulation of metagenomic sequence data provide new opportunities to assess polyketide biosynthetic potential across biomes. Here, we used the webtool NaPDoS2 to assess type I PKS diversity and distributions by detecting and classifying KS domains across 137 metagenomes. We show that biomes are differentially enriched in KS domain classes, providing a roadmap for future biodiscovery strategies. Further, KS phylogenies reveal both biome-specific clades that do not include biochemically characterized PKSs, highlighting the biosynthetic potential of poorly explored environments. The large metagenome-derived KS dataset allowed us to identify regions of commonly used type I KS PCR primers that could be modified to capture a larger extent of KS diversity. These results facilitate both the search for novel polyketides and our understanding of the biogeographical distribution of PKSs across earth's major biomes.

The Natural Product Domain Seeker version 2 (NaPDoS2) webtool relates ketosynthase phylogeny to biosynthetic function.

The Natural Product Domain Seeker (NaPDoS) webtool detects and classifies ketosynthase (KS) and condensation domains from genomic, metagenomic, and amplicon sequence data. Unlike other tools, a phylogeny-based classification scheme is used to make broader predictions about the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes in which these domains are found. NaPDoS is particularly useful for the analysis of incomplete biosynthetic genes or gene clusters, as are often observed in poorly assembled genomes and metagenomes, or when loci are not clustered, as in eukaryotic genomes. To help support the growing interest in sequence-based analyses of natural product biosynthetic diversity, here we introduce version 2 of the webtool, NaPDoS2, available at http://napdos.ucsd.edu/napdos2. This update includes the addition of 1417 KS sequences, representing a major expansion of the taxonomic and functional diversity represented in the webtool database. The phylogeny-based KS classification scheme now recognizes 41 class and subclass assignments, including new type II PKS subclasses. Workflow modifications accelerate run times, allowing larger datasets to be analyzed. In addition, default parameters were established using statistical validation tests to maximize KS detection and classification accuracy while minimizing false positives. We further demonstrate the applications of NaPDoS2 to assess PKS biosynthetic potential using genomic, metagenomic, and PCR amplicon datasets. These examples illustrate how NaPDoS2 can be used to predict biosynthetic potential and detect genes involved in the biosynthesis of specific structure classes or new biosynthetic mechanisms.

Microbe Profile: Salinispora tropica: natural products and the evolution of a unique marine bacterium.

Salinispora tropica was originally cultured from tropical marine sediments and described as the first obligate marine actinomycete genus. Soon after its discovery, it yielded the potent proteasome inhibitor salinosporamide A, a structurally novel natural product that is currently in phase III clinical trials for the treatment of cancer. If approved, it will be the first natural product derived from a cultured marine microbe to achieve clinical relevance. S. tropica produces many other biologically active natural products, including some linked to chemical defence, thus providing ecological context for their production. However, genomic analyses reveal that most natural product biosynthetic gene clusters remain orphan, suggesting that more compounds await discovery. The abundance of biosynthetic gene clusters in S. tropica supports the concept that the small molecules they encode serve important ecological functions, while their evolutionary histories suggest a potential role in promoting diversification. Better insights into the ecological functions of microbial natural products will help inform future discovery efforts.

Structure and Candidate Biosynthetic Gene Cluster of a Manumycin-Type Metabolite from Salinispora pacifica.

A new manumycin-type natural product named pacificamide (1) and its candidate biosynthetic gene cluster (pac) were discovered from the marine actinobacterium Salinispora pacifica CNT-855. The structure of the compound was determined using NMR, electronic circular dichroism, and bioinformatic predictions. The pac gene cluster is unique to S. pacifica and found in only two of the 119 Salinispora genomes analyzed across nine species. Comparative analyses of biosynthetic gene clusters encoding the production of related manumycin-type compounds revealed genetic differences in accordance with the unique pacificamide structure. Further queries of manumycin-type gene clusters from public databases revealed their limited distribution across the phylum Actinobacteria and orphan diversity that suggests additional products remain to be discovered in this compound class. Production of the known metabolite triacsin D is also reported for the first time from the genus Salinispora. This study adds two classes of compounds to the natural product collective isolated from the genus Salinispora, which has proven to be a useful model for natural product research.

