Production of polyhydroxyalkanoates by three novel species of Marinobacterium.

Several species of novel marine bacteria from the genus Marinobacterium, including M. nitratireducens, M. sediminicola, and M. zhoushanense were found to be capable of producing polyhydroxyalkanoates (PHA) using sugars and volatile fatty acids (VFAs) as the carbon source. M. zhoushanense produced poly-3-hydroxybutytate (PHB) from sucrose, achieving a product titer and PHB content of 2.89 g/L and 64.05 wt%, respectively. By contrast, M. nitratireducens accumulated 3.38 g/L PHB and 66.80 wt% polymer content using butyrate as the substrate. A third species, M. sediminicola showed favorable tolerance to propionate, butyrate, and valerate. The use of 10 g/L valerate yielded 3.37 g/L poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), with a 3-hydroxyvalerate (3 HV) monomer content of 94.75 mol%. Moreover, M. sediminicola could be manipulated to produce PHBV with changeable polymer compositions by feeding different mixtures of VFAs. Our results indicate that M. sediminicola is a promising halophilic bacterium for the production of PHA.

Protein expression in the obligate hydrocarbon-degrading psychrophile Oleispira antarctica RB-8 during alkane degradation and cold tolerance.

In cold marine environments, the obligate hydrocarbon-degrading psychrophile Oleispira antarctica RB-8, which utilizes aliphatic alkanes almost exclusively as substrates, dominates microbial communities following oil spills. In this study, LC-MS/MS shotgun proteomics was used to identify changes in the proteome induced during growth on n-alkanes and in cold temperatures. Specifically, proteins with significantly higher relative abundance during growth on tetradecane (n-C14 ) at 16°C and 4°C have been quantified. During growth on n-C14 , O. antarctica expressed a complete pathway for the terminal oxidation of n-alkanes including two alkane monooxygenases, two alcohol dehydrogenases, two aldehyde dehydrogenases, a fatty-acid-CoA ligase, a fatty acid desaturase and associated oxidoreductases. Increased biosynthesis of these proteins ranged from 3- to 21-fold compared with growth on a non-hydrocarbon control. This study also highlights mechanisms O. antarctica may utilize to provide it with ecological competitiveness at low temperatures. This was evidenced by an increase in spectral counts for proteins involved in flagella structure/output to overcome higher viscosity, flagella rotation to accumulate cells and proline metabolism to counteract oxidative stress, during growth at 4°C compared with 16°C. Such species-specific understanding of the physiology during hydrocarbon degradation can be important for parameterizing models that predict the fate of marine oil spills.

Analysis of allosteric communication in a multienzyme complex by ancestral sequence reconstruction.

Tryptophan synthase (TS) is a heterotetrameric αßßα complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the ß-subunit TrpB via a complex allosteric network. We have analyzed this allosteric network by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins. Previously, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) have been computed by means of ASR and characterized. LBCA-TS is similar to modern TS by forming a αßßα complex with indole channeling taking place. However, LBCA-TrpA allosterically decreases the activity of LBCA-TrpB, whereas, for example, the modern ncTrpA from Neptuniibacter caesariensis allosterically increases the activity of ncTrpB. To identify amino acid residues that are responsible for this inversion of the allosteric effect, all 6 evolutionary TrpA and TrpB intermediates that stepwise link LBCA-TS with ncTS were characterized. Remarkably, the switching from TrpB inhibition to TrpB activation by TrpA occurred between 2 successive TS intermediates. Sequence comparison of these 2 intermediates and iterative rounds of site-directed mutagenesis allowed us to identify 4 of 413 residues from TrpB that are crucial for its allosteric activation by TrpA. The effect of our mutational studies was rationalized by a community analysis based on molecular dynamics simulations. Our findings demonstrate that ancestral sequence reconstruction can efficiently identify residues contributing to allosteric signal propagation in multienzyme complexes.

Metagenomic and metatranscriptomic responses of natural oil degrading bacteria in the presence of dispersants.

Oil biodegradation has been extensively studied in the wake of the deepwater horizon spill, but the application of dispersant to oil spills in marine environments remains controversial. Here, we report metagenomic (MG) and metatranscriptomic (MT) data mining from microcosm experiments investigating the oil degrading potential of Canadian west and east coasts to estimate the gene abundance and activity of oil degrading bacteria in the presence of dispersant. We found that the addition of dispersant to crude oil mainly favours the abundance of Thalassolituus in the summer and Oleispira in the winter, two key natural oil degrading bacteria. We found a high abundance of genes related not only to n-alkane and aromatics degradation but also associated with transporters, two-component systems, bacterial motility, secretion systems and bacterial chemotaxis.

