Capsular polysaccharide from the marine bacterium Cobetia marina induces apoptosis via both caspase-dependent and mitochondria-mediated pathways in HL-60 cells.

In the present study, we investigated the antiproliferative effect of the capsular polysaccharide (CPS) from marine Gram-negative bacterium Cobetia marina (formerly C. pacifica) KMM 3878 against human leukemia cells in vitro and the potential molecular mechanism underlying this activity. Our results showed that the CPS could inhibit the proliferation of HL-60 cells in a dose-dependent manner with no effect on normal PBMC cells. HL-60 cells treated with the CPS exhibited typical morphologic and biochemical signs of apoptosis. We found that the CPS caused the collapse of mitochondrial transmembrane potential (ΔΨm), activated caspases-8,-9, and - 3, decreased the ratio of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins, increased ROS production and TNF-α secretion, and stimulated phosphorylation of p38 MAPK and p53 in HL-60 cells. Taken together, these data suggest that both extracellular and intracellular signaling pathways contribute to the CPS-induced apoptosis in HL-60 cells.

Complete genome sequence of Sagittula stellata strain E-37 reveals a plasmid-encoded type six secretion system.

We announce the complete genome sequence of Sagittula stellata strain E-37. The hybrid assembly of long and short reads revealed one chromosome and four plasmids. Furthermore, the genome analysis showed that the plasmid-encoded type six secretion system is linked to plasmid replication genes that may be common to Roseobacters.

Effect of bacterial dissociation on lipopolysaccharide structure: A study of O-polysaccharide from the marine bacterium Pseudoalteromonas agarivorans KMM 232 (O-form).

The lipopolysaccharide (LPS) was obtained from a bacterium Pseudoalteromonas agarivorans KMM 232 (O-form) isolated from a seawater sample collected at a depth of 500 m. The O-polysaccharide (OPS) was isolated by mild acid degradation of the LPS and studied by chemical methods along with 1D and 2D 1H and 13C NMR spectroscopy, including 1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY and 1H,13C HSQC, and 1H,13C HMBC experiments. The following new structure of the OPS from P. agarivorans KMM 232 (O-form) containing 2-acetamido-2-deoxy-d-glucose (D-GlcNAc), d-glucose (D-Glc), d-glucuronic acid (D-GlcA), 4,6-O-[(R)-1-carboxyethylidene]-d-galactose [D-Galp4,6 (R-Pyr)] and two residues of d-galactose (D-Gal) was established.

Structure and in vitro antiproliferative activity against breast cancer cells of the cell-wall polysaccharide from the marine bacterium Kangiella japonica KMM 3899T.

Kangiella japonica KMM 3899T is a Gram-negative bacterium isolated from a sandy sediment sample collected from the Sea of Japan. Here the results of the structure and the biological activity against breast cancer cells of the cell-wall polysaccharide from K. japonica KMM 3899T have been described. The structure of the repeating unit of the polysaccharide was elucidated using chemical analysis and NMR spectroscopy: →4)-α-L-GalpNAc3AcA-(1 â†’ 3)-α-D-GlcpNAc-(1 â†’ 4)-ß-D-GlcpNAc3NAcAN-(1→. The cell-wall polysaccharide had an antiproliferative effect against T-47D cells. Flow cytometric and Western blot analysis revealed that the polysaccharide induced S phase arrest and mitochondrial-dependent apoptosis.

Growth-dependent cr(VI) reduction by Alteromonas sp. ORB2 under haloalkaline conditions: toxicity, removal mechanism and effect of heavy metals.