Specialized Metabolite-Mediated Predation Defense in the Marine Actinobacterium Salinispora.

The obligate marine actinobacterial genus Salinispora has become a model organism for natural product discovery, yet little is known about the ecological functions of the compounds produced by this taxon. The aims of this study were to assess the effects of live cultures and culture extracts from two Salinispora species on invertebrate predators. In choice-based feeding experiments using the bacterivorous nematode Caenorhabditis elegans, live cultures of both Salinispora species were less preferred than Escherichia coli. When given a choice between the two species, C. elegans preferred S. areniolca over S. tropica. Culture extracts from S. tropica deterred C. elegans, while those from S. arenicola did not, suggesting that compounds produced by S. tropica account for the feeding deterrence. Bioactivity-guided isolation linked compounds in the lomaiviticin series to the deterrent activity. Additional assays using the marine polychaete Ophryotrocha siberti and marine nematodes further support the deterrent activity of S. tropica against potential predators. These results provide evidence that Salinispora natural products function as a defense against predation and that the strategies of predation defense differ between closely related species. IMPORTANCE Bacteria inhabiting marine sediments are subject to predation by bacterivorous eukaryotes. Here, we test the hypothesis that sediment-derived bacteria in the genus Salinispora produce biologically active natural products that function as a defense against predation. The results reveal that cultures and culture extracts of S. tropica deter feeding by Caenorhabditis elegans and negatively affect the habitat preference of a marine annelid (Ophryotrocha siberti). These activities were linked to the lomaiviticins, a series of cytotoxic compounds produced by S. tropica. Microbial natural products that function as a defense against predation represent a poorly understood trait that can influence community structure in marine sediments.

Vertical Inheritance Facilitates Interspecies Diversification in Biosynthetic Gene Clusters and Specialized Metabolites.

While specialized metabolites are thought to mediate ecological interactions, the evolutionary processes driving chemical diversification, particularly among closely related lineages, remain poorly understood. Here, we examine the evolutionary dynamics governing the distribution of natural product biosynthetic gene clusters (BGCs) among 118 strains representing all nine currently named species of the marine actinobacterial genus Salinispora. While much attention has been given to the role of horizontal gene transfer (HGT) in structuring BGC distributions, we find that vertical descent facilitates interspecies BGC diversification over evolutionary timescales. Moreover, we identified a distinct phylogenetic signal among Salinispora species at both the BGC and metabolite level, indicating that specialized metabolism represents a conserved phylogenetic trait. Using a combination of genomic analyses and liquid chromatography-high-resolution tandem mass spectrometry (LC-MS/MS) targeting nine experimentally characterized BGCs and their small molecule products, we identified gene gain/loss events, constrained interspecies recombination, and other evolutionary processes associated with vertical inheritance as major contributors to BGC diversification. These evolutionary dynamics had direct consequences for the compounds produced, as exemplified by species-level differences in salinosporamide production. Together, our results support the concept that specialized metabolites, and their cognate BGCs, can represent phylogenetically conserved functional traits with chemical diversification proceeding in species-specific patterns over evolutionary time frames. IMPORTANCE Microbial natural products are traditionally exploited for their pharmaceutical potential, yet our understanding of the evolutionary processes driving BGC evolution and compound diversification remain poorly developed. While HGT is recognized as an integral driver of BGC distributions, we find that the effects of vertical inheritance on BGC diversification had direct implications for species-level specialized metabolite production. As such, understanding the degree of genetic variation that corresponds to species delineations can enhance natural product discovery efforts. Resolving the evolutionary relationships between closely related strains and specialized metabolism can also facilitate our understanding of the ecological roles of small molecules in structuring the environmental distribution of microbes.

Microbial diversity in tropical marine sediments assessed using culture-dependent and culture-independent techniques.

The microbial communities associated with marine sediments are critical for ecosystem function yet remain poorly characterized. While culture-independent (CI) techniques capture the broadest perspective on community composition, culture-dependent (CD) methods can select for low abundance taxa that are missed using CI approaches. This study aimed to assess microbial diversity in tropical marine sediments at five shallow-water sites in Belize using both CD and CI techniques. The CD methods captured approximately 3% of the >800 genera detected across all sites using the CI approach. Additionally, 39 genera were only detected in culture, revealing rare taxa that were missed with the CI approach. Significantly different communities were detected across sites, with rare taxa playing an important role in distinguishing among communities. This study provides important baseline data describing shallow-water sediment microbial communities, evidence that standard cultivation techniques may be more effective than previously recognized, and the first steps towards identifying new taxa that are amenable to agar plate cultivation.

