Antibacterial activity of antagonistic bacterium Bacillus subtilis DJM-51 against phytopathogenic Clavibacter michiganense subsp. michiganense ATCC 7429 in vitro.

To investigate antibacterial activity against the tomato pathogen Clavibacter michiganense subsp. michiganense ATCC 7429 (Cmm ATCC 7429), Bacillus subtilis DJM-51 was isolated from rhizosphere soil. For isolation of bacteria, samples were taken from rhizosphere soil. The isolate, DJA-51, had strong antagonistic ability against Tomato pathogen Cmm ATCC 7429 on nutrient-broth yeast extract agar (NBYA) as indicated by inhibition zones around colonies. On the basis of the nucleotide sequence of a conserved segment of the 16S rRNA gene, the bacterium has been identified as B. subtilis DJM-51. The growth of Cmm ATCC 7429 on NBYA plates was inhibited by culture broth of B. subtilis DJM-51 including cells, by the supernatant of culture broth of B. subtilis DJM-51, and by the liquid material resulting from butanol extract of bacterial cultures. The OD value in co-culture mixture was lower than the control throughout the entire incubation period. Antibiotics obtained from B. subtilis DJM-51 inhibited the growth of Tomato pathogen Cmm ATCC 7429. These results provide potentially information about the protection of tomato from pathogen Cmm ATCC 7429 under greenhouse conditions in Quebec.

The COSPAR planetary protection policy for missions to Icy Worlds: A review of history, current scientific knowledge, and future directions.

Recent discoveries related to the habitability and astrobiological relevance of the outer Solar System have expanded our understanding of where and how life may have originated. As a result, the Icy Worlds of the outer Solar System have become among the highest priority targets for future spacecraft missions dedicated to astrobiology-focused and/or direct life detection objectives. This, in turn, has led to a renewed interest in planetary protection concerns and policies for the exploration of these worlds and has been a topic of discussion within the COSPAR (Committee on Space Research) Panel on Planetary Protection. This paper summarizes the results of those discussions, reviewing the current knowledge and the history of planetary protection considerations for Icy Worlds as well as suggesting ways forward. Based on those discussions, we therefore suggest to (1) Establish a new definition for Icy Worlds for Planetary Protection that captures the outer Solar System moons and dwarf planets like Pluto, but excludes more primitive bodies such as comets, centaurs, and asteroids: Icy Worlds in our Solar System are defined as all bodies with an outermost layer that is believed to be greater than 50 % water ice by volume and have enough mass to assume a nearly round shape. (2) Establish indices for the lower limits of Earth life with regards to water activity (LLAw) and temperature (LLT) and apply them into all areas of the COSPAR Planetary Protection Policy. These values are currently set at 0.5 and -28 °C and were originally established for defining Mars Special Regions; (3) Establish LLT as a parameter to assign categorization for Icy Worlds missions. The suggested categorization will have a 1000-year period of biological exploration, to be applied to all Icy Worlds and not just Europa and Enceladus as is currently the case. (4) Have all missions consider the possibility of impact. Transient thermal anomalies caused by impact would be acceptable so long as there is less than 10-4 probability of a single microbe reaching deeper environments where temperature is >LLT in the period of biological exploration. (5) Restructure or remove Category II* from the policy as it becomes largely redundant with this new approach, (6) Establish that any sample return from an Icy World should be Category V restricted Earth return.

Hydrocarbon-degrading potential of microbial communities from Arctic plants.

