Mar1, a high mobility group box protein, regulates n-alkane adsorption and cell morphology of the dimorphic yeast Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica possesses an excellent ability to utilize n-alkane as a sole carbon and energy source. Although there are detailed studies on the enzymes that catalyze the reactions in the metabolic processes of n-alkane in Y. lipolytica, the molecular mechanism underlying the incorporation of n-alkane into the cells remains to be elucidated. Because Y. lipolytica adsorbs n-alkane, we postulated that Y. lipolytica incorporates n-alkane through direct interaction with it. We isolated and characterized mutants defective in adsorption to n-hexadecane. One of the mutants harbored a nonsense mutation in MAR1 (Morphology and n-alkane Adsorption Regulator 1) encoding a protein containing a high mobility group box. The deletion mutant of MAR1 exhibited defects in adsorption to n-hexadecane and filamentous growth on solid media, whereas the strain that overexpressed MAR1 exhibited hyperfilamentous growth. Fluorescence microscopic observations suggested that Mar1 localizes in the nucleus. RNA-sequencing analysis revealed the alteration of the transcript levels of several genes, including those encoding transcription factors and cell surface proteins, by the deletion of MAR1. These findings suggest that MAR1 is involved in the transcriptional regulation of the genes required for n-alkane adsorption and cell morphology transition.IMPORTANCEYarrowia lipolytica, a dimorphic yeast capable of assimilating n-alkane as a carbon and energy source, has been extensively studied as a promising host for bioconversion of n-alkane into useful chemicals and bioremediation of soil and water contaminated by petroleum. While the metabolic pathway of n-alkane in this yeast and the enzymes involved in this pathway have been well characterized, the molecular mechanism to incorporate n-alkane into the cells is yet to be fully understood. Due to the ability of Y. lipolytica to adsorb n-alkane, it has been hypothesized that Y. lipolytica incorporates n-alkane through direct interaction with it. In this study, we identified a gene, MAR1, which plays a crucial role in the transcriptional regulation of the genes necessary for the adsorption to n-alkane and the transition of the cell morphology in Y. lipolytica. Our findings provide valuable insights that could lead to advanced applications of Y. lipolytica in n-alkane bioconversion and bioremediation.

AlmA involved in the long-chain n-alkane degradation pathway in Acinetobacter baylyi ADP1 is a Baeyer-Villiger monooxygenase.

Many Acinetobacter species can grow on n-alkanes of varying lengths (≤C40). AlmA, a unique flavoprotein in these Acinetobacter strains, is the only enzyme proven to be required for the degradation of long-chain (LC) n-alkanes, including C32 and C36 alkanes. Although it is commonly presumed to be a terminal hydroxylase, its role in n-alkane degradation remains elusive. In this study, we conducted physiological, biochemical, and bioinformatics analyses of AlmA to determine its role in n-alkane degradation by Acinetobacter baylyi ADP1. Consistent with previous reports, gene deletion analysis showed that almA was vital for the degradation of LC n-alkanes (C26-C36). Additionally, enzymatic analysis revealed that AlmA catalyzed the conversion of aliphatic 2-ketones (C10-C16) to their corresponding esters, but it did not conduct n-alkane hydroxylation under the same conditions, thus suggesting that AlmA in strain ADP1 possesses Baeyer-Villiger monooxygenase (BVMO) activity. These results were further confirmed by bioinformatics analysis, which revealed that AlmA was closer to functionally identified BVMOs than to hydroxylases. Altogether, the results of our study suggest that LC n-alkane degradation by strain ADP1 possibly follows a novel subterminal oxidation pathway that is distinct from the terminal oxidation pathway followed for short-chain n-alkane degradation. Furthermore, our findings suggest that AlmA catalyzes the third reaction in the LC n-alkane degradation pathway.IMPORTANCEMany microbial studies on n-alkane degradation are focused on the genes involved in short-chain n-alkane (≤C16) degradation; however, reports on the genes involved in long-chain (LC) n-alkane (>C20) degradation are limited. Thus far, only AlmA has been reported to be involved in LC n-alkane degradation by Acinetobacter spp.; however, its role in the n-alkane degradation pathway remains elusive. In this study, we conducted a detailed characterization of AlmA in A. baylyi ADP1 and found that AlmA exhibits Baeyer-Villiger monooxygenase activity, thus indicating the presence of a novel LC n-alkane biodegradation mechanism in strain ADP1.