Production of indoleacetic acid by strains of the epiphytic bacteria Neptunomonas spp. isolated from the red alga Pyropia yezoensis and the seagrass Zostera marina.

Neptunomonas sp. BPy-1 is an epiphytic bacterium isolated from in vitro culture of the red alga Pyropia yezoensis. It uses ethanol as a sole carbon source and promotes the growth of host alga. A related bacterium, Neptunomonas sp. BZm-1, was isolated from leaves of Zostera marina found in the Yatsushiro Sea (Japan). BZm-1 showed 99% 16S rRNA sequence identity with Neptunomonas sp. BPy-1. Similar to BPy-1, BZm-1 grew in artificial seawater (ASW) medium containing ethanol or butanol. When thalli were treated with a multi-enzyme cleaner, the growth of treated thalli was retarded, but the addition of BZm-1 to the medium promoted growth. To explore the benefits of epiphytic bacteria, indoleacetic acid (IAA) production by isolated bacteria was examined under conditions of limited nutrients. Salkowski assays and GC-MS analysis revealed that both BZm-1 and BPy-1 excreted IAA during growth in ASW medium containing glucose or ethanol in the presence of tryptophan. In ASW medium containing tryptophan but lacking a carbon source, neither isolate grow, but produced IAA. ASW medium includes nitrate as the sole nitrogen source. In the absence of carbon source, different nitrogen forms in the presence of tryptophan did not affect IAA production by the two isolates. These findings indicate that IAA production by the two isolates is strictly dependent on tryptophan but less affected by carbon and nitrogen sources. Based on the different origins of BPy-1 and BZm-1, this mode of IAA production seems to be conserved among relatives of BPy-1.

Complete characterization of new isolates of Neptunomonas phycophila leads to emend its description and opens possibilities of biotechnological applications.

Five strains were isolated from gonad of Great scallop (Pecten maximus) broodstock in a Norwegian hatchery. The study of 16S rRNA gene sequences showed that these isolates belong to Neptunomonas phycophila, a bacterium originally isolated from a symbiont of the anemone Aiptasia tagetes from Puerto Rico. The gyrB and rpoB genes sequences confirmed the affiliation of the scallop isolates to this species. Phenotypic characterization was performed and some differences between the Norwegian isolates and the type strain of N. phycophila were detected, such as ranges of temperature, pH, and tolerance to salinity or the use of several substrates as sole carbon source which lead to an emended description of the species. The strain 3CM2.5 showed phosphatidylethanolamine and phosphatidylglycerol as the major polar lipids. The whole genomes of the scallop strain 3CM2.5 and type strain of the species CECT 8716T were obtained and the annotation of these genomes revealed the presence of genes involved in degradation of aromatic compounds in both strains. Results obtained not only widen the geographical and host ranges of N. phycophila, but also point out possible biotechnological applications for this bacterial species.

Comparative metabolomic studies of Alkanivorax xenomutans showing differential power output in a three chambered microbial fuel cell.

Metabolomic study of electrogenic bacteria is a necessity to understand the extent of complex organic matter degradation and to invent new co-culture techniques to achieve complete degradation. In this study, we have subjected Alkanivorax xenomutans (KCTC 23751T; NBRC 108843T), a bacterium capable for biodegradation of complex hydrocarbons, to oxic and anoxic conditions in a three chambered microbial fuel cell. In an attempt to understand the molecular mechanisms during the electrogenic processes of A. xenomutans, intra cellular (endo metabolome or the fingerprint) and exo metabolome (extracellular metabolome or the foot print) were analyzed under oxic and anoxic conditions, using FTIR and GC-MS. Interpretation of the data revealed higher number of metabolites in the anoxic fraction as compared to oxic fraction. In addition, expression of putative metabolites that influence electron transfer like flavins, fumarate and quinones were found to be predominant in the organisms when grown in anoxic conditions. Hence, the presence of anoxic conditions governed the electrogenic bacteria to produce enhanced power output by modulating differential metabolomic profiling, compared to the culture grown in oxic conditions.

Comparative proteomics reveals signature metabolisms of exponentially growing and stationary phase marine bacteria.

