Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora

Here we report associations between secondary metabolite production and phylogenetically distinct but closely related marine actinomycete species belonging to the genus Salinispora. The pattern emerged in a study that included global collection sites, and it indicates that secondary metabolite production can be a species-specific, phenotypic trait associated with broadly distributed bacterial populations. Associations between actinomycete phylotype and chemotype revealed an effective, diversity-based approach to natural product discovery that contradicts the conventional wisdom that secondary metabolite production is strain specific. The structural diversity of the metabolites observed, coupled with gene probing and phylogenetic analyses, implicates lateral gene transfer as a source of the biosynthetic genes responsible for compound production. These results conform to a model of selection-driven pathway fixation occurring subsequent to gene acquisition and provide a rare example in which demonstrable physiological traits have been correlated to the fine-scale phylogenetic architecture of an environmental bacterial community.     

Competitive strategies differentiate closely related species of marine actinobacteria

Although competition, niche partitioning, and spatial isolation have been used to describe the ecology and evolution of macro-organisms, it is less clear to what extent these principles account for the extraordinary levels of bacterial diversity observed in nature. Ecological interactions among bacteria are particularly challenging to address due to methodological limitations and uncertainties over how to recognize fundamental units of diversity and link them to the functional traits and evolutionary processes that led to their divergence. Here we show that two closely related marine actinomycete species can be differentiated based on competitive strategies. Using a direct challenge assay to investigate inhibitory interactions with members of the bacterial community, we observed a temporal difference in the onset of inhibition. The majority of inhibitory activity exhibited by Salinispora arenicola occurred early in its growth cycle and was linked to antibiotic production. In contrast, most inhibition by Salinispora tropica occurred later in the growth cycle and was more commonly linked to nutrient depletion or other sources. Comparative genomics support these differences, with S. arenicola containing nearly twice the number of secondary metabolite biosynthetic gene clusters as S. tropica, indicating a greater potential for secondary metabolite production. In contrast, S. tropica is enriched in gene clusters associated with the acquisition of growth-limiting nutrients such as iron. Coupled with differences in growth rates, the results reveal that S. arenicola uses interference competition at the expense of growth, whereas S. tropica preferentially employs a strategy of exploitation competition. The results support the ecological divergence of two co-occurring and closely related species of marine bacteria by providing evidence they have evolved fundamentally different strategies to compete in marine sediments.     

Genome mining of Streptomyces ambofaciens

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.     

Phylogenetic and Chemical Diversity of a Hybrid-Isoprenoid-Producing Streptomycete Lineage

Streptomyces species dedicate a large portion of their genomes to secondary metabolite biosynthesis. A diverse and largely marine-derived lineage within this genus has been designated MAR4 and identified as a prolific source of hybrid isoprenoid (HI) secondary metabolites. These terpenoid-containing compounds are common in nature but rarely observed as bacterial secondary metabolites. To assess the phylogenetic diversity of the MAR4 lineage, complementary culture-based and culture-independent techniques were applied to marine sediment samples collected off the Channel Islands, CA. The results, including those from an analysis of publically available sequence data and strains isolated as part of prior studies, placed 40 new strains in the MAR4 clade, of which 32 originated from marine sources. When combined with sequences cloned from environmental DNA, 28 MAR4 operational taxonomic units (0.01% genetic distance) were identified. Of these, 82% consisted exclusively of either cloned sequences or cultured strains, supporting the complementarity of these two approaches. Chemical analyses of diverse MAR4 strains revealed the production of five different HI structure classes. All 21 MAR4 strains tested produced at least one HI class, with most strains producing from two to four classes. The two major clades within the MAR4 lineage displayed distinct patterns in the structural classes and the number and amount of HIs produced, suggesting a relationship between taxonomy and secondary metabolite production. The production of HI secondary metabolites appears to be a phenotypic trait of the MAR4 lineage, which represents an emerging model with which to study the ecology and evolution of HI biosynthesis.     

Distribution of live and dead cells in pellets of an actinomycete Amycolatopsis balhimycina and its correlation with balhimycin productivity

Secondary metabolites such as antibiotics are typically produced by actinomycetes as a response to growth limiting stress conditions. Several studies have shown that secondary metabolite production is correlated with changes observed in actinomycete pellet morphology. Therefore, we investigated the correlation between the production of balhimycin and the spatio-temporal distribution of live and dead cells in pellets of Amycolatopsis balhimycina in submerged cultures. To this end, we used laser scanning confocal microscopy to analyze pellets from balhimycin producing and nonproducing media containing 0.2 and 1.0 g l^?1 of potassium di-hydrogen phosphate, respectively. We observed a substantially higher fraction of live cells in pellets from cultures yielding larger amounts of balhimycin. Moreover, in media that resulted in no balhimycin production, the pellets exhibit an initial death phase which commences from the centre of the pellet and extends in the radial direction. A second growth phase was observed in these pellets, where live mycelia are seen to appear in the dead core of the pellets. This secondary growth was absent in pellets from media producing higher amounts of balhimycin. These results suggest that distribution of live and dead cells and its correlation with antibiotic production in the non-sporulating A. balhimycina differs markedly than that observed in Streptomycetes.     