AIMS: To explore rhizospheric microbial communities from Arctic native plant species evaluating their bacterial hydrocarbon-degrading capacities. METHODS AND RESULTS: Eriophorum scheuchzeri, Potentilla cf. rubricaulis, Oxyria digyna, Salix arctica and Puccinellia angustata plant species were collected at Eureka (Canadian high Arctic) along with their rhizospheric soil samples. Their bacterial community fingerprints (16S rRNA gene, DGGE) were distinctive encompassing members from the phyla: Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria. Isolated diesel-degrading bacteria belonged to the phyla Actinobacteria and Proteobacteria. Strains of Mycobacterium, Nocardia, Rhodococcus, Intrasporangiaceae, Leifsoni and Arthrobacter possessed alkB and Pseudomonas possessed both ndoB and xylE gene sequences. Two Rhodococcus strains mineralized [1-(14) C] hexadecane at 5 and -5°C. From the rhizosphere of P. angustata, larger numbers of hydrocarbon-degrading bacteria were isolated than from other plant rhizosphere samples and all three genes alkB (from Actinobacteria), ndoB and xylE (from Pseudomonas) were detected by PCR. CONCLUSIONS: (i) Arctic plants have unique rhizospheric bacterial communities. (ii) P. angustata has potential for phytoremediation research at high Arctic soils. (iii) Isolated bacteria mineralized hydrocarbons at ambient low temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first rhizospheric exploration examining the phytoremediation potential of five Arctic plants and evaluating their microbial hydrocarbon-degrading capacities.

Lichen-associated microbial members are prevalent in the snow microbiome of a sub-arctic alpine tundra.

Snow is the largest component of the cryosphere, with its cover and distribution rapidly decreasing over the last decade due to climate warming. It is imperative to characterize the snow (nival) microbial communities to better understand the role of microorganisms inhabiting these rapidly changing environments. Here, we investigated the core nival microbiome, the cultivable microbial members, and the microbial functional diversity of the remote Uapishka mountain range, a massif of alpine sub-arctic tundra and boreal forest. Snow samples were taken over a two-month interval along an altitude gradient with varying degree of anthropogenic traffic and vegetation cover. The core snow alpine tundra/boreal microbiome, which was present across all samples, constituted of Acetobacterales, Rhizobiales and Acidobacteriales bacterial orders, and of Mycosphaerellales and Lecanorales fungal orders, with the dominant fungal taxa being associated with lichens. The snow samples had low active functional diversity, with Richness values ranging from 0 to 19.5. The culture-based viable microbial enumeration ranged from 0 to 8.05 × 103 CFUs/mL. We isolated and whole-genome sequenced five microorganisms which included three fungi, one alga, and one potentially novel bacterium of the Lichenihabitans genus; all of which appear to be part of lichen-associated taxonomic clades.

Novel Antarctic yeast adapts to cold by switching energy metabolism and increasing small RNA synthesis.

The novel extremophilic yeast Rhodotorula frigidialcoholis, formerly R. JG1b, was isolated from ice-cemented permafrost in University Valley (Antarctic), one of coldest and driest environments on Earth. Phenotypic and phylogenetic analyses classified R. frigidialcoholis as a novel species. To characterize its cold-adaptive strategies, we performed mRNA and sRNA transcriptomic analyses, phenotypic profiling, and assessed ethanol production at 0 and 23 °C. Downregulation of the ETC and citrate cycle genes, overexpression of fermentation and pentose phosphate pathways genes, growth without reduction of tetrazolium dye, and our discovery of ethanol production at 0 °C indicate that R. frigidialcoholis induces a metabolic switch from respiration to ethanol fermentation as adaptation in Antarctic permafrost. This is the first report of microbial ethanol fermentation utilized as the major energy pathway in response to cold and the coldest temperature reported for natural ethanol production. R. frigidialcoholis increased its diversity and abundance of sRNAs when grown at 0 versus 23 °C. This was consistent with increase in transcription of Dicer, a key protein for sRNA processing. Our results strongly imply that post-transcriptional regulation of gene expression and mRNA silencing may be a novel evolutionary fungal adaptation in the cryosphere.

Variability in carbon uptake and (re)cycling in Antarctic cryptoendolithic microbial ecosystems demonstrated through radiocarbon analysis of organic biomarkers.