Bacterial reduction of hexavalent chromium (VI) to chromium (III) is a sustainable bioremediation approach. However, the Cr(VI) containing wastewaters are often characterized with complex conditions such as high salt, alkaline pH and heavy metals which severely impact the growth and Cr(VI) reduction potential of microorganisms. This study investigated Cr(VI) reduction under complex haloalkaline conditions by an Alteromonas sp. ORB2 isolated from aerobic granular sludge cultivated from the seawater-microbiome. Optimum growth of Alteromonas sp. ORB2 was observed under haloalkaline conditions at 3.5-9.5% NaCl and pH 7-11. The bacterial growth in normal culture conditions (3.5% NaCl; pH 7.6) was not inhibited by 100 mg/l Cr(VI)/ As(V)/ Pb(II), 50 mg/l Cu(II) or 5 mg/l Cd(II). Near complete reduction of 100 mg/l Cr(VI) was achieved within 24 h at 3.5-7.5% NaCl and pH 8-11. Cr(VI) reduction by Alteromonas sp. ORB2 was not inhibited by 100 mg/L As(V), 100 mg/L Pb(II), 50 mg/L Cu(II) or 5 mg/L Cd(II). The bacterial cells grew in the medium with 100 mg/l Cr(VI) contained lower esterase activity and higher reactive oxygen species levels indicating toxicity and oxidative stress. In-spite of toxicity, the cells grew and reduced 100 mg/l Cr(VI) completely within 24 h. Cr(VI) removal from the medium was driven by bacterial reduction to Cr(III) which remained in the complex medium. Cr(VI) reduction was strongly linked to aerobic growth of Alteromonas sp. The Cr(VI) reductase activity of cytosolic protein fraction was pronounced by supplementing with NADPH in vitro assays. This study demonstrated a growth-dependent aerobic Cr(VI) reduction by Alteromonas sp. ORB2 under complex haloalkaline conditions akin to wastewaters.

A Noble Extract of Pseudomonas sp. M20A4R8 Efficiently Controlling the Influenza Virus-Induced Cell Death.

Epidemic diseases that arise from infectious RNA viruses, particularly influenza viruses, pose a constant threat to the global economy and public health. Viral evolution has undermined the efficacy of acquired immunity from vaccines and the antiviral effects of FDA-approved drugs. As such, there is an urgent need to develop new antiviral lead agents. Natural compounds, owing to their historical validation of application and safety, have become a promising solution. In this light, a novel marine bacterium, Pseudomonas sp. M20A4R8, has been found to exhibit significant antiviral activity [half maximal inhibitory concentration (IC50) = 1.3 µg/mL, selectivity index (SI) = 919.4] against influenza virus A/Puerto Rico/8/34, surpassing the activity of chloroquine. The antiviral response via M20A4R8 extract was induced during post-entry stages of the influenza virus, indicating suitability for post-application after the establishment of viral infection. Furthermore, post-treatment with M20A4R8 extract protected the host from virus-induced apoptosis, suggesting its potential use in acute respiratory disease complexes resulting from immune effectors' overstimulation and autophagy-mediated self-apoptosis. The extract demonstrated an outstanding therapeutic index against influenza virus A/Wisconsin/15/2009 (IC50 = 8.1 µg/mL, SI = 146.2) and B/Florida/78/2015 Victoria lineage (IC50 = 3.5 µg/mL, SI = 343.8), indicating a broad anti-influenza virus activity with guaranteed safety and effectiveness. This study provides a new perspective on mechanisms for preventing a broad spectrum of viral infections through antiviral agents from novel and natural origins. Future studies on a single or combined compound from the extract hold promise, encouraging its use in preclinical challenge tests with various influenza virus strains.

Cloning and Characterization of a Novel N-Acetyl-D-galactosamine-4-O-sulfate Sulfatase, SulA1, from a Marine Arthrobacter Strain.

Sulfation is gaining increased interest due to the role of sulfate in the bioactivity of many polysaccharides of marine origin. Hence, sulfatases, enzymes that control the degree of sulfation, are being more extensively researched. In this work, a novel sulfatase (SulA1) encoded by the gene sulA1 was characterized. The sulA1-gene is located upstream of a chondroitin lyase encoding gene in the genome of the marine Arthrobacter strain (MAT3885). The sulfatase was produced in Escherichia coli. Based on the primary sequence, the enzyme is classified under sulfatase family 1 and the two catalytic residues typical of the sulfatase 1 family-Cys57 (post-translationally modified to formyl glycine for function) and His190-were conserved. The enzyme showed increased activity, but not improved stability, in the presence of Ca2+, and conserved residues for Ca2+ binding were identified (Asp17, Asp18, Asp277, and Asn278) in a structural model of the enzyme. The temperature and pH activity profiles (screened using p-nitrocatechol sulfate) were narrow, with an activity optimum at 40-50 °C and a pH optimum at pH 5.5. The Tm was significantly higher (67 °C) than the activity optimum. Desulfation activity was not detected on polymeric substrates, but was found on GalNAc4S, which is a sulfated monomer in the repeated disaccharide unit (GlcA-GalNAc4S) of, e.g., chondroitin sulfate A. The position of the sulA1 gene upstream of a chondroitin lyase gene and combined with the activity on GalNAc4S suggests that there is an involvement of the enzyme in the chondroitin-degrading cascade reaction, which specifically removes sulfate from monomeric GalNAc4S from chondroitin sulfate degradation products.

