Isolation of alkane‐degrading bacteria from deep‐sea Mediterranean sediments

Aims: To isolate and identify alkane‐degrading bacteria from deep‐sea superficial sediments sampled at a north‐western Mediterranean station. Methods and Results: Sediments from the water/sediment interface at a 2400 m depth were sampled with a multicorer at the ANTARES site off the French Mediterranean coast and were promptly enriched with Maya crude oil as the sole source of carbon and energy. Alkane‐degrading bacteria belonging to the genera Alcanivorax, Pseudomonas, Marinobacter, Rhodococcus and Clavibacter‐like were isolated, indicating that the same groups were potentially involved in hydrocarbon biodegradation in deep sea as in coastal waters. Conclusions: These results confirm that members of Alcanivorax are important obligate alkane degraders in deep‐sea environments and coexist with other degrading bacteria inhabiting the deep‐subsurface sediment of the Mediterranean. Significance and Impact of the Study: The results suggest that the isolates obtained have potential applications in bioremediation strategies in deep‐sea environments and highlight the need to identify specific piezophilic hydrocarbon‐degrading bacteria (HCB) from these environments.     

Microbial community of freshwater sponges in Lake Baikal

The microbial community of Baikal sponges has been studied in five species belonging to the genera Swartschewskia, Baicalospongia, and Lubomirskia of the endemic family Lubomirskiidae. The results show that the total numbers of bacteria and bacterioplankton production have an effect on the growth of L. baicalensis body. Bacteria of the genera Pseudomonas, Bacillus, Micrococcus, Sarcina, Flavobacterium, Arthrobacter, and Acinetobacter living in the sponges are representatives of the Baikal bacterioplankton. Actinomycetes of the genera Streptomyces and Micromonospora are a permanent component of the cultivable sponge microbial community. The numbers and enzyme activities of heterotrophic, oligotrophic, and psychrophilic bacteria isolated from different sponge species and from the ambient water in autumn and in winter have been estimated.     

Cultivable Bacterial Community from South China Sea Sponge as Revealed by DGGE Fingerprinting and 16S rDNA Phylogenetic Analysis

The cultivable bacterial communities associated with four South China Sea sponges—Stelletta tenuis, Halichondria rugosa, Dysidea avara, and Craniella australiensis in mixed cultures—were investigated by microbial community DNA-based DGGE fingerprinting and 16S rDNA phylogenetic analysis. Diverse bacteria such as α-, γ-, δ-Proteobacteria, Bacteroidetes, and Firmicutes were cultured, some of which were previously uncultivable bacteria, potential novel strains with less than 95% similarity to their closest relatives and sponge symbionts growing only in the medium with the addition of sponge extract. According to 16S rDNA BLAST analysis, most of the bacteria were cultured from sponge for the first time, although similar phyla of bacteria have been previously recognized. The selective pressure of sponge extract on the cultured bacterial species was suggested, although the effect of sponge extract on bacterial community in high nutrient medium is not significant. Although α- and γ-Proteobacteria appeared to form the majority of the dominant cultivable bacterial communities of the four sponges, the composition of the cultivable bacterial community in the mixed culture was different, depending on the medium and sponge species. Greater bacterial diversity was observed in media C and CS for Stelletta tenuis, in media F and FS for Halichondria rugosa and Craniella australiensis. S. tenuis was found to have the highest cultivable bacterial diversity including α-, γ-, δ-Proteobacteria, Bacteroidetes, and Firmicutes, followed by sponge Dysidea avara without δ-Proteobacteria, sponge Halichondria rugosa with only α-, γ-Proteobacteria and Bacteroidetes, and sponge C. australiensis with only α-, γ-Proteobacteria and Firmicutes. Based on this study, by the strategy of mixed cultivation integrated with microbial community DNA-based DGGE fingerprinting and phylogenetic analysis, the cultivable bacterial community of sponge could be revealed effectively.     