Cyclic-AMP inhibition of fimbriae and prodigiosin production by Serratia marcescens is strain-dependent

The cyclic-nucleotide 3′,5′-cyclic AMP (cAMP) is an ancient and widespread regulatory molecule. Previous studies have shown that fimbria production and secondary metabolite production are inhibited by cAMP in the prokaryote Serratia marcescens. This study used genetic manipulations to test the strain specificity of cAMP–cyclic-AMP receptor protein regulation of fimbria production and of the red pigment, prodigiosin. A surprising amount of variation was observed, as multicopy expression of the cAMP-phosphodiesterase gene, cpdS, conferred either an increase or decrease in fimbriae-dependent yeast agglutination and prodigiosin production depending upon the strain background. Mutation of crp, the gene coding for the cAMP-receptor protein, similarly conferred strain-dependent phenotypes. This study shows that three distinct biological properties, modulated by a conserved genetic regulatory molecule, can vary significantly among strains. Such variation can complicate the functional analysis of bacterial phenotypic properties which are dependent upon global genetic regulators such as cAMP.     

Pleiotropic regulatory genes bldA,adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production—bldA, adpA and absB—exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNA^Leu_UAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs—that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.     

Unique Actinomycetes from Marine Caves and Coral Reef Sediments Provide Novel PKS and NRPS Biosynthetic Gene Clusters

In the ever-expanding search for novel bioactive molecules and enzymes, marine actinomycetes have proven to be a productive source. While open reef sediment and sponge-associated actinomycetes have been extensively examined, their marine cave counterparts remain unevaluated. Anchialine cave systems in the Bahamas offered an ideal setting to evaluate the occurrence and variation within sediment-associated actinomycete communities. While in close geographical proximity to open reef environments, these systems provide a specialized environmental niche devoid of light and direct exposure to nutrient input. In the present study, selective isolation techniques and molecular methods were used to test the hypothesis that variable distribution of actinomycetes and secondary metabolite gene clusters occur between open reef and marine cave systems. The results indicated that differences exist within the culturable sediment-associated actinomycete communities between marine caves and open reef systems, with members of the genus Streptomyces dominating cultures from open reef sediments and a more diverse suite of actinomycetes isolated from marine cave sediment samples. Within the cave isolates, members of the proposed genus Solwaraspora were the most represented. Based on PKS- and NRPS-gene-targeted PCR amplification and sequencing, geographic variation in the occurrence of these biosynthetic pathways was also observed. These findings indicate that marine cave systems are a lucrative source in the search for novel secondary metabolite producers with biotechnological applications and that environmental and geographic factors likely affect the occurrence of these biosynthetic pathways.     

Improved oxytetracycline production in Streptomyces rimosus M4018 by metabolic engineering of the G6PDH gene in the pentose phosphate pathway

The aromatic polyketide antibiotic, oxytetracycline (OTC), is produced by Streptomyces rimosus as an important secondary metabolite. High level production of antibiotics in Streptomycetes requires precursors and cofactors which are derived from primary metabolism; therefore it is exigent to engineer the primary metabolism. This has been demonstrated by targeting a key enzyme in the oxidative pentose phosphate pathway (PPP) and nicotinamide adenine dinucleotide phosphate (NADPH) generation, glucose-6-phosphate dehydrogenase (G6PDH), which is encoded by zwf1 and zwf2. Disruption of zwf1 or zwf2 resulted in a higher production of OTC. The disrupted strain had an increased carbon flux through glycolysis and a decreased carbon flux through PPP, as measured by the enzyme activities of G6PDH and phosphoglucose isomerase (PGI), and by the levels of ATP, which establishes G6PDH as a key player in determining carbon flux distribution. The increased production of OTC appeared to be largely due to the generation of more malonyl-CoA, one of the OTC precursors, as observed in the disrupted mutants. We have studied the effect of zwf modification on metabolite levels, gene expression, and secondary metabolite production to gain greater insight into flux distribution and the link between the fluxes in the primary and secondary metabolisms.     

