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Volatile organic compounds from arctic bacteria of the Cytophaga-Flavobacterium-Bacteroides group: a retrobiosynthetic approach in chemotaxonomic investigations.

Volatile organic compounds emitted by different marine arctic strains of the Cytophaga-Flavobacterium-Bacteroides group were investigated by using a modified closed-loop stripping apparatus (CLSA). Seven of nine strains emitted volatiles, dominated by methyl ketones, in specific patterns. The methyl ketones were aliphatic saturated, or unsaturated, and comprised 12 to 18 C-atoms, sometimes with terminal Me branches. They were identified by GC/MS, retention-index calculations, derivatization with dimethyl disulfide for C=C bond location, and GC/FTIR to elucidate their uniform (Z)-configuration. The proposed structures of all methyl ketones were subsequently confirmed by synthesis, while the absolute configuration of chiral volatiles was elucidated by stereoselective synthesis. From retrobiosynthetic considerations, it was found that strain ARK10267 uses mainly valine, and strain ARK10063 mainly isoleucine for formation of starters for the ketone biosynthesis, which is correlated to fatty acid biosynthesis. Four strains (ARK10223, ARK10044, ARK10141, and ARK10146) use leucine. These separations are supported by phylogenetic affiliations based on 16S rRNA. Strain ARK10255b, in the course of this study found to be not a member of the Cytophaga-Flavobacterium-Bacteroides phylum, did not emit aliphatic ketones of medium chain length, but methionine-derived 4-(methylsulfanyl)butan-2-one and corresponding 4-(methylsulfanyl)butan-2-ol. Most of the compounds described have not been reported previously from nature.

Arctic Regions↗

Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice.

Extracellular degradative enzymes released by psychrophilic marine bacteria (growing optimally at or below 15 degrees C and maximally at 20 degrees C) typically express activity optima at temperatures well above the upper growth limit of the producing strain. In the present study, we investigated whether or not near-zero Arctic environments contain extracellular enzymes with activity optimized to temperatures lower than previously reported. By applying fluorescently tagged substrate analogues to measure leucine-aminopeptidase and chitobiase activity, the occurrence of extracellular enzymatic activity (EEA) with remarkably low temperature optima (15 degrees C) was documented in sea-ice samples. An extremely psychrophilic bacterial isolate, strain 34H, yielded an extract of cell-free protease with activity optimized at 20 degrees C, the lowest optimum yet reported for cell-free EEA from a pure culture. The use of zymogram gels revealed the presence of three proteolytic bands (between 37 and 45 kDa) in the extract and the release of the greatest quantities of the proteases when the strain was grown at -1 degrees C, suggesting a bacterial strategy for counteracting the effects of very cold temperatures on the catalytic efficiency of released enzymes. The detection of unusually cold-adapted EEA in environmental samples has ramifications not only to polar ecosystems and carbon cycling but also to protein evolution, biotechnology and bioremediation.

Acetylglucosaminidase↗

Effect of temperature on sulphate reduction, growth rate and growth yield in five psychrophilic sulphate-reducing bacteria from Arctic sediments.

Five psychrophilic sulphate-reducing bacteria (strains ASv26, LSv21, PSv29, LSv54 and LSv514) isolated from Arctic sediments were examined for their adaptation to permanently low temperatures. All strains grew at -1.8 degrees C, the freezing point of sea water, but their optimum temperature for growth (T(opt)) were 7 degrees C (PSv29), 10 degrees C (ASv26, LSv54) and 18 degrees C (LSv21, LSv514). Although T(opt) was considerably above the in situ temperatures of their habitats (-1.7 degrees C and 2.6 degrees C), relative growth rates were still high at 0 degrees C, accounting for 25-41% of those at T(opt). Short-term incubations of exponentially growing cultures showed that the highest sulphate reduction rates occurred 2-9 degrees C above T(opt). In contrast to growth and sulphate reduction rates, growth yields of strains ASv26, LSv54 and PSv29 were almost constant between -1.8 degrees C and T(opt). For strains LSv21 and LSv514, however, growth yields were highest at the lowest temperatures, around 0 degrees C. The results indicate that psychrophilic sulphate-reducing bacteria are specially adapted to permanently low temperatures by high relative growth rates and high growth yields at in situ conditions.

Arctic Regions↗

Comparison of psychro-active arctic marine bacteria and common mesophillic bacteria using surface-enhanced Raman spectroscopy.