Grincamycins P-T: Rearranged Angucyclines from the Marine Sediment-Derived Streptomyces sp. CNZ-748 Inhibit Cell Lines of the Rare Cancer Pseudomyxoma Peritonei.

While marine natural products have been investigated for anticancer drug discovery, they are barely screened against rare cancers. Thus, in our effort to discover potential drug leads against the rare cancer pseudomyxoma peritonei (PMP), which currently lacks effective drug treatments, we screened extracts of marine actinomycete bacteria against the PMP cell line ABX023-1. This effort led to the isolation of nine rearranged angucyclines from Streptomyces sp. CNZ-748, including five new analogues, namely, grincamycins P-T (1-5). The chemical structures of these compounds were unambiguously established based on spectroscopic and chemical analyses. Particularly, grincamycin R (3) possesses an S-containing α-l-methylthio-aculose residue, which was discovered in nature for the first time. All of the isolated compounds were evaluated against four PMP cell lines and some exhibited low micromolar inhibitory activities. To identify a candidate biosynthetic gene cluster (BGC) encoding the grincamycins, we sequenced the genome of the producing strain, Streptomyces sp. CNZ-748, and compared the BGCs detected with those linked to the production of angucyclines with different aglycon structures.

Verrucosamide, a Cytotoxic 1,4-Thiazepane-Containing Thiodepsipeptide from a Marine-Derived Actinomycete.

A new cytotoxic thiodepsipeptide, verrucosamide (1), was isolated along with the known, related cyclic peptide thiocoraline, from the extract of a marine-derived actinomycete, a Verrucosispora sp., our strain CNX-026. The new peptide, which is composed of two rare seven-membered 1,4-thiazepane rings, was elucidated by a combination of spectral methods and the absolute configuration was determined by a single X-ray diffraction study. Verrucosamide (1) showed moderate cytotoxicity and selectivity in the NCI 60 cell line bioassay. The most susceptible cell lines were MDA-MB-468 breast carcinoma with an LD50 of 1.26 µM, and COLO 205 colon adenocarcinoma with an LD50 of 1.4 µM. Also isolated along with verrucosamide were three small 3-hydroxy(alkoxy)-quinaldic acid derivatives that appear to be products of the same biosynthetic pathway.

Expansion of Gamma-Butyrolactone Signaling Molecule Biosynthesis to Phosphotriester Natural Products.

Bacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species-specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their antimalarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the salinipostins and newly identified natural A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed among many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.

Six novel species of the obligate marine actinobacterium Salinispora, Salinispora cortesiana sp. nov., Salinispora fenicalii sp. nov., Salinispora goodfellowii sp. nov., Salinispora mooreana sp. nov., Salinispora oceanensis sp. nov. and Salinispora vitiensis sp. nov., and emended description of the genus Salinispora.

Ten representative actinobacterial strains isolated from marine sediments collected worldwide were studied to determine their taxonomic status. The strains were previously identified as members of the genus Salinispora and shared >99 % 16S rRNA gene sequence similarity to the three currently recognized Salinispora species. Comparative genomic analyses resulted in the delineation of six new species based on average nucleotide identity and digital DNA-DNA hybridization values below 95 and 70 %, respectively. The species status of the six new groups was supported by a core-genome phylogeny reconstructed from 2106 orthologs detected in 118 publicly available Salinispora genomes. Chemotaxonomic and physiological studies were used to complete the phenotypic characterization of the strains. The fatty acid profiles contained the major components iso-C16 : 0, C15 : 0, iso-17 : 0 and anteiso C17 : 0. Galactose and xylose were common in all whole-sugar patterns but differences were found between the six groups of strains. Polar lipid compositions were also unique for each species. Distinguishable physiological and biochemical characteristics were also recorded. The names proposed are Salinispora cortesiana sp. nov., CNY-202T (=DSM 108615T=CECT 9739T); Salinispora fenicalii sp. nov., CNT-569T (=DSM 108614T=CECT 9740T); Salinispora goodfellowii sp. nov., CNY-666T (=DSM 108616T=CECT 9738T); Salinispora mooreana sp. nov., CNT-150T (=DSM 45549T=CECT 9741T); Salinispora oceanensis sp. nov., CNT-138T (=DSM 45547T=CECT 9742T); and Salinispora vitiensis sp. nov., CNT-148T (=DSM 45548T=CECT 9743T).

