The dual role of TonB genes in turnerbactin uptake and carbohydrate utilization in the shipworm symbiont Teredinibacter turnerae.

IMPORTANCE: This study highlights diversity in iron acquisition and regulation in bacteria. The mechanisms of iron acquisition and its regulation in Teredinibacter turnerae, as well as its connection to cellulose utilization, a hallmark phenotype of T. turnerae, expand the paradigm of bacterial iron acquisition. Two of the four TonB genes identified in T. turnerae exhibit functional redundancy and play a crucial role in siderophore-mediated iron transport. Unlike typical TonB genes in bacteria, none of the TonB genes in T. turnerae are clearly iron regulated. This unusual regulation could be explained by another important finding in this study, namely, that the two TonB genes involved in iron transport are also essential for cellulose utilization as a carbon source, leading to the expression of TonB genes even under iron-rich conditions.

Resistance mechanisms for Gram-negative bacteria-specific lipopeptides, turnercyclamycins, differ from that of colistin.

IMPORTANCE: Bacterial resistance to antibiotics is a crisis. Acinetobacter baumannii is among the CDC urgent threat pathogens in part for this reason. Lipopeptides known as turnercyclamycins are produced by symbiotic bacteria that normally live in marine mollusks, where they may be involved in shaping their symbiotic niche. Turnercyclamycins killed Gram-negative pathogens including drug-resistant Acinetobacter, but how do the mechanisms of resistance compare to other lipopeptide drugs? Here, we define resistance from a truncation of MlaA, a protein involved in regulating bacterial membrane phospholipids. Intriguingly, this resistance mechanism only affected one turnercyclamycin variant, which differed only in two atoms in the lipid tail of the compounds. We could not obtain significant resistance to the second turnercyclamycin variant, which was also effective in an infection model. This study reveals an unexpected subtlety in resistance to lipopeptide antibiotics, which may be useful in the design and development of antibiotics to combat drug resistance.

The dual role of TonB genes in turnerbactin uptake and carbohydrate utilization in the shipworm symbiont Teredinibacter turnerae.

Teredinibacter turnerae is an intracellular bacterial symbiont that resides in the gills of shipworms, wood-eating bivalve mollusks. This bacterium produces a catechol siderophore, turnerbactin, required for the survival of this bacterium under iron limiting conditions. The turnerbactin biosynthetic genes are contained in one of the secondary metabolite clusters conserved among T. turnerae strains. However, Fe(III)-turnerbactin uptake mechanisms are largely unknown. Here, we show that the first gene of the cluster, fttA a homologue of Fe(III)-siderophore TonB-dependent outer membrane receptor (TBDR) genes is indispensable for iron uptake via the endogenous siderophore, turnerbactin, as well as by an exogenous siderophore, amphi-enterobactin, ubiquitously produced by marine vibrios. Furthermore, three TonB clusters containing four tonB genes were identified, and two of these genes, tonB1b and tonB2, functioned not only for iron transport but also for carbohydrate utilization when cellulose was a sole carbon source. Gene expression analysis revealed that none of the tonB genes and other genes in those clusters were clearly regulated by iron concentration while turnerbactin biosynthesis and uptake genes were up-regulated under iron limiting conditions, highlighting the importance of tonB genes even in iron rich conditions, possibly for utilization of carbohydrates derived from cellulose.

A Conserved Biosynthetic Gene Cluster Is Regulated by Quorum Sensing in a Shipworm Symbiont.