Low-cost and efficient strategy for brown algal hydrolysis: Combination of alginate lyase and cellulase.

Brown algae are rich in biostimulants that not only stimulate the overall development and growth of plants but also have great beneficial effects on the whole soil-plant system. However, alginate, the major component of brown algae, is comparatively difficult to degrade. The cost of preparing alginate oligosaccharides (AOSs) is still too high to produce seaweed fertilizer. In this work, the marine bacterium Vibrio sp. B1Z05 is found to be capable of efficient alginate depolymerization and harbors an extended pathway for alginate metabolism. The B1Z05 extracellular cell-free supernatant exhibited great potential for AOS production at low cost, which, together with cellulase, can efficiently hydrolyze seaweed. The brown algal hydrolysis rates were significantly greater than those of the commercial alginate lyase product CE201, and the obtained seaweed extracts were rich in phytohormones. This work provides a low-cost but efficient strategy for the sustainable production of desirable AOSs and seaweed fertilizer.

Shewanella goraebulensis sp. nov., isolated from sea water.

A novel Gram-stain-negative, facultatively anaerobic and rod-shaped bacterial strain, designated as DAU312T, was isolated from the sea water of the eastern coast of the Republic of Korea. Optimal growth was observed at 25 °C, pH 7.0-8.0 and with NaCl concentrations of 2.0 % (w/v). Catalase and oxidase activities were detected. On the basis of 16S rRNA gene sequences, strain DAU312T showed the highest similarity (99.2 %) to the type strain Shewanella electrodiphila MAR441T. The complete genome sequence of strain DAU312T contains 4 893 483 bp and 40.5 mol% G+C. Phylogenetic analyses based on 16S rRNA gene sequences and the up-to-date bacterial core genes showed that strain DAU312T, S. electrodiphila MAR441T and S. olleyana were all part of the same monophyletic clade. Their average nucleotide identity, digital DNA-DNA hybridization and two-way average amino acid identity values with each other and type strains of close Shewanella species were 83.4-77.5 %, 27.3-22.0 % and 89.8-81.2 %, respectively. The major cellular fatty acids (>10 %) were iso-C15 : 0, summed feature 3 (C16 : 1 ω7с and/or C16 : 1 ω6с) and C16 : 0. Phosphatidylethanolamine and phosphatidylglycerol were the main polar lipids. The respiratory quinones were Q-7, Q-8, MK-7 and MMK-7. Based on these polyphasic taxonomic findings, the name Shewanella goraebulensis sp. nov. is suggested for strain DAU312T, which is considered to represent a novel species of the genus Shewanella. The type strain is DAU312T (=KCTC 72427 T=JCM 35744T=KCCM 43478T).

The antibiofilm potential of a heteropolysaccharide produced and characterized from the isolated marine bacterium Glutamicibacter nicotianae BPM30.

Biofilms are the significant causes of 80% of chronic infections in the oral cavity, urinary tract, biliary tube, lungs, gastrointestinal tract, and so on to the general public. Treatment of pathogenic biofilm using bacterial exopolysaccharides (EPS) is an effective and promising strategy. In the present work, a marine bacterium was isolated, studied for exopolysaccharide production, and tested for its antibiofilm activity. Approximately 1.31 ± 0.07 g/L of a purified extracellular polysaccharide was produced and characterized from the isolated marine bacterium Glutamicibacter nicotianae BPM30. The hydrolyzed EPS contains multiple monosaccharides such as rhamnose, fructose, glucose, and galactose. The EPS demonstrated potential antibiofilm activity on four tested pathogens in a concentration-dependent mode. The antibiofilm activity of the purified EPS was studied by crystal violet assay and fluorescence staining method. Comparative inhibition results obtained for the tested strains are 93.25% ± 5.25 and 88.56% ± 2.25 for K. pneumoniae; 92.65% ± 7.6 and 98.33% ± 0.85 for P. aeruginosa; 90.36% ± 6.3 and 52.08% ± 7.74 for S. typhi; 84.62% ± 5.6 and 77.90% ± 5.90 for S. dysenteriae. The results of the present work demonstrated the antibiofilm potential of EPS, which could be helpful in the invention of novel curative approaches in battling bacterial biofilm-related medical complications.

