The complete alk sequences of Rhodococcus erythropolis from Lake Baikal
© Likhoshvay et al.; licensee Springer. 2014
Received: 13 February 2014
Accepted: 9 October 2014
Published: 21 October 2014
Rhodococci are bacteria able to degrade a wide range of hydrocarbons, including the alkanes present in crude oil, due to alk genes in their genomes.
Genome sequencing of DNA from Rhodococcus erythropolis strain 4 (obtained from a deep-water bitumen mound) revealed four alk genes, and the predicted amino acid sequences coded by these genes were highly conserved, having sections up to 11 amino acid residues.
Obtained four genes from Rhodococcus erythropolis were similar to corresponding genes from other bacteria collected from other environments, including marine sources. This indicated a large-scale horizontal alk gene transfer between bacteria from different subgenera.
Alkanes may constitute up to 88% of the volume present in natural oil, and due to their high toxicity can serve as a convenient source of energy only for oil-degrading microorganisms (van Beilen et al. 2003).
To overcome this obstacle, bacteria have learned to synthesize specific enzymes for extracting energy from n-alkanes. The initial step in n-alkane oxidation is catalyzed by a monooxygenase complex composed of an alkane hydroxylase (alkB), rubredoxin reductase (alkT) and rubredoxin (alkG, an electron carrier), which are known to play an important role in oil bioremediation (Kloos et al. 2006; van Beilen et al. 2003; van van Beilen and Funhoff 2007). Biodegradation starts with cleavage of the C-H bond, catalysed by the oxygenase-group enzyme alkane hydroxylase, which inserts an atom of oxygen from O2 into the hydrocarbon molecule. Prior to this catalysis, the enzyme must be activated by NADH (nicotinamide adenine dinucleotide) which transfers a pair of electrons from FAD (flavin adenine dinucleotide) to rubredoxin. One pair of electrons is transferred to alkane hydroxylase leading to the formation of primary or secondary alcohols [R-CH3 + O2 + NAH(P)H + H+ → R-CH2OH + NAD(P)+ +H2O], which are further converted to dicarboxylic acids (van Beilen et al. 2003).
This enzyme system was originally discovered in Pseudomonas putida and further research found that the genes encoding for alkane-degrading enzymes (alk-genes, rubredoxin, rubredoxin reductase) are located on a plasmid or chromosomes (van Beilen et al. 2001). Bacteria from different genera, including Rhodococcus, possess similar enzyme systems and alk-genes (Whyte et al. 1998; Whyte et al. 2002). Members of the genus Rhodococcus seem to play significant role in bioremediation of oil spills (Whyte et al. 2002) and are recognized as ideal candidates for the biodegradation of hydrocarbons due to their ability to degrade a wide range of organic compounds (Beard and Page 1998), their hydrophobic cell surface and the production of biosurfactants as well as their ubiquity and robustness in the environment (Larkin et al. 1998; Warhurst and Fewson 1994).
Lake Baikal, the deepest (1637 m) and oldest (25 mln y) lacustrine reservoir on Earth, is located in the middle of Eurasia. During a 2008 exploration using Mir submersibles, natural oil seepages surrounded with “bitumen mounds” were discovered on the lake bottom (Khlystov et al. 2009). These structures are stable, inhabited by living creatures and persist even if the source of oil is depleted. One of these bitumen mounds (No. 8) contained 148 mg/g of aliphatic C22-C34n-alkanes (primarily C25) where several strains of bacteria were isolated and later identified as Rhodococcus erythropolis (Likhoshvay et al. 2013) by means of 16S rRNA analysis.
This study aimed to identify alk genes in the genome of one of these isolates (strain 4) by nucleotide and complete genome sequence analysis. Strain 4, identified as R. erythropolis, has four alk-genes which differed from each other, but were similar to corresponding genes in bacteria from other habitats.
Materials and methods
R. erythropolis strain 4 (Acc. No HQ702471), isolated from bitumen mound 8 at the natural oil seep near Cape of Gorevoi Utes (10 km offshore, depth 900 m, Central Baikal) (Likhoshvay et al. 2013).
DNA extraction and sequencing analysis
DNA was extracted by the method of Sambrook et al. (1989) with minor modifications - enzymatic lysis followed by phenol-chloroform extraction.
Complete genome sequencing of DNA was carried out according to the manual/protocol provided with the Illumina GAIIx (India). Number of readings equalled to approximately 10 Mbp. Reassembling of individual nucleotide sequences by Velvet_1.1.02 resulted to 3897 contigs with an average length of 1.8 Kbp and a total length of 6.9 Mbp. The nucleotide sequences of the alkB genes were translated into amino acid sequences by the Expasy Translate Tool (http://web.expasy.org/tools/translate/) and uploaded to the NCBI data base with the following accession numbers: alkane hydroxylase 1 (KF498365), alkane hydroxylase 2 (KF498366), alkane hydroxylase 3 (KF498367), alkane hydroxylase 4 (KF498368).
Homology between the four sequences was estimated by BLASTX (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi) where the nucleotide sequences and inferred amino acid sequences were aligned with homologous sequences retrieved from GenBank using the CLUSTAL W software. A phylogenetic tree for the genes was constructed by the neighbor-joining method (Saitou and Nei 1987) using the MEGA4 program (Tamura et al. 2007). The relative synonymous codon usage (RSCU) was computed for the alkB genes and correspondence analysis was performed using CODONW software.
Comparative analysis of a wide range of homologous eubacterial sequences (NCBI) revealed that the genome of R. erythropolis strain 4 contained highly conserved areas. In the sequence of the fourth alkane hydroxylase (BAH36166) – EHNFGHH – polar histidine (H) with basic properties was substituted for a nonpolar hydrophobic phenylalanine (F). The same substitutions were only found in corresponding sequences of R. erythropolis SK121 (ZP04385381) and R. erythropolis PR4 (YP002768905).
