Revisiting the metal sites of nitrous oxide reductase in a low-dose structure from Marinobacter nauticus.

Copper-containing nitrous oxide reductase catalyzes a 2-electron reduction of the green-house gas N2O to yield N2. It contains two metal centers, the binuclear electron transfer site CuA, and the unique, tetranuclear CuZ center that is the site of substrate binding. Different forms of the enzyme were described previously, representing variations in oxidation state and composition of the metal sites. Hypothesizing that many reported discrepancies in the structural data may be due to radiation damage during data collection, we determined the structure of anoxically isolated Marinobacter nauticus N2OR from diffraction data obtained with low-intensity X-rays from an in-house rotating anode generator and an image plate detector. The data set was of exceptional quality and yielded a structure at 1.5 Å resolution in a new crystal form. The CuA site of the enzyme shows two distinct conformations with potential relevance for intramolecular electron transfer, and the CuZ cluster is present in a [4Cu:2S] configuration. In addition, the structure contains three additional types of ions, and an analysis of anomalous scattering contributions confirms them to be Ca2+, K+, and Cl-. The uniformity of the present structure supports the hypothesis that many earlier analyses showed inhomogeneities due to radiation effects. Adding to the earlier description of the same enzyme with a [4Cu:S] CuZ site, a mechanistic model is presented, with a structurally flexible CuZ center that does not require the complete dissociation of a sulfide prior to N2O binding.

Marinobacter azerbaijanicus sp. nov., a moderately halophilic bacterium from Urmia Lake, Iran.

A novel moderately halophilic, Gram-stain-negative and facultatively anaerobic bacterium, designated as strain TBZ242T, was isolated from water of Urmia Lake in the Azerbaijan region of Iran. The cells were found to be rod-shaped and motile by a single polar flagellum, producing circular and yellowish colonies. The strain could grow in the presence of 0.5-10 % (w/v) NaCl (optimum, 2.5-5 %). The temperature and pH ranges for growth were 15-45 °C (optimum 30 °C) and pH 7.0-11.0 (optimum pH 8.0) on marine agar. The 16S rRNA gene sequence analysis revealed that strain TBZ242T belonged to the genus Marinobacter, showing the highest similarities to Marinobacter algicola DG893T (98.8 %), Marinobacter vulgaris F01T (98.8 %), Marinobacter salarius R9SW1T (98.5 %), Marinobacter panjinensis PJ-16T (98.4 %), Marinobacter orientalis W62T (98.0 %) and Marinobacter denitrificans JB2H27T (98.0 %). The 16S rRNA and core-genome phylogenetic trees showed that strain TBZ242T formed a distinct branch, closely related to a subclade accommodating M. vulgaris, M. orientalis, M. panjinensis, M. denitrificans, M. algicola, M. salarius and M. iranensis, within the genus Marinobacter. Average nucleotide identity and digital DNA-DNA hybridization values between strain TBZ242T and the type strains of the related species of Marinobacter were ≤85.0 and 28.6 %, respectively, confirming that strain TBZ242T represents a distinct species. The major cellular fatty acids of strain TBZ242T were C16 : 0 and C16 : 1 ω7c/C16 : 1 ω6c and the quinone was ubiquinone Q-9. The genomic DNA G+C content of strain TBZ242T is 57.2 mol%. Based on phenotypic, chemotaxonomic and genomic data, strain TBZ242T represents a novel species within the genus Marinobacter, for which the name Marinobacter azerbaijanicus sp. nov. is proposed. The type strain is TBZ242T (= CECT 30649T = IBRC-M 11466T). Genomic fragment recruitment analysis showed that this species prefers aquatic saline environments with intermediate salinities, being detected on metagenomic databases of Lake Meyghan (Iran) with 5 and 18 % salinity, respectively.

Massilia litorea sp. nov., Marinobacter salinisoli sp. nov. and Rhodobacter xanthinilyticus sp. nov., isolated from coastal environments.