Phosphatidylserine synthase plays a critical role in the utilization of n-alkanes in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica can assimilate n-alkane as a carbon and energy source. To elucidate the significance of phosphatidylserine (PS) in the utilization of n-alkane in Y. lipolytica, we investigated the role of the Y. lipolytica ortholog (PSS1) of Saccharomyces cerevisiae PSS1/CHO1, which encodes a PS synthase. The PSS1 deletion mutant (pss1Δ) of Y. lipolytica could not grow on minimal medium in the absence of ethanolamine and choline but grew when either ethanolamine or choline was supplied to synthesize phosphatidylethanolamine and phosphatidylcholine. The pss1Δ strain exhibited severe growth defects on media containing n-alkanes even in the presence of ethanolamine and choline. In the pss1Δ strain, the transcription of ALK1, which encodes a primary cytochrome P450 that catalyses the hydroxylation of n-alkanes in the endoplasmic reticulum, was upregulated by n-alkane as in the wild-type strain. However, the production of functional P450 was not detected, as indicated by the absence of reduced CO-difference spectra in the pss1Δ strain. PS was undetectable in the lipid extracts of the pss1Δ strain. These results underscore the critical role of PSS1 in the biosynthesis of PS, which is essential for the production of functional P450 enzymes involved in n-alkane hydroxylation in Y. lipolytica.

Seasonal variations in the distribution of aliphatic hydrocarbons in surface sediments from the Selangor River, Peninsular Malaysia's West Coast.

The seasonal variation of petroleum pollution including n-alkanes in surface sediments of the Selangor River in Malaysia during all four climatic seasons was investigated using GC-MS. The concentrations of n-alkanes in the sediment samples did not significantly correlate with TOC (r = 0.34, p > 0.05). The concentrations of the 29 n-alkanes in the Selangor River ranged from 967 to 3711 µg g-1 dw, with higher concentrations detected during the dry season. The overall mean per cent of grain-sized particles in the Selangor River was 85.9 ± 2.85% sand, 13.5 ± 2.8% clay, and 0.59 ± 0.34% gravel, respectively. n-alkanes are derived from a variety of sources, including fresh oil, terrestrial plants, and heavy/degraded oil in estuaries. The results of this study highlight concerns and serve as a warning that hydrocarbon contamination is affecting human health. As a result, constant monitoring and assessment of aliphatic hydrocarbons in coastal and riverine environments are needed.

Homologue Composition of Technical Chlorinated Paraffins Used in Several Countries over the Last 50 Years─SCCPs Are Still Out There.

Chlorinated paraffins (CPs) are widely produced chemicals, with certain CP subgroups facing global restrictions due to their environmental dispersion, persistence, bioaccumulation, and toxicity. To evaluate the effectiveness of these international restrictions, we assessed the homologue group contribution and the mass fraction of short-chain CPs (SCCPs: C10-C13), medium-chain CPs (MCCPs: C14-C17), and long-chain CPs (LCCPs: ≥C18) in 36 technical CP mixtures used worldwide over the last 50 years. Using low-resolution mass spectrometry (LC-ESI-MS/MS), we quantified 74 CP homologue groups (C10Cl4-C20Cl10). Additionally, high-resolution mass spectrometry (LC-ESI-QTOF-MS) screening was employed to identify unresolved CP contents, covering 375 CP homologue groups (C6Cl4-C30Cl30). Overall, 1 sample was mainly composed of

Chemical Composition of Essential Oil from Mosses from the Brazilian Atlantic Forest.

