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Living deep-water coral assemblages inhabiting the Mediterranean Sea, off the Cape of Santa Maria di Leuca (Apulian platform, Ionian Sea), namely Lophelia pertusa ( L. pertusa) are studied. It is found that living specimens of L. pertusa possess a specific microbial community different from that of dead corals and sediment samples.

[Display omitted] According to current estimates, the annual volume of crude oil entering the ocean due to both anthropogenic activities and naturally occurring seepages reaches approximately 8.3 million metric tons. Huge discharges from accidents have caused large-scale environmental disasters with extensive damage to the marine ecosystem. The natural clean-up of petroleum spills in marine environments is carried out primarily by naturally occurring obligate hydrocarbonoclastic bacteria (OHCB). The natural hosts of OHCB include a range of marine primary producers, unicellular photosynthetic eukaryotes and cyanobacteria, which have been documented as both, suppliers of hydrocarbon-like compounds that fuel the 'cryptic' hydrocarbon cycle and as a source of isolation of new OHCB. A very new body of evidence suggests that OHCB are not only the active early stage colonizers of plastics and hence the important component of the ocean's 'plastisphere' but also encode an array of enzymes experimentally proven to act on petrochemical and bio-based polymers. [ABSTRACT FROM AUTHOR]

A microbiological survey of the Priolo Bay (eastern coast of Sicily, Ionian Sea), a chronically polluted marine coastal area, was carried out in order to discern its intrinsic bioremediation potential. Microbiological analysis, 16S rDNA-based DGGE fingerprinting and PLFAs analysis were performed on seawater and sediment samples from six stations on two transects. Higher diversity and variability among stations was detected by DGGE in sediment than in water samples although seawater revealed higher diversity of culturable hydrocarbon-degrading bacteria. The most polluted sediment hosted higher total bacterial diversity and higher abundance and diversity of culturable HC degraders. Alkane- and PAH-degrading bacteria were isolated from all stations and assigned to Alcanivorax , Marinobacter , Thalassospira , Alteromonas and Oleibacter (first isolation from the Mediterranean area). High total microbial diversity associated to a large selection of HC degraders is believed to contribute to natural attenuation of the area, provided that new contaminant contributions are avoided. [ABSTRACT FROM AUTHOR]

The real-time polymerase chain reaction (RT-PCR) was used to follow changes in the proportion of hydrocarbonoclastic bacteria in the marine microbial community in oil polluted mesocosms during bioremediation field trial. Assay for alk-B1 of Alcanivorax borkumensis and alk-BT of Thalassolituus oleivorans were validated and found to be both sensitive and reproducible. Quantification of alk-B1 from A. borkumensis SK2 in mesocosms show that in single bioaugmentation mesocosm (M1) this gene has high quantity in fifth day of sampling but in biostimulating mesocosm (M2) and consortium bioaugmentation mesocosm (M3) the high quantity of this gene was in tenth day of sampling. The comparison between expression of alk-BT and alk-B1 in M3 mesocosm show that alk-B1 copy number was more than alk-BT. The proportion of alk-B1 or alk-BT containing bacteria was positively correlated to the concentration of crude oil in the mesocosms. After the concentration of crude oil in the mesocosms decreased the gene copy number of alkane monooxygenase genes also decreased. [ABSTRACT FROM AUTHOR]

Abstract: Microcosms experiments were carried out to evaluate the effect of bioemulsificant exopolysaccharide (EPS2003) on microbial community dynamics. An experimental seawater microcosm, supplemented with crude oil and EPS2003 (SW+OIL+EPS2003), was monitored for 15days and compared to control microcosm (only oil-polluted seawater, SW+OIL). Determination of bacterial abundance, heterotrophic cultivable and hydrocarbon-degrading bacteria were carried out during all experimentation period. The microbial community dynamic was monitored by isolation of total RNA, RT-PCR amplification of 16S rRNA, cloning and sequencing. Oil degradation was monitored by GC–MS analysis. Bioemulsificant addition stimulated an increase of the total bacterial abundance, change in the community structure and activity. The bioemulsificant also increased of 5 times the oil biodegradation rate. The data obtained from microcosm experiment indicated that EPS2003 could be used for the dispersion of oil slicks and could stimulate the selection of marine hydrocarbon degraders thus increasing bioremediation process. [Copyright &y& Elsevier]