New phylogenetic lineages of the <Emphasis Type="Italic">Spirochaetes</Emphasis> phylum associated with <Emphasis Type="Italic">Clathrina</Emphasis> species (Porifera)

Though spirochetes have been repeatedly found in marine sponges and other invertebrates, little attention has been paid to the specificity of this association. This study demonstrates that different genoand morphotypes of spirochetes can reside within the same sponge individual and develop in considerable numbers. Specimens of the calcareous sponge Clathrina clathrus collected from the Adriatic Sea off Rovinj (Croatia) were found to harbor spirochete-like bacteria, which were characterized by scanning electron microscopy (SEM), 16S rRNA gene analysis, and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Two novel spirochete sequence types related to the Brachyspiraceae could be retrieved. By use of specifically designed CARD-FISH probes, the C. clathrus-associated sequences could be assigned to a linear and a helical spirochete morphotype. Both were located within the sponge mesohyl and resembled the spirochete-like cells identified by SEM. In addition, from a Clathrina sp., most likely C. coriacea, that originated from Indonesian coastal waters, four different spirochete type sequences were recovered. Two of these also affiliated with the Brachyspiraceae, the other two were found associated with the Spirochaetaceae, one with the genera Borrelia and Cristispira.     

First evidences of surfactant biodegradation by marine sponges (Porifera): an experimental study with a linear alkylbenzenesulfonate

The main objective of this work was to better appreciate the role of benthic macro-organisms in the biodegradation of detergents in the marine environment. Sponges, which could be highly resistant to pollution and which are highly active filter-feeders, appear as interesting organisms in this topic. An experimental study conducted in aquarium with seawater enriched in a pure linear alkylbenzenesulfonate (LAS), namely 1-(p-Sulfophenyl)nonane, has shown that the primary degradation was 10 times more rapid in the presence of the sponge Spongia officinalis than in the presence of only marine bacteria. The main degradation metabolites, 3-(p-sulfophenyl)propionic acid and p-sulfobenzoic acid were produced in greater amounts in the presence of sponge. The very rapid degradation kinetics observed in this study may be due to the symbiotic microbial community present in S. officinalis. The sponge may also have promoted the activity of marine bacteria by transforming the original molecule in a more available secondary product. This study demonstrates for the first time that benthic macro-organisms can play an important role in the transformation of biodegradable contaminants, such as LAS.     

Exploration,antifungal and antiaflatoxigenic activity of halophilic bacteria communities from saline soils of Howze-Soltan playa in Iran

In the present study, halophilic bacteria communities were explored in saline soils of Howze-Soltan playa in Iran with special attention to their biological activity against an aflatoxigenic Aspergillus parasiticus NRRL 2999. Halophilic bacteria were isolated from a total of 20 saline soils using specific culture media and identified by 16S rRNA sequencing in neighbor-joining tree analysis. Antifungal and antiaflatoxigenic activities of the bacteria were screened by a nor-mutant A. parasiticus NRRL 2999 using visual agar plate assay and confirmed by high-performance liquid chromatography. Among a total of 177 halophilic bacteria belonging to 11 genera, 121 isolates (68.3%) inhibited A. parasiticus growth and/or aflatoxin production. The most potent inhibitory bacteria of the genera Bacillus, Paenibacillus and Staphylococcus were distributed in three main phylogenetic clusters as evidenced by 16S rRNA sequence analysis. A. parasiticus growth was inhibited by 0.7–92.7%, while AFB₁ and AFG₁ productions were suppressed by 15.1–98.9 and 57.0–99.6%, respectively. Taken together, halophilic bacteria identified in this study may be considered as potential sources of novel bioactive metabolites as well as promising candidates to develop new biocontrol agents for managing toxigenic fungi growth and subsequent aflatoxin contamination of food and feed in practice.