Psychro-active bacteria, important constituents of polar ecosystems, have a unique ability to remain active at temperatures below 0 degrees C, yet it is not known to what extent the composition of their outer cell surfaces aids in their low-temperature viability. In this study, aqueous suspensions of five strains of Arctic psychro-active marine bacteria (PAMB) (mostly sea-ice isolates), were characterized by surface-enhanced Raman spectroscopy (SERS) and compared with SERS spectra from E. coli and P. aerigunosa. We find the SERS spectra of the five psychro-active bacterial strains are similar within experimental reproducibility. However, these spectra are significantly different from the spectra of P. aeruginosa and E. coli. We find that the relative intensities of many of the common peaks show the largest differences reported so far for bacterial samples. An indication of a peak was found in the PAMB spectra that has been identified as characteristic of unsaturated fatty acids and suggests that the outer membranes of the PAMB may contain unsaturated fatty acids. We find that using suspensions of silver colloid particles greatly intensifies the Raman peaks and quenches the fluorescence from bacterial samples. This technique is useful for examination of specific biochemical differences among bacteria.

Adaptation, Physiological↗

Community size and metabolic rates of psychrophilic sulfate-reducing bacteria in Arctic marine sediments.

The numbers of sulfate reducers in two Arctic sediments with in situ temperatures of 2.6 and -1.7 degrees C were determined. Most-probable-number counts were higher at 10 degrees C than at 20 degrees C, indicating the predominance of a psychrophilic community. Mean specific sulfate reduction rates of 19 isolated psychrophiles were compared to corresponding rates of 9 marine, mesophilic sulfate-reducing bacteria. The results indicate that, as a physiological adaptation to the permanently cold Arctic environment, psychrophilic sulfate reducers have considerably higher specific metabolic rates than their mesophilic counterparts at similarly low temperatures.

Arctic Regions↗

Psychrotolerant bacteria isolated from arctic soil that degrade polychlorinated biphenyls at low temperatures

Psychrotolerant polychlorinated biphenyl (PCB)-degrading bacteria were isolated at 7 degreesC from PCB-contaminated Arctic soil by using biphenyl as the sole organic carbon source. These isolates were distinguished from each other by differences in substrates that supported growth and substrates that were oxidized. 16S ribosomal DNA sequences suggest that these isolates are most closely related to the genus Pseudomonas. Total removal of Aroclor 1242, and rates of removal of selected PCB congeners, by cell suspensions of Arctic soil isolates and the mesophile Burkholderia cepacia LB400 were determined at 7, 37, and 50 degreesC. Total removal values of Aroclor 1242 at 7 degreesC by LB400 and most Arctic soil isolates were similar (between 2 and 3.5 &mgr;g of PCBs per mg of cell protein). However the rates of removal of some individual PCB congeners by Arctic isolates were up to 10 times higher than corresponding rates of removal by LB400. Total removal of Aroclor 1242 and the rates of removal of individual congeners by the Arctic soil bacteria were higher at 37 degreesC than at 7 degreesC but as much as 90% lower at 50 degreesC than at 37 degreesC. In contrast, rates of PCB removal by LB400 were higher at 50 degreesC than at 37 degreesC. In all cases, temperature did not affect the congener specificity of the bacteria. These observations suggest that the PCB-degrading enzyme systems of the bacteria isolated from Arctic soil are cold adapted.

Journal Article↗

Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice, Spitzbergen.

The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4 degrees C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses alpha and gamma of Proteobacteria, the Bacillus-Clostridium group, the order Actinomycetales, and the Cytophaga-Flexibacter-Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria gamma subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25 degrees C. Only a few strains were psychrophilic, with an optimal growth at 10-15 degrees C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4 degrees C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, alpha-amylase and beta-galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10 degrees C, and almost no production was detected at higher temperatures (20-30 degrees C). Catalytic activity was detected even below the freezing point of water (at -5 degrees C), demonstrating the unique properties of these enzymes.

Actinomycetales↗

Community structure, cellular rRNA content, and activity of sulfate-reducing bacteria in marine arctic sediments.

The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. The SRB community was dominated by members of the Desulfosarcina-Desulfococcus group. This group accounted for up to 73% of the SRB detected and up to 70% of the SRB rRNA detected. The predominance was shown to be a common feature for different stations along the coast of Svalbard. In a top-to-bottom approach we aimed to further resolve the composition of this large group of SRB by using probes for cultivated genera. While this approach failed, directed cloning of probe-targeted genes encoding 16S rRNA was successful and resulted in sequences which were all affiliated with the Desulfosarcina-Desulfococcus group. A group of clone sequences (group SVAL1) most closely related to Desulfosarcina variabilis (91.2% sequence similarity) was dominant and was shown to be most abundant in situ, accounting for up to 54. 8% of the total SRB detected. A comparison of the two methods used for quantification showed that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of Desulfosarcina-Desulfococcus cells were highest in the first 5 mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface. Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell(-1) day(-1)), decreased by a factor of 3 within the first 2 cm, and were relatively constant in deeper layers.