Abstract: The autotrophic and ammonia-oxidizing crenarchaeal assemblage at offshore site located in the deep Mediterranean (Tyrrhenian Sea, depth 3000m) water was studied by PCR amplification of the key functional genes involved in energy (ammonia mono-oxygenase alpha subunit, amoA) and central metabolism (acetyl-CoA carboxylase alpha subunit, accA). Using two recently annotated genomes of marine crenarchaeons, an initial set of primers targeting archaeal accA-like genes was designed. Approximately 300 clones were analyzed, of which 100% of amoA library and almost 70% of accA library were unambiguously related to the corresponding genes from marine Crenarchaeota. Even though the acetyl-CoA carboxylase is phylogenetically not well conserved and the remaining clones were affiliated to various bacterial acetyl-CoA/propionyl-CoA carboxylase genes, the pool of archaeal sequences was applied for development of quantitative PCR analysis of accA-like distribution using TaqMan® methodolgy. The archaeal accA gene fragments, together with alignable gene fragments from the Sargasso Sea and North Pacific Subtropical Gyre (ALOHA Station) metagenome databases, were analyzed by multiple sequence alignment. Two accA-like sequences, found in ALOHA Station at the depth of 4000m, formed a deeply branched clade with 64% of all archaeal Tyrrhenian clones. No close relatives for residual 36% of clones, except of those recovered from Eastern Mediterranean, was found, suggesting the existence of a specific lineage of the crenarchaeal accA genes in deep Mediterranean water. Alignment of Mediterranean amoA sequences defined four cosmopolitan phylotypes of Crenarchaeota putative ammonia mono-oxygenase subunit A gene occurring in the water sample from the 3000m depth. Without exception all phylotypes fell into Deep Marine Group I cluster that contain the vast majority of known sequences recovered from global deep-sea environment. Remarkably, three phylotypes accounted for 91% of all Mediterranean amoA clones and corresponded to the sequences retrieved from the less deep compartments of the world''s ocean, most likely reflecting the higher temperature at the depth of the Mediterranean Sea. In order to verify whether these phylotypes might represent important Crenarchaeota in the functioning of the Mediterranean bathypelagic ecosystem, expression of crenarchaeal amoA gene was monitored by direct RNA retrieval and following analysis of amoA-related mRNA transcripts. Surprisingly, all mRNA-derived sequences formed a tight monophyletic group, which fell into large Shallow Marine Group I cluster with sequences retrieved from shallow (up to 200m) waters, sediments and corals. This group was not detected in DNA-based clone library, obviously, due to an overwhelming dominance of the Deep Marine Group I. The failure to recover the amoA transcripts, related to Deep Marine Group I of Crenarchaeota, was unanticipated and likely resulted from the physiology of these strongly adapted deep-sea organisms. As far as all seawater samples were treated on-board under atmospheric pressure conditions and sunlight, the decompression and/or photoinhibition likely affected their metabolic activity, followed by the strong decay of gene expression. [Copyright &y& Elsevier]

Nine pure cultures of neutrophilic haloaloarchaea capable of anaerobic growth by carbohydrate-dependent sulfur respiration were isolated from hypersaline lakes in southwestern Siberia and southern Russia. According to phylogenomic analysis the isolates were closely related to each other and formed a new species within the genus Halapricum (family Haloarculaceae). They have three types of catabolism: fermentative, resulting in H 2 formation; anaerobic respiration using sulfur compounds as e -acceptors and aerobic respiration. Apart from elemental sulfur, all isolates can also use three different sulfoxides as acceptors and the type strain also grows with thiosulfate, reducing it partially to sulfide and sulfite. All strains utilized sugars and glycerol as the e -donors and C source for anaerobic growth and some can also grow with alpha-glucans, such as starch and dextrins. The major respiratory menaquinones are MK-8:8 and MK-8:7, but 5–19% consists of "thermoplasmata" quinones (MMK-8:8 and MMK-8:7), whose occurrence in haloarchaea is unprecedented. On the basis of their unique physiological properties and results of phylogenomic analysis, the isolates are suggested to be classified into a novel species Halapricum desulfuricans sp. nov. (type strain HSR12-2T = JCM 34032T = UNIQEM U1001T). [ABSTRACT FROM AUTHOR]

