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Lipopeptide natural products are essential agents against multidrug-resistant bacteria, but their clinical utility is often constrained by toxicity and resistance. Here, we compare the mechanisms of action of two superficially similar lipopeptide antibiotics: colistin, a last-line treatment for Gram-negative infections, and turnercyclamycins, a new class active against certain colistin-resistant strains. Both antibiotics require lipopolysaccharide (LPS) biosynthesis, even when LPS transport to the outer membrane (OM) is impaired. Colistin rapidly disrupts both the OM and the cytoplasmic membrane (CM), causing swift bacterial death. Turnercyclamycins, by contrast, act independently of the CM, with delayed OM disruption. Unlike colistin, which binds LPS directly to damage membranes, turnercyclamycins show no measurable LPS binding by calorimetry. Instead, their activity is modulated by different phospholipids, as confirmed by phospholipidomic profiling on whole cells, which identifies alterations in bacterial lipid biosynthesis and membrane homeostasis. These findings support a mechanistically distinct mode of action for turnercyclamycins, which we propose to correlate with their different pharmacological properties and potential therapeutic applications. Our results highlight how subtle structural differences between lipopeptides can lead to major functional divergence, offering a framework for the rational design of next-generation antibiotics with improved safety and efficacy profiles. Lipopeptides are used to combat drug-resistant infections, but drawbacks limit their application. Here, Lim et al . contrast the mechanisms of lipopeptides that are superficially similar yet disrupt membranes in distinctly different ways.

Secondary metabolites are ubiquitous in bacteria, but by definition, they are thought to be nonessential. Highly toxic secondary metabolites such as patellazoles have been isolated from marine tunicates, where their exceptional potency and abundance implies a role in chemical defense, but their biological source is unknown. Here, we describe the association of the tunicate Lissoclinum patella with a symbiotic α-proteobacterium, Candidatus Endolissoclinum faulkneri, and present chemical and biological evidence that the bacterium synthesizes patellazoles. We sequenced and assembled the complete Ca . E. faulkneri genome, directly from metagenomic DNA obtained from the tunicate, where it accounted for 0.6% of sequence data. We show that the large patellazoles biosynthetic pathway is maintained, whereas the remainder of the genome is undergoing extensive streamlining to eliminate unneeded genes. The preservation of this pathway in streamlined bacteria demonstrates that secondary metabolism is an essential component of the symbiotic interaction.

Significance In animals, gut microbes are essential for digestion. Here, we show that bacteria outside the gut can also play a critical role in digestion. In shipworms, wood-eating marine bivalves, endosymbiotic bacteria are found within specialized cells in the gills. We show that these endosymbionts produce wood-degrading enzymes that are selectively transported to the shipworm’s bacteria-free gut, where wood digestion occurs. Because only selected wood-degrading enzymes are transported, the shipworm system naturally identifies those endosymbiont enzymes most relevant to lignocellulose deconstruction without interference from other microbial proteins. Thus, this work expands the known biological repertoire of bacterial endosymbionts to include digestion of food and identifies previously undescribed enzymes and enzyme combinations of potential value to biomass-based industries, such as cellulosic biofuel production. Bacteria play many important roles in animal digestive systems, including the provision of enzymes critical to digestion. Typically, complex communities of bacteria reside in the gut lumen in direct contact with the ingested materials they help to digest. Here, we demonstrate a previously undescribed digestive strategy in the wood-eating marine bivalve Bankia setacea , wherein digestive bacteria are housed in a location remote from the gut. These bivalves, commonly known as shipworms, lack a resident microbiota in the gut compartment where wood is digested but harbor endosymbiotic bacteria within specialized cells in their gills. We show that this comparatively simple bacterial community produces wood-degrading enzymes that are selectively translocated from gill to gut. These enzymes, which include just a small subset of the predicted wood-degrading enzymes encoded in the endosymbiont genomes, accumulate in the gut to the near exclusion of other endosymbiont-made proteins. This strategy of remote enzyme production provides the shipworm with a mechanism to capture liberated sugars from wood without competition from an endogenous gut microbiota. Because only those proteins required for wood digestion are translocated to the gut, this newly described system reveals which of many possible enzymes and enzyme combinations are minimally required for wood degradation. Thus, although it has historically had negative impacts on human welfare, the shipworm digestive process now has the potential to have a positive impact on industries that convert wood and other plant biomass to renewable fuels, fine chemicals, food, feeds, textiles, and paper products.