Arctic Regions↗

Desulfuromonas svalbardensis sp. nov. and Desulfuromusa ferrireducens sp. nov., psychrophilic, Fe(III)-reducing bacteria isolated from Arctic sediments, Svalbard.

Two psychrophilic, Gram-negative, rod-shaped, motile bacteria (strains 112T and 102T) that conserved energy from dissimilatory Fe(III) reduction concomitant with acetate oxidation were isolated from permanently cold Arctic marine sediments. Both strains grew at temperatures down to -2 degrees C, with respective temperature optima of 14 degrees C and 14-17 degrees C for strains 112T and 102T. The isolated strains reduced Fe(III) using common fermentation products such as acetate, lactate, propionate, formate or hydrogen as electron donors, and they also grew with fumarate as the sole substrate. As alternatives to Fe(III), they reduced fumarate, S0 and Mn(IV). Based on 16S rRNA gene sequence similarity, strain 112T was most closely related to Desulfuromonas acetoxidans (97.0 %) and Desulfuromonas thiophila NZ27T (95.5 %), and strain 102T to Malonomonas rubra Gra Mal 1T (96.3 %) and Desulfuromusa succinoxidans GylacT (95.9 %) within the Deltaproteobacteria. Strains 112T and 102T therefore represent novel species, for which the names Desulfuromonas svalbardensis sp. nov. (type strain 112T=DSM 16958T=JCM 12927T) and Desulfuromusa ferrireducens sp. nov. (type strain 102T=DSM 16956T=JCM 12926T) are proposed.

Acetic Acid↗

Molecular diversity of nifH genes from bacteria associated with high arctic dwarf shrubs.

Biological nitrogen fixation is the primary source of new N in terrestrial arctic ecosystems and is fundamental to the long-term productivity of arctic plant communities. Still, relatively little is known about the nitrogen-fixing microbes that inhabit the soils of many dominant vegetation types. Our objective was to determine which diazotrophs are associated with three common, woody, perennial plants in an arctic glacial lowland. Dryas integrifolia, Salix arctica, and Cassiope tetragona plants in soil were collected at Alexandra Fiord, Ellesmere Island, Canada. DNA was extracted from soil and root samples and a 383-bp fragment of the nifH gene amplified by the polymerase chain reaction. Cloned genotypes were screened for similarity by restriction fragment length polymorphism (RFLP) analysis. Nine primary RFLP phylotypes were identified and 42 representative genotypes selected for sequencing. Majority of sequences (33) were type I nitrogenases, whereas the remaining sequences belonged to the divergent, homologous, type IV group. Within the type I nitrogenases, nifH genes from posited members of the Firmicutes were most abundant, and occurred in root and soil samples from all three plant species. nifH genes from posited Pseudomonads were found to be more closely associated with C. tetragona, whereas nifH genes from putative alpha-Proteobacteria were more commonly associated with D. integrifolia and S. arctica. In addition, 12 clones likely representing a unique clade within the type I nitrogenases were identified. To our knowledge, this study is the first to report on the nifH diversity of arctic plant-associated soil microbes.

Arctic Regions↗

Thiomicrospira arctica sp. nov. and Thiomicrospira psychrophila sp. nov., psychrophilic, obligately chemolithoautotrophic, sulfur-oxidizing bacteria isolated from marine Arctic sediments.