     

Analysis of bacterial communities in sponges and coral inhabiting Red Sea,using barcoded 454 pyrosequencing

Microbial communities are linked with marine sponge are diverse in their structure and function. Our understanding of the sponge-associated microbial diversity is limited especially from Red Sea in Saudi Arabia where few species of sponges have been studied. Here we used pyrosequencing to study two marine sponges and coral species sampled from Obhur region from Red sea in Jeddah. A total of 168 operational taxonomic units (OTUs) were identified from Haliclona caerulea, Stylissa carteri and Rhytisma fulvum. Taxonomic identification of tag sequences of 16S ribosomal RNA revealed 6 different bacterial phyla and 9 different classes. A proportion of unclassified reads were was also observed in sponges and coral sample. We found diverse bacterial communities associated with two sponges and a coral sample. Diversity and richness estimates based on OUTs revealed that sponge H. caerulea had significantly high bacterial diversity. The identified OTUs showed unique clustering in three sponge samples as revealed by Principal coordinate analysis (PCoA). Proteobacteria (88–95%) was dominant phyla alonwith Bacteroidetes, Planctomycetes, Cyanobacteria, Firmicutes and Nitrospirae. Seventeen different genera were identified where genus Pseudoalteromonas was dominant in all three samples. This is first study to assess bacterial communities of sponge and coral sample that have never been studied before to unravel their microbial communities using 454-pyrosequencing method.     

广西蚕沙细菌组成多样性解析和VBNC菌群的发掘

【目的】解析广西蚕沙中细菌群落组成与多样性,为蚕沙中菌种资源发掘和蚕沙的综合利用提供科学依据。【方法】通过高通量测序技术研究细菌群落组成特征,同时利用常规稀释涂布平板法和基于复苏促进因子(Rpf)的MPN培养系统解析并筛选蚕沙中可培养和活的非可培养(VBNC)状态的优势菌群,并经16SrRNA基因测序对筛选得到的菌株作初步分类鉴定。【结果】高通量测序表明,广西蚕沙样品中细菌归属于10个门、18个纲、27个目、57个科、96个属,其中4个属的丰度达1%以上,优势菌群为变形菌门(Proteobacteria)肠杆菌属(Enterobacter);通过稀释涂布平板法共获得14个属的33株可培养细菌,其中4个属(Citrobacter、Weissella、Chitinophaga、Pseudoclavibacter)在高通量测序中未被检测到;而在MPN培养系统中,基于复苏促进因子的处理组细菌总数最大检出丰度提高了100倍,并从中共检出21株对Rpf敏感的VBNC菌株,其中6个属(Paenibacillus、Caulobacter、Roseomonas、Pantoea、Erwinia、Acine...     

Culturable heterotrophic bacteria from the marine sponge <Emphasis Type="Italic">Dendrilla</Emphasis> <Emphasis Type="Italic">nigra</Emphasis>: isolation and phylogenetic diversity of actinobacteria

Culturable heterotrophic bacterial composition of marine sponge Dendrilla nigra was analysed using different enrichments. Five media compositions including without enrichment (control), enriched with sponge extract, with growth regulator (antibiotics), with autoinducers, and complete enrichment containing sponge extract, antibiotics, and autoinducers were developed. DNA hybridization assay was performed to explore host specific bacteria and ecotypes of culturable sponge-associated bacteria. Enrichment with selective inducers (AHLs and sponge extract) and regulators (antibiotics) considerably enhanced the cultivation potential of sponge-associated bacteria. It was found that Marinobacter (MSI032), Micromonospora (MSI033), Streptomyces (MSI051), and Pseudomonas (MSI057) were sponge-associated obligate symbionts. The present findings envisaged that “Micromonospora–Saccharomonospora–Streptomyces” group was the major culturable actinobacteria in the marine sponge D. nigra. The DNA hybridization assay was a reliable method for the analysis of culturable bacterial community in marine sponges. Based on the culturable community structure, the sponge-associated bacteria can be grouped (ecotypes) as general symbionts, specific symbionts, habitat flora, and antagonists.     

