Two novel alkane hydroxylase-rubredoxin fusion genes isolated from a Dietzia bacterium and the functions of fused rubredoxin domains in long-chain n-alkane degradation

Two alkane hydroxylase-rubredoxin fusion gene homologs (alkW1 and alkW2) were cloned from a Dietzia strain, designated DQ12-45-1b, which can grow on crude oil and n-alkanes ranging in length from 6 to 40 carbon atoms as sole carbon sources. Both AlkW1 and AlkW2 have an integral-membrane alkane monooxygenase (AlkB) conserved domain and a rubredoxin (Rd) conserved domain which are fused together. Phylogenetic analysis showed that these two AlkB-fused Rd domains formed a novel third cluster with all the Rds from the alkane hydroxylase-rubredoxin fusion gene clusters in Gram-positive bacteria and that this third cluster was distant from the known AlkG1- and AlkG2-type Rds. Expression of the alkW1 gene in DQ12-45-1b was induced when cells were grown on C(8) to C(32) n-alkanes as sole carbon sources, but expression of the alkW2 gene was not detected. Functional heterologous expression in an alkB deletion mutant of Pseudomonas fluorescens KOB2Δ1 suggested the alkW1 could restore the growth of KOB2Δ1 on C(14) and C(16) n-alkanes and induce faster growth on C(18) to C(32) n-alkanes than alkW1ΔRd, the Rd domain deletion mutant gene of alkW1, which also caused faster growth than KOB2Δ1 itself. In addition, the artificial fusion of AlkB from the Gram-negative P. fluorescens CHA0 and the Rds from both Gram-negative P. fluorescens CHA0 and Gram-positive Dietzia sp. DQ12-45-1b significantly increased the degradation of C(32) alkane compared to that seen with AlkB itself. In conclusion, the alkW1 gene cloned from Dietzia species encoded an alkane hydroxylase which increased growth on and degradation of n-alkanes up to C(32) in length, with its fused rubredoxin domain being necessary to maintain the functions. In addition, the fusion of alkane hydroxylase and rubredoxin genes from both Gram-positive and -negative bacteria can increase the degradation of long-chain n-alkanes (such as C(32)) in the Gram-negative bacterium.     

Degradation of petroleum hydrocarbons (C6-C40) and crude oil by a novel Dietzia strain

A novel bacterial strain, DQ12-45-1b, was isolated from the production water of a deep subterranean oil-reservoir. Morphological, physiological and phylogenetic analyses indicated that the strain belonged to the genus Dietzia with both alkB (coding for alkane monooxygenase) and CYP153 (coding for P450 alkane hydroxylase of the cytochrome CYP153 family) genes and their induction detected. It was capable of utilizing a wide range of n-alkanes (C6-C40), aromatic compounds and crude oil as the sole carbon sources for growth. In addition, it preferentially degraded short-chain hydrocarbons (?C25) in the early cultivation phase and accumulated hydrocarbons with chain-lengths from C23 to C27 during later cultivation stage with crude oil as the sole carbon source. This is the first study to report the different behaviors of a bacterial species toward crude oil degradation as well as a species of Dietzia degrading a wide range of hydrocarbons.     

红灯食烷菌(Alcanivorax hongdengensis)黄素结合单加氧酶(AlmA)的基因克隆及其烷烃诱导表达

【目的】研究海洋烷烃降解菌新种模式菌株Alcanivorax hongdengensis A-11-3降解长链烷烃的分子机制。【方法】PCR克隆编码黄素结合单加氧酶的基因序列,利用生物信息学软件对序列进行分析,运用RT-PCR和实时荧光定量PCR技术分析基因在不同烷烃诱导下的表达水平。【结果】从菌株A-11-3中克隆获得了两个黄素结合单加氧酶基因片段(almA1和almA2)。它们编码的氨基酸序列与菌株Acinetobacter sp.DSM17874的AlmA同源性分别为58.6%和53.2%。实时荧光定量PCR分析表明,almA1基因只在长链烷烃(C28-C32)的诱导下上调表达,而almA2基因中能在更宽范围的长链烷烃(C24-C34)和支链烷烃诱导下上调表达。两者均在C9-C22的烷烃诱导下没有上调表达。【结论】黄素结合单加氧酶可能是A-11-3降解长链烷烃和支链烷烃的关键酶。     

