Petrobactin, a siderophore produced by Alteromonas, mediates community iron acquisition in the global ocean.

It is now widely accepted that siderophores play a role in marine iron biogeochemical cycling. However, the mechanisms by which siderophores affect the availability of iron from specific sources and the resulting significance of these processes on iron biogeochemical cycling as a whole have remained largely untested. In this study, we develop a model system for testing the effects of siderophore production on iron bioavailability using the marine copiotroph Alteromonas macleodii ATCC 27126. Through the generation of the knockout cell line ΔasbB::kmr, which lacks siderophore biosynthetic capabilities, we demonstrate that the production of the siderophore petrobactin enables the acquisition of iron from mineral sources and weaker iron-ligand complexes. Notably, the utilization of lithogenic iron, such as that from atmospheric dust, indicates a significant role for siderophores in the incorporation of new iron into marine systems. We have also detected petrobactin, a photoreactive siderophore, directly from seawater in the mid-latitudes of the North Pacific and have identified the biosynthetic pathway for petrobactin in bacterial metagenome-assembled genomes widely distributed across the global ocean. Together, these results improve our mechanistic understanding of the role of siderophore production in iron biogeochemical cycling in the marine environment wherein iron speciation, bioavailability, and residence time can be directly influenced by microbial activities.

Complexation of europium(III) with exopolysaccharides from a marine bacterium envisaged as luminescent probe in a theranostic approach.

Exopolysaccharide (EPS) derivatives, produced by Alteromonas infernus bacterium, showed anti-metastatic properties in osteosarcoma (bone tumor). These EPSs could be employed as new drug delivery systems for therapeutic uses. They may represent a new class of ligands to be combined in a theranostic approach with fluorescent metals, such as Eu(III), to serve as imaging probe. The goal of this work was to investigate the feasibility of such coupling by time-resolved laser-induced fluorescence spectroscopy (TRLFS). Since these EPSs are polyelectrolytes their conformation could affect the complexation properties. Thus, viscosimetric measurements were performed as a function of their concentration as well as the background electrolyte concentration. Polysaccharides conformation exhibited a lower hydrodynamic volume for the highest ionic strengths. The resulting random-coiled conformation could affect the complexation with metal for high concentration but no change was evidenced when increasing europium concentration. Two sites of complexation of Eu(III) were evidenced by TRLFS in heparin, whereas only one site was evidenced in two modified EPSs produced from Alteromonas infernus.

Antiproliferative Properties of Scandium Exopolysaccharide Complexes on Several Cancer Cell Lines.

Antimetastatic properties on both murine and human osteosarcoma cell lines (POS-1 and KHOS) have been evidenced using exopolysaccharide (EPS) derivatives, produced by Alteromonas infernus bacterium. These derivatives had no significant effect on the cell cycle neither a pro-apoptotic effect on osteosarcoma cells. Based on this observation, these EPSs could be employed as new drug delivery systems for therapeutic uses. A theranostic approach, i.e., combination of a predictive biomarker with a therapeutic agent, has been developed notably by combining with true pair of theranostic radionuclides, such as scandium 47Sc/44Sc. However, it is crucial to ensure that, once complexation is done, the biological properties of the vector remain intact, allowing the molecular tropism of the ligand to recognize its molecular target. It is important to assess if the biological properties of EPS evidenced on osteosarcoma cell lines remain when scandium is complexed to the polymers and can be extended to other cancer cell types. Scandium-EPS complexes were thus tested in vitro on human cell lines: MNNG/HOS osteosarcoma, A375 melanoma, A549 lung adenocarcinoma, U251 glioma, MDA231 breast cancer, and Caco2 colon cancer cells. An xCELLigence Real Cell Time Analysis (RTCA) technology assay was used to monitor for 160 h, the proliferation kinetics of the different cell lines. The tested complexes exhibited an anti-proliferative effect, this effect was more effective compared to EPS alone. This increase of the antiproliferative properties was explained by a change in conformation of EPS complexes due to their polyelectrolyte nature that was induced by complexation. Alterations of both growth factor-receptor signaling, and transmembrane protein interactions could be the principal cause of the antiproliferative effect. These results are very promising and reveal that EPS can be coupled to scandium for improving its biological effects and also suggesting that no major structural modification occurs on the ligand.

Metabolomic Comparison and Assessment of Co-cultivation and a Heat-Killed Inducer Strategy in Activation of Cryptic Biosynthetic Pathways.