Over the past few years, a new and ecophysiologically unusual group of marine hydrocarbon-degrading bacteria – the obligate hydrocarbonoclastic bacteria (OHCB) – has been recognized and shown to play a significant role in the biological removal of petroleum hydrocarbons from polluted marine waters. The introduction of oil or oil constituents into seawater leads to successive blooms of a relatively limited number of indigenous marine bacterial genera —Alcanivorax, Marinobacter, Thallassolituus, Cycloclasticus, Oleispira and a few others (the OHCB) — which are present at low or undetectable levels before the polluting event. The types of OHCB that bloom depend on the latitude/temperature, salinity, redox and other prevailing physical-chemical factors. These blooms result in the rapid degradation of many oil constituents, a process that can be accelerated further by supplementation with limiting nutrients. Genome sequencing and functional genomic analysis of Alcanivorax borkumensis, the paradigm of OHCB, has provided significant insights into the genomic basis of the efficiency and versatility of its hydrocarbon utilization, the metabolic routes underlying its special hydrocarbon diet, and its ecological success. These and other studies have revealed the potential of OHCB for multiple biotechnological applications that include not only oil pollution mitigation, but also biopolymer production and biocatalysis. [Copyright &y& Elsevier]

Predation by grazing protists in aquatic habitats can influence prokaryotic community structure and provides a source of new, labile organic matter. Due to methodological difficulties associated with studies of deep-sea (below photic zone) microbiota, trophic interactions between eukaryotes and prokaryotes in mesopelagic and bathypelagic realms are largely obscured. Further complicating matters, examinations of trophic interactions using water samples that have been exposed to upwards of hundreds of atmospheres of pressure change prior to initiating experiments can potentially introduce significant artifacts. Here we present results of the first study of protistan grazing in water layers ranging from the euphotic zone to the bathypelagic, utilizing the Microbial Sampler-Submersible Incubation Device (MS-SID) that makes possible in situ studies of microbial activities. Protistan grazing in the mesopelagic and bathypelagic realm of the East Mediterranean Sea was quantified using fluorescently labeled prokaryotes (FLP) prepared from the naturally-occurring prokaryotic assemblages. These studies reveal daily prokaryotic removal due to grazing ranging from 31.3±5.9% at 40 m depth to 0.5±0.3% at 950 m. At 3540 m depth, where a chemocline habitat exists with abundant and active prokaryotes above Urania basin, the daily consumption of prokaryotes by protists was 19.9±6.6% of the in situ abundance. [ABSTRACT FROM AUTHOR]

A collection of forty-two (42) strains was obtained during microbiological screening of a Membrane Bioreactor (MBR) system developed for the treatment of saline oily waste originated from marine transportation. The diversity of the bacterial collection was analyzed by amplification and sequencing of the 16S rRNA gene. Taxonomic analysis showed high level of identity with recognized sequences of seven (7) distinct bacterial genera (Alcanivorax , Erythrobacter , Marinobacter , Microbacterium , Muricauda , Rhodococcus and Rheinheimera ). The biotechnological potential of the isolates was screened considering an important factor such as the biosurfactant production. In particular fourteen (14) biosurfactant producing bacteria were selected and further tested, for growth on crude oil and hydrocarbon degradation. Data obtained from this study confirmed the high activity of bacteria related to genera Alcanivorax (isolates MBR-B11 and MBR-G10), Rheinheimera (isolates MBR-H02 and MBR-H05), Rhodococcus (isolates MBR-F04 and MBR-G05) and Muricauda (isolate MBR-G04) and underline the possible application of these bacteria in remediation of saline oily waste water. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to assess medium-term toxicity of weathered oil on European seabass. A mesocosm system reproducing an oil spill at sea was applied. Fish were collected after 48 h, 7, 30 and 60 days. Cyp1a gene transcription, EROD and UDPGT activities, bile PAHs metabolites and micronuclei frequency were investigated. A progressive disappearance of low molecular weight n-alkanes and PAHs in the water of the mesocosm occurred during the experimentation. Fishes exposed to oil displayed a significant increase of cyp1a expression and EROD activity during the entire experiment as well as higher concentrations of PAHs metabolites in bile. Micronulei frequency resulted significantly higher during all experiment in oil exposed sea bass compared to controls. The results highlight the environmental risk associated with the release of oil products at sea and confirm the adopted parameters as useful tools for studying the impact of accidental oil spills on fish. [Copyright &y& Elsevier]