Membrane vesicles from a Dietzia bacterium containing multiple cargoes and their roles in iron delivery

Membrane vesicles (MVs) released from bacteria act as extracellular vehicles carrying various functional cargoes between cells. MVs with different cargoes play multiple roles in stress adaptation, nutrient acquisition and microbial interactions. However, previous studies have primarily focused on MVs from Gram-negative bacteria, while the characteristics of cargoes in MVs from Gram-positive bacteria and their involvement in microbial interactions remain to be elucidated. Here, we used a Gram-positive strain, Dietzia sp. DQ12-45-1b from Corynebacteriales, to analyse the characteristics and functions of MVs. We identified the ‘antioxidant’ canthaxanthin is stored within MVs by LC–MS/MS. In addition, nearly the entire genomic content of strain DQ12-45-1b are evenly distributed in MVs, suggesting that MVs from DQ12-45-1b might involve in horizontal gene transfer. Finally, the mycobactin-type siderophores were detected in MVs. The iron-loaded MVs effectively mediate iron binding and delivery to homologous bacteria from the order Corynebacteriales, but not to more distantly related species from the orders Pseudomonadales, Bacillales and Enterobacterales. These results revealed that the iron-loaded MVs are shared between homologous species. Together, we report the Gram-positive bacterium Dietzia sp. DQ12-45-1b released MVs that contain canthaxanthin, DNA and siderophores and prove that MVs act as public goods between closely related species.     

Identification of alkane hydroxylase genes in Rhodococcus sp. strain TMP2 that degrades a branched alkane

Rhodococcus sp. TMP2 is an alkane-degrading strain that can grow with a branched alkane as a sole carbon source. TMP2 degrades considerable amounts of pristane at 20 degrees C but not at 30 degrees C. In order to gain insights into microbial alkane degradation, we characterized one of the key enzymes for alkane degradation. TMP2 contains at least five genes for membrane-bound, non-heme iron, alkane hydroxylase, known as AlkB (alkB1-5). Phylogenetical analysis using bacterial alkB genes indicates that TMP2 is a close relative of the alkane-degrading bacteria, such as Rhodococcus erythropolis NRRL B-16531 and Q15. RT-PCR analysis showed that expressions of the genes for AlkB1 and AlkB2 were apparently induced by the addition of pristane at a low temperature. The results suggest that TMP2 recruits certain alkane hydroxylase systems to utilize a branched alkane under low temperature conditions.     

Biodegradation of chlorinated paraffins and long-chain chloroalkanes by Rhodococcus sp. S45-1

A strain of the genus Rhodococcus, designated isolate S45-1, was isolated from an environmental water sample by enrichment, using the chlorinated paraffin Cereclor S45 as the sole carbon and energy source. This is the first report of microbial utilisation of chlorinated paraffins as sole source of carbon and energy. Biochemical studies of isolate S45-1 revealed little similarity with other Rhodococcus species. Isolate S45-1 was able to utilise 1-chloroalkanes of chain-length 12–18C as sole source of carbon and energy. Gas chromatography-mass spectrometry of the reaction medium indicated that γ-butyrolactone was formed as a product of 1-chlorotetradecane metabolism.     

Monitoring of alkane-degrading bacteria in a sea-water microcosm during crude oil degradation by polymerase chain reaction based on alkane-catabolic genes

Behaviour of microbial populations responsible for degrading n-alkanes, a major component of crude oil, was monitored during crude oil degradation in a sea-water microcosm by both traditional colony culturing and molecular techniques. A DNA extraction method applicable to crude oil-amended sea-water samples was developed to obtain DNA applicable to most probable number (MPN) polymerase chain reaction (PCR). The population of alkane-degrading bacteria responsible for degradation of n-alkanes in a crude oil-amended microcosm altered, so that shorter alkanes were degraded first by alkane-degrading bacteria possessing alkane hydroxylase genes from group I (Kohno et al., 2002, Microb Environ 17: 114-121) and longer ones afterwards by those possessing alkane hydroxylase genes from group II. Thus, the degradation mechanism of the n-alkanes can be clarified during crude oil degradation. Application of the method of detecting different types of alkane-catabolic genes, as shown in the present study, enabled bacterial groups preferring alkanes of either shorter or longer chain lengths to be enumerated selectively.     