This study aimed to report the unprecedented volatile composition of the mosses Phyllogonium viride BRID, Orthotichella rigida (MÜLL.HAL.) B. H. ALLEN & MAGILL and Schlotheimia rugifolia (HOOK.) SCHWÄGR occurring in the Brazilian Atlantic Forest, in order to elucidate the chemical composition of these species and enrich the chemotaxonomic knowledge of mosses. 28 compounds were identified, the major constituent being hexadecanoic acid, also known as palmitic acid, specifically P. viride com (38.55 %), O. rigida com (17.17 %) and S. rugifolia com (24.94 %), followed by phytol, P. viride com (3.92 %), O. rigida com (28.57 %) and S. rugifolia com (36.13 %). In addition, there was a prevalence of aliphatic hydrocarbons (25 %) and fatty acids (17.8 %) in the evaluated samples. These data contribute to the generation of new scientific information about the chemical constitution of mosses, still little studied, enriching the chemotaxonomic collection of the taxon.

Short-term microbial effects on n-alkane during the early phase degradation and consequential modification of biomarkers in a lowland subtropical rainforest in southern Taiwan: A litterbag experiment.

Accurate reconstructions of past environments are critical and urgent because they can help understand how modern environments might respond to current climatic and land-use changes. However, the effect of microbial degradation and consequential modification in plant-derived-biomarkers during the early degradation phase is not yet apparent, that might bias the paleoenvironmental investigation. In this regard, a litterbag experiment was conducted to reveal the microbial effects on n-alkane-associated biomarker changes associated with three habitats (ravine, windward, and leeward) in a lowland subtropical rainforest in southern Taiwan. Freshly collected leaves of plant species Iles rotunda, Ficus benjamina, and Castanopsis carlesii were distributed in the habitat leaf litterbag experiment for 15 and 75 days incubation, respectively. The results revealed that the average leaf decomposition rate was 19.4% ± 6.4% during the first 15 days and 39% ± 11% within 75 days incubation for all leaves. The overall leaf mass degradation of I. rotunda, F. benjamina and C. carlesii in the ravine after 75 days was 58%, 51% and 41%, respectively, which were higher than those in the windward (28%, 36% and 38%) and leeward habitats (35%, 26% and 42%, respectively) indicating higher decomposition rate in the ravine habitat than the others. The predominant n-alkanes in I. rotunda were C31 and C29, whereas in F. benjamina these were C31, C29, and C33, and in C. carlesii it was C31. After 75 days, the ravine habitat showed a 60% decrease in the total n-alkane concentration compared to windward and leeward habitats, suggesting the microbial community associated with the ravine habitat has a higher efficiency of degrading n-alkanes. However, the biomarkers such as carbon preference index (CPI), average carbon length (ACL) and the C31/C29 ratio did not show statistical difference in all habitats from 15 to 75 days incubation. The next-generation sequencing revealed that microbial communities changed significantly from 15 to 75 days in all habitats. The alkB gene-containing bacteria and their family lineages increased substantially during the first 15 days incubation in all habitats. Furthermore, several bacterial genera were exclusively present in the ravine habitat, whereas some were only in the leeward and windward habitats. Despite the heterogeneity of microbial proliferation, difference in biomass and n-alkane degradation among the three habitats, most of the n-alkane-associated biomarkers remained the same. Therefore, we concluded that the microbial effects on n-alkane degradation during the early phase in plant leaves had little influence on the results of most n-alkane biomarkers.

Utilization of n-alkane and roles of lipid transfer proteins in Yarrowia lipolytica.

Yarrowia lipolytica, a dimorphic yeast belonging to the Ascomycota, has potent abilities to utilize hydrophobic compounds, such as n-alkanes and fatty acids, as carbon and energy sources. Yarrowia lipolytica can synthesize and accumulate large amounts of lipids, making it a promising host to produce various lipids and convert n-alkanes to useful compounds. For advanced use of Y. lipolytica in these applications, it is necessary to understand the metabolism of these hydrophobic compounds in this yeast and the underlying molecular mechanisms. In this review, current knowledge on the n-alkane metabolism and how this is regulated in Y. lipolytica is summarized. Furthermore, recent studies revealed that lipid transfer proteins are involved in the utilization of n-alkanes and the regulation of cell morphology in response to n-alkanes. This review discusses the roles of membrane lipids in these processes in Y. lipolytica.