Co-cultivation has been used as a promising tool to turn on or up-regulate cryptic biosynthetic pathways for microbial natural product discovery. Recently, a modified culturing strategy similar to co-cultivation was investigated, where heat-killed inducer cultures were supplemented to the culture medium of producer fermentations to induce cryptic pathways. In the present study, the repeatability and effectiveness of both methods in turning on cryptic biosynthetic pathways were unbiasedly assessed using UHPLC-HRESIMS-based metabolomics analysis. Both induction methods had good repeatability, and they resulted in very different induced metabolites from the tested producers. Co-cultivation generated more induced mass features than the heat-killed inducer cultures, while both methods resulted in the induction of mass features not observed using the other induction method. As examples, pathways leading to two new natural products, N-carbamoyl-2-hydroxy-3-methoxybenzamide (1) and carbazoquinocin G (5), were induced and up-regulated through co-culturing a producer Streptomyces sp. RKND-216 with inducers Alteromonas sp. RKMC-009 and M. smegmatis ATCC 120515, respectively.

A hybrid NRPS-PKS gene cluster related to the bleomycin family of antitumor antibiotics in Alteromonas macleodii strains.

Although numerous marine bacteria are known to produce antibiotics via hybrid NRPS-PKS gene clusters, none have been previously described in an Alteromonas species. In this study, we describe in detail a novel hybrid NRPS-PKS cluster identified in the plasmid of the Alteromonasmacleodii strain AltDE1 and analyze its relatedness to other similar gene clusters in a sequence-based characterization. This is a mobile cluster, flanked by transposase-like genes, that has even been found inserted into the chromosome of some Alteromonasmacleodii strains. The cluster contains separate genes for NRPS and PKS activity. The sole PKS gene appears to carry a novel acyltransferase domain, quite divergent from those currently characterized. The predicted specificities of the adenylation domains of the NRPS genes suggest that the final compound has a backbone very similar to bleomycin related compounds. However, the lack of genes involved in sugar biosynthesis indicates that the final product is not a glycopeptide. Even in the absence of these genes, the presence of the cluster appears to confer complete or partial resistance to phleomycin, which may be attributed to a bleomycin-resistance-like protein identified within the cluster. This also suggests that the compound still shares significant structural similarity to bleomycin. Moreover, transcriptomic evidence indicates that the NRPS-PKS cluster is expressed. Such sequence-based approaches will be crucial to fully explore and analyze the diversity and potential of secondary metabolite production, especially from increasingly important sources like marine microbes.

Alteromonas as a key agent of polycyclic aromatic hydrocarbon biodegradation in crude oil-contaminated coastal sediment.

Following the 2007 oil spill in South Korean tidal flats, we sought to identify microbial players influencing the environmental fate of released polycyclic aromatic hydrocarbons (PAHs). Two years of monitoring showed that PAH concentrations in sediments declined substantially. Enrichment cultures were established using seawater and modified minimal media containing naphthalene as sole carbon source. The enriched microbial community was characterized by 16S rRNA-based DGGE profiling; sequencing selected bands indicated Alteromonas (among others) were active. Alteromonas sp. SN2 was isolated and was able to degrade naphthalene, phenanthrene, anthracene, and pyrene in laboratory-incubated microcosm assays. PCR-based analysis of DNA extracted from the sediments revealed naphthalene dioxygenase (NDO) genes of only two bacterial groups: Alteromonas and Cycloclasticus, having gentisate and catechol metabolic pathways, respectively. However, reverse transcriptase PCR-based analysis of field-fixed mRNA revealed in situ expression of only the Alteromonas-associated NDO genes; in laboratory microcosms these NDO genes were markedly induced by naphthalene addition. Analysis by GC/MS showed that naphthalene in tidal-flat samples was metabolized predominantly via the gentisate pathway; this signature metabolite was detected (0.04 µM) in contaminated field sediment. A quantitative PCR-based two-year data set monitoring Alteromonas-specific 16S rRNA genes and NDO transcripts in sea-tidal flat field samples showed that the abundance of bacteria related to strain SN2 during the winter season was 20-fold higher than in the summer season. Based on the above data, we conclude that strain SN2 and its relatives are site natives--key players in PAH degradation and adapted to winter conditions in these contaminated sea-tidal-flat sediments.

Effects of a sulfated exopolysaccharide produced by Altermonas infernus on bone biology.

The growth and differentiation of bone cells is controlled by various factors, which can be modulated by heparan sulfates. Here, we investigated the effects of an oversulfated exopolysaccharide (OS-EPS) on the bone. We compared the effect of this compound with that of a native EPS. Long-term administration of OS-EPS causes cancellous bone loss in mice due, in part, to an increase in the number of osteoclasts lining the trabecular bone surface. No significant difference in cancellous bone volume was found between EPS-treated mice and age-matched control mice, underlying the importance of sulfation in trabecular bone loss. However, the mechanism sustaining this osteoporosis was unclear. To clarify OS-EPS activities, we investigated the effect of OS-EPS on osteogenesis. Our results demonstrated that OS-EPS inhibited osteoclastogenesis in two cell models. Using the surface plasmon resonance technique, we revealed that OS-EPS can form a hetero-molecular complex OS-EPS/receptor activator of NF-κB ligand (RANKL)/RANK and that RANK had a higher affinity for RANKL pre-incubated with OS-EPS than for RANKL alone, which would be in favor of an increase in bone resorption. However, in vitro, OS-EPS inhibited the early steps of osteoclast precursor adhesion and therefore inhibited the cell fusion step. In addition, we showed that OS-EPS reduced proliferation and accelerated osteoblastic differentiation, leading to strong inhibition of mineralized nodule formation, which would be in favor of an increase in bone resorption. Taken together, these data show different levels of bone resorption regulation by EPSs, most of them leading to proresorptive effects.