Identification of novel genes involved in long-chain n-alkane degradation by Acinetobacter sp. strain DSM 17874

Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C10H22) to that of tetracontane (C40H82) as a sole carbon source. Two genes encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30 degrees C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C32H66) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains.     

Predominant role of hydrocarbon solubilization in the microbial uptake of hydrocarbons

Using EDTA and proteolytic enzymes to suppress hydrocarbon solubilization, direct evidence is presented in support of the mechanism of liquid hydrocarbon uptake by microbial cells predominantly from the solubilized or accommodated substrate. EDTA (2-5mM) strongly inhibited growth of three yeast species and one bacterial species on n-hexadecane and the inhibition was removed by surfactant-emulsified and surfactant-solubilized alkane and also by excess addition of Ca(2+). EDTA had no inhibitory effect on the growth of the organisms on soluble substrates such as sodium acetate and nutrient broth or on n-pentane, a volatile alkane which was primarily transported by diffusion from gas phase. EDTA was shown to have no significant effect on the adsorption of cells on alkane drops. EDTA inhibition of growth was considered to be due to suppression of alkane solubilization, brought about by the solubilizing factor(s) produced by cells. It was shown that this chelating agent did not inhibit the growth of yeast on solubilized alkane but strongly inhibited its growth on alkane drops. It was demonstrated that adherent capacity of microbial cell to oil phase was closely related to the state of hydrocarbon emulsification and had no relationship to the ability of organisms to grow on hydrocarbon. Certain proteolytic enzymes inhibited the growth of yeast on alkane, presumably by digesting the alkane solubilizing protein, but not on glucose, and the inhibition was removed by a supply of surfactant-emulsified and surfactant-solubilized alkane. Specific solubilization of various hydrocarbon types during growth of the prokaryotic bacterial strain was demonstrated. The specific solubilization of hydrocarbon was strongly inhibited strain was demonstrated. The specific solubilization of hydrocarbon was strongly inhibited by EDTA, and the inhibition was removed by excess Ca(2+). It was concluded that specific solubilization of hydrocarbons is an important mechanism in the microbial uptake of hydrocarbons.     

n-Alkane Chain Length Alters Dietzia sp. Strain DQ12-45-1b Biosurfactant Production and Cell Surface Activity

Upon growth on n-hexadecane (C₁₆), n-tetracosane (C₂₄), and n-hexatriacontane (C₃₆), Dietzia sp. strain DQ12-45-1b could produce different glycolipids, phospholipids, and lipopeptides. Interestingly, cultivation with C₃₆ increased cell surface hydrophobic activity, which attenuated the negative effect of the decline of the emulsification activity. These results suggest that the mechanisms of biosurfactant production and cell surface hydrophobicity are dependent upon the chain lengths of the n-alkanes used as carbon sources.     

Biodegradation of fuel oil hydrocarbons by a mixed bacterial consortium in sandy and loamy soils

The aerobic degradation of light fuel oil in sandy and loamy soils by an environmental bacterial consortium was investigated. Soils were spiked with 1 or 0.1% of oil per dry weight of soil. Acetone extracts of dried soils were analyzed by GC and the overall degradation was calculated by comparison with hydrocarbon recovery from uninoculated soils. In sandy soils, the sum of alkanes n-C(12) to n-C(23) was degraded to about 45% within 6 days at 20 degrees C and to 27-31% within 28 days, provided that moisture and nutrients were replenished. Degradation in loamy soil was about 12% lower. The distribution of recovered alkanes suggested a preferential degradation of shorter chain molecules (n-C(12) to n-C(16)) by the bacterial consortium. Partial 16S rDNA sequences indicated the presence of strains of Pseudomonas aeruginosa, Pseudomonas citronellolis, and Stenotrophomonas maltophilia. Toxicity tests using commercial standard procedures showed a moderate inhibition of bacterial activity. The study showed the applicability of a natural microbial community for the degradation of oil spills into soils at ambient temperatures.     