Orthologs of Saccharomyces cerevisiae SFH2, genes encoding Sec14 family proteins, implicated in utilization of n-alkanes and filamentous growth in response to n-alkanes in Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica has an ability to assimilate n-alkanes as carbon and energy sources. In this study, the roles of orthologs of Saccharomyces cerevisiae SEC14 family gene SFH2, which we named SFH21, SFH22, SFH23 and SFH24, of Y. lipolytica were investigated. The transcript levels of SFH21, SFH22 and SFH23, determined by RNA-seq analysis, qRT-PCR analysis and northern blot analysis, were found to increase in the presence of n-alkanes. The deletion mutant of SFH21, but not that of SFH22, SFH23 or SFH24, showed defects in growth in the media containing n-alkanes and in filamentous growth on the solid media containing n-alkanes. Additional deletions of SFH22 and SFH23 significantly exaggerated the defect in filamentous growth of the deletion mutant of SFH21, and expression of SFH22 or SFH24 using the SFH21 promoter partially suppressed the growth defect of the deletion mutant of SFH21 on n-alkanes. These results suggest that SFH2 orthologs are involved in the utilization of n-alkanes and filamentous growth in response to n-alkanes in Y. lipolytica.

Degradation of long-chain n-alkanes by a novel thermal-tolerant Rhodococcus strain.

A novel bacterial strain, CH91, was isolated from a high-temperature oil reservoir. Morphological characterization, phylogenetic analyses of 16S rRNA gene sequence and genome relatedness indicated that the strain is a potential new species in the genus Rhodococcus. Strain CH91 could grow in the temperature range of 25-50 °C (optimally at 37 °C) and utilize a broad range of long-chain n-alkanes from hexadecane to hexatriacontane. The utilization of the n-alkanes mixture of strain CH91 revealed that the degradation rate was correlated to the length of the carbon chain. Two novel alkB genes encoding alkane 1-monooxygenase were found in the genome of this strain. The protein sequences of both alkane 1-monooxygenases showed a remarkable phylogenetic distance to other reported AlkB protein sequences. These results would help broaden our knowledge about alkane degradation by Rhodocuccus and its potential ecological role. The ability of the strain in the long-chain alkane degradation and thermal tolerance could also be further exploited for bioremediation of oil contaminations and microbial enhanced oil recovery.

Unravelling the role of GntR on the regulation of alkane hydroxylase AlkB2 in Pseudomonas aeruginosa DN1 based on transcriptome analysis.

AIMS: The purpose of this study is to acquire a comprehensive understanding of the involvement of the gene alkB2 in alkane degradation. METHODS AND RESULTS: The changes of gene expression in the wild-type and alkB2 knockout strains of Pseudomonas aeruginosa DN1 were characterized based on transcriptional profiling, when grown in a medium containing eicosane (C20 n-alkane) as the sole carbon source. Compared to wild-type, approximately 7% of the genes in the knockout mutant was significantly differentially expressed, including 344 upregulated genes and 78 downregulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that numerous differentially expressed genes (DEGs) were potentially associated with degradation or physiological response to n-alkane, including genes encoding methyl-accepting chemotaxis proteins (MCPs), an outer membrane fatty acid transport protein (FadL), a membrane receptor protein (FptA), oprin and transcriptional regulators. Notably, the transcriptional regulator gene gntR (RS18845) located upstream of alkB2 (RS18850) was upregulated. The possible regulatory function of this transcriptional regulator on alkB2 was investigated using a gene knockout approach and quantitative reverse transcriptase PCR (RT-qPCR) combined with electrophoretic mobility shift assay (EMSA) experiments. The RT-qPCR results showed that in the gntR mutant, alkB2 expression was independent of the presence of eicosane, while its expression was significantly induced by the substrate when GntR was produced. Based on the EMSA analysis, the palindromic DNA motif 5'-ATTGTCAGACAAT-3' was verified as being recognized by GntR, and two copies of GntR were able to bind this sequence. However, the interaction between GntR and DNA was altered in the presence of eicosane, suggesting that GntR could bind with eicosane to regulate the expression of alkB2 . CONCLUSION: These findings indicate that GntR plays a key role in the transcriptional regulation of alkB2 , which affects the degradation of C20 n-alkane in P. aeruginosa DN1. SIGNIFICANCE AND IMPACT OF THE STUDY: This report presents insights into the significance of GntR in the regulation of alkane degradation by alkB2 , and increases our understanding of the complex regulatory network involved in alkane degradation.