Biodiversity of Actinomycetes Associated with Caribbean Sponges and Their Potential for Natural Product Discovery

Marine actinomycetes provide a rich source of structurally unique and bioactive secondary metabolites. Numerous genera of marine actinomycetes have been isolated from marine sediments as well as several sponge species. In this study, 16 different species of Caribbean sponges were collected from four different locations in the coastal waters off Puerto Rico in order to examine diversity and bioactive metabolite production of marine actinomycetes in Caribbean sponges. Sediments were also collected from each location, in order to compare actinomycete communities between these two types of samples. A total of 180 actinomycetes were isolated and identified based on 16S rRNA gene analysis. Phylogenetic analysis revealed the presence of at least 14 new phylotypes belonging to the genera Micromonospora, Verruscosispora, Streptomyces, Salinospora, Solwaraspora, Microbacterium and Cellulosimicrobium. Seventy-eight of the isolates (19 from sediments and 59 from sponges) shared 100 % sequence identity with Micromonospora sp. R1. Despite having identical 16S rRNA sequences, the bioactivity of extracts and subsequent fractions generated from the fermentation of both sponge- and sediment-derived isolates identical to Micromonospora sp. R1 varied greatly, with a marked increase in antibiotic metabolite production in those isolates derived from sponges. These results indicate that the chemical profiles of isolates with high 16S rRNA sequence homology to known strains can be diverse and dependent on the source of isolation. In addition, seven previously reported dihydroquinones produced by five different Streptomyces strains have been purified and characterized from one Streptomyces sp. strain isolated in this study from the Caribbean sponge Agelas sceptrum.     

Strain improvement in actinomycetes in the postgenomic era

With the recent advances in DNA sequencing technologies, it is now feasible to sequence multiple actinomycete genomes rapidly and inexpensively. An important observation that emerged from early Streptomyces genome sequencing projects was that each strain contains genes that encode 20 or more potential secondary metabolites, only a fraction of which are expressed during fermentation. More recently, this observation has been extended to many other actinomycetes with large genomes. The discovery of a wealth of orphan or cryptic secondary metabolite biosynthetic gene clusters has suggested that sequencing large numbers of actinomycete genomes may provide the starting materials for a productive new approach to discover novel secondary metabolites. The key issue for this approach to be successful is to find ways to turn on or turn up the expression of cryptic or poorly expressed pathways to provide material for structure elucidation and biological testing. In this review, I discuss several genetic approaches that are potentially applicable to many actinomycetes for this application.     

Sponge-associated sp. RM66 metabolome induction with N-acetylglucosamine: Antibacterial,antifungal and anti-trypanosomal activities

The marine sponge Amphimedon sp., collected from Hurghada (Egypt) was investigated for its sponge-derived actinomycetes diversity. Nineteen actinomycetes were cultivated and phylogenetically identified using 16S rDNA gene sequencing were carried out. The strains belong to genera Kocuria, Dietzia, Micrococcus, Microbacterium and Streptomyces. Many silent biosynthetic genes clusters were investigated using genome sequencing of actinomycete strains and has revealed in particular the genus Streptomyces that has indicated their exceptional capacity for the secondary metabolites production that not observed under classical cultivation conditions. In this study, the effect of N-acetylglucosamine on the metabolome of Streptomyces sp. RM66 was investigated using three actinomycetes media (ISP2, M1 and MA). In total, twelve extracts were produced using solid and liquid fermentation approaches. Liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data were analysed using metabolomics tools to compare natural product production across all crude extracts. Our study highlighted the elicitation effect of N-acetylglucosamine on the secondary metabolite profiles of Streptomyces sp. RM66. These results highlight the of N-acetylglucosamine application as an elicitor to induce the cryptic metabolites and for increasing the chemical diversity. All the twelve extracts were tested for their antibacterial activity was tested against Staphylococcus aureus NCTC 8325, antifungal activity against Candida albicans 5314 (ATCC 90028) and anti-trypanosomal activity against Trypanosoma brucei brucei. Extract St1 showed the most potent one with activities 2.3, 3.2 and 4.7 ug/ml as antibacterial, antifungal and anti-trypanosomal, respectively.     

Identification of Unique Type II Polyketide Synthase Genes in Soil

Many bacteria, particularly actinomycetes, are known to produce secondary metabolites synthesized by polyketide synthases (PKS). Bacterial polyketides are a particularly rich source of bioactive molecules, many of which are of potential pharmaceutical relevance. To directly access PKS gene diversity from soil, we developed degenerate PCR primers for actinomycete type II KS_α (ketosynthase) genes. Twenty-one soil samples were collected from diverse sources in New Jersey, and their bacterial communities were compared by terminal restriction fragment length polymorphism (TRFLP) analysis of PCR products generated using bacterial 16S rRNA gene primers (27F and 1525R) as well as an actinomycete-specific forward primer. The distribution of actinomycetes was highly variable but correlated with the overall bacterial species composition as determined by TRFLP. Two samples were identified to contain a particularly rich and unique actinomycete community based on their TRFLP patterns. The same samples also contained the greatest diversity of KS_α genes as determined by TRFLP analysis of KS_α PCR products. KS_α PCR products from these and three additional samples with interesting TRFLP pattern were cloned, and seven novel clades of KS_α genes were identified. Greatest sequence diversity was observed in a sample containing a moderate number of peaks in its KS_α TRFLP. The nucleotide sequences were between 74 and 81% identical to known sequences in GenBank. One cluster of sequences was most similar to the KS_α involved in ardacin (glycopeptide antibiotic) production by Kibdelosporangium aridum. The remaining sequences showed greatest similarity to the KS_α genes in pathways producing the angucycline-derived antibiotics simocyclinone, pradimicin, and jasomycin.     