Bacterial symbionts often provide critical functions for their hosts. For example, wood-boring bivalves called shipworms rely on cellulolytic endosymbionts for wood digestion. However, how the relationship between shipworms and their bacterial symbionts is formed and maintained remains unknown. Quorum sensing (QS) often plays an important role in regulating symbiotic relationships. We identified and characterized a QS system found in Teredinibacter sp. strain 2052S, a gill isolate of the wood-boring shipworm Bactronophorus cf. thoracites. We determined that 2052S produces the signal N-decanoyl-l-homoserine lactone (C10-HSL) and that this signal controls the activation of a biosynthetic gene cluster colocated in the symbiont genome that is conserved among all symbiotic Teredinibacter isolates. We subsequently identified extracellular metabolites associated with the QS regulon, including ones linked to the conserved biosynthetic gene cluster, using mass spectrometry-based molecular networking. Our results demonstrate that QS plays an important role in regulating secondary metabolism in this shipworm symbiont. This information provides a step toward deciphering the molecular details of the relationship between these symbionts and their hosts. Furthermore, because shipworm symbionts harbor vast yet underexplored biosynthetic potential, understanding how their secondary metabolism is regulated may aid future drug discovery efforts using these organisms. IMPORTANCE Bacteria play important roles as symbionts in animals ranging from invertebrates to humans. Despite this recognized importance, much is still unknown about the molecular details of how these relationships are formed and maintained. One of the proposed roles of shipworm symbionts is the production of bioactive secondary metabolites due to the immense biosynthetic potential found in shipworm symbiont genomes. Here, we report that a shipworm symbiont uses quorum sensing to coordinate activation of its extracellular secondary metabolism, including the transcriptional activation of a biosynthetic gene cluster that is conserved among many shipworm symbionts. This work is a first step toward linking quorum sensing, secondary metabolism, and symbiosis in wood-boring shipworms.

Halogenated Metal-Binding Compounds from Shipworm Symbionts.

Bacteria use small molecules to impose strict regulation over the acquisition, uptake, and sequestration of transition metal ions. Low-abundance nutrient metals, such as Fe(III), need to be scavenged from the environment by high-affinity chelating molecules called siderophores. Conversely, metal ions that become toxic at high concentrations need to be sequestered and detoxified. Often, bacteria produce a suite of compounds that bind various metal ions at different affinities in order to maintain homeostasis. Turnerbactin, a triscatecholate siderophore isolated from the intracellular shipworm symbiont Teredinibacter turnerae T7901, is responsible for iron regulation and uptake. Herein, another series of compounds are described that complex with iron, copper, and molybdenum in solution. Teredinibactins belong to a class of metal-binding molecules that utilize a phenolate-thiazoline moiety in the coordination of metal ions. In contrast to other compounds in this class, such as yersiniabactin, the phenyl ring is decorated with a 2,4-dihydroxy-3-halo substitution pattern. UV-vis absorption spectroscopy based titration experiments with CuCl2 show the formation of an intermediate complex at substoichiometric concentrations and conversion to a copper-bound complex at 1:1 molar equiv.

Shipworm symbiosis ecology-guided discovery of an antibiotic that kills colistin-resistant Acinetobacter.

Teredinibacter turnerae is an intracellular bacterial symbiont in the gills of wood-eating shipworms, where it is proposed to use antibiotics to defend itself and its animal host. Several biosynthetic gene clusters are conserved in T. turnerae and their host shipworms around the world, implying that they encode defensive compounds. Here, we describe turnercyclamycins, lipopeptide antibiotics encoded in the genomes of all sequenced T. turnerae strains. Turnercyclamycins are bactericidal against challenging Gram-negative pathogens, including colistin-resistant Acinetobacter baumannii. Phenotypic screening identified the outer membrane as the likely target. Turnercyclamycins and colistin operate by similar cellular, although not necessarily molecular, mechanisms, but turnercyclamycins kill colistin-resistant A. baumannii, potentially filling an urgent clinical need. Thus, by exploring environments that select for the properties we require, we harvested the fruits of evolution to discover compounds with potential to target unmet health needs. Investigating the symbionts of shipworms is a powerful example of this principle.

Teredinibacter haidensis sp. nov., Teredinibacter purpureus sp. nov. and Teredinibacter franksiae sp. nov., marine, cellulolytic endosymbiotic bacteria isolated from the gills of the wood-boring mollusc Bankia setacea (Bivalvia: Teredinidae) and emended description of the genus Teredinibacter.