Recovery and Phylogenetic Diversity of Culturable Fungi Associated with Marine Sponges <Emphasis Type="Italic">Clathrina luteoculcitella</Emphasis> and <Emphasis Type="Italic">Holoxea</Emphasis> sp. in the South China Sea

Sponge-associated fungi represent an important source of marine natural products, but little is known about the fungal diversity and the relationship of sponge–fungal association, especially no research on the fungal diversity in the South China Sea sponge has been reported. In this study, a total of 111 cultivable fungi strains were isolated from two South China Sea sponges Clathrina luteoculcitella and Holoxea sp. using eight different media. Thirty-two independent representatives were selected for analysis of phylogenetic diversity according to ARDRA and morphological characteristics. The culturable fungal communities consisted of at least 17 genera within ten taxonomic orders of two phyla (nine orders of the phylum Ascomycota and one order of the phylum Basidiomycota) including some potential novel marine fungi. Particularly, eight genera of Apiospora, Botryosphaeria, Davidiella, Didymocrea, Lentomitella, Marasmius, Pestalotiopsis, and Rhizomucor were isolated from sponge for the first time. Sponge C. luteoculcitella has greater culturable fungal diversity than sponge Holoxea sp. Five genera of Aspergillus, Davidiella, Fusarium, Paecilomyces, and Penicillium were isolated from both sponges, while 12 genera of Apiospora, Botryosphaeria, Candida, Marasmius, Cladosporium, Didymocrea, Hypocrea, Lentomitella, Nigrospora, Pestalotiopsis, Rhizomucor, and Scopulariopsis were isolated from sponge C. luteoculcitella only. Order Eurotiales especially genera Penicillium, Aspergillus, and order Hypocreales represented the dominant culturable fungi in these two South China Sea sponges. Nigrospora oryzae strain PF18 isolated from sponge C. luteoculcitella showed a strong and broad spectrum antimicrobial activities suggesting the potential for antimicrobial compounds production.     

Exploration of cultivable fungal communities in deep coal‐bearing sediments from ∼1.3 to 2.5 km below the ocean floor

Although subseafloor sediments are known to harbour a vast number of microbial cells, the distribution, diversity, and origins of fungal populations remain largely unexplored. In this study, we cultivated fungi from 34 of 47 deep coal‐associated sediment samples collected at depths ranging from 1289 to 2457 m below the seafloor (mbsf) off the Shimokita Peninsula, Japan (1118 m water depth). We obtained a total of 69 fungal isolates under strict contamination controls, representing 61 Ascomycota (14 genera, 23 species) and 8 Basidiomycota (4 genera, 4 species). Penicillium and Aspergillus relatives were the most dominant genera within the Ascomycetes, followed by the members of genera Cladosporium, Hamigera, Chaetomium, Eutypella, Acremonium, Aureobasidium, Candida, Eurotium, Exophiala, Nigrospora, Bionectria and Pseudocercosporella. Four Basidiomycota species were identified as genera Schizophyllum, Irpex, Bjerkandera and Termitomyces. Among these isolates, Cladosporium sphaerospermum and Aspergillus sydowii relatives were isolated from a thin lignite coal‐sandstone formation at 2457 mbsf. Our results indicate that these cultivable fungal populations are indigenous, originating from past terrigenous environments, which have persisted, possibly as spores, through ～20 million years of depositional history.     

Antibiotic Susceptibility as a Taxonomic Characteristic of Proteobacteria of the Genera Alteromonas, Pseudoalteromonas, Marinomonas, and Marinobacter

Type strains of bacteria of the genera Alteromonas, Pseudoalteromonas, Marinomonas, and Marinobacterand symbiotrophic bacteria isolated from the Far Eastern bivalve mollusks Crenomytilus grayanusand Mizuhopecten yessoensisand identified as Pseudoalteromonas nigrifaciensand P. citreawere tested for susceptibility to antibiotics. Of the ten antibiotics used in this study, benzylpenicillin, polymyxin, lincomycin, and oleandomycin were valuable for the polyphasic taxonomy of marine aerobic proteobacteria. Marine bacteria of the genera studied exhibited characteristic antibiotic susceptibility profiles, which were useful for the differentiation of the genera.     

