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

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

[1]  R. Crawford,et al.  Extraction and purification of microbial DNA from petroleum‐contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis , 1995, Molecular ecology.

[2]  I. Banat,et al.  The degradation of n-hexadecane in soil by thermophilic geobacilli. , 2006, FEMS microbiology ecology.

[3]  Roger C. Prince,et al.  Bioremediation of Marine Oil Spills , 1997, Consequences of Microbial Interactions with Hydrocarbons, Oils, and Lipids: Biodegradation and Bioremediation.

[4]  Franz F. Roos,et al.  Identification of an Amino Acid Position That Determines the Substrate Range of Integral Membrane Alkane Hydroxylases , 2005, Journal of bacteriology.

[5]  M. Wubbolts,et al.  Genetics of alkane oxidation byPseudomonas oleovorans , 1994, Biodegradation.

[6]  F. Rojo,et al.  Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis. , 2004, Environmental microbiology.

[7]  R. Atlas,et al.  Frequency of genes in aromatic and aliphatic hydrocarbon biodegradation pathways within bacterial populations from Alaskan sediments. , 1994, Canadian journal of microbiology.

[8]  S. Harayama,et al.  Cloning and functional analysis of alkB genes in Alcanivorax borkumensis SK2. , 2004, Environmental microbiology.

[9]  U. Klinner,et al.  Distribution of alkB genes within n‐alkane‐degrading bacteria , 2000, Journal of applied microbiology.

[10]  M. Schloter,et al.  A new method for the detection of alkane-monooxygenase homologous genes (alkB) in soils based on PCR-hybridization. , 2006, Journal of microbiological methods.

[11]  B. Witholt,et al.  Diversity of Alkane Hydroxylase Systems in the Environment , 2003 .

[12]  B. Witholt,et al.  Alkane hydroxylase systems in Pseudomonas aeruginosa strains able to grow on n-octane , 1998 .

[13]  T. Maier,et al.  Molecular characterization of the 56-kDa CYP153 from Acinetobacter sp. EB104. , 2001, Biochemical and biophysical research communications.

[14]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[15]  H. Kleber,et al.  Occurrence of cytochrome P‐450 in Acinetobacter strains after growth on N‐hexadecane , 1981 .

[16]  C. Vetriani,et al.  Salinisphaera hydrothermalis sp. nov., a mesophilic, halotolerant, facultatively autotrophic, thiosulfate-oxidizing gammaproteobacterium from deep-sea hydrothermal vents, and emended description of the genus Salinisphaera. , 2009, International journal of systematic and evolutionary microbiology.

[17]  A. Halpern,et al.  The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific , 2007, PLoS biology.

[18]  T. Ellingsen,et al.  Utilization of n-alkanes by a newly isolated strain of Acinetobacter venetianus: the role of two AlkB-type alkane hydroxylases , 2006, Applied Microbiology and Biotechnology.

[19]  E. G. Funhoff,et al.  Alkane hydroxylases involved in microbial alkane degradation , 2007, Applied Microbiology and Biotechnology.

[20]  K. Timmis,et al.  Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis , 2006, Nature Biotechnology.

[21]  J. Kingma,et al.  The Pseudomonas oleovorans alkane hydroxylase gene. Sequence and expression. , 1989, The Journal of biological chemistry.

[22]  O. White,et al.  Environmental Genome Shotgun Sequencing of the Sargasso Sea , 2004, Science.

[23]  G. Sayler,et al.  Assessment of the Microbiological Potential for the Natural Attenuation of Petroleum Hydrocarbons in a Shallow Aquifer System , 1998, Microbial Ecology.

[24]  B. Witholt,et al.  Functional Analysis of Alkane Hydroxylases from Gram-Negative and Gram-Positive Bacteria , 2002, Journal of bacteriology.

[25]  N. Misawa,et al.  Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784 , 2006, Applied Microbiology and Biotechnology.

[26]  E. G. Funhoff,et al.  Cytochrome P450 Alkane Hydroxylases of the CYP153 Family Are Common in Alkane-Degrading Eubacteria Lacking Integral Membrane Alkane Hydroxylases , 2006, Applied and Environmental Microbiology.