Cryptoendolithic lichens and cyanobacteria living in porous sandstone in the high-elevation McMurdo Dry Valleys are purported to be among the slowest growing organisms on Earth with cycles of death and regrowth on the order of 103 -104  years. Here, organic biomarker and radiocarbon analysis were used to better constrain ages and carbon sources of cryptoendoliths in University Valley (UV; 1,800 m.a.s.l) and neighboring Farnell Valley (FV; 1,700 m.a.s.l). Δ14 C was measured for membrane component phospholipid fatty acids (PLFA) and glycolipid fatty acids, as well as for total organic carbon (TOC). PLFA concentrations indicated viable cells comprised a minor (<0.5%) component of TOC. TOC Δ14 C values ranged from -272‰ to -185‰ equivalent to calibrated ages of 1,100-2,550 years old. These ages may be the result of fractional preservation of biogenic carbon and/or sudden large-scale community death and extended period(s) of inactivity prior to slow recolonization and incorporation of 14 C-depleted fossil material. PLFA Δ14 C values were generally more modern than the corresponding TOC and varied widely between sites; the FV PLFA Δ14 C value (+40‰) was consistent with modern atmospheric CO2 , while UV values ranged from -199‰ to -79‰ (calibrated ages of 1,665-610 years). The observed variability in PLFA Δ14 C depletions is hypothesized to reflect variations in the extent of fixation of modern atmospheric CO2 and the preservation and recycling of older organic carbon by the community in various stages of sandstone recolonization. PLFA profiles and microbial community compositions as determined by molecular genetic characterizations and microscopy differed between the two valleys (e.g., predominance of biomarker 18:2 [>50%] in FV compared to UV), representing microbial communities that may reflect distinct stages of sandstone recolonization and/or environmental conditions. It is thus proposed that Dry Valley cryptoendolithic microbial communities are faster growing than previously estimated.

An active atmospheric methane sink in high Arctic mineral cryosols.

Methane (CH4) emission by carbon-rich cryosols at the high latitudes in Northern Hemisphere has been studied extensively. In contrast, data on the CH4 emission potential of carbon-poor cryosols is limited, despite their spatial predominance. This work employs CH4 flux measurements in the field and under laboratory conditions to show that the mineral cryosols at Axel Heiberg Island in the Canadian high Arctic consistently consume atmospheric CH4. Omics analyses present the first molecular evidence of active atmospheric CH4-oxidizing bacteria (atmMOB) in permafrost-affected cryosols, with the prevalent atmMOB genotype in our acidic mineral cryosols being closely related to Upland Soil Cluster α. The atmospheric (atm) CH4 uptake at the study site increases with ground temperature between 0 °C and 18 °C. Consequently, the atm CH4 sink strength is predicted to increase by a factor of 5-30 as the Arctic warms by 5-15 °C over a century. We demonstrate that acidic mineral cryosols are a previously unrecognized potential of CH4 sink that requires further investigation to determine its potential impact on larger scales. This study also calls attention to the poleward distribution of atmMOB, as well as to the potential influence of microbial atm CH4 oxidation, in the context of regional CH4 flux models and global warming.

Rapid, direct extraction of DNA from soils for PCR analysis using polyvinylpolypyrrolidone spin columns.

Polyvinylpolypyrrolidone spin columns were used to rapidly purify crude soil DNA extracts from humic materials for polymerase chain reaction (PCR) analysis. The PCR detection limit for the tfdC gene, encoding chlorocatechol dioxygenase from the 2,4-dichlorophenoxyacetic acid degradation pathway, was 10(1)-10(2) cells/g soil in inoculated soils. The procedure could be applied to the amplification of biodegradative genes from indigenous microbial populations from a wide variety of soil types, and the entire analysis could be performed within 8 h.

Methanogen community composition and rates of methane consumption in Canadian High Arctic permafrost soils.