Gene analysis,optimized production and property of marine lipase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> B106 associated with South China Sea sponge <Emphasis Type="Italic">Halichondria rugosa</Emphasis>

Gene cloning, optimized production and property of marine lipase from Bacillus pumilus B106 associated with South China Sea sponge Halichondria rugosa were investigated in this paper. A lipase gene with whole ORF encoding 215 amino acids was obtained by PCR, protein domain prediction suggested that the deduced lipase belongs to α/β hydrolases family. Based on single factor Seriatim-Factorial test and Plackett–Burman experimental design, the optimal medium consisted of (per l) 12.5 ml maize oil, 5.0 g beef extract, 2.0 g PO₄ ³⁻ (0.6 g KH₂PO₄, 1.4 g K₂HPO₄), 17.15 g Mg²⁺, 5.0 g yeast extract, 2.282 g CaCl₂ and 5.0 ml Tween80 with artificial sea water. Using this optimum medium, lipase activity and cell concentration were increased by 3.54- and 1.31-fold over that of the basal medium, respectively. This lipase showed tolerance to high salinity, pH and temperature. About 10–20% methanol exhibited a stimulatory effect on the lipase activity, while activity was inhibited by 30–40% methanol, 2-propanol, DMSO, and ethanol. This study provides a valuable resource for marine lipase production and extends our understanding of the possible role of sponge-associated bacteria in the biotransformation of chemical compounds for the sponge host.     

Microbial community structure analysis of produced water from a high-temperature North Sea oil-field

Molecular and culture-based methods were used to investigate the microbial diversity in produced water obtained from the high-temperature Troll oil formation in the North Sea. 16S rRNA gene libraries were generated from total community DNA, using universal archaeal or bacterial oligonucleotide primer sets. Sequence analysis of 88 clones in the bacterial library indicated that they originated from members of Firmicutes (48 sequences), Bacteroidetes (17 sequences), δ-Proteobacteria (15 sequences), Spirochaetes (5 sequences), Thermotogales (2 sequences) and γ-Proteobacteria (1 sequence). Twenty-two sequences in the archaeal library were close relatives to members of the genera Methanococcus (18 sequences), Methanolobus (3 sequences) and Thermococcus (1 sequence). Most of the bacterial sequences shared less than 95% identity with their closest match in GenBank, indicating that the produced water harbours a unique community of novel bacterial species or genera. Members of the thermophilic genera Thermosipho, Thermotoga, Anaerophaga and Thermovirga were isolated. The Troll formations are not injected with sea water. Thus, dramatic changes of the in situ conditions have been avoided, and a common source of continuous contamination from injection water can be excluded. However, the majority of the organisms detected in the gene libraries were most closely related to cultivated organisms with optimum temperatures for growth well below the in situ reservoir temperature (70°C), indicating that produced water from the Troll platform harbours a substantial amount of non-indigenous organisms. This was confirmed by the isolation of a number of mesophilic and moderately thermophilic organisms that were unable to grow at reservoir temperature.     

Isolation of faecal coliform bacteria from the American alligator (Alligator mississippiensis)

Aims: To determine whether American alligators (Alligator mississippiensis) are an unrecognized poikilothermic source of faecal coliform and/or potential pathogenic bacteria in South Carolina’s coastal waters. Methods and Results: Bacteria from the cloaca of American alligators, as well as bacteria from surface water samples from their aquatic habitat, were isolated and identified. The predominant enteric bacteria identified from alligator samples using biochemical tests included Aeromonas hydrophila, Citrobacter braakii, Edwardsiella tarda, Escherichia coli, Enterobacter cloacae, Plesiomonas shigelloides and putative Salmonella, and these were similar to bacteria isolated from the surface waters in which the alligators inhabited. Based on most‐probable‐number enumeration estimates from captive alligator faeces, faecal coliform bacteria numbered 8·0 × 10⁹ g^?1 (wet weight) of alligator faecal material, a much higher concentration than many other documented endothermic animal sources. Conclusions: A prevalence of enteric bacteria, both faecal coliforms and potential pathogens, was observed in American alligators. The high faecal coliform bacterial density of alligator faeces may suggest that alligators are a potential source of bacterial contamination in South Carolina coastal waters. Significance and Impact of the Study: These findings help to increase our understanding of faecal coliform and potential pathogenic bacteria from poikilothermic reptilian sources, as there is the potential for these sources to raise bacterial water quality levels above regulatory thresholds.     