Abstract: Two samples of the same bathypelagic bacterioplankton community (Tyrrhenian Sea at the depth of 3000m) were collected during single cast by both traditional sampling with Niskin bottle and using a high pressure-maintaining HPSS sampler. Total RNA was isolated, reverse transcribed, amplified with Archaea-specific primers and cloned. Riboclones were sequenced, 117 riboclones from decompressed library and 104 rRNA-based riboclones retrieved from HPSS sampler. Both RNA libraries were additionally compared with 115 riboclones based on DNA obtained from the decompressed sample (16S rRNA genes). In terms of repetitive riboclones, obtained in all three libraries, the bathypelagic community was dominated by Crenarchaeota Marine Group I (52–64%). The combined analysis led to a characterization of sampling-specific phylotypes otherwise uncharacterized if only one type of library had been analyzed alone. Of the DNA riboclones, 22% were found only in this library and no close relatives of Euryarchaeota Marine Group II were detected in both RNA-based libraries, suggesting the dormant state of these organisms in deep-sea environment. For clones from the RNA libraries, one Crenarchaeota MG I phylotype did not indicate close relatives in the DNA library. In general, among 10 archaeal phylotypes recovered in total, 8, 7 and 6 of them were faced in DNA, RNA_HPSS and RNA decompressed libraries, respectively. Based on comparisons between all three libraries, our data indicate that (i) the short-term decompression of seawater samples during cast recovery and following treatments caused only slight changes in the total rRNA diversity and (ii) rRNA-based analysis is likely affected the characterization of phylotypes, present in deep-sea environment as the dormant cells, detected only on the DNA level. [Copyright &y& Elsevier]

Abstract: The present study is focused on the development of a cultivation-independent molecular approach for specific detection of bioluminescent bacteria within microbial communities by direct amplification of luxA gene from environmental DNA. A new set of primers, specifically targeting free-living bioluminescent bacteria, was designed on the base of luxA sequences available from the public database. Meso- and bathypelagic seawater samples were collected from two stations in Tyrrhenian Sea at the depths of 500 and 2750m. The same seawater samples also were used to isolate bioluminescent bacteria that were further subjected to luxA and 16S rRNA gene sequencing. PCR products obtained by amplification with designed primers were cloned, and the phylogenetic affiliation of 40 clones was determined. All of them were clustered into three groups, only distantly related to the Photobacterium phosphoreum and Photobacterium kishitanii clades. The half of all clones formed a tight monophyletic clade, while the rest of clones were organized in “compartment”-specific, meso- and bathypelagic ecotypes. No matches with luxA gene sequences of four bioluminescent strains, isolated from the same seawater samples, were observed. These findings indicate that the PCR-based approach developed in present manuscript, allowed us to detect the novel, “yet to be cultivated” lineages of bioluminescent bacteria, which are likely specific for distinct warm bathypelagic realms of Mediterranean Sea. [Copyright &y& Elsevier]

Summary: Mesocosm experiments were performed to study the changes on bacterial community composition following oil spill in marine environment. The analysis of 16S crDNA revealed a shift in the structure of initial bacterial population that was drastically different from that one measured after 15 days. The results showed that, after 15 days, bacteria closely related to the genus Alcanivorax became the dominant group of bacterial community in petroleum-contaminated sea water nitrogen and phosphorus amended. This suggested that these bacteria played the most important role in the process of bioremediation of oil-contaminated marine environments. [Copyright &y& Elsevier]

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coil revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) ⪢ phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 °C, pH 4.5, yielded values of 26, 0.43, and 0.45 μ and 18, 660, and 1175 s-1, respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases. [ABSTRACT FROM AUTHOR]