Increasing permafrost thaw, driven by climate change, has the potential to result in organic carbon stores being mineralized into carbon dioxide (CO2) and methane (CH4) through microbial activity. This study examines the effect of increasing temperature on community structure and metabolic activity of methanogens from the Canadian High Arctic, in an attempt to predict how warming will affect microbially controlled CH4 soil flux. In situ CO2 and CH4 flux, measured in 2010 and 2011 from ice-wedge polygons, indicate that these soil formations are a net source of CO2 emissions, but a CH4 sink. Permafrost and active layer soil samples were collected at the same sites and incubated under anaerobic conditions at warmer temperatures, with and without substrate amendment. Gas flux was measured regularly and indicated an increase in CH4 flux after extended incubation. Pyrosequencing was used to examine the effects of an extended thaw cycle on methanogen diversity and the results indicate that in situ methanogen diversity, based on the relative abundance of the 16S ribosomal ribonucleic acid (rRNA) gene associated with known methanogens, is higher in the permafrost than in the active layer. Methanogen diversity was also shown to increase in both the active layer and permafrost soil after an extended thaw. This study provides evidence that although High Arctic ice-wedge polygons are currently a sink for CH4, higher arctic temperatures and anaerobic conditions, a possible result of climate change, could result in this soil becoming a source for CH4 gas flux.

A mineralogical characterization of biogenic calcium carbonates precipitated by heterotrophic bacteria isolated from cryophilic polar regions.

Precipitation of calcium carbonate (CaCO3(s) ) can be driven by microbial activity. Here, a systematic approach is used to identify the morphological and mineralogical characteristics of CaCO3(s) precipitated during the heterotrophic growth of micro-organisms isolated from polar environments. Focus was placed on establishing mineralogical features that are common in bioliths formed during heterotrophic activity, while in parallel identifying features that are specific to bioliths precipitated by certain microbial phylotypes. Twenty microbial isolates that precipitated macroscopic CaCO3(s) when grown on B4 media supplemented with calcium acetate or calcium citrate were identified. A multimethod approach, including scanning electron microscopy, high-resolution transmission electron microscopy, and micro-X-ray diffraction (µ-XRD), was used to characterize CaCO3(s) precipitates. Scanning and transmission electron microscopy showed that complete CaCO3(s) crystal encrustation of Arthrobacter sp. cells was common, while encrustation of Rhodococcus sp. cells did not occur. Several euhedral and anhedral mineral formations including disphenoid-like epitaxial plates, rhomboid-like aggregates with epitaxial rhombs, and spherulite aggregates were observed. While phylotype could not be linked to specific mineral formations, isolates tended to precipitate either euhedral or anhedral minerals, but not both. Three anhydrous CaCO3(s) polymorphs (calcite, aragonite, and vaterite) were identified by µ-XRD, and calcite and aragonite were also identified based on TEM lattice-fringe d value measurements. The presence of certain polymorphs was not indicative of biogenic origin, although several mineralogical features such as crystal-encrusted bacterial cells, or casts of bacterial cells embedded in mesocrystals are an indication of biogenic origin. In addition, some features such as the formation of vaterite and bacterial entombment appear to be linked to certain phylotypes. Identifying phylotypes consistent with certain mineralogical features is the first step toward discovering a link between these crystal features and the precise underlying molecular biology of the organism precipitating them.

Fluorescence microscopy as a tool for in situ life detection.

The identification of extant and, in some cases, extinct bacterial life is most convincingly and efficiently performed with modern high-resolution microscopy. Epifluorescence microscopy of microbial autofluorescence or in conjunction with fluorescent dyes is among the most useful of these techniques. We explored fluorescent labeling and imaging of bacteria in rock and soil in the context of in situ life detection for planetary exploration. The goals were two-fold: to target non-Earth-centric biosignatures with the greatest possible sensitivity and to develop labeling procedures amenable to robotic implementation with technologies that are currently space qualified. A wide panel of commercially available dyes that target specific biosignature molecules was screened, and those with desirable properties (i.e., minimal binding to minerals, strong autofluorescence contrast, no need for wash steps) were identified. We also explored the potential of semiconductor quantum dots (QDs) as bacterial and space probes. A specific instrument for space implementation is suggested and discussed.