In vitro human cell-based TTR-TRß CALUX assay indicates thyroid hormone transport disruption of short-chain, medium-chain, and long-chain chlorinated paraffins.

Over the last decades, short-chain chlorinated paraffins (SCCPs), medium-chain chlorinated paraffins (MCCPs), and long-chain chlorinated paraffins (LCCPs) have become the most heavily produced monomeric organohalogen compound class of environmental concern. However, knowledge about their toxicology is still scarce, although SCCPs were shown to have effects on the thyroid hormone system. The lack of data in the case of MCCPs and LCCPs and the structural similarity with perfluoroalkyl substances (PFAS) prompted us to test CPs in the novel TTR-TR CALUX assay for their thyroid hormone transport disrupting potential. Four self-synthesized and additionally purified single chain length CP mixtures (C10-CPs, C11-CPs, C14-CPs and C16-CPs) and two each of industrial MCCP and LCCP products were tested in parallel with PFOA. All CP mixtures influenced the TTR binding of T4, giving activities of 1,300 to 17,000 µg/g PFOA equivalents and lowest observable effect concentrations (LOELs) of 0.95 to 0.029 mM/L incubate. Highest activities and lowest LOELs were observed for C16-CPs (48.3% Cl content, activity 17,000, LOEL 0.047 mM/L) and a LCCP mixture (71.7% Cl content; activity 10,000; LOEL 0.029 mM/L). A trend of higher activities and lower LOELs towards longer chains and higher chlorination degrees was implied, but could not be statistically confirmed. Irrespectively, the less well examined and current-use LCCPs showed the highest response in the TTR-TRß CALUX assay.

Relative contributions of selected multigeneration products to chamber SOA formed from photooxidation of a range (C10-C17) of n-alkanes under high NO x conditions.

A series of chamber experiments was conducted to investigate the composition of secondary organic aerosol (SOA) following oxidation of a range of parent n-alkanes (C10-C17) in the presence of NO x . The relative contribution of selected species representing first, second, and higher generation products to SOA mass was measured using a high-resolution aerosol mass spectrometer. Gas chromatography was also used for a limited set of amenable species. Relative contributions varied substantially across the range of investigated alkanes reflecting slight changes in SOA composition. The contribution of first-generation cyclic hemiacetal is minimal toward the small end of the investigated range and gradually increase with n-alkane size. The relative contribution of second generation and higher nitrate-containing species, in contrast, decrease with an increased alkane size. A similar trend is observed for relative contribution of organonitrates to SOA. Finally, SOA yield and composition are sensitive to water vapor concentrations. This sensitivity is limited to a narrow range (dry to ~15% RH) with little, if any, impact above 15% suggesting that this impact may be negligible under ambient conditions. The impact of water vapor also appears to decrease with increasing alkane carbon number.

Chlorinated paraffins in hinges of kitchen appliances.

Chlorinated paraffins (CPs) are anthropogenic pollutants of growing environmental concern. These highly complex mixtures of thousands of homologs and congeners are usually applied as additives in lubricants or as flame retardants and plasticizers in polymers and paints. Recent studies indicated the presence of high amounts of CPs in the kitchen environment whose sources could not be unequivocally identified. One option was the use of CPs as or in lubricants of hinges. To test this hypothesis, we performed wipe tests on lubricants on 29 hinges of different types of kitchen appliances (refrigerators, baking ovens, dishwashers, freezers, microwave oven, pasta machine, food processor, steam cooker) and analyzed them for short-chain CPs (SCCPs) and medium-chain CPs (MCCPs). CPs were detected in 21 samples (72%). Per wipe, SCCP concentrations ranged between 0.02 and 10 µg (median 0.23 µg), while MCCPs ranged from 0.09 to 750 µg (median 1.0 µg). Highest MCCP amounts (380 and 750 µg per wipe, respectively) were determined in new and unused appliances. A medium correlation between SCCP content and appliance age was observed, but no additional statistic correlation between SCCP/MCCP amount and appliance type or manufacturer could be observed. CPs released from hinges by volatilization, abrasion, and cleaning processes could enter the environment and come in contact with persons living in the corresponding households.