Teredinid bivalves, commonly referred to as shipworms, are known for their propensity to inhabit, bioerode, and digest woody substrates across a range of brackish and fully marine settings. Shipworm body fossils and/or their borings, which are most allied with the ichnotaxon Teredolites longissimus, are found in wood preserved in sedimentary sequences ranging in age from Early Cretaceous to Recent and traditionally they have been regarded as evidence of marginal marine or marine depositional environments. Recent studies associated with the Philippine Mollusk Symbiont International Collaboration Biodiversity Group (PMS-ICBG) expedition on the island of Bohol, Philippines, have identified a new shipworm taxon (Lithoredo abatanica) that is responsible for macrobioerosion of a moderately indurated Neogene foraminiferal packstone cropping out along a freshwater reach of the Abatan River. In the process of drilling into and ingesting the limestone, these shipworms produce elongate borings that expand in diameter very gradually toward distal termini, exhibit sinuous or highly contorted axes and circular transverse outlines, and are lined along most of their length by a calcite tube. Given their strong resemblance to T. longissimus produced in wood but their unusual occurrence in a lithic substrate, these shipworm borings can be regarded as incipient Gastrochaenolites or, alternatively, as Apectoichnus. The alternate names reflect that the borings provide a testbed for ideas of the appropriateness of substrate as an ichnotaxobasis. The discovery of previously unrecognized shipworm borings in lithic substrates and the co-occurrence of another shipworm (Nausitora) in submerged logs in the same freshwater setting have implications for interpreting depositional conditions based on fossil teredinids or their ichnofossils. Of equal significance, the Abatan River study demonstrates that macrobioerosion in freshwater systems may be just as important as it is in marine systems with regard to habitat creation and landscape development. L. abatanica serve as ecosystems engineers in the sense that networks of their abandoned borings provide habitats for a variety of nestling invertebrates, and associated bioerosion undoubtedly enhances rates of mechanical and chemical degradation, thus influencing the Abatan River profile.

Prochloron spp. are obligate cyanobacterial symbionts of many didemnid family ascidians. It has been proposed that the cyclic peptides of the patellamide class found in didemnid extracts are synthesized by Prochloron spp., but studies in which host and symbiont cells are separated and chemically analyzed to identify the biosynthetic source have yielded inconclusive results. As part of the Prochloron didemni sequencing project, we identified patellamide biosynthetic genes and confirmed their function by heterologous expression of the whole pathway in Escherichia coli. The primary sequence of patellamides A and C is encoded on a single ORF that resembles a precursor peptide. We propose that this prepatellamide is heterocyclized to form thiazole and oxazoline rings, and the peptide is cleaved to yield the two cyclic patellamides, A and C. This work represents the full sequencing and functional expression of a marine natural-product pathway from an obligate symbiont. In addition, a related cluster was identified in Trichodesmium erythraeum IMS101, an important bloom-forming cyanobacterium.

Shipworms are marine bivalve mollusks (Family Teredinidae) that use wood for shelter and food. They harbor a group of closely related, yet phylogenetically distinct, bacterial endosymbionts in bacteriocytes located in the gills. This endosymbiotic community is believed to support the host's nutrition in multiple ways, through the production of cellulolytic enzymes and the fixation of nitrogen. The genome of the shipworm endosymbiont Teredinibacter turnerae T7901 was recently sequenced and in addition to the potential for cellulolytic enzymes and diazotrophy, the genome also revealed a rich potential for secondary metabolites. With nine distinct biosynthetic gene clusters, nearly 7% of the genome is dedicated to secondary metabolites. Bioinformatic analyses predict that one of the gene clusters is responsible for the production of a catecholate siderophore. Here we describe this gene cluster in detail and present the siderophore product from this cluster. Genes similar to the entCEBA genes of enterobactin biosynthesis involved in the production and activation of dihydroxybenzoic acid (DHB) are present in this cluster, as well as a two-module non-ribosomal peptide synthetase (NRPS). A novel triscatecholate siderophore, turnerbactin, was isolated from the supernatant of iron-limited T. turnerae T7901 cultures. Turnerbactin is a trimer of N-(2,3-DHB)-L-Orn-L-Ser with the three monomeric units linked by Ser ester linkages. A monomer, dimer, dehydrated dimer, and dehydrated trimer of 2,3-DHB-L-Orn-L-Ser were also found in the supernatant. A link between the gene cluster and siderophore product was made by constructing a NRPS mutant, TtAH03. Siderophores could not be detected in cultures of TtAH03 by HPLC analysis and Fe-binding activity of culture supernatant was significantly reduced. Regulation of the pathway by iron is supported by identification of putative Fur box sequences and observation of increased Fe-binding activity under iron restriction. Evidence of a turnerbactin fragment was found in shipworm extracts, suggesting the production of turnerbactin in the symbiosis.