Utilization of fluorescent microspheres and a green fluorescent protein-marked strain for assessment of microbiological contamination of permafrost and ground ice core samples from the Canadian High Arctic.

Fluorescent microspheres were applied in a novel fashion during subsurface drilling of permafrost and ground ice in the Canadian High Arctic to monitor the exogenous microbiological contamination of core samples obtained during the drilling process. Prior to each drill run, a concentrated fluorescent microsphere (0.5-microm diameter) solution was applied to the interior surfaces of the drill bit, core catcher, and core tube and allowed to dry. Macroscopic examination in the field demonstrated reliable transfer of the microspheres to core samples, while detailed microscopic examination revealed penetration levels of less than 1 cm from the core exterior. To monitor for microbial contamination during downstream processing of the permafrost and ground ice cores, a Pseudomonas strain expressing the green fluorescent protein (GFP) was painted on the core exterior prior to processing. Contamination of the processed core interiors with the GFP-expressing strain was not detected by culturing the samples or by PCR to detect the gfp marker gene. These methodologies were quick, were easy to apply, and should help to monitor the exogenous microbiological contamination of pristine permafrost and ground ice samples for downstream culture-dependent and culture-independent microbial analyses.

Biodegradation of petroleum hydrocarbons by psychrotrophic Pseudomonas strains possessing both alkane (alk) and naphthalene (nah) catabolic pathways.

Three hydrocarbon-degrading psychrotrophic bacteria were isolated from petroleum-contaminated Arctic soils and characterized. Two of the strains, identified as Pseudomonas spp., degraded C5 to C12 n-alkanes, toluene, and naphthalene at both 5 and 25 degrees C and possessed both the alk catabolic pathway for alkane biodegradation and the nah catabolic pathway for polynuclear aromatic hydrocarbon biodegradation. One of these strains contained both a plasmid slightly smaller than the P. oleovorans OCT plasmid, which hybridized to an alkB gene probe, and a NAH plasmid similar to NAH7, demonstrating that both catabolic pathways, located on separate plasmids, can naturally coexist in the same bacterium.

Assessment of the biodegradation potential of psychrotrophic microorganisms.

Bioremediation of polluted temperate and cold temperature environments may require the activity of psychrotrophic bacteria, because their low temperature growth range parallels the ambient temperatures encountered in these environments. In the present study, 135 psychrotrophic microorganisms isolated from a variety of ecosystems in Canada were examined for their ability to mineralize 14C-labelled toluene, naphthalene, dodecane, hexadecane, 2-chlorobiphenyl, and pentachlorophenol. A number of the psychrotrophic strains mineralized toluene, naphthalene, dodecane, and hexadecane. None of the psychrotrophs were capable of mineralizing 2-chlorobiphenyl or pentachlorophenol. Those strains demonstrating mineralization activity were subsequently screened by the polymerase chain reaction (PCR) and Southern hybridization of PCR products for the presence of catabolic genes (alkB, ndoB, todCl, and xylE) involved in known bacterial biodegradative pathways for these compounds. Some of the psychrotrophs able to mineralize toluene and naphthalene possessed catabolic genes that hybridized to xylE or todCl, and ndoB, respectively. The alkB PCR fragments obtained from the strains that mineralized dodecane and hexadecane did not hybridize to an alkB gene probe derived from Pseudomonas oleovorans. Psychrotrophic strain Q15, identified as a Rhodococcus sp., also mineralized the C28 n-paraffin octacosane. A gene probe constructed from the "alkB" PCR fragment from strain Q15 did hybridize with the alkB PCR fragments from most of the psychrotrophic alkane biodegraders, indicating that the alkB primers may be amplifying another gene(s), perhaps with low homology to P. oleovorans alkB, which may be involved in the biodegradation of both short chain (dodecane) and longer chain alkanes (hexadecane, octacosane). All of the psychrotrophic biodegradative isolates examined were capable of mineralization activity at both 23 and 5 degrees C, indicating their potential for low temperature bioremediation of petroleum hydrocarbon contaminated sites.