Extending the Salinilactone Family.

Five new members of the salinilactone family, salinilactones D-H, are reported. These bicyclic lactones are produced by Salinispora bacteria and display extended or shortened alkyl side chains relative to the recently reported salinilactones A-C. They were identified by GC/MS, gas chromatographic retention index, and comparison with synthetic samples. We further investigated the occurrence of salinilactones across six newly proposed Salinispora species to gain insight into how compound production varies among taxa. The growth-inhibiting effect of this compound family on multiple biological systems including non-Salinispora actinomycetes was analyzed. Additionally, we found strong evidence for significant cytotoxicity of the title compounds.

Detection of Natural Products and Their Producers in Ocean Sediments.

Thousands of natural products have been identified from cultured microorganisms, yet evidence of their production in the environment has proven elusive. Technological advances in mass spectrometry, combined with public databases, now make it possible to address this disparity by detecting compounds directly from environmental samples. Here, we used adsorbent resins, tandem mass spectrometry, and next-generation sequencing to assess the metabolome of marine sediments and its relationship to bacterial community structure. We identified natural products previously reported from cultured bacteria, providing evidence they are produced in situ, and compounds of anthropogenic origin, suggesting this approach can be used as an indicator of environmental impact. The bacterial metabolite staurosporine was quantified and shown to reach physiologically relevant concentrations, indicating that it may influence sediment community structure. Staurosporine concentrations were correlated with the relative abundance of the staurosporine-producing bacterial genus Salinispora and production confirmed in strains cultured from the same location, providing a link between compound and candidate producer. Metagenomic analyses revealed numerous biosynthetic gene clusters related to indolocarbazole biosynthesis, providing evidence for noncanonical sources of staurosporine and a path forward to assess the relationships between natural products and the organisms that produce them. Untargeted environmental metabolomics circumvents the need for laboratory cultivation and represents a promising approach to understanding the functional roles of natural products in shaping microbial community structure in marine sediments.IMPORTANCE Natural products are readily isolated from cultured bacteria and exploited for useful purposes, including drug discovery. However, these compounds are rarely detected in the environments from which the bacteria are obtained, thus limiting our understanding of their ecological significance. Here, we used environmental metabolomics to directly assess chemical diversity in marine sediments. We identified numerous metabolites and, in one case, isolated strains of bacteria capable of producing one of the compounds detected. Coupling environmental metabolomics with community and metagenomic analyses provides opportunities to link compounds and producers and begin to assess the complex interactions mediated by specialized metabolites in marine sediments.

Diversity and distribution of the bmp gene cluster and its Polybrominated products in the genus Pseudoalteromonas.

The production of pentabromopseudilin and related brominated compounds by Pseudoalteromonas spp. has recently been linked to the bmp biosynthetic gene cluster. This study explored the distribution and evolutionary history of this gene cluster in the genus Pseudoalteromonas. A phylogeny of the genus revealed numerous clades that do not contain type strains, suggesting considerable species level diversity has yet to be described. Comparative genomics revealed four distinct versions of the gene cluster distributed among 19 of the 101 Pseudoalteromonas genomes examined. These were largely localized to the least inclusive clades containing the Pseudoalteromonas luteoviolacea and Pseudoalteromonas phenolica type strains and show clear evidence of gene and gene cluster loss in certain lineages. Bmp gene phylogeny is largely congruent with the Pseudoalteromonas species phylogeny, suggesting vertical inheritance within the genus. However, the gene cluster is found in three different genomic environments suggesting either chromosomal rearrangement or multiple acquisition events. Bmp conservation within certain lineages suggests the encoded products are highly relevant to the ecology of these bacteria.