Much of the phenotype of a microorganism consists of its repertoire of metabolisms and how and when its proteins are deployed under different growth conditions. Hence, analyses of protein expression could provide important understanding of how bacteria adapt to different environmental settings. To characterize the flexibility of proteomes of marine bacteria, we investigated protein profiles of three important marine bacterial lineages - Oceanospirillaceae (Neptuniibacter caesariensis strain MED92), Roseobacter (Phaeobacter sp. MED193) and Flavobacteria (Dokdonia sp. MED134) - during transition from exponential to stationary phase. As much as 59-80% of each species' total proteome was expressed. Moreover, all three bacteria profoundly altered their expressed proteomes during growth phase transition, from a dominance of proteins involved in translation to more diverse proteomes, with a striking appearance of enzymes involved in different nutrient-scavenging metabolisms. Whereas the three bacteria shared several overarching metabolic strategies, they differed in important details, including distinct expression patterns of membrane transporters and proteins in carbon and phosphorous metabolism and storage compounds. These differences can be seen as signature metabolisms - metabolisms specific for lineages. These findings suggest that quantitative proteomics can inform about the divergent ecological strategies of marine bacteria in adapting to changes in environmental conditions.

Bacterioplanes sanyensis gen. nov., sp. nov., a PHB-accumulating bacterium isolated from a pool of Spirulina platensis cultivation.

A Gram-negative, poly-3-hydroxybutyrate-accumulating rod bacterium, strain GYP-2(T), was isolated from a pool of marine Spirulina platensis cultivation, Sanya, China. Growth was observed at 10-45 °C and pH 6-10 in the presence of 1-10 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the new isolate belonged to Gammaproteobacteria and displayed 93.8-95.3 % 16S rRNA gene sequences similarities to members of the genera Thalassolituus, Oleibacter, and Oceanobacter, but house-keeping gene gyrB (encode DNA gyrase beta subunit) demonstrated that the new isolate was distantly related to Thalassolituus, Oleibacter, and Oceanobacter species (only 77-83 % gene gyrB sequences similarities).The G+C content of genomic DNA was 55 mol%. The major respiratory quinone was Q-9, while that for Oceanobacter kriegii LMG 6238(T) was Q-8. Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. On the basis of its physiological, chemotaxonomic, and molecular properties, strain GYP-2(T) is suggested to represent a novel species of a new genus in Gammaproteobacteria, for which the name Bacterioplanes sanyensis gen. nov., sp. nov. is proposed. The type strain is GYP-2(T) (=CGMCC 1.12392(T)=KCTC 32220(T)).

Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: a mesocosm simulation study.

Bioaugmentation (amendment with selected bacterial strains) and/or biostimulation (nutrients addition and/or air supply) are relatively new fields in environmental microbiology for preventing pollution and cleanup contamination. In this study, the efficiency of application of bioaugmentation/biostimulation treatments, for recovery of crude oil-polluted seawater, was evaluated. Three different series of experiments were performed in a "Mesocosm Facility" (10.000 L). Natural seawater was artificially polluted with crude oil (1000 ppm) and was amended with inorganic nutrients (Mesocosm 1, M1), inorganic nutrient and an inoculum of Alcanivorax borkumensis SK2(T) (Mesocosm 2, M2) and inorganic nutrient and an inoculum of A. borkumensis SK2(T) and Thalassolituus oleivorans MIL-1(T) (Mesocosm 3, M3), respectively. During the experimental period (20 days) bacterial abundance (DAPI count), culturable heterotrophic bacteria (CFU count), MPN, microbial metabolic activity [Biochemical Oxygen Demand and enzymatic activity (leucine aminopeptidase LAP, ß-glucosidase BG, alkaline phosphatase AP)] and quali-, quantitative analysis of the composition of total extracted and resolved hydrocarbons and their derivates (TERHCs) were carried out. The microbiological and physiological analysis of marine microbial community found during the three different biostimulation and bioaugmentation assays performed in mesocosms show that the load of crude oil increases total microbial abundance, inhibits the activity of some enzymes such as LAP while stimulates both AP and BG activities. The biodegradation results show that bioaugmentation with A. borkumensis SK2(T) alone is able to produce the highest percentage of degradation (95%) in comparison with the biostimulation treatment (80%) and bioaugmentation using an Alcanivorax-Thalassolituus bacterial consortium (70%). This result highlights the reduced biodegradation capability of the consortium used in this study, suggesting an unfavourable interaction between the two bacterial genera.

Identification of acetate-oxidizing bacteria in a coastal marine surface sediment by RNA-stable isotope probing in anoxic slurries and intact cores.