Three bacterial strains, namely LPB0304T, LPB0319T and LPB0142T, were isolated from coastal environments. The 16S rRNA gene sequences of the three isolates were found to show the highest sequence similarities to Massilia litorea (98.44 %), Marinobacter salinisoli (97.55 %) and Rhodobacter lacus (97.60 %), respectively. The low (<98.7 %) sequence similarities and tree topologies implied the novelty of the three isolates, representing novel genomic species of the genus Massilia, Marinobacter and Rhodobacter. Numerous biochemical and physiological features also supported the distinctiveness of the isolates from previously known species. Based on the phenotypic and phylogenetic data presented in this study, three novel species are suggested with the following names: Massilia litorea sp. nov. (LPB0304T=KACC 21523T=ATCC TSD-216T), Marinobacter salinisoli sp. nov. (LPB0319T=KACC 21522T=ATCC TSD-218T) and Rhodobacter xanthinilyticus sp. nov. (LPB0142T=KACC 18892T=JCM 31567T).

Marinobacter qingdaonensis sp. nov., a moderately halotolerant bacterium isolated from intertidal sediment.

A Gram-stain-negative, aerobic, rod-shaped and halotolerant bacterium, designated as strain ASW11-75T, was isolated from intertidal sediments in Qingdao, PR China, and identified using a polyphasic taxonomic approach. Growth of strain ASW11-75T occurred at 10-45 °C (optimum, 37 °C), pH 6.5-9.0 (optimum, pH 8.0) and 0.5-18.0 % NaCl concentrations (optimum, 2.5 %). Phylogenetic analyses based on 16S rRNA gene sequences and 1179 single-copy orthologous clusters indicated that strain ASW11-75T is affiliated with the genus Marinobacter. Strain ASW11-75T showed highest 16S rRNA gene sequence similarity to 'Marinobacter arenosus' CAU 1620T (98.5 %). The digital DNA-DNA hybridization and average nucleotide identity values between strain ASW11-75T and its closely related strains (Marinobacter salarius R9SW1T, Marinobacter similis A3d10T, 'Marinobacter arenosus' CAU 1620T, Marinobacter sediminum R65T, Marinobacter salinus Hb8T, Marinobacter alexandrii LZ-8T and Marinobacter nauticus ATCC 49840T) were 19.8-24.5 % and 76.6-80.7 %, respectively. The predominant cellular fatty acids were C16 : 0, C18 : 1 ω9c and C16 : 0 N alcohol. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminophospholipid and two unidentified lipids. The major isoprenoid quinone was ubiquinone-9. The genomic DNA G+C content was 62.2 mol%. Based on genomic and gene function analysis, strain ASW11-75T had lower protein isoelectric points with higher ratios of acidic residues to basic residues and possessed genes related to ion transport and organic osmoprotectant uptake, implying its potential tolerance to salt. The results of polyphasic characterization indicated strain ASW11-75T represents a novel Marinobacter species, for which the name Marinobacter qingdaonensis sp. nov. with the type strain ASW11-75T is proposed. The type strain is ASW11-75T (=KCTC 82497T=MCCC 1K05587T).

Marinobacter albus sp. nov., Isolated from Sand Sediment in a Coastal Intertidal Zone.

A Gram-stain-negative bacterium with a rod-to-ovoid shape, named strain M216T, was isolated from sand sediment from the coastal intertidal zone of Huludao, Liaoning Province, China. Growth was observed at 8-40 °C (optimal, 30 °C), pH 5.5-9.5 (optimal, pH 6.5) and 0.5-14.0% (w/v) NaCl (optimal, 6%). Strain M216T possessed ubiquinone-9 as its sole respiratory quinone and phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, one unidentified aminophosphoglycolipid, one unidentified aminophospholipid, two unidentified phosphoglycolipids, three unidentified phospholipids and three unidentified glycolipids as the main polar lipids. C12:0, C16:0, C12:0 3-OH, C16:1 ω9c, C18:1 ω9c and summed features 3 (C16:1 ω7c and/or C16:1 ω6c) were the major fatty acids (> 5%). The 16S rRNA gene sequence of strain M216T exhibited high similarity to those of 'Marinobacter arenosus' CAU 1620T and Marinobacter adhaerens HP15T (99.3% and 98.5%, respectively) and less than 98.5% similarity to those of the other type strains. The ANI and dDDH values between the strain M216T and 'Marinobacter arenosus' CAU 1620T were 87.4% and 33.3%, respectively; these values were the highest among the other type strains but lower than the species threshold. The G+C content of strain M216T was 58.3%. Genomic analysis revealed that strain M216T harbors the major CAZymes of GH13, GH23, GH73, and PL5, which are responsible for polysaccharide degradation and the potential ability to reduce nitrate to ammonia. Through phenotypic, genotypic, and chemotaxonomic analyses, we proposed the name Marinobacter albus sp. nov., a novel species in the genus Marinobacter, with its type strain M216T (= MCCC 1K08600T = KCTC 82894T).