Characterization of Yarrowia lipolytica mutants affected in hydrophobic substrate utilization

In order to get deeper insights into oxidative degradation of the hydrophobic substrates (HS) triglycerides and alkanes by yeasts, tagged mutants affected in these pathways were generated by random insertion of a mutagenesis cassette MTC into the genome of Yarrowia lipolytica. About 9.600 Ura+ transformants were screened in plate tests for utilization of alkanes (C10, C16), oleic acid and tributyrin. HS degradation mutants were recovered as unable to grow on alkane or on intermediates of the pathway (AlkA-AlkE phenotype classes). To identify the disrupted genes, insertion points of the MTC were sequenced using convergent and divergent PCR. Sequence analysis evidenced both known and new genes required for HS utilization, e.g. for AlkD/E mutants MTC insertion had occurred in genes of thioredoxin reductase, peroxines PEX14 and PEX20, succinate-fumarate carrier SFC1, and isocitrate lyase ICL1. Several mutants were affected in alkane utilization depending on chain length. Mutant Z110 (AlkAb: C10- C16+) was shown to be disrupted for ANT1 encoding a peroxisomal membrane localized adenine nucleotide transporter protein, providing ATP for the activation of short-chain fatty acids by acyl-CoA synthetase II in peroxisomes. Mutants N046 and B095 (AlkAc: C10+ C16-) were disrupted for the ABC transporter encoded by ABC1 gene, thus providing first evidence for its participation in chain length dependent alkane transport processes.     

Microbial population dynamics associated with crude-oil biodegradation in diverse soils

Soil bacterial population dynamics were examined in several crude-oil-contaminated soils to identify those organisms associated with alkane degradation and to assess patterns in microbial response across disparate soils. Seven soil types obtained from six geographically distinct areas of the United States (Arizona, Oregon, Indiana, Virginia, Oklahoma, and Montana) were used in controlled contamination experiments containing 2% (wt/wt) crude oil spiked with [1-(14)C]hexadecane. Microbial populations present during hydrocarbon degradation were analyzed using both 16S rRNA gene sequence analysis and by traditional methods for cultivating hydrocarbon-oxidizing bacteria. After a 50-day incubation, all seven soils showed comparable hydrocarbon depletion, where >80% of added crude oil was depleted and approximately 40 to 70% of added [(14)C]hexadecane was converted to (14)CO(2). However, the initial rates of hydrocarbon depletion differed up to 10-fold, and preferential utilization of shorter-chain-length n-alkanes relative to longer-chain-length n-alkanes was observed in some soils. Distinct microbial populations developed, concomitant with crude-oil depletion. Phylogenetically diverse bacterial populations were selected across different soils, many of which were identical to hydrocarbon-degrading isolates obtained from the same systems (e.g., Nocardioides albus, Collimonas sp., and Rhodococcus coprophilus). In several cases, soil type was shown to be an important determinant, defining specific microorganisms responding to hydrocarbon contamination. However, similar Rhodococcus erythropolis-like populations were observed in four of the seven soils and were the most common hydrocarbon-degrading organisms identified via cultivation.     