Characterization of hydrocarbon-degrading microbial populations in contaminated and pristine Alpine soils.

Biodegradation of petroleum hydrocarbons in cold environments, including Alpine soils, is a result of indigenous cold-adapted microorganisms able to degrade these contaminants. In the present study, the prevalence of seven genotypes involved in the degradation of n-alkanes (Pseudomonas putida GPo1 alkB; Acinetobacter spp. alkM; Rhodococcus spp. alkB1, and Rhodococcus spp. alkB2), aromatic hydrocarbons (P. putida xylE), and polycyclic aromatic hydrocarbons (P. putida ndoB and Mycobacterium sp. strain PYR-1 nidA) was determined in 12 oil-contaminated (428 to 30,644 mg of total petroleum hydrocarbons [TPH]/kg of soil) and 8 pristine Alpine soils from Tyrol (Austria) by PCR hybridization analyses of total soil community DNA, using oligonucleotide primers and DNA probes specific for each genotype. The soils investigated were also analyzed for various physical, chemical, and microbiological parameters, and statistical correlations between all parameters were determined. Genotypes containing genes from gram-negative bacteria (P. putida alkB, xylE, and ndoB and Acinetobacter alkM) were detected to a significantly higher percentage in the contaminated (50 to 75%) than in the pristine (0 to 12.5%) soils, indicating that these organisms had been enriched in soils following contamination. There was a highly significant positive correlation (P < 0.001) between the level of contamination and the number of genotypes containing genes from P. putida and Acinetobacter sp. but no significant correlation between the TPH content and the number of genotypes containing genes from gram-positive bacteria (Rhodococcus alkB1 and alkB2 and Mycobacterium nidA). These genotypes were detected at a high frequency in both contaminated (41.7 to 75%) and pristine (37.5 to 50%) soils, indicating that they are already present in substantial numbers before a contamination event. No correlation was found between the prevalence of hydrocarbon-degradative genotypes and biological activities (respiration, fluorescein diacetate hydrolysis, lipase activity) or numbers of culturable hydrocarbon-degrading soil microorganisms; there also was no correlation between the numbers of hydrocarbon degraders and the contamination level. The measured biological activities showed significant positive correlation with each other, with the organic matter content, and partially with the TPH content and a significant negative correlation with the soil dry-mass content (P < 0.05 to 0.001).

A survey of indigenous microbial hydrocarbon degradation genes in soils from Antarctica and Brazil.

Total community DNA from 29 noncontaminated soils and soils impacted by petroleum hydrocarbons and chloro-organics from Antarctica and Brazil were screened for the presence of nine catabolic genes, encoding alkane monooxygenase or aromatic dioxygenases, from known bacterial biodegradation pathways. Specific primers and probes targeting alkane monooxygenase genes were derived from Pseudomonas putida ATCC 29347 (Pp alkB), Rhodococcus sp. strain Q15 (Rh alkB1, Rh alkB2), and Acinetobacter sp. ADP-1 (Ac alkM). In addition, primers and probes detecting aromatic dioxygenase genes were derived from P. putida ATCC 17484 (ndoB), P. putida F1 (todC1), P. putida ATCC 33015 (xylE and cat23), and P. pseudoalcaligenes KF707 (bphA). The primers and probes were used to analyze total community DNA extracts by using PCR and hybridization analysis. All the catabolic genes, except the Ac alkM, were detected in contaminated and control soils from both geographic regions, with a higher frequency in the Antarctic soils. The alkane monooxygenase genes, Rh alkB1 and Rh alkB2, were the most frequently detected alk genes in both regions, while Pp alkB was not detected in Brazil soils. Genes encoding the aromatic dioxygenases toluene dioxygenase (todC1) and biphenyl dioxygenase (bphA) were the most frequently detected in Antarctica, and todC1 and catechol-2,3-dioxygenase (cat23) were the most frequent in Brazil soils. Hybridization analysis confirmed the PCR results, indicating that the probes used had a high degree of homology to the genes detected in the soil extracts and were effective in detecting biodegradative potential in the indigenous microbial population.