Marinobacter sediminicola sp. nov. and Marinobacter xiaoshiensis sp. nov., Isolated from Coastal Sediment.

Two Gram-stain-negative, facultative anaerobic, rod-shaped, motile bacterial strains, designated F26243T and F60267T were isolated from coastal sediment in Weihai, China. Strains F26243T and F60267T were grown at 4-40 °C (optimum 33 °C), pH 7.0-9.5 and pH 6.5-9.5 (optimum at pH 7.0), in the presence of 1.0-7.0% (w/v) NaCl (optimum 2.5%) and 1.0-12.0% (w/v) NaCl (optimum 2.0%), respectively. The 16S rRNA gene sequences phylogenetic analysis showed that strains F26243T and F60267T are closely related to the genus Marinobacter and exhibited the highest sequence similarities to Marinobacter salexigens HJR7T (97.7% and 98.0%, respectively), the similarity between two isolates was 96.7%. Strains F26243T and F60267T displayed genomic DNA G + C content of 53.6% and 53.8%, respectively. When compared to the M. salexigens HJR7T, the average nucleotide identity (ANI) values were 83.7% and 84.1%, and the percentage of conserved proteins (POCP) values were 79.9% and 84.6%, respectively. Ubiquinone 9 (Q-9) was the only respiratory quinone detected in both isolates. The major cellular fatty acids (> 10.0%) were summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c), C16:0 and C18:1ω9c. The polar lipid profiles of strains F26243T and F60267T contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidyldimethylethanolamine, phosphatidylglycerol, aminophospholipid and one unidentified phospholipid. Based on genomic characteristics, phenotypic and chemotaxonomic, strains F26243T and F60267T represent two novel species of the genus Marinobacter, for which the names Marinobacter sediminicola sp. nov. and Marinobacter xiaoshiensis sp. nov. are proposed, the type strains are F26243T (= KCTC 92640T = MCCC 1H01345T) and F60267T (= KCTC 92638T = MCCC 1H01346T).

The Description of Pseudoalteromonas apostichopi sp. nov., Vibrio apostichopi sp. nov., and Marinobacter apostichopi sp. nov. from the Fertilized Eggs and Larvae of Apostichopus japonicus.

Three novel bacterial strains, FE4T, FE10T, and LA51T, which are phylogenetically affiliated to the genera Pseudoalteromonas, Vibrio, or Marinobacter, respectively, isolated from fertilized eggs and juveniles of sea cucumber Apostichopus japonicus were characterized by a genome-based taxonomical approach including multilocus sequence analysis (MLSA) combined with classical phenotypic and chemotaxonomic characterizations. A molecular network reconstructed on the basis of nucleotide sequences of four phylogenetic maker protein genes revealed that the strains FE4T, FE10T, and LA51T were closely related to Pseudoalteromonas shioyasakiensis, Vibrio lentus, and Marinobacter similis, respectively. Average nucleotide identity (ANI) comparisons against phylogenetically related species to FE4T, FE10T, and LA51T demonstrated that each newly described strain could not be identified as any previously described species within each genus showing < 95% ANI: 91.3% of FE4T against P. shioyasakiensis JCM 18891 T, 92.6% of FE10T against "V. bathopelagicus" Sal10, and 92.6% of LA51T against M. similis A3d10T, in maximum, respectively. Here, we show molecular phylogenetic, genomic, phenotypic, and chemotaxonomic features of the newly described species FE4T, FE10T, and LA51T. We also propose Pseudoalteromonas apostichopi sp. nov. with FE4T (JCM 36173 T = LMG 33143 T) as the type strain, Vibrio apostichopi sp. nov. with FE10T (JCM 36174 T = LMG 33144 T) as the type strain, and Marinobacter apostichopi sp. nov. with LA51T (JCM 36175 T = LMG 33145 T) as the type strain.