[27]  T. Uchiyama,et al.  Isolation and Functional Analysis of Cytochrome P450 CYP153A Genes from Various Environments , 2005, Bioscience, biotechnology, and biochemistry.

[28]  Chenli Liu,et al.  Alcanivorax dieselolei sp. nov., a novel alkane-degrading bacterium isolated from sea water and deep-sea sediment. , 2005, International journal of systematic and evolutionary microbiology.

[29]  Peter N Golyshin,et al.  Obligate oil-degrading marine bacteria. , 2007, Current opinion in biotechnology.

[30]  L. Whyte,et al.  A survey of indigenous microbial hydrocarbon degradation genes in soils from Antarctica and Brazil. , 2004, Canadian journal of microbiology.

[31]  F. Rojo,et al.  The Alkane Hydroxylase Gene of Burkholderia cepacia RR10 Is under Catabolite Repression Control , 2001, Journal of bacteriology.

[32]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[33]  K. Timmis,et al.  Proteomic Insights into Metabolic Adaptations in Alcanivorax borkumensis Induced by Alkane Utilization , 2006, Journal of bacteriology.

[34]  W. Hillen,et al.  Alkane Hydroxylase from Acinetobactersp. Strain ADP1 Is Encoded by alkM and Belongs to a New Family of Bacterial Integral-Membrane Hydrocarbon Hydroxylases , 1998, Applied and Environmental Microbiology.

[35]  Robert Huber,et al.  Salinisphaerashabanensis gen. nov., sp. nov., a novel, moderately halophilic bacterium from the brine–seawater interface of the Shaban Deep, Red Sea , 2003, Extremophiles.

[36]  Tianfeng Tan,et al.  A pyrene-degrading consortium from deep-sea sediment of the West Pacific and its key member Cycloclasticus sp. P1. , 2008, Environmental microbiology.

[37]  Z. Rao,et al.  Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase. , 2008, Journal of molecular biology.

[38]  F. Rojo.  Degradation of alkanes by bacteria. , 2009, Environmental microbiology.

[39]  V. Pellizari,et al.  New alk genes detected in Antarctic marine sediments. , 2009, Environmental microbiology.

[40]  B. Witholt,et al.  Functional characterization of genes involved in alkane oxidation by Pseudomonas aeruginosa , 2003 .

[41]  Myong-Ok Park,et al.  New Pathway for Long-Chain n-Alkane Synthesis via 1-Alcohol in Vibrio furnissii M1 , 2005, Journal of bacteriology.

[42]  A. Karagouni,et al.  Biodegradation of Crude Oil by Thermophilic Bacteria Isolated from a Volcano Island , 2006, Biodegradation.

[43]  K. Timmis,et al.  Alcanivorax borkumensis gen. nov., sp. nov., a new, hydrocarbon-degrading and surfactant-producing marine bacterium. , 1998, International journal of systematic bacteriology.

[44]  B. Cho,et al.  Salinisphaera dokdonensis sp. nov., isolated from surface seawater. , 2010, International journal of systematic and evolutionary microbiology.

[45]  B. Witholt,et al.  Gene Cloning and Characterization of Multiple Alkane Hydroxylase Systems in Rhodococcus Strains Q15 and NRRL B-16531 , 2002, Applied and Environmental Microbiology.

[46]  K. Timmis,et al.  Genome sequence completed of Alcanivorax borkumensis, a hydrocarbon-degrading bacterium that plays a global role in oil removal from marine systems. , 2003, Journal of biotechnology.

[47]  B. Witholt,et al.  Molecular screening for alkane hydroxylase genes in Gram-negative and Gram-positive strains. , 1999, Environmental microbiology.

[48]  B. Witholt,et al.  Alkane hydroxylase homologues in Gram-positive strains. , 2002, Environmental microbiology.

[49]  Chenli Liu,et al.  Alcanivorax hongdengensis sp. nov., an alkane-degrading bacterium isolated from surface seawater of the straits of Malacca and Singapore, producing a lipopeptide as its biosurfactant. , 2009, International journal of systematic and evolutionary microbiology.

[50]  W. Röling,et al.  Marine microorganisms make a meal of oil , 2006, Nature Reviews Microbiology.