The relationship between tunicates and the uncultivated cyanobacterium Prochloron didemni has long provided a model symbiosis. P. didemni is required for survival of animals such as Lissoclinum patella and also makes secondary metabolites of pharmaceutical interest. Here, we present the metagenomes, chemistry, and microbiomes of four related L. patella tunicate samples from a wide geographical range of the tropical Pacific. The remarkably similar P. didemni genomes are the most complex so far assembled from uncultivated organisms. Although P. didemni has not been stably cultivated and comprises a single strain in each sample, a complete set of metabolic genes indicates that the bacteria are likely capable of reproducing outside the host. The sequences reveal notable peculiarities of the photosynthetic apparatus and explain the basis of nutrient exchange underlying the symbiosis. P. didemni likely profoundly influences the lipid composition of the animals by synthesizing sterols and an unusual lipid with biofuel potential. In addition, L. patella also harbors a great variety of other bacterial groups that contribute nutritional and secondary metabolic products to the symbiosis. These bacteria possess an enormous genetic potential to synthesize new secondary metabolites. For example, an antitumor candidate molecule, patellazole, is not encoded in the genome of Prochloron and was linked to other bacteria from the microbiome. This study unveils the complex L. patella microbiome and its impact on primary and secondary metabolism, revealing a remarkable versatility in creating and exchanging small molecules.

Shipworms are marine wood-boring bivalve mollusks (family Teredinidae) that harbor a community of closely related Gammaproteobacteria as intracellular endosymbionts in their gills. These symbionts have been proposed to assist the shipworm host in cellulose digestion and have been shown to play a role in nitrogen fixation. The genome of one strain of Teredinibacter turnerae , the first shipworm symbiont to be cultivated, was sequenced, revealing potential as a rich source of polyketides and nonribosomal peptides. Bioassay-guided fractionation led to the isolation and identification of two macrodioloide polyketides belonging to the tartrolon class. Both compounds were found to possess antibacterial properties, and the major compound was found to inhibit other shipworm symbiont strains and various pathogenic bacteria. The gene cluster responsible for the synthesis of these compounds was identified and characterized, and the ketosynthase domains were analyzed phylogenetically. Reverse-transcription PCR in addition to liquid chromatography and high-resolution mass spectrometry and tandem mass spectrometry revealed the transcription of these genes and the presence of the compounds in the shipworm, suggesting that the gene cluster is expressed in vivo and that the compounds may fulfill a specific function for the shipworm host. This study reports tartrolon polyketides from a shipworm symbiont and unveils the biosynthetic gene cluster of a member of this class of compounds, which might reveal the mechanism by which these bioactive metabolites are biosynthesized.

A large family of cytotoxic cyclic peptides exemplified by the patellamides has been isolated from ascidians harboring the obligate cyanobacterial symbionts Prochloron spp. 1 , 2 , 3 , 4 , 5 . Genome sequence analysis of these symbionts has revealed that Prochloron spp. synthesize patellamides by a ribosomal pathway 6 . To understand how this pathway evolved to produce a suite of related metabolites, we analyzed 46 prochloron-containing ascidians from the tropical Pacific Ocean for the presence of patellamide biosynthetic genes and taxonomic markers. Here, we show that Prochloron spp. generate a diverse library of patellamides using small, hypervariable cassettes within a conserved genetic background. Each symbiont strain contains a single pathway, and mixtures of symbionts within ascidians lead to the accumulation of chemical libraries. We used this information to engineer the production of a new cyclic peptide in Escherichia coli , thereby demonstrating the power of comparative analysis of closely related symbiotic pathways to direct the genetic synthesis of new molecules.

The emergence of antibiotic resistance necessitates not only the identification of new compounds with antimicrobial properties, but also new strategies and combination therapies to circumvent this growing problem. Here, we report synergistic activity against methicillin-resistant Staphylococcus aureus (MRSA) of the β-lactam antibiotic oxacillin combined with 7,8-dideoxygriseorhodin C in vitro. Ongoing efforts to identify antibiotics from marine mollusk-associated bacteria resulted in the isolation of 7,8-dideoxygriseorhodin C from a Streptomyces sp. strain cultivated from a marine gastropod tissue homogenate. Despite the long history of 7,8-dideoxygriseorhodin C in the literature, the absolute configuration has never been previously reported. A comparison of measured and calculated ECD spectra resolved the configuration of the spiroketal carbon C6, and 2D ROESY NMR spectroscopy established the absolute configuration as 6s,6aS. The compound is selective against Gram-positive bacteria including MRSA and Enterococcus faecium with an MIC range of 0.125–0.5 μg ml−1. Moreover, the compound synergizes with oxacillin against MRSA as observed in the antimicrobial microdilution and time-kill assays. Simultaneous treatment of the compound with oxacillin resulted in an approximately tenfold decrease in MIC with a combination index of <0.5, indicating synergistic anti-MRSA activity.