Here, we describe three endosymbiotic bacterial strains isolated from the gills of the shipworm, Bankia setacea (Teredinidae: Bivalvia). These strains, designated as Bs08T, Bs12T and Bsc2T, are Gram-stain-negative, microaerobic, gammaproteobacteria that grow on cellulose and a variety of substrates derived from lignocellulose. Phenotypic characterization, phylogeny based on 16S rRNA gene and whole genome sequence data, amino acid identity and percentage of conserved proteins analyses, show that these strains are novel and may be assigned to the genus Teredinibacter. The three strains may be differentiated and distinguished from other previously described Teredinibacter species based on a combination of four characteristics: colony colour (Bs12T, purple; others beige to brown), marine salt requirement (Bs12T, Bsc2T and Teredinibacter turnerae strains), the capacity for nitrogen fixation (Bs08T and T. turnerae strains) and the ability to respire nitrate (Bs08T). Based on these findings, we propose the names Teredinibacter haidensis sp. nov. (type strain Bs08T=ATCC TSD-121T=KCTC 62964T), Teredinibacter purpureus sp. nov. (type strain Bs12T=ATCC TSD-122T=KCTC 62965T) and Teredinibacter franksiae sp. nov. (type strain Bsc2T=ATCC TSD-123T=KCTC 62966T).

Secondary Metabolism in the Gill Microbiota of Shipworms (Teredinidae) as Revealed by Comparison of Metagenomes and Nearly Complete Symbiont Genomes.

Shipworms play critical roles in recycling wood in the sea. Symbiotic bacteria supply enzymes that the organisms need for nutrition and wood degradation. Some of these bacteria have been grown in pure culture and have the capacity to make many secondary metabolites. However, little is known about whether such secondary metabolite pathways are represented in the symbiont communities within their hosts. In addition, little has been reported about the patterns of host-symbiont co-occurrence. Here, we collected shipworms from the United States, the Philippines, and Brazil and cultivated symbiotic bacteria from their gills. We analyzed sequences from 22 shipworm gill metagenomes from seven shipworm species and from 23 cultivated symbiont isolates. Using (meta)genome sequencing, we demonstrate that the cultivated isolates represent all the major bacterial symbiont species and strains in shipworm gills. We show that the bacterial symbionts are distributed among shipworm hosts in consistent, predictable patterns. The symbiotic bacteria harbor many gene cluster families (GCFs) for biosynthesis of bioactive secondary metabolites, only <5% of which match previously described biosynthetic pathways. Because we were able to cultivate the symbionts and to sequence their genomes, we can definitively enumerate the biosynthetic pathways in these symbiont communities, showing that ∼150 of ∼200 total biosynthetic gene clusters (BGCs) present in the animal gill metagenomes are represented in our culture collection. Shipworm symbionts occur in suites that differ predictably across a wide taxonomic and geographic range of host species and collectively constitute an immense resource for the discovery of new biosynthetic pathways corresponding to bioactive secondary metabolites.IMPORTANCE We define a system in which the major symbionts that are important to host biology and to the production of secondary metabolites can be cultivated. We show that symbiotic bacteria that are critical to host nutrition and lifestyle also have an immense capacity to produce a multitude of diverse and likely novel bioactive secondary metabolites that could lead to the discovery of drugs and that these pathways are found within shipworm gills. We propose that, by shaping associated microbial communities within the host, the compounds support the ability of shipworms to degrade wood in marine environments. Because these symbionts can be cultivated and genetically manipulated, they provide a powerful model for understanding how secondary metabolism impacts microbial symbiosis.

Boholamide A, an APD-Class, Hypoxia-Selective Cyclodepsipeptide.

Calcium homeostasis is implicated in some cancers, leading to the possibility that selective control of calcium might lead to new cancer drugs. On the basis of this idea, we designed an assay using a glioblastoma cell line and screened a collection of 1000 unique bacterial extracts. Isolation of the active compound from a hit extract led to the identification of boholamide A (1), a 4-amido-2,4-pentadieneoate (APD)-class peptide. Boholamide A (1) applied in the nanomolar range induces an immediate influx of Ca2+ in glioblastoma and neuronal cells. APD-class natural products are hypoxia-selective cytotoxins that primarily target mitochondria. Like other APD-containing compounds, 1 is hypoxia selective. Since APD natural products have received significant interest as potential chemotherapeutic agents, 1 provides a novel APD scaffold for the development of new anticancer compounds.

Teredinibacter waterburyi sp. nov., a marine, cellulolytic endosymbiotic bacterium isolated from the gills of the wood-boring mollusc Bankia setacea (Bivalvia: Teredinidae) and emended description of the genus Teredinibacter.