Marinobacter iranensis sp. nov., a slightly halophilic bacterium from a hypersaline lake.

A novel halophilic bacterium, strain 71-iT, was isolated from Inche-Broun hypersaline lake in Golestan province, in the north of Iran. It was a Gram-stain-negative, non-endospore forming, rod-shaped bacterium. It grew at 4-40 °C (optimum 30 °C), pH 6.0-11.0 (optimum pH 7.5) and with 0.5-15 % (w/v) NaCl [optimum 3 % (w/v) NaCl]. The results of phylogenetic analyses based on the 16S rRNA gene sequence comparison indicated its affiliation to the genus Marinobacter and the low percentage of identity with the most closely related species (97.5 %), indicated its placement as a novel species within this genus. Digital DNA-DNA hybridization (dDDH) values and average nucleotide identity (ANI) analyses of this strain against closely related species confirmed its condition of novel taxon. On the other hand, the percentage of the average amino acid identity (AAI) affiliated strain 71-iT within the genus Marinobacter. The DNA G+C content of this isolate was 57.7 mol%. The major fatty acids were C16 : 0 and C16 : 1ω7c and/or C16 : 1 ω6c. Ubiquinone-9 was the major isoprenoid quinone and diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) were the main polar lipids of this strain. On the basis of the phylogenomic and phenotypic (including chemotaxonomic) features, we propose strain 71-iT (= IBRC M 11023T = CECT 30160T = LMG 29252T) as the type strain of a novel species within the genus Marinobacter, with the name Marinobacter iranensis sp. nov. Genomic detections of this strain in various metagenomic databases indicate that it is a relatively abundant species in environments with low salinities (approximately 5 % salinity), but not in hypersaline habitats with high salt concentrations.

Marinobacter panjinensis sp. nov., a moderately halophilic bacterium isolated from sea tidal flat environment.

Two moderately halotolerant bacterium strains, designated PJ-16T and PJ-38, were isolated from a tidal flat of the red beach in Panjin City, Liaoning Province, PR China. Cells were found to be Gram-stain-negative, aerobic, motile, rod-shaped with a single polar flagellum. Optimum growth of strain PJ-16T occurred at 30 °C, pH 7.0 and 0.2-8.0  % (w/v) NaCl, and strain PJ-38 at 30 °C, pH 6.0-7.0 and 0.2-8.0  % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PJ-16T was most closely related to Marinobacter denitrificans KCTC 62941T (99.2 % 16S rRNA gene sequence similarity), Marinobacter algicola DSM 16394T (98.6 %), Marinobacter salarius JCM 19399T (98.4 %) and Marinobacter confluentis KCTC 42705T (98.2 %), and strain PJ-38 was most closely related to M. denitrificans KCTC 62941T (99.1 %), M. algicola DSM 16394T (98.6 %), M. salarius JCM 19399T (98.4 %) and M. confluentis KCTC 42705T (98.1 %). The G+C content of the genomic DNA of strain PJ-16T based on its draft genomic sequence was 57.4 mol%. The major cellular fatty acids of strain PJ-16T were C16 : 0, C16 : 1 ω7c/C16 : 1 ω6c and C18 : 1 ω9c. The major respiratory quinone of PJ-16T was ubiquinone-9 and the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The results of the phenotypic, phylogenetic and genomic analyses revealed that strains PJ-16T and PJ-38 represent a novel species of the genus Marinobacter, and the name Marinobacter panjinensis sp. nov. is proposed. The type strain is PJ-16T (= CGMCC 1.13694T= KCTC 72023T).

Influence of Cupric (Cu2+) Ions on the Iron Oxidation Mechanism by DNA-Binding Protein from Starved Cells (Dps) from Marinobacter nauticus.