Reduction of selenite and tellurite by a highly metal-tolerant marine bacterium.

Selenium (Se) and tellurium (Te) contaminations in soils and water bodies have been widely reported in recent years. Se(IV) and Te(IV) were regarded as their most dangerous forms. Microbial treatments of Se(IV)- and Te(IV)-containing wastes are promising approaches because of their environmentally friendly and sustainable advantages. However, the salt-tolerant microbial resources that can be used for selenium/tellurium pollution control are still limited since industrial wastewaters usually contain a large number of salts. In this study, a marine Shewanella sp. FDA-1 (FDA-1) was reported for efficient Se(IV) and Te(IV) reduction under saline conditions. Process and product analyses were performed to investigate the bioreduction processes of Se(IV) and Te(IV). The results showed that FDA-1 can effectively reduce Se(IV) and Te(IV) to Se0 and Te0 Se(IV)/Te(IV) to Se0/Te0 in 72 h, which were further confirmed by XRD and XPS analyses. In addition, enzymatic and RT‒qPCR assays showed that flavin-related proteins, reductases, dehydrogenases, etc., could be involved in the bioreduction of Se(IV)/Te(IV). Overall, our results demonstrate the ability of FDA-1 to reduce high concentrations of Se(IV)/or Te(IV) to Se0/or Te0 under saline conditions and thus provide efficient microbial candidate for controlling Se and Te pollution.

Cell-cycle arrest and mitochondria-dependent apoptosis induction in T-47D cells by the capsular polysaccharide from the marine bacterium Kangiella japonica KMM 3897.

In this study, we reported the in vitro mechanisms of antiproliferative activity of capsular polysaccharide derived from marine Gram-negative bacteria Kangiella japonica KMM 3897 in human breast сarcinoma T-47D cells. Flow cytometric and Western blot analysis revealed that capsular polysaccharide effectively suppressed T-47D cell proliferation by inducing G0/G1 phase arrest and mitochondrial-dependent apoptosis. Moreover, polysaccharide influenced the ERK1/2 and p38 signaling pathways. The results of this study would enrich our understanding of the molecular mechanism of the anti-cancer activity of sulfated polysaccharides from marine Gram-negative bacteria.

Complete genome sequence of pectin-degrading Flavobacteriaceae bacterium GSB9.

Pectic oligosaccharides, which are considered to be potential prebiotics, may be generated by pectin-degrading enzymes. Here, we report the complete genome sequence of the pectin-degrading marine bacterium, Flavobacteriaceae bacterium GSB9, which was isolated from seawater of South Korea. The complete genome sequence revealed that the chromosome was 3,630,376 bp in size, had a G + C content of 36.6 mol%, and was predicted to encode 3100 protein-coding sequences (CDSs), 40 tRNAs, and six 16S-23S-5S rRNAs. Genome sequence analysis revealed that this strain possesses multiple genes predicted to encode pectin-degrading enzymes. Our analysis may facilitate the future application of this strain against pectin in various industries.

Purification and Characterization of a DegP-Type Protease from the Marine Bacterium Cobetia amphilecti KMM 296.