Two psychrophilic, chemolithoautotrophic, sulfur-oxidizing bacteria were isolated from marine Arctic sediments sampled off the coast of Svalbard with thiosulfate as the electron donor and CO(2) as carbon source. Comparative analysis of 16S rRNA gene sequences suggested that the novel strains, designated SVAL-D(T) and SVAL-E(T), represent members of the genus Thiomicrospira. Further genotypic (DNA-DNA relatedness, DNA G+C content) and phenotypic characterization revealed that the strains represent members of two novel species. Both organisms are obligately autotrophic and strictly aerobic. Nitrate was not used as an electron acceptor. Chemolithoautotrophic growth was observed with thiosulfate, tetrathionate and sulfur. The temperature limits for growth of both strains were between -2 degrees C and 20.8 degrees C, with optima of 11.5-13.2 degrees C (SVAL-E(T)) and 14.6-15.4 degrees C (SVAL-D(T)), which is about 13-15 degrees C lower than the optima of all other recognized Thiomicrospira species. The maximum growth rate on thiosulfate at 14 degrees C was 0.14 h(-1) for strain SVAL-E(T) and 0.2 h(-1) for strain SVAL-D(T). Major fatty acids of SVAL-D(T) are C(16 : 1), C(18 : 0) and C(16 : 0), and those of SVAL-E(T) are C(16 : 1), C(18 : 1), C(16 : 0) and C(14 : 1). Cells of SVAL-D(T) and SVAL-E(T) are rods, like those of their closest relatives. To our knowledge the novel strains are the first psychrophilic, chemolithoautotrophic, sulfur-oxidizing bacteria so far described. The names Thiomicrospira arctica sp. nov. and Thiomicrospira psychrophila sp. nov. are proposed for SVAL-E(T) (=ATCC 700955(T)=DSM 13458(T)) and SVAL-D(T) (=ATCC 700954(T)=DSM 13453(T)), respectively.

Arctic Regions↗

Desulfovibrio frigidus sp. nov. and Desulfovibrio ferrireducens sp. nov., psychrotolerant bacteria isolated from Arctic fjord sediments (Svalbard) with the ability to reduce Fe(III).

Strains 18T, 61T and 77 were isolated from two permanently cold fjord sediments on the west coast of Svalbard. The three psychrotolerant strains, with temperature optima at 20-23 degrees C, were able to grow at the freezing point of sea water, -2 degrees C. The strains oxidized important fermentation products such as hydrogen, formate and lactate with sulfate as the electron acceptor. Sulfate could be replaced by sulfite, thiosulfate or elemental sulfur. Poorly crystalline and soluble Fe(III) compounds were reduced in sulfate-free medium, but no growth occurred under these conditions. In the absence of electron acceptors, fermentative growth was possible. The pH optimum for the strains was around 7.1. The DNA G+C contents were 43.3 and 42.0 mol% for strains 18T and 61T, respectively. Strains 18T, 61T and 77 were most closely related to Desulfovibrio hydrothermalis (95.0-95.7 % 16S rRNA gene sequence similarity). Strains 18T and 77, exhibiting 99.9 % sequence similarity, represent a novel species for which the name Desulfovibrio frigidus sp. nov. is proposed. The type strain is strain 18T (=DSM 17176T = JCM 12924T). Strain 61T was closely related to strains 18T and 77 (97.6 and 97.5 % 16S rRNA gene sequence similarity), but on the basis of DNA-DNA hybridization strain 61T represents a novel species for which the name Desulfovibrio ferrireducens sp. nov. is proposed. The type strain is strain 61T (=DSM 16995T = JCM 12925T).

Arctic Regions↗

New method for rapid and sensitive quantification of sulphide-producing bacteria in fish from arctic and temperate waters.

The offensive, fishy, rotten H2S-off-odours in spoiled, aerobically and cold stored fish from arctic and temperate waters are generally caused by sulphide-producing bacteria (SPB), mainly Shewanella putrefaciens. In the present work, a new, rapid, simple and accurate method for estimation of the SPB content in fish from these areas is described. The quantification is based on the formation rate of iron sulphide during growth of SPBs incubated at 30 degrees C in a liquid growth medium containing cysteine, sodium thiosulphate and iron(III)citrate as specific substrates for iron sulphide formation. The iron sulphide turns the medium grey and masks the background fluorescence in the medium when the SPB content in the assay is approximately 10(9) cfu/ml. The fluorescence change could be detected instrumentally and the colour change visually. The method was developed and evaluated in tests with S. putrefaciens CCUG 13452 DT as well as naturally occurring SPBs in cod, salmon, wolf fish and coal fish. A linear correlation between the SPB count and detection time was obtained over the entire range from 1 to 10(9) cfu SPB/g, corresponding to detection times 17 and 1 h, respectively. The correlation is described by the equation: log cfu/g fish= -0.59(+/- 0.17) x DT+ 9.65(+/- 0.09), where DT is the detection time in hours. The model was valid for all the tested fish species and all tested naturally occurring SPBs in these species. The regression coefficients (R2) for cod, coal fish, wolf fish and salmon were 0.99, 0.92, 0.97 and 0.97, respectively. The detection level of the method is 1 SPB per sample tube, corresponding to 16 cfu/g fish. The method could be used to predict the remaining shelf life of the fish for different markets, even when the time-temperature history during storage of the fish is unknown.

Animals↗