Alteromonas marina sp. nov., isolated from sea water of the East Sea in Korea.

Two Gram-negative, motile, non-spore-forming and moderately halophilic rods (strains SW-47(T) and SW-49) were isolated from sea water of the East Sea in Korea and subjected to a polyphasic taxonomic study. The two strains grew optimally between 30 and 37 degrees C, and grew at 4 and 44 degrees C but not at temperatures above 45 degrees C. They grew optimally in the presence of 2-5 % (w/v) NaCl, but did not grow in the absence of NaCl. Strains SW-47(T) and SW-49 had ubiquinone-8 (Q-8) as the predominant respiratory lipoquinone and C(16 : 1) omega7c and/or iso-C(15 : 0) 2OH, C(16 : 0) and C(18 : 1) omega7c as the major fatty acids, which are consistent with the corresponding data for Alteromonas macleodii. The DNA G+C contents of strains SW-47(T) and SW-49 were 45 and 44 mol%, respectively. Strains SW-47(T) and SW-49 showed a high level of 16S rDNA sequence similarity (99.9 %) and a mean level of DNA-DNA relatedness of 96.5 %. Phylogenetic analyses based on 16S rDNA sequences showed that the two strains form a coherent cluster with A. macleodii. Strains SW-47(T) and SW-49 exhibited levels of 16S rDNA sequence similarity of 99.3 and 99.1 %, respectively, with A. macleodii DSM 6062(T) and of less than 89.4 % with other species used in the phylogenetic analyses. Alteromonas fuliginea CIP 105339(T) was found to be more closely related to the genus Pseudoalteromonas than to the genus ALTEROMONAS: On the basis of phenotypic properties and phylogenetic and genomic data, strains SW-47(T) and SW-49 represent a new species of the genus Alteromonas, for which the name Alteromonas marina (type strain SW-47(T)=KCCM 41638(T)=JCM 11804(T)) is proposed.

Assignment of 'Alteromonas marinoglutinosa' NCIMB 1770 to Pseudoalteromonas mariniglutinosa sp. nov., nom. rev., comb. nov.

The taxonomic position of the marine bacterium 'Alteromonas marinoglutinosa' NCIMB 1770 was investigated in a polyphasic study. Analysis of 16S rDNA sequence and DNA-DNA reassociation values confirmed the phylogenetic position of strain NCIMB 1770 within the genus Pseudoalteromonas as a separate species, distinct from all Pseudoalteromonas species with validly described names. On the basis of physiological and molecular properties, it is proposed that strain NCIMB 1770 is classified as Pseudoalteromonas mariniglutinosa sp. nov., nom. rev., comb. nov., with the type strain NCIMB 1770T (=KMM 3635T).

Extracellular polysaccharides of Rhodococcus rhodochrous S-2 stimulate the degradation of aromatic components in crude oil by indigenous marine bacteria.

Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing D-glucose, D-galactose, D-mannose, D-glucuronic acid, and lipids, which is important to the tolerance of this strain to an aromatic fraction of (AF) Arabian light crude oil (N. Iwabuchi, N. Sunairi, H. Anzai, M. Nakajima, and S. Harayama, Appl. Environ. Microbiol. 66:5073-5077, 2000). In the present study, we examined the effects of S-2 EPS on the growth of indigenous marine bacteria on AF. Indigenous bacteria did not grow significantly in seawater containing AF even when nitrogen, phosphorus, and iron nutrients were supplemented. The addition of S-2 EPS to seawater containing nutrients and AF resulted in the emulsification of AF, promotion of the growth of indigenous bacteria, and enhancement of the degradation of AF by the bacteria. PCR-denaturing gradient gel electrophoresis analyses show that addition of S-2 EPS to the seawater containing nutrients and AF changed the composition of the bacterial populations in the seawater and that bacteria closely related to the genus Cycloclasticus became the major population. These results suggest that Cycloclasticus was responsible for the degradation of hydrocarbons in AF. The effects of 15 synthetic surfactants on the degradation of AF by indigenous marine bacteria were also examined, but enhancement of the degradation of AF was not significant. S-2 EPS was hence the most effective of the surfactants tested in promoting the biodegradation of AF and may thus be an attractive agent to use in the bioremediation of oil-contaminated marine environments.