Laboratory Evolution of Escherichia coli K-12 under Aromatic Acid Stress: Adaptation in a Stressful World

The intestinal bacterium Escherichia coli K-12 can survive and protect its cell contents from a wide range of acidic and basic conditions ranging from low pH in the stomach to higher pH levels in the small intestine. We initiated an adaptive laboratory evolution procedure that consisted of daily dilutions in increasing concentrations of benzoic acid for 2,000 generations. Fitness increased step-wise over subsequent generations up to the final 2,000th generation. Sixteen unique clones from 12 of the 24-permeant aromatic acid-adapted populations grew significantly better in a lethal concentration of benzoate and salicylate compared to the ancestral strain. The genomes of the 16 isolates were whole genome sequenced and analyzed to find over 100 mutations including SNPs, large deletions, and transposable mobile elements. Some isolates acquired large deletions in the benzoate- induced multiple antibiotic resistance (mar) regulon and thus showed increased sensitivity to the antibiotic chloramphenicol. All isolates grew similarly to the ancestral wild type strain in various external pH conditions and in the presence of permeant aliphatic acids. However, the isolates showed profiles of adaption in salicylate that resembled the effects of benzoate, thus suggesting that the isolates had increased fitness specifically to permeant aromatic acids such as benzoate and salicylate. We predict that evolution in benzoic acid appears to downregulate regulons induced by aromatic acids and this project likely uncovered novel genes and their products that contribute to fitness, resistance, and growth in the presence of benzoate, the aspirin salicylate, and the antibiotic chloramphenicol

Phylogenetic analysis of the salinipostin γ-butyrolactone gene cluster uncovers new potential for bacterial signalling-molecule diversity

Bacteria communicate by small-molecule chemicals that facilitate intra- and inter-species interactions. These extracellular signalling molecules mediate diverse processes including virulence, bioluminescence, biofilm formation, motility and specialized metabolism. The signalling molecules produced by members of the phylum Actinobacteria generally comprise γ-butyrolactones, γ-butenolides and furans. The best-known actinomycete γ-butyrolactone is A-factor, which triggers specialized metabolism and morphological differentiation in the genus Streptomyces . Salinipostins A–K are unique γ-butyrolactone molecules with rare phosphotriester moieties that were recently characterized from the marine actinomycete genus Salinispora . The production of these compounds has been linked to the nine-gene biosynthetic gene cluster (BGC) spt. Critical to salinipostin assembly is the γ-butyrolactone synthase encoded by spt9. Here, we report the surprising distribution of spt9 homologues across 12 bacterial phyla, the majority of which are not known to produce γ-butyrolactones. Further analyses uncovered a large group of spt-like gene clusters outside of the genus Salinispora , suggesting the production of new salinipostin-like diversity. These gene clusters show evidence of horizontal transfer and location-specific recombination among Salinispora strains. The results suggest that γ-butyrolactone production may be more widespread than previously recognized. The identification of new γ-butyrolactone BGCs is the first step towards understanding the regulatory roles of the encoded small molecules in Actinobacteria

Metagenomic Data Reveal Type I Polyketide Synthase Distributions Across Biomes

UNLABELLED: 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

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

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

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

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

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