Dps proteins (DNA-binding proteins from starved cells) are multifunctional stress defense proteins from the Ferritin family expressed in Prokarya during starvation and/or acute oxidative stress. Besides shielding bacterial DNA through binding and condensation, Dps proteins protect the cell from reactive oxygen species by oxidizing and storing ferrous ions within their cavity, using either hydrogen peroxide or molecular oxygen as the co-substrate, thus reducing the toxic effects of Fenton reactions. Interestingly, the interaction between Dps and transition metals (other than iron) is a known but relatively uncharacterized phenomenon. The impact of non-iron metals on the structure and function of Dps proteins is a current topic of research. This work focuses on the interaction between the Dps from Marinobacter nauticus (a marine facultative anaerobe bacterium capable of degrading petroleum hydrocarbons) and the cupric ion (Cu2+), one of the transition metals of greater biological relevance. Results obtained using electron paramagnetic resonance (EPR), Mössbauer and UV/Visible spectroscopies revealed that Cu2+ ions bind to specific binding sites in Dps, exerting a rate-enhancing effect on the ferroxidation reaction in the presence of molecular oxygen and directly oxidizing ferrous ions when no other co-substrate is present, in a yet uncharacterized redox reaction. This prompts additional research on the catalytic properties of Dps proteins.

Marinobacter arenosus sp. nov., a halotolerant bacterium isolated from a tidal flat.

A Gram-negative, rod-shaped, motile bacterium, designated strain CAU 1620T, was isolated from a tidal flat sediment in Incheon, Republic of Korea. Strain CAU 1620T grew optimally at 30 °C and pH 8.0 in the presence of 6.0% (w/v) NaCl. The results of 16S rRNA gene sequence analysis revealed that strain CAU 1620T showed the highest similarity to Marinobacter adhaerens DSM 23420T (98.5%), followed by Marinobacter algicola DSM 16394T (98.3%) and Marinobacter maroccanus LMG 30465T (98.2%). The average nucleotide identity and digital DNA-DNA hybridisation values between strain CAU 1620T and related strains were estimated as 75.6-78.1% and 19.5-20.9%, respectively. The DNA G + C content based on the draft genome sequence was 59.2%, and the major respiratory quinone was ubiquinone-9. The predominant cellular fatty acids were C12:0, C16:0, C18:1 ω9c, and C12:0 3OH. The predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. According to phenotypic, genotypic, and chemotaxonomic analyses, strain CAU 1620T represents a novel species of the genus Marinobacter, for which the name Marinobacter arenosus sp. nov. is proposed. The type strain is CAU 1620T (= KCTC 82431T = MCCC 1K06079T).

Immunostimulant properties of full-length and truncated Marinobacter algicola flagellins, and their effects against viral hemorrhagic septicemia virus (VHSV) in trout.

Adjuvants that would help optimize fish vaccines against bacterial and viral pathogens are highly demanded by the aquaculture sector. Flagellin has been proposed as an immunostimulant and an adjuvant for more than a decade. However, the adjuvant ability of flagellins with hypervariable region deleted is still unclear in fish. In this study, we evaluated the immune-stimulating capacity of two recombinant flagellins, the wild-type flagellin F from Marinobacter algicola and a version with the hypervariable region deleted (FredV2), to induce the transcription of a wide range of immune genes using two rainbow trout cell lines: a monocyte/macrophage-cell line (RTS-11) and an epithelial cell line from intestine (RTgutGC). Additionally, we studied the capacity of both flagellins to limit the replication of viral hemorrhagic septicemia virus (VHSV) on the RTgutGC cell line. Our results demonstrated that both recombinant flagellins can significantly increase the transcription of IL-1ß1, IL-6, and IL-8 in both cell lines. However, other cytokines such as IFNγ1, and TNFα or antimicrobial peptides such as hepcidin were induced by both flagellins in RTgutGC but not in RTS-11 cells. Furthermore, both flagellins were capable of reducing the replication of VHSV in RTgutGC cells. Although the immunostimulatory and the antiviral capacities exerted by F were slightly more potent than those obtained with FredV2, the effects were retained after losing the hypervariable region. Our results provide new information on the immunostimulating and antiviral capacities of flagellins that point to their potential as suitable adjuvants for the future optimization of vaccines in aquaculture.

Sulfide removal characteristics, pathways and potential application of a novel chemolithotrophic sulfide-oxidizing strain, Marinobacter sp. SDSWS8.