Efficient newton-raphson/singular value decomposition-based optimization scheme with dynamically updated critical condition number for rapid convergence of weighted histogram analysis method equations.

Selection of the successful optimization strategy is an essential part of solving numerous practical problems yet often is a nontrivial task, especially when a function to be optimized is multidimensional and involves statistical data. Here we propose a robust optimization scheme, referred to as NR/SVD-Cdyn, which is based on a combination of the Newton-Raphson (NR) method along with singular value decomposition (SVD), and demonstrate its performance by numerically solving a system of the weighted histogram analysis method equations. Our results show significant improvement over the direct iteration and conventional NR optimization methods. The proposed scheme is universal and could be used for solving various optimization problems in the field of computational chemistry such as parameter fitting for the methods of molecular mechanics and semiempirical quantum-mechanical methods. © 2019 Wiley Periodicals, Inc.

Elucidation of multiple alkane hydroxylase systems in biodegradation of crude oil n-alkane pollution by Pseudomonas aeruginosa DN1.

AIMS: The purpose of this study was to elucidate the characteristics of multiple alkane hydroxylase systems in Pseudomonas aeruginosa DN1, including two homologues of AlkB (AlkB1 and AlkB2 ), a CYP153 homologue (P450), and two homologues of Alm-like (AlmA1 and AlmA2 ). METHODS AND RESULTS: DN1 was capable of utilizing diverse n-alkanes with chain lengths from 8 to 40 C atoms as the sole carbon source, and displayed high degradation efficiency (＞85%) of crude oil and a majority of n-alkanes using gas chromatography method. RT-qPCR analysis showed that the five enzyme genes could be induced by n-alkanes ranging from medium-chain length to long-chain length which indicated the dissimilarity of expression between those genes when grown on different n-alkanes. Notably, the expression of alkB2 gene was upregulated in the presence of all of the tested n-alkanes, particularly responded to long-chain n-alkanes like C20 and C32 . Meanwhile, long-chain n-alkanes (C20 -C36 ) significantly elevated cyp153 expression level, and the expression of two almA genes was only upregulated in the presence of n-alkanes with chain lengths of 20C's and longer. Furthermore, the disruption of those genes demonstrated that AlkB2 appeared to play a key role in the biodegradation of substrates of a broad-chain length ranges, besides other alkane hydroxylase systems ensured the utilization of n-alkanes with chain lengths of from 20 to 40 C atoms. CONCLUSION: The five functional alkane hydroxylase genes make DN1 an attractive option for its versatile alkane degradation, which is primarily dependent on the expression of alkB2 . SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggest that P. aeruginosa DN1 is a predominately potential long-chain n-alkane-degrading bacterium with multiple alkane hydroxylase systems in crude oil-contaminated environment.

Source tracing of n-alkanes in Songhua Lake, based on correspondence analysis and geochemical index.

This paper analyzes the source of n-alkanes in sediments spanning 63 years since 1943 from Songhua Lake, using the method of correspondence analysis and geochemical index. The area of Songhua Lake is 554 km2, and its ecological problems affect hundreds of thousands of people in the basin. The concentration of total n-alkanes (C13-C36) in the sediments ranged from 20.39 to 168.35 µg/g, averaged at 72.58 µg/g, indicating that the Songhua Lake has an ecological risk of n-alkanes and should be taken seriously and protected. The analysis of geochemical indexes, including main carbon distributions (MH), LMW/HMW ratio, Paq, average chain length and so on, shows the n-alkanes in the Songhua Lake sediments are mainly derived from modern higher terrestrial plants, followed by human activities, and a small part from aquatic vascular plants and algae. Moreover, the source and sedimentary history of n-alkanes in Songhua Lake were carried out by corresponding analysis. And the correspondence analysis could be a feasible and effective means of source tracing.