Prevalence of alkane monooxygenase genes in Arctic and Antarctic hydrocarbon-contaminated and pristine soils.

Abstract The prevalence of four alkane monooxygenase genotypes (Pseudomonas putida GPo1, Pp alkB; Rhodococcus sp. strain Q15, Rh alkB1 and Rh alkB2; and Acinetobacter sp. strain ADP-1, Ac alkM) in hydrocarbon-contaminated and pristine soils from the Arctic and Antarctica were determined by both culture-independent (PCR hybridization analyses) and culture-dependent (colony hybridization analyses) molecular methods, using oligonucleotide primers and DNA probes specific for each of the alk genotypes. PCR hybridization of total soil community DNA detected the rhodococcal alkB genotypes in most of the contaminated (Rh alkB1, 18/20 soils; Rh alkB2, 13/20) and many pristine soils (Rh alkB1, 9/10 soils; Rh alkB2, 7/10), while Pp alkB was generally detected in the contaminated soils (15/20) but less often in pristine soils (5/10). Ac alkM was rarely detected in the soils (1/30). The colony hybridization technique was used to determine the prevalence of each of the alk genes and determine their relative abundance in culturable cold-adapted (5 degrees C) and mesophilic populations (37 degrees C) from eight of the polar soils. The cold-adapted populations, in general, possessed relatively higher percentages of the Rh alkB genotypes (Rh alkB1, 1.9% (0.55); Rh alkB2, 2.47% (0.89)), followed by the Pp alkB (1.13% (0.50)), and then the Ac alkM (0.53% (0.36)). The Rh alkB1 genotype was clearly more prevalent in culturable cold-adapted bacteria (1.9% (0.55)) than in culturable mesophiles (0.41 (0.55)), suggesting that cold-adapted bacteria are the predominant organisms possessing this genotype. Overall, these results indicated that (i) Acinetobacter spp. are not predominant members of polar alkane degradative microbial communities, (ii) Pseudomonas spp. may become enriched in polar soils following contamination events, and (iii) Rhodococcus spp. may be the predominant alkane-degradative bacteria in both pristine and contaminated polar soils.

Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp.

The psychorotrophic Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5 degrees C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C10 to C21 alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5 degrees C. Mineralization of hexadecane at 5 degrees C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-dodecanol and 2-dodecanone, respectively) by solid-phase microextraction-gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis the A gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25 degrees C.

Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531.

The alkane hydroxylase systems of two Rhodococcus strains (NRRL B-16531 and Q15, isolated from different geographical locations) were characterized. Both organisms contained at least four alkane monooxygenase gene homologs (alkB1, alkB2, alkB3, and alkB4). In both strains, the alkB1 and alkB2 homologs were part of alk gene clusters, each encoding two rubredoxins (rubA1 and rubA2; rubA3 and rubA4), a putative TetR transcriptional regulatory protein (alkU1; alkU2), and, in the alkB1 cluster, a rubredoxin reductase (rubB). The alkB3 and alkB4 homologs were found as separate genes which were not part of alk gene clusters. Functional heterologous expression of some of the rhodococcal alk genes (alkB2, rubA2, and rubA4 [NRRL B-16531]; alkB2 and rubB [Q15]) was achieved in Escherichia coli and Pseudomonas expression systems. Pseudomonas recombinants containing rhodococcal alkB2 were able to mineralize and grow on C(12) to C(16) n-alkanes. All rhodococcal alkane monooxygenases possessed the highly conserved eight-histidine motif, including two apparent alkane monooxygenase signature motifs (LQRH[S/A]DHH and NYXEHYG[L/M]), and the six hydrophobic membrane-spanning regions found in all alkane monooxygenases related to the Pseudomonas putida GPo1 alkane monooxygenase. The presence of multiple alkane hydroxylases in the two rhodococcal strains is reminiscent of other multiple-degradative-enzyme systems reported in Rhodococcus.