Nutrients Can Enhance the Abundance and Expression of Alkane Hydroxylase CYP153 Gene in the Rhizosphere of Ryegrass Planted in Hydrocarbon-Polluted Soil

Plant-bacteria partnership is a promising strategy for the remediation of soil and water polluted with hydrocarbons. However, the limitation of major nutrients (N, P and K) in soil affects the survival and metabolic activity of plant associated bacteria. The objective of this study was to explore the effects of nutrients on survival and metabolic activity of an alkane degrading rhizo-bacterium. Annual ryegrass (Lolium multiflorum) was grown in diesel-contaminated soil and inoculated with an alkane degrading bacterium, Pantoea sp. strain BTRH79, in greenhouse experiments. Two levels of nutrients were applied and plant growth, hydrocarbon removal, and gene abundance and expression were determined after 100 days of sowing of ryegrass. Results obtained from these experiments showed that the bacterial inoculation improved plant growth and hydrocarbon degradation and these were further enhanced by nutrients application. Maximum plant biomass production and hydrocarbon mineralization was observed by the combined use of inoculum and higher level of nutrients. The presence of nutrients in soil enhanced the colonization and metabolic activity of the inoculated bacterium in the rhizosphere. The abundance and expression of CYP153 gene in the rhizosphere of ryegrass was found to be directly associated with the level of applied nutrients. Enhanced hydrocarbon degradation was associated with the population of the inoculum bacterium, the abundance and expression of CYP153 gene in the rhizosphere of ryegrass. It is thus concluded that the combination between vegetation, inoculation with pollutant-degrading bacteria and nutrients amendment was an efficient approach to reduce hydrocarbon contamination.     

Protein expression in the obligate hydrocarbon-degrading psychrophile Oleispira antarctica RB-8 during alkane degradation and cold tolerance

In cold marine environments, the obligate hydrocarbon-degrading psychrophile Oleispira antarctica RB-8, which utilizes aliphatic alkanes almost exclusively as substrates, dominates microbial communities following oil spills. In this study, LC–MS/MS shotgun proteomics was used to identify changes in the proteome induced during growth on n-alkanes and in cold temperatures. Specifically, proteins with significantly higher relative abundance during growth on tetradecane (n-C₁₄) at 16°C and 4°C have been quantified. During growth on n-C₁₄, O. antarctica expressed a complete pathway for the terminal oxidation of n-alkanes including two alkane monooxygenases, two alcohol dehydrogenases, two aldehyde dehydrogenases, a fatty-acid-CoA ligase, a fatty acid desaturase and associated oxidoreductases. Increased biosynthesis of these proteins ranged from 3- to 21-fold compared with growth on a non-hydrocarbon control. This study also highlights mechanisms O. antarctica may utilize to provide it with ecological competitiveness at low temperatures. This was evidenced by an increase in spectral counts for proteins involved in flagella structure/output to overcome higher viscosity, flagella rotation to accumulate cells and proline metabolism to counteract oxidative stress, during growth at 4°C compared with 16°C. Such species-specific understanding of the physiology during hydrocarbon degradation can be important for parameterizing models that predict the fate of marine oil spills.     

Metabolic and phylogenetic analysis of microbial communities during phytoremediation of soil contaminated with weathered hydrocarbons and heavy metals

In the current study, the microbial ecology of weathered hydrocarbon and heavy metal contaminated soil undergoing phytoremediation was studied. The relationship of functional diversity, measured as carbon source utilisation in Biolog plates and extracellular enzymatic activities, and genetic diversity of bacteria was evaluated. Denaturing gradient gel electrophoresis was used for community analyses at the species level. Bulk soil and rhizosphere soil from pine and poplar plantations were analysed separately to determine if the plant rhizosphere impacted hydrocarbon degradation. Prevailing microbial communities in the field site were both genetically and metabolically diverse. Furthermore, both tree rhizosphere and fertilisation affected the compositions of these communities and increased activities of extracellular aminopeptidases. In addition, the abundance of alkane hydroxylase and naphthalene dioxygenase genes in the communities was low, but the prevalence of these genes was increased by the addition of bioavailable hydrocarbons. Tree rhizosphere communities had greater hydrocarbon degradation potential than those of bulk soil. Hydrocarbon utilising communities were dominated generally by the species Ralstonia eutropha and bacteria belonging to the genus Burkholderia. Despite the presence of viable hydrocarbon-degrading microbiota, decomposition of hydrocarbons from weathered hydrocarbon contaminated soil over four years, regardless of the presence of vegetation, was low in unfertilised soil. Compost addition enhanced the removal of hydrocarbons.