Antiangiogenic, Antimicrobial, and Cytotoxic Potential of Sponge-Associated Bacteria

The bacteria associated with marine invertebrates are a rich source of bioactive metabolites. In the present study bacteria associated with the sponge Suberites domuncula and its primmorphs (3-dimensional aggregates containing proliferating cells) were isolated and cultured. These bacteria were extracted, and the extracts were assayed for antiangiogenic, hemolytic, antimicrobial, and cytotoxic activities. Our studies revealed that extract obtained from the bacterium (PB2) isolated from sponge primmorphs is a potent angiogenesis inhibitor. In the chick chorio-allantoic membrane (CAM) assay, it showed 50% activity at 5 μg ml⁻¹ and 100% activity at 10 and 20 μg ml⁻¹ concentrations. Extracts obtained from 5 bacterial strains isolated from sponge and its primmorphs showed hemolytic activity. The sponge-associated bacteria belonging to the α subdivision of Proteobacteria and the primmorph-associated bacterium identified as a possible novel Pseudomonas sp. displayed remarkable antimicrobial activity. It is important to note that these bacterial extracts were strongly active against multidrug-resistant clinical strains such as Staphylococcus aureus and Staphylococcus epidermidis, isolated from hospital patients. The bacterial extracts having antimicrobial activity also showed cytotoxicity against HeLa and PC12 cells. In summary, this investigation explores the importance of sponge-associated bacteria as a valuable resource for the discovery of novel bioactive molecules.     

Assessment of Antibacterial Activity of Colebrookia oppositifolia against Waterborne Pathogens Isolated from Drinking Water of the Pothwar Region in Pakistan

An attempt was made to control waterborne pathogens by using medicinal plant extracts. One hundred and twenty-six water samples from filtration plants, tube wells, and water supplies were collected and analyzed for total and faecal Coliform bacteria as well as for total viable count. Results showed that waterborne pathogens were numerous and significantly higher than the World Health Organization's recommended guidelines. The methanolic and aqueous extracts of different parts of Colebrookia oppositifolia (Labiateae) were examined for antibacterial activities in vitro by an agar diffusion method. Antibacterial activity of leaves, shoots, and roots of Colebrookia oppositifolia was assessed against Gram positive and Gram negative bacteria that were isolated and identified from water samples by the API 20E method. Extract of roots showed more antibacterial activities against Staph. aureus and B. cereus var. mycoides, Pseudomonas aeruginosa, Klebsiella pneumonia, and Shigella flexneri at 37°C, than extracts from leaves and shoots. The lowest MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) were observed in roots as compared to shoots and leaves. These results suggest that there is an urgent need for improvement in existing water quality treatment. Secondly, the fruit extract can be practical for protection and to avoid risk of contamination by waterborne pathogens and to promote indigenous solutions for disease-control and environmental management.     