We investigated the terminal electron-accepting pathways and the acetate-oxidizing bacteria in surface sediment (0-5 mm depth) of Aarhus Bay, Denmark, in anoxic slurry and intact core incubations. In the intact cores, oxygen, nitrate, oxides of manganese and iron, and sulfate were all available and likely all used as electron acceptors by the microbial community, whereas microbial iron and sulfate reduction dominated in the slurries. The availability of electron acceptors clearly affected which organisms were labeled by 16S rRNA-stable isotope probing (SIP). Members of the Oceanospirillaceae were identified as (13) C-acetate oxidizers in both types of incubations, but bacteria related to Colwellia and Arcobacter oxidized acetate in the intact core, while members of the Desulfuromonadales and Acidithiobacillaceae did so in the slurry incubation. Desulfuromonadales sequences also dominated 16S rRNA gene clone libraries from the highest positive dilution of the acetate-oxidizing most probable number cultures with manganese and iron oxides. Thus, members of Desulfuromonadales are likely important for acetate oxidation coupled to iron and manganese reduction in situ, while the identified Gammaproteobacteria and affiliates of Arcobacter may utilize oxygen, nitrate and manganese oxides. Our study further highlights some of the biases that are associated with the use of RNA-SIP as well as slurry and intact core incubations.

Thalassolituus marinus sp. nov., a hydrocarbon-utilizing marine bacterium.

Gram-negative strains, motile by a single polar flagellum, non-pigmented and with a curved rod-shaped morphology, designated IMCC1826(T) and IMCC1883, were isolated from a surface seawater sample from the Yellow Sea. The two strains shared 99.9% 16S rRNA gene sequence similarity and showed 92% DNA-DNA relatedness, suggesting that they belonged to the same genomic species. Phylogenetic analysis based on 16S rRNA gene sequences showed that the two isolates were related most closely to the type strain of Thalassolituus oleivorans with a sequence similarity of 96.4% and formed a robust phyletic lineage with T. oleivorans. DNA-DNA relatedness between the two strains and T. oleivorans DSM 14913(T) was 8.7-11.6%. A putative alkane hydroxylase (alkB) gene was detected in strain IMCC1826(T) by PCR, but the amino acid sequence of the gene was distantly related to that of the AlkB homologue of T. oleivorans DSM 14913(T). As expected from the presence of the alkB gene, the new strains utilized n-tetradecane and n-hexadecane as a carbon source. The DNA G+C content was 54.6-56.0 mol% and the main isoprenoid quinone detected was Q-9. Polar lipids of strain IMCC1826(T) included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and amino-group-containing lipids. On the basis of taxonomic data obtained in this study, strains IMCC1826(T) and IMCC1883 represent a novel species of the genus Thalassolituus, for which the name Thalassolituus marinus sp. nov. is proposed, with IMCC1826(T) (=KCTC 23084(T)=NBRC 107590(T)) as the type strain.

Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to Deepwater Horizon oil spill.

The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

Three manganese oxide-rich marine sediments harbor similar communities of acetate-oxidizing manganese-reducing bacteria.

Dissimilatory manganese reduction dominates anaerobic carbon oxidation in marine sediments with high manganese oxide concentrations, but the microorganisms responsible for this process are largely unknown. In this study, the acetate-utilizing manganese-reducing microbiota in geographically well-separated, manganese oxide-rich sediments from Gullmar Fjord (Sweden), Skagerrak (Norway) and Ulleung Basin (Korea) were analyzed by 16S rRNA-stable isotope probing (SIP). Manganese reduction was the prevailing terminal electron-accepting process in anoxic incubations of surface sediments, and even the addition of acetate stimulated neither iron nor sulfate reduction. The three geographically distinct sediments harbored surprisingly similar communities of acetate-utilizing manganese-reducing bacteria: 16S rRNA of members of the genera Colwellia and Arcobacter and of novel genera within the Oceanospirillaceae and Alteromonadales were detected in heavy RNA-SIP fractions from these three sediments. Most probable number (MPN) analysis yielded up to 10(6) acetate-utilizing manganese-reducing cells cm(-3) in Gullmar Fjord sediment. A 16S rRNA gene clone library that was established from the highest MPN dilutions was dominated by sequences of Colwellia and Arcobacter species and members of the Oceanospirillaceae, supporting the obtained RNA-SIP results. In conclusion, these findings strongly suggest that (i) acetate-dependent manganese reduction in manganese oxide-rich sediments is catalyzed by members of taxa (Arcobacter, Colwellia and Oceanospirillaceae) previously not known to possess this physiological function, (ii) similar acetate-utilizing manganese reducers thrive in geographically distinct regions and (iii) the identified manganese reducers differ greatly from the extensively explored iron reducers in marine sediments.