Sulfide generally exists in wastewater, black and odor river, as well as aquaculture water, and give rise to adverse effect on ecological stability and biological safety, due to the toxicity, corrosivity and malodor of sulfide. In the present study, a chemolithotrophic sulfide-oxidizing bacteria (SOB) was isolated and identified as Marinobacter maroccanus strain SDSWS8. And it produced no hemolysin and was susceptible to most antibiotics. There were no accumulation of sulfide, sulfate and thiosulfate during the sulfide removal process. The optimum conditions of sulfide removal were temperature 15-40 °C, initial pH value 4.5-9.5, salinity 10-40‰, C/N ratio 0-20 and sulfide concentration 25-150 mg/L. The key genes of sulfide oxidation, Sox system (soxB, soxX, soxA, soxZ, soxY, soxD, soxC), dissimilatory sulfur oxidation (dsrA, aprA and sat) and sqr, were successfully amplified and expressed, indicating the three pathways coordinated to complete the sulfide oxidation. Besides, strain SDSWS8 had inhibitory effect on four pathogen Vibrio (V. harveyi, V. parahaemolyticus, V. anguillarum and V. splendidus). Furthermore, efficient removal of sulfide from real aquaculture water and sludge mixture could be accomplished by strain SDSWS8. This study may provide a promising candidate strain for sulfide-rich water treatment.

Characterization and Expression Analysis of Extradiol and Intradiol Dioxygenase of Phenol-Degrading Haloalkaliphilic Bacterial Isolates.

Haloalkophilic bacteria have a potential advantage as a bioremediation organism of high oil-polluted and industrial wastewater. In the current study, Haloalkaliphilic isolates were obtained from Hamralake, Wadi EL-Natrun, Egypt. The phenotype script, biochemical characters, and sequence analysis of bacterial-16S rRNA were used to identify the bacterial isolates; Halomonas HA1 and Marinobacter HA2. These strains required high concentrations of NaCl to ensure bacterial growth, especially Halomonas HA1 strain. Notably, both isolates can degrade phenol at optimal pH values, between 8 and 9, with the ability to grow in pH levels up to 11, like what was seen in the Halomonas HA1 strain. Moreover, both isolates represent two different mechanistic pathways for phenol degradation. Halomonas HA1 exploits the 1,2 phenol meta-cleavage pathway, while Marinobacter HA2 uses the 2,3 ortho-cleavage pathway as indicated by universal primers for 1,2 and 2,3 CTD genes. Interestingly, Marinobacter HA2 isolate eliminated the added phenol within an incubation period of 72 h, while the Halomonas HA1 isolate invested 96 h in degrading 84% of the same amount of phenol. Phylogenetic analysis of these 1,2 CTD (catechol dioxygenase) sequences clearly showed an evolutionary relationship between 1,2 dioxygenases of both Halomonadaceae and Pseudomonadaceae. In comparison, 2,3 CTD of Marinobacter HA2 shared the main domains of the closely related species. Furthermore, semi-quantitative RT-PCR analysis proved the constitutive expression pattern of both dioxygenase genes. These findings provide new isolates of Halomonas sp. and Marinobacter sp. that can degrade phenol at high salt and pH conditions via two independent mechanisms.

Differing Escape Responses of the Marine Bacterium Marinobacter adhaerens in the Presence of Planktonic vs. Surface-Associated Protist Grazers.

Protist grazing pressure plays a major role in controlling aquatic bacterial populations, affecting energy flow through the microbial loop and biogeochemical cycles. Predator-escape mechanisms might play a crucial role in energy flow through the microbial loop, but are yet understudied. For example, some bacteria can use planktonic as well as surface-associated habitats, providing a potential escape mechanism to habitat-specific grazers. We investigated the escape response of the marine bacterium Marinobacter adhaerens in the presence of either planktonic (nanoflagellate: Cafeteria roenbergensis) or surface-associated (amoeba: Vannella anglica) protist predators, following population dynamics over time. In the presence of V. anglica, M. adhaerens cell density increased in the water, but decreased on solid surfaces, indicating an escape response towards the planktonic habitat. In contrast, the planktonic predator C. roenbergensis induced bacterial escape to the surface habitat. While C. roenbergensis cell numbers dropped substantially after a sharp initial increase, V. anglica exhibited a slow, but constant growth throughout the entire experiment. In the presence of C. roenbergensis, M. adhaerens rapidly formed cell clumps in the water habitat, which likely prevented consumption of the planktonic M. adhaerens by the flagellate, resulting in a strong decline in the predator population. Our results indicate an active escape of M. adhaerens via phenotypic plasticity (i.e., behavioral and morphological changes) against predator ingestion. This study highlights the potentially important role of behavioral escape mechanisms for community composition and energy flow in pelagic environments, especially with globally rising particle loads in aquatic systems through human activities and extreme weather events.