Whole-genome sequencing of the endemic Antarctic fungus Antarctomyces pellizariae reveals an ice-binding protein,a scarce set of secondary metabolites gene clusters and provides insights on Thelebolales phylogeny

The snow-covered surfaces of Antarctica comprise an extreme environment that favors the development of life forms with adaptations to adverse low-temperature habitats. The ability to survive and such temperatures might involve the production of antifreeze proteins and ice-binding proteins that attenuate the effects of intense cold temperatures. He, we sequenced and reconstructed the nuclear and mitochondrial genomes of the endemic Antarctic fungus Antarctomyces pellizariae UFMGCB 12416. We then have identified a putative ice-binding protein-coding gene, mapped the presence of secondary metabolite gene clusters, and reconstructed the phylogenetic relationships of a A. pellizariae with others Leotiomycetes from the alignment of hundreds of orthologous single-copy proteins. Our results will deepen the understanding of microbial ice-binding proteins and the genomic aspects of psychrophilic fungi.DatasetThe GenBank/EMBL/DDBJ accession number for the gene sequence of ice-binding protein from A. pellizariae determined in this study is MN867686. The Whole Genome Shotgun project of strain A. pellizariae UFMGCB 12416 has been deposited at DDBJ/ENA/GenBank under accession WCAA00000000. The version described in this paper is version WCAA01000000. The mitochondrial genome has been deposited under accession MT197497.     

A novel taxonomic marker that discriminates between morphologically complex actinomycetes

In the era when large whole genome bacterial datasets are generated routinely, rapid and accurate molecular systematics is becoming increasingly important. However, 16S ribosomal RNA sequencing does not always offer sufficient resolution to discriminate between closely related genera. The SsgA-like proteins are developmental regulatory proteins in sporulating actinomycetes, whereby SsgB actively recruits FtsZ during sporulation-specific cell division. Here, we present a novel method to classify actinomycetes, based on the extraordinary way the SsgA and SsgB proteins are conserved. The almost complete conservation of the SsgB amino acid (aa) sequence between members of the same genus and its high divergence between even closely related genera provides high-quality data for the classification of morphologically complex actinomycetes. Our analysis validates Kitasatospora as a sister genus to Streptomyces in the family Streptomycetaceae and suggests that Micromonospora, Salinispora and Verrucosispora may represent different clades of the same genus. It is also apparent that the aa sequence of SsgA is an accurate determinant for the ability of streptomycetes to produce submerged spores, dividing the phylogenetic tree of streptomycetes into liquid-culture sporulation and no liquid-culture sporulation branches. A new phylogenetic tree of industrially relevant actinomycetes is presented and compared with that based on 16S rRNA sequences.     

Ecological role of a seaweed secondary metabolite for a colonizing bacterial community

Bacteria associated with seaweeds can both harm and benefit their hosts. Many seaweed species are known to produce compounds that inhibit growth of bacterial isolates, but the ecological role of seaweed metabolites for the associated bacterial community structure is not well understood. In this study the response of a colonizing bacterial community to the secondary metabolite (1,1,3,3-tetrabromo-2-heptanone) from the red alga Bonnemaisonia hamifera was investigated by using field panels coated with the metabolite at a range of concentrations covering those measured at the algal surface. The seaweed metabolite has previously been shown to have antibacterial effects. The metabolite significantly affected the natural fouling community by (i) altering the composition, (ii) altering the diversity by increasing the evenness and (iii) decreasing the density, as measured by terminal restriction fragment length polymorphism in conjunction with clone libraries of the 16S rRNA genes and by bacterial enumeration. No single major bacterial taxon (phylum, class) was particularly affected by the metabolite. Instead changes in community composition were observed at a more detailed phylogenetic level. This indicates a broad specificity of the seaweed metabolite against bacterial colonization, which is supported by the observation that the bacterial density was significantly affected at a lower concentration (0.02 μg cm^?2) than the composition (1–2.5 μg cm^?2) and the evenness (5 μg cm^?2) of the bacterial communities. Altogether, the results emphasize the role of secondary metabolites for control of the density and structure of seaweed-associated bacterial communities.