Kinetic consequences of replacing the internucleotide phosphorus atoms in DNA with arsenic.

It was claimed in a recent publication that a strain of Halomonadacea bacteria (GFAJ-1) isolated from the arsenic-rich waters of Mono Lake, California is able to substitute arsenic for phosphorus in its macromolecules and small molecule metabolites. In this short Perspective, we consider chemical and biochemical issues surrounding the central claim that Halomonadacea GFAJ-1 is able to survive while incorporating kinetically labile arsenodiester linkages into the backbone of its DNA. Chemical precedents suggest that arsenodiester linkages in the putative arsenic-containing DNA of GFAJ-1 would undergo very rapid hydrolytic cleavage in water at 25 °C with an estimated half-life of 0.06 s. In contrast, the phosphodiester linkages of native DNA undergo spontaneous hydrolysis with a half-life of approximately 30,000,000 y at 25 °C. Overcoming such dramatic kinetic instability in its genetic material would present serious challenges to Halomonadacea GFAJ-1.

Deep-sea oil plume enriches indigenous oil-degrading bacteria.

The biological effects and expected fate of the vast amount of oil in the Gulf of Mexico from the Deepwater Horizon blowout are unknown owing to the depth and magnitude of this event. Here, we report that the dispersed hydrocarbon plume stimulated deep-sea indigenous γ-Proteobacteria that are closely related to known petroleum degraders. Hydrocarbon-degrading genes coincided with the concentration of various oil contaminants. Changes in hydrocarbon composition with distance from the source and incubation experiments with environmental isolates demonstrated faster-than-expected hydrocarbon biodegradation rates at 5°C. Based on these results, the potential exists for intrinsic bioremediation of the oil plume in the deep-water column without substantial oxygen drawdown.

Cloning and nucleotide sequences of carbazole degradation genes from marine bacterium Neptuniibacter sp. strain CAR-SF.

The marine bacterium Neptuniibacter sp. strain CAR-SF utilizes carbazole as its sole carbon and nitrogen sources. Two sets of clustered genes related to carbazole degradation, the upper and lower pathways, were obtained. The marine bacterium genes responsible for the upper carbazole degradation pathway, carAa, carBa, carBb, and carC, encode the terminal oxygenase component of carbazole 1,9a-dioxygenase, the small and large subunits of the meta-cleavage enzyme, and the meta-cleavage compound hydrolase, respectively. The genes involved in the lower degradation pathway encode the anthranilate dioxygenase large and small subunit AntA and AntB, anthranilate dioxygenase reductase AntC, 4-oxalocrotonate tautomerase, and catechol 2,3-dioxygenase. Reverse transcription-polymerase chain reaction confirmed the involvement of the isolated genes in carbazole degradation. Escherichia coli cells transformed with the CarAa of strain CAR-SF required ferredoxin and ferredoxin reductase for biotransformation of carbazole. Although carAc, which encodes the ferredoxin component of carbazole 1,9a-dioxygenase, was not found immediately downstream of carAaBaBbC, the carAc-like gene may be located elsewhere based on Southern hybridization. This is the first report of genes involved in carbazole degradation isolated from a marine bacterium.

Neptunomonas antarctica sp. nov., isolated from marine sediment.

A Gram-negative, motile, oxidase- and catalase-positive and facultatively aerobic bacterium, designated S3-22T, was isolated from marine sediment of the Nella Fjord, Antarctica. Strain S3-22T reduced nitrate to nitrite and grew at pH 6.0-8.0, at 4-25 degrees C and with 0.5-5% (w/v) NaCl. It contained Q-8 as the only respiratory quinone and summed feature 3 (C16:1omega7c and/or iso-C15:0 2-OH), C16:0 and C18:1omega7c as the major cellular fatty acids. The genomic DNA G+C content was 45.6 mol%. Phylogenetic analyses of 16S rRNA gene sequences showed that strain S3-22T was affiliated with the genus Neptunomonas, with 97.1% sequence similarity to Neptunomonas japonica JAMM 0745T and 94.8% to Neptunomonas naphthovorans NAG-2N-126T, the type strains of the only two recognized Neptunomonas species. DNA-DNA relatedness between strain S3-22T and N. japonica JCM 14595T was 20.4%. Strain S3-22T could be distinguished from the type strains of Neptunomonas species by several phenotypic properties. Based on the evidence from our polyphasic study, strain S3-22T represents a novel Neptunomonas species, for which the name Neptunomonas antarctica sp. nov. is proposed. The type strain is S3-22T (=CCTCC AB 209086T =KACC 14056T).