A new member of the DegP-type periplasmic serine endoproteases of the S1C family from the marine bacterium Cobetia amphilecti KMM 296 (CamSP) was expressed in Escherichia coli cells. The calculated molecular weight, number of amino acids, and isoelectric point (pI) of the mature protein CamSP are 69.957 kDa, 666, and 4.84, respectively. The proteolytic activity of the purified recombinant protease CamSP was 2369.4 and 1550.9 U/mg with the use of 1% bovine serum albumin (BSA) and casein as the substrates, respectively. The enzyme CamSP exhibited maximum activity at pH 6.0-6.2, while it was stable over a wide pH range from 5.8 to 8.5. The optimal temperature for the CamSP protease activity was 50 °C. The enzyme required NaCl or KCl at concentrations of 0.3 and 0.5 M, respectively, for its maximum activity. The Michaelis constant (Km) and Vmax for BSA were determined to be 41.7 µg/mL and 0.036 µg/mL min-1, respectively. The metal ions Zn2+, Cu2+, Mn2+, Li2+, Mg2+, and Ca2+ slightly activated CamSP, while the addition of CoCl2 to the incubation mixture resulted in a twofold increase in its protease activity. Ethanol, isopropanol, glycerol, and Triton-X-100 increased the activity of CamSP from two- to four-times. The protease CamSP effectively degraded the wheat flour proteins but had no proteolytic activity towards soybean, corn, and the synthetic substrates, α-benzoyl-Arg-p-nitroanilide (BAPNA) and N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide (SAPNA).

A blue enzyme from marine bacterium for green technological applications.

Laccases are the green tools that can find potential applications in various industries. There are many reports available on laccases from plants and fungal sources but very few reports are available on bacterial laccases. Bacterial laccases show broad range of substrate specificity and it is easy to isolate and purify the bacterial extracellular laccases as compared to fungal laccases. Therefore, there are many advantages of bacterial laccases over fungal laccases.

Characterization of a Novel Alginate Lyase with Two Alginate Lyase Domains from the Marine Bacterium Vibrio sp. C42.

Alginate is abundant in the cell walls of brown algae. Alginate lyases can degrade alginate, and thus play an important role in the marine carbon cycle and industrial production. Currently, most reported alginate lyases contain only one functional alginate lyase domain. AlyC8 is a putative alginate lyase with two alginate lyase domains (CD1 and CD2) from the marine alginate-degrading strain Vibrio sp. C42. To characterize AlyC8 and its two catalytic domains, AlyC8 and its two catalytic domain-deleted mutants, AlyC8-CD1 and AlyC8-CD2, were expressed in Escherichia coli. All three proteins have noticeable activity toward sodium alginate and exhibit optimal activities at pH 8.0-9.0 and at 30-40 °C, demonstrating that both CD1 and CD2 are functional. However, CD1 and CD2 showed opposite substrate specificity. The differences in substrate specificity and degradation products of alginate between the mutants and AlyC8 demonstrate that CD1 and CD2 can act synergistically to enable AlyC8 to degrade various alginate substrates into smaller oligomeric products. Moreover, kinetic analysis indicated that AlyC8-CD1 plays a major role in the degradation of alginate by AlyC8. These results demonstrate that AlyC8 is a novel alginate lyase with two functional catalytic domains that are synergistic in alginate degradation, which is helpful for a better understanding of alginate lyases and alginate degradation.

Synergy of the Two Alginate Lyase Domains of a Novel Alginate Lyase from Vibrio sp. NC2 in Alginate Degradation.

Alginate lyases play a vital role in the degradation of alginate, an important marine carbon source. Alginate is a complex macromolecular substrate, and the synergy of alginate lyases is important for the alginate utilization by microbes and the application of alginate lyases in biotechnology. Although many studies have focused on the synergy between different alginate lyases, the synergy between two alginate lyase domains of one alginate lyase has not been reported. Here, we report the synergism between the two catalytic domains of a novel alginate lyase, AlyC6', from the marine alginate-degrading bacterium Vibrio sp. NC2. AlyC6' contains two PL7 catalytic domains (CD1 and CD2) that have no sequence similarity. While both CD1 and CD2 are endo-lyases with the highest activity at 30°C, pH 8.0, and 1.0 M NaCl, they also displayed some different properties. CD1 was PM-specific, but CD2 was PG-specific. Compared with CD2, CD1 had higher catalytic efficiency, but lower substrate affinity. In addition, CD1 had a smaller minimal substrate than CD2, and the products from CD2 could be further degraded by CD1. These distinctions between the two domains enable them to synergize intramolecularly in alginate degradation, resulting in efficient and complete degradation of various alginate substrates. The bioinformatics analysis revealed that diverse alginate lyases have multiple catalytic domains, which are widespread, especially abundant in Flavobacteriaceae and Alteromonadales, which may secret multimodular alginate lyases for alginate degradation. This study provides new insight into bacterial alginate lyases and alginate degradation and is helpful for designing multimodular enzymes for efficient alginate depolymerization. IMPORTANCE Alginate is a major component in the cell walls of brown algae. Alginate degradation is carried out by alginate lyases. Until now, while most characterized alginate lyases contain one single catalytic domain, only a few have been shown to contain two catalytic domains. Furthermore, the synergy of alginate lyases has attracted increasing attention since it plays important roles in microbial alginate utilization and biotechnological applications. Although many studies have focused on the synergy between different alginate lyases, the synergy between two catalytic domains of one alginate lyase has not been reported. Here, a novel alginate lyase, AlyC6', with two functional alginate lyase domains was biochemically characterized. Moreover, the synergism between the two domains of AlyC6' was revealed. Additionally, the distribution of the alginate lyases with multiple alginate lyase domains was investigated based on the bioinformatics analysis. This study provides new insight into bacterial alginate lyases and alginate degradation.