Oligo-heterotrophic Activity of Marinobacter subterrani Creates an Indirect Fe(II) Oxidation Phenotype in Gradient Tubes.

Autotrophic bacteria utilizing Fe(II) as their energy and electron sources for growth affect multiple biogeochemical cycles. Some chemoheterotrophic bacteria have also been considered to exhibit an Fe(II) oxidation phenotype. For example, several Marinobacter strains have been reported to oxidize Fe(II) based on formation of oxidized iron bands in semi-solid gradient tubes that produce opposing concentration gradients of Fe(II) and oxygen. While gradient tubes are a simple and visually compelling method to test for Fe(II) oxidation, this method alone cannot confirm if, and to what extent, Fe(II) oxidation is linked to metabolism in chemoheterotrophic bacteria. Here we probe the possibility of protein-mediated and metabolic by-product-mediated Fe(II) oxidation in Marinobacter subterrani JG233, a chemoheterotroph previously proposed to oxidize Fe(II). Results from conditional and mutant studies, along with measurements of Fe(II) oxidation rates, suggest M. subterrani is unlikely to facilitate Fe(II) oxidation under microaerobic conditions. We conclude that the Fe(II) oxidation phenotype observed in gradient tubes inoculated with M. subterrani JG233 is a result of oligo-heterotrophic activity, shifting the location where oxygen dependent chemical Fe(II) oxidation occurs, rather than a biologically mediated process. IMPORTANCE Gradient tubes are the most commonly used method to isolate and identify neutrophilic Fe(II)-oxidizing bacteria. The formation of oxidized iron bands in gradient tubes provides a compelling assay to ascribe the ability to oxidize Fe(II) to autotrophic bacteria whose growth is dependent on Fe(II) oxidation. However, the physiological significance of Fe(II) oxidation in chemoheterotrophic bacteria is less well understood. Our work suggests that oligo-heterotrophic activity of certain bacteria may create a false-positive phenotype in gradient tubes by altering the location of the abiotic, oxygen-mediated oxidized iron band. Based on the results and analysis presented here, we caution against utilizing gradient tubes as the sole evidence for the capability of a strain to oxidize Fe(II) and that additional experiments are necessary to ascribe this phenotype to new isolates.

Dps-DNA interaction in Marinobacter hydrocarbonoclasticus protein: effect of a single-charge alteration.

DNA-binding proteins from starved cells (Dps) are members of the ferritin family of proteins found in prokaryotes, with hollow rounded cube-like structures, composed of 12 equal subunits. These protein nanocages are bifunctional enzymes that protect the cell from the harmful reaction of iron and peroxide (Fenton reaction), thus preventing DNA damage by oxidative stress. Ferrous ions are oxidized at specific iron-binding sites in the presence of the oxidant and stored in its cavity that can accommodate up to ca. 500 iron atoms. DNA-binding properties of Dps are associated with the N-terminal, positive charge rich, extensions that can promote DNA binding and condensation, apparently by a cooperative binding mechanism. Here, we describe the binding and protection activities of Marinobacter hydrocarbonoclasticus Dps using Electrophoretic Mobility Shift Essays (EMSA), and synchrotron radiation circular dichroism (SRCD) spectroscopy. While no DNA condensation was observed in the tested conditions, it was possible to determine a Dps-DNA complex formation with an apparent dissociation constant of 6.0 ± 1.0 µM and a Hill coefficient of 1.2 ± 0.1. This interaction is suppressed by the inclusion of a single negative charge in the N-terminal region by point mutation. In Dps proteins containing a ferric mineral core (above 96 Fe/protein), DNA binding was impaired. SRCD data clearly showed that no significant modification existed either in secondary structure or protein stability of WT, Q14E variant and core containing proteins. It was, however, interesting to note that, in our experimental conditions, thermal denaturation induced protein aggregation that caused artifacts in thermal denaturation curves, which were dependent on radiation flux and vertical arrangement of the CD cell.