A cellulolytic, aerobic, gammaproteobacterium, designated strain Bs02T, was isolated from the gills of a marine wood-boring mollusc, Bankia setacea (Bivalvia: Teredinidae). The cells are Gram-stain-negative, slightly curved motile rods (2-5×0.4-0.6 µm) that bear a single polar flagellum and are capable of heterotrophic growth in a simple mineral medium supplemented with cellulose as a sole source of carbon and energy. Cellulose, carboxymethylcellulose, xylan, cellobiose and a variety of sugars also support growth. Strain Bs02T requires combined nitrogen for growth. Temperature, pH and salinity optima (range) for growth were 20 °C (range, 10-30 °C), 8.0 (pH 6.5-8.5) and 0.5 M NaCl (range, 0.0-0.8 M), respectively when grown on 0.5 % (w/v) galactose. Strain Bs02T does not require magnesium and calcium ion concentrations reflecting the proportions found in seawater. The genome size is approximately 4.03 Mbp and the DNA G+C content of the genome is 47.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequences, and on conserved protein-coding sequences, show that strain Bs02T forms a well-supported clade with Teredinibacter turnerae. Average nucleotide identity and percentage of conserved proteins differentiate strain Bs02T from Teredinibacter turnerae at threshold values exceeding those proposed to distinguish bacterial species but not genera. These results indicate that strain Bs02T represents a novel species in the previously monotypic genus Teredinibacter for which the name Teredinibacter waterburyi sp. nov. is proposed. The strain has been deposited under accession numbers ATCC TSD-120T and KCTC 62963T.

Synergistic anti-methicillin-resistant Staphylococcus aureus (MRSA) activity and absolute stereochemistry of 7,8-dideoxygriseorhodin C.

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 ß-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.

Inhibition of Biofilm Formation by Modified Oxylipins from the Shipworm Symbiont Teredinibacter turnerae.

The bioactivity-guided purification of the culture broth of the shipworm endosymbiont Teredinibacter turnerae strain 991H.S.0a.06 yielded a new fatty acid, turneroic acid (1), and two previously described oxylipins (2-3). Turneroic acid (1) is an 18-carbon fatty acid decorated by a hydroxy group and an epoxide ring. Compounds 1-3 inhibited bacterial biofilm formation in Staphylococcus epidermidis, while only 3 showed antimicrobial activity against planktonic S. epidermidis. Comparison of the bioactivity of 1-3 with structurally related compounds indicated the importance of the epoxide moiety for selective and potent biofilm inhibition.

Shipworm bioerosion of lithic substrates in a freshwater setting, Abatan River, Philippines: Ichnologic, paleoenvironmental and biogeomorphical implications.

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.

A rock-boring and rock-ingesting freshwater bivalve (shipworm) from the Philippines.

Shipworms are a group of wood-boring and wood-feeding bivalves of extraordinary economic, ecological and historical importance. Known in the literature since the fourth century BC, shipworms are both destructive pests and critical providers of ecosystem services. All previously described shipworms are obligate wood-borers, completing all or part of their life cycle in wood and most are thought to use wood as a primary source of nutrition. Here, we report and describe a new anatomically and morphologically divergent species of shipworm that bores in carbonate limestone rather than in woody substrates and lacks adaptations associated with wood-boring and wood digestion. The species is highly unusual in that it bores by ingesting rock and is among the very few known freshwater rock-boring macrobioeroders. The calcareous burrow linings of this species resemble fossil borings normally associated with bivalve bioerosion of wood substrates (ichnospecies Teredolites longissimus) in marginal and fully marine settings. The occurrence of this newly recognized shipworm in a lithic substrate has implications for teredinid phylogeny and evolution, and interpreting palaeoenvironmental conditions based on fossil bioerosion features.

Tamilokus mabinia, a new, anatomically divergent genus and species of wood-boring bivalve from the Philippines.