Pseudovibrio flavus sp. nov. isolated from the sea sponge Verongula gigantea.

A Gram-negative, motile, rod-shaped marine bacterium, designated RKSG542T, was isolated from the sea sponge Verongula gigantea collected at a depth of 20 m off the west coast of San Salvador, The Bahamas. Phylogenetic analyses based on 16S rRNA gene and genome sequences place RKSG542T in a monophyletic clade with members of the genus Pseudovibrio. Strain RKSG542T shared <96.7 % 16S rRNA gene sequence similarity,<72.2 % average nucleotide identity,<66.7 % average amino acid identity, and <24.8 % digital DNA-DNA hybridization with type strains of the family Stappiaceae. Growth occurred at 22-37 °C (22-30 °C optimum), at pH 7-9 (pH 7 optimum), and with 0.5-5 % (w/v) NaCl (2 % optimum). The predominant fatty acids (>10 %) were summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c), C18 : 0 and C16 : 0, and the respiratory lipoquinone was Q-10. The polar lipid composition comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, three unknown aminolipids, six unknown phospholipids and four unknown lipids. The DNA G+C content of the genome sequence was 52.5 mol%. Based on the results of biochemical, phylogenetic and genomic analyses, RKSG542T (=TSD-76T=LMG 29867T) is presented here as the type strain of a novel species within the genus Pseudovibrio (family Stappiaceae, order Hyphomicrobiales, class Alphaproteobacteria), for which the name Pseudovibrio flavus sp. nov. is proposed.

Genome-Scale Mutational Analysis of Cathode-Oxidizing Thioclava electrotropha ElOx9T.

Extracellular electron transfer (EET) - the process by which microorganisms transfer electrons across their membrane(s) to/from solid-phase materials - has implications for a wide range of biogeochemically important processes in marine environments. Though EET is thought to play an important role in the oxidation of inorganic minerals by lithotrophic organisms, the mechanisms involved in the oxidation of solid particles are poorly understood. To explore the genetic basis of oxidative EET, we utilized genomic analyses and transposon insertion mutagenesis screens (Tn-seq) in the metabolically flexible, lithotrophic Alphaproteobacterium Thioclava electrotropha ElOx9T. The finished genome of this strain is 4.3 MB, and consists of 4,139 predicted ORFs, 54 contain heme binding motifs, and 33 of those 54 are predicted to localize to the cell envelope or have unknown localizations. To begin to understand the genetic basis of oxidative EET in ElOx9T, we constructed a transposon mutant library in semi-rich media which was comprised of >91,000 individual mutants encompassing >69,000 unique TA dinucleotide insertion sites. The library was subjected to heterotrophic growth on minimal media with acetate and autotrophic oxidative EET conditions on indium tin oxide coated glass electrodes poised at -278 mV vs. SHE or un-poised in an open circuit condition. We identified 528 genes classified as essential under these growth conditions. With respect to electrochemical conditions, 25 genes were essential under oxidative EET conditions, and 29 genes were essential in both the open circuit control and oxidative EET conditions. Though many of the genes identified under electrochemical conditions are predicted to be localized in the cytoplasm and lack heme binding motifs and/or homology to known EET proteins, we identified several hypothetical proteins and poorly characterized oxidoreductases that implicate a novel mechanism(s) for EET that warrants further study. Our results provide a starting point to explore the genetic basis of novel oxidative EET in this marine sediment microbe.