Structural features and stability of apo- and holo-forms of a simple iron-sulfur protein.

Iron-sulfur centers are widespread in living organisms, mostly performing electron transfer functions, either in electron transfer chains or as part of multi-enzymatic complexes, while being also present in enzyme active sites, handling substrate catalysis. Rubredoxin is the simplest iron-sulfur containing protein constituted by a single polypeptide chain of 50 to 60 amino acids, of which four cysteine residues are responsible for metal binding in a tetrahedral coordination sphere. In this manuscript we explore the structure and stability of both apo- and holo-forms of a Rubredoxin from Marinobacter hydrocarbonoclasticus using Synchrotron Radiation Circular Dichroism (SRCD) in combination with other biochemical and spectroscopic techniques. The results are consistent with a holo-protein form containing a monomeric iron center with UV-visible maxima at 760, 578, 494, 386, 356 and 279 nm, an intense EPR resonance with a g value around 4.3 and Mössbauer spectroscopy parameters of δ equal to 0.69 mm/s and ΔEQ equal to 3.25 mm/s, for the ferrous reconstituted state. SRCD data, obtained for the first time for the apo-form, show a quite defined structure with ∆ε maximum at 191 nm and minima at 203 and 231 nm. Most significantly, the presence of isosbestic points at 189 and 228 nm made the interconversion between the two stable apo- and holo-form solution structures clear. SRCD temperature dependence data shows that for both forms the denaturation process proceeds through an intermediate species.

Marinobacter mangrovi sp. nov., isolated from mangrove sediment.

A novel marine bacterium, designated strain CHFG3-1-5T, was isolated from mangrove sediment sampled at Jiulong River estuary, Fujian, PR China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CHFG3-1-5T belonged to the genus Marinobacter, with the highest sequence similarity to Marinobacter segnicrescens SS011B1-4T (97.6%), followed by Marinobacter nanhaiticus D15-8WT (97.5%), Marinobacter bohaiensis T17T (97.1%) and Marinobacter hydrocarbonoclasticus SP.17T (90.6%). The bacterium was Gram-stain-negative, facultative anaerobic, oxidase- and catalase-positive, rod-shaped and motile with a polar flagellum. Strain CHFG3-1-5T grew optimally at 32-37 °C, pH 6.0-8.0 and in the presence of 2.0-3.0% (w/v) NaCl. The G+C content of the chromosomal DNA was 61.1 mol%. The major respiratory quinone was determined to be Q-9. The principal fatty acids were C16 : 0, summed feature 3 (C16 : 1 ω7c/ω6c), C12 : 0, summed feature 9 (C17 : 1 iso ω9c and/or C16 : 0 10-methyl), C12 : 0 3-OH and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, three phospholipids, one glycolipid and two aminolipids. The average nucleotide identity and digital DNA-DNA hybridization values among the genomes of strain CHFG3-1-5T and the reference strains were 73.4-79.4 and 19.6-22.4%, respectively. Like many other species reported in the genus Marinobacter, strain CHFG3-1-5T was able to oxidise iron. The combined genotypic and phenotypic data showed that strain CHFG3-1-5T represents a novel species within the genus Marinobacter, for which the name Marinobacter mangrovi sp. nov. is proposed, with the type strain CHFG3-1-5T (=MCCC 1A18306T=KCTC 82398T).

Bacterial aggregation assisted by anionic surfactant and calcium ions.

We identify factors leading to aggregation of bacteria in the presence of a surfactant using absorbance and microscopy. Two marine bacteria, Marinobacter hydrocarbonoclasticus SP17 and Halomonas titanicae Bead 10BA, formed aggregates of a broad size distribution in synthetic sea water in the presence of an anionic surfactant, dioctyl sodium sulfosuccinate (DOSS). Both DOSS at high concentrations and calcium ions were necessary for aggregate formation, but DOSS micelles were not required for aggregation. Addition of proteinase K but not DNase1 eliminated aggregate formation over two hours. Finally, swimming motility also enhanced aggregate formation.