Here we describe an anatomically divergent wood-boring bivalve belonging to the family Teredinidae. Specimens were collected off the coast of Mabini, Batangas, Philippines, in February 2018, from sunken driftwood at a depth of less than 2 m. A combination of characteristics differentiates these specimens from members of previously named teredinid genera and species. Most notable among these include: an enlarged cephalic hood which extends across the posterior slope of the shell valves and integrates into the posterior adductor muscle; a unique structure, which we term the 'cephalic collar', formed by protruding folds of the mantle immediately ventral to the foot and extending past the posterior margin of the valves; a large globular stomach located entirely posterior to the posterior adductor muscle and extending substantially beyond the posterior gape of the valves; an elongate crystalline style and style sac extending from the base of the foot, past the posterior adductor muscle, to the posteriorly located stomach; calcareous pallets distinct from those of described genera; a prominently flared mantle collar which extends midway along the stalk of the pallets; and, separated siphons that bear a pigmented pinstripe pattern with highly elaborate compound papillae on the incurrent siphon aperture. We used Micro-Computed Tomography (Micro-CT) to build a virtual 3D anatomical model of this organism, confirming the spatial arrangement of the structures described above. Phylogenetic analysis of the small (18S) and large (28S) nuclear rRNA gene sequences, place this bivalve within the Teredindae on a branch well differentiated from previously named genera and species. We propose the new genus and species Tamilokus mabinia to accommodate these organisms, raising the total number of genera in this economically and environmentally important family to 17. This study demonstrates the efficacy of Micro-CT for anatomical description of a systematically challenging group of bivalves whose highly derived body plans are differentiated predominantly by soft tissue adaptations rather than features of calcareous hard-parts.

Mindapyrroles A-C, Pyoluteorin Analogues from a Shipworm-Associated Bacterium.

Three new pyoluteorin analogues, mindapyrroles A-C (1-3), were purified from Pseudomonas aeruginosa strain 1682U.R.0a.27, a gill-associated bacterium isolated from the tissue homogenate of the giant shipworm Kuphus polythalamius. Mindapyrroles B and C inhibit the growth of multiple pathogenic bacteria, with mindapyrrole B (2) showing the most potent antimicrobial activity and widest selectivity index over mammalian cells. Preliminary structure-activity relationship analysis showed that dimerization of the pyoluteorin moiety through a C-C linkage is detrimental to the antimicrobial activity, but addition of an aerugine unit in the methylene bridge is favorable for both the antimicrobial activity and selectivity index.

Complete Genome Sequence of Vibrio campbellii DS40M4.

We present the complete genome sequence of Vibrio campbellii DS40M4, assembled from Illumina and Oxford Nanopore data. This effort improves upon a previous draft assembly to resolve this organism's two-chromosome and one-plasmid genetic structure and to provide valuable context for evaluating the gene arrangement and evolution of this species.

Thiosocius teredinicola gen. nov., sp. nov., a sulfur-oxidizing chemolithoautotrophic endosymbiont cultivated from the gills of the giant shipworm, Kuphus polythalamius.

A chemolithoautotrophic sulfur-oxidizing, diazotrophic, facultatively heterotrophic, endosymbiotic bacterium, designated as strain 2141T, was isolated from the gills of the giant shipworm Kuphus polythalamius (Teredinidae: Bivalvia). Based on its 16S rRNA sequence, the endosymbiont falls within a clade that includes the as-yet-uncultivated thioautotrophic symbionts of a marine ciliate and hydrothermal vent gastropods, uncultivated marine sediment bacteria, and a free-living sulfur-oxidizing bacterium ODIII6, all of which belong to the Gammaproteobacteria. The endosymbiont is Gram-negative, rod-shaped and has a single polar flagellum when grown in culture. This bacterium can be grown chemolithoautotrophically on a chemically defined medium supplemented with either hydrogen sulfide, thiosulfate, tetrathionate or elemental sulfur. The closed-circular genome has a DNA G+C content of 60.1 mol% and is 4.79 Mbp in size with a large nitrogenase cluster spanning nearly 40 kbp. The diazotrophic capability was confirmed by growing the strain on chemolithoautotrophic thiosulfate-based medium without a combined source of fixed nitrogen. The bacterium is also capable of heterotrophic growth on organic acids such as acetate and propionate. The pH, temperature and salinity optima for chemolithoautotrophic growth on thiosulfate were found to be 8.5, 34 °C and 0.2 M NaCl, respectively. To our knowledge, this is the first report of pure culture of a thioautotrophic animal symbiont. The type strain of Thiosocius teredinicola is PMS-2141T.STBD.0c.01aT (=DSM 108030T).

Correction to: Amphi­enterobactin commonly produced among Vibrio campbellii and Vibrio harveyi strains can be taken up by a novel outer membrane protein FapA that also can transport canonical Fe(III)­enterobactin.

In the original publication, third author's name was incorrectly published as Aneta L. Jelowicki.