Antimicrobial Activity of Rosmarinus officinalis against Oral Pathogens: Relevance of Carnosic Acid and Carnosol

The in vitro inhibitory activity of crude EtOH/H₂O extracts from the leaves and stems of Rosmarinus officinalis L. was evaluated against the following microorganisms responsible for initiating dental caries: Streptococcus mutans, S. salivarius, S. sobrinus, S. mitis, S. sanguinis, and Enterococcus faecalis. Minimum inhibitory concentrations (MIC) were determined with the broth microdilution method. The bioassay‐guided fractionation of the leaf extract, which displayed the higher antibacterial activity than the stem extract, led to the identification of carnosic acid ( 2 ) and carnosol ( 3 ) as the major compounds in the fraction displaying the highest activity, as identified by HPLC analysis. Rosmarinic acid ( 1 ), detected in another fraction, did not display any activity against the selected microorganisms. HPLC Analysis revealed the presence of low amounts of ursolic acid ( 4 ) and oleanolic acid ( 5 ) in the obtained fractions. The results suggest that the antimicrobial activity of the extract from the leaves of R. officinalis may be ascribed mainly to the action of 2 and 3 .     

Gene diversity of CYP153A and AlkB alkane hydroxylases in oil‐degrading bacteria isolated from the Atlantic Ocean

Alkane hydroxylases, including the integral‐membrane non‐haem iron monooxygenase (AlkB) and cytochrome P450 CYP153 family, are key enzymes in bacterial alkane oxidation. Although both genes have been detected in a number of bacteria and environments, knowledge about the diversity of these genes in marine alkane‐degrading bacteria is still limited, especially in pelagic areas. In this report, 177 bacterial isolates, comprising 43 genera, were obtained from 18 oil‐degrading consortia enriched from surface seawater samples collected from the Atlantic Ocean. Many isolates were confirmed to be the first oil‐degraders in their affiliated genera including Brachybacterium, Idiomarina, Leifsonia, Martelella, Kordiimonas, Parvibaculum and Tistrella. Using degenerate PCR primers, alkB and CYP153A P450 genes were surveyed in these bacteria. In total, 82 P450 and 52 alkB gene fragments were obtained from 80 of the isolates. These isolates mainly belonged to Alcanivorax, Bacillus, Erythrobacter, Martelella, Parvibaculum and Salinisphaera, some of which were reported, for the first time, to encode alkane hydroxylases. Phylogenetic analysis showed that both genes were quite diverse and formed several clusters, most of which were generated from various Alcanivorax bacteria. Noticeably, some sequences, such as those from the Salinisphaera genus, were grouped into a distantly related novel cluster. Inspection of the linkage between gene and host revealed that alkB and P450 tend to coexist in Alcanivorax and Salinisphaera, while in all isolates of Parvibaculum, only P450 genes were found, but of multiple homologues. Multiple homologues of alkB mostly cooccurred in Alcanivorax isolates. Conversely, distantly related isolates contained similar or even identical sequences. In summary, various oil‐degrading bacteria, which harboured diverse P450 and alkB genes, were found in the surface water of Atlantic Ocean. Our results help to show the diversity of P450 and alkB genes in prokaryotes, and to portray the geographic distribution of oil‐degrading bacteria in marine environments.     

Microbial Diversity of the Freshwater Sponge Spongilla lacustris

To provide insight into the phylogenetic bacterial diversity of the freshwater sponge Spongilla lacustris, a 16S rRNA gene libraries were constructed from sponge tissues and from lake water. Restriction fragment length polymorphism (RFLP) analysis of >190 freshwater sponge-derived clones resulted in six major restriction patterns, from which 45 clones were chosen for sequencing. The resulting sequences were affiliated with the Alphaproteobacteria (n = 19), the Actinobacteria (n = 15), the Betaproteobacteria (n = 2), and the Chloroflexi (n = 2) lineages. About half of the sequences belonged to previously described actinobacterial (hgc-I) and betaproteobacterial (beta-II) sequence clusters of freshwater bacteria that were also present in the lake water 16S rRNA gene library. At least two novel, deeply rooting alphaproteobacterial lineages were recovered from S. lacustris that showed <89% sequence similarity to known phylogenetic groups. Electron microscopical observations revealed that digested bacterial remnants were contained within food vacuoles of sponge archaeocytes, whereas the extracellular matrix was virtually free of bacteria. This study is the first molecular diversity study of a freshwater sponge and adds to a growing database on the diversity and community composition of sponge-associated microbial consortia.