Osh6p, a homologue of the oxysterol-binding protein, is involved in production of functional cytochrome P450 belonging to CYP52 family in n-alkane-assimilating yeast Yarrowia lipolytica.

In this study, we investigated the role of OSH6, which encodes a homolog of the oxysterol-binding protein, in the assimilation of n-alkanes in the yeast Yarrowia lipolytica. The deletion mutant of OSH6 showed growth defects on n-alkanes of 10-16 carbons. In the deletion mutant, production of the functional cytochrome P450 was not observed. However, transcription of ALK1, encoding a major P450 belonging to the CYP52 family that plays a critical role in n-alkane hydroxylation, and further translation of its transcript were noted in the deletion mutant as well as in the wild-type strain. The phospholipid composition was altered and, the ratio of phosphatidylserine (PS) was reduced by the deletion of OSH6. Residues involved in the transport of PS and phosphatidylinositol-4-phosphate in Osh6 of Saccharomyces cerevisiae are conserved in Y. lipolytica Osh6p and substitutions of these residues resulted in a defect in the n-alkane assimilation by Y. lipolytica. From these results, we propose a hypothesis that Osh6p provides an ideal endoplasmic reticulum membrane environment for Alk proteins to have a functional conformation via lipid transport activity in Y. lipolytica.

Ketobacter alkanivorans gen. nov., sp. nov., an n-alkane-degrading bacterium isolated from seawater.

Strain GI5T was isolated from a surface seawater sample collected from Garorim Bay (West Sea, Republic of Korea). The isolated strain was aerobic, Gram-stain-negative, rod-shaped, motile by means of a polar flagellum, negative for catalase and weakly positive for oxidase. The optimum growth pH, salinity and temperature were determined to be pH 7.5-8.0, 3 % NaCl (w/v) and 25 °C, respectively; the growth ranges were pH 6.0-9.0, 1-7 % NaCl (w/v) and 18-40 °C. The results of phylogenetic analysis of 16S rRNA gene sequences indicated that GI5T clustered within the family Alcanivoracaceae, and most closely with Alcanivorax dieseloleiB-5T and Alcanivorax marinusR8-12T (91.9 % and 91.6 % similarity, respectively). The major cellular fatty acids in GI5T were C18 : 1ω7c/C18 : 1ω6c (44.45 %), C16 : 1ω6c/C16 : 1ω7c (14.17 %) and C16 : 0 (10.19 %); this profile was distinct from those of the closely related species. The major respiratory quinone of GI5T was Q-8. The main polar lipids were phosphatidylethanolamine and phosphatidylglycerol. Two putative alkane hydroxylase (alkB) genes were identified in GI5T. The G+C content of the genomic DNA of GI5T was determined to be 51.2 mol%. On the basis of the results of phenotypic, chemotaxonomic and phylogenetic studies, strain GI5T represents a novel species of a novel genus of the family Alcanivoracaceae, for which we propose the name Ketobacter alkanivorans gen. nov., sp. nov.; the type strain is GI5T (=KCTC 52659T=JCM 31835T).

Bioprospecting for Genes Encoding Hydrocarbon-Degrading Enzymes from Metagenomic Samples Isolated from Northern Adriatic Sea Sediments.

Three metagenomic libraries were constructed using surface sediment samples from the northern Adriatic Sea. Two of the samples were taken from a highly polluted and an unpolluted site respectively. The third sample from a polluted site had been enriched using crude oil. The results of the metagenome analyses were incorporated in the REDPET relational database (http://redpet.bioinfo.pbf.hr/REDPET), which was generated using the previously developed MEGGASENSE platform. The database includes taxonomic data to allow the assessment of the biodiversity of metagenomic libraries and a general functional analysis of genes using hidden Markov model (HMM) profiles based on the KEGG database. A set of 22 specialised HMM profiles was developed to detect putative genes for hydrocarbon-degrading enzymes. Use of these profiles showed that the metagenomic library generated after selection on crude oil had enriched genes for aerobic n-alkane degradation. The use of this system for bioprospecting was exemplified using potential alkB and almA genes from this library.