AlkB genes in the genomes of Gram-positive and Gram-negative alkane-degrading bacteria are usually present as several individual copies (van Beilen et al. 2003). In particular, R. erythropolis NRRL B-16531 and R. erythropolis Q15 possess four alkB homologues and suggests these bacteria tend to have several alkB-genes encoding for alkane hydroxylase (Whyte et al. 2002). In the genome of the R. erythropolis strain 4, 4 nucleotide sequences for (oxygenase group) alkane hydroxylases were identified. A 5th alkB gene has also been identified which encoded for rubredoxin reductase, but did not cluster the other 4 and will be discussed in later articles.
The alkane hydroxylase amino acid sequence homologies between R. erythropolis strain 4, R. erythropolis SK121 and R. erythropolis PR4 are remarkable for the following reasons: strain SK121 (Hamamura et al. 2008) was isolated from oil contaminated soil and tends to utilise aromatic hydrocarbons. Strain PR4 was isolated at a depth of 1 km from the Pacific Ocean and is unable to utilise arenes, but does use n-alkanes with chain length of C8-C20 as the sole energy source (Sekine et al. 2006). The R. erythropolis strain 4 was isolated from the inner part of bitumen mound, located on the bottom of Lake Baikal and tends to utilise n-alkanes with a broader chain length (C12-C29). This adaptation could be explained by the composition of the bitumen mound 8, which included n-alkanes with chain lengths of C22-C34 (Likhoshvay et al. 2013). However, as a final product of alkanes biodegradation serve fatty acids with chain length of C16-C18. These substances could further be degraded during phospholipid synthesis (Alvarez 2010).
Homologue sequence analysis (NCBI) of the 4 amino acid sequences from R. erythropolis strain 4 revealed that the 4 alkane hydroxylases were highly divergent, however each enzyme was similar to the corresponding homologue from Rhodococcus. The absence of other bacterial genera in the analyses suggested this was an enzyme system specific to rhodococci, based on the differences in alkane hydroxylases.
All alkane-degrading bacteria have alkane hydroxylases containing the following three sequences: (numbering from Pseudomonas putida GPo1): H138E[L/M]xHK143, E167HxxGHH173 and L309QRH[S/A]DHHA317. According to van Beilen et al. (2005), a histidine in the second and third sequences may affect enzyme activity. Furthermore, the histidine residues in these conserved sequences bind two atoms of Fe(II) in the alkane hydroxylase (Whyte et al. 1999; van Beilen et al. 2005). The longest sequence, L309QRH[S/A]DHHA317, was present in the alkane hydroxylase sequences of most hydrocarbon-oxidising bacteria, including R. opacus B4, which was initially isolated from oil contaminated soil and metabolized a wide range of arenes and aliphatics. The genes coding for these enzymes were located in (at least) six replicons: a large linear chromosome of 7,913,450 bp, two linear plasmids - pROB01 (558,192 bp) and pROB02 (244,997 bp), and three circular ones – pKNR (111,160 bp), pKNR01 (4,367 bp) pKNR02 (2,773 bp). Originally isolated at a depth of 1 km in Pacific Ocean, R. erythrypolis PR4 had a circular chromosome of 6,516,310 bp, a separate linear plasmid - pREL1: 271,577 bp and two circular plasmids – pREC1 - 104,014 bp and pREC2 - 3,637 bp. The first two code for most of the genes responsible for alkane metabolism. Obviously, plasmid alk genes could be transferred between bacteria by horizontal gene transfer (Turova et al. 2008). Hence, bacteria of the Geobacillus genus could obtain alk genes from Rhodococcus.
The least ones could be found everywhere and in different climatic zones and they have enormous biodegradation potential to utilise widest range of organic substrates. The structure of alk genes apparently has an adaptive character and encodes alkane hydroxylase. This might be necessary for R. erythropolis strain 4 to degrade of heavy n-alkanes, which are present in bitumen mound 8 at low temperture (3.5°С) and high pressure (90 atm).
We would like to thank Genotypic (Bangolore, India) and Dr. Roja C. Mugasimangalam personally for assistance in sequencing on the Illumina GAIIx; Dr. T.A. Scherbakova for consulting assistance and Dr. Y.P. Galachyants for assembling the contigs. The work was supported by grants from Integration Project SB RAS 82 and Programme of RAS Presidium 23.8.
- Alvarez HM: Biology of Rhodococcus. Springer, London, New York; 2010.View ArticleGoogle Scholar
- Beard TM, Page MI: Enantioselective biotransformations using rhodococci. Antonie Leeuwenhoek 1998, 74: 99-106. 10.1023/A:1001712230455View ArticleGoogle Scholar
- Hamamura N, Fukui M, Ward DM, Inskeep WP: Assessing soil microbial populations responding to crude-oil amendment at different temperatures using phylogenetic, functional gene ( alkB ) and physiological analyses. Environ Sci Technol 2008, 42(20):7580-7586. 10.1021/es800030fView ArticleGoogle Scholar
- Khlystov OM, Zemskaya TI, Sitnikova TYA, Mekhanikova IV, Kaigorodova IA, Gorshkov AG, Timoshkin OA, Shubenkova OV, Chernitsyna SM, Lomakina AV, Likhoshvai AV, Sagalevich AM, Moskvin VI, Peresypkin VI, Belyaev NA, Slipenchuk MV, Tulokhonov AK, Grachev MA: Bottom bituminous constructions and biota inhabiting them according to investigation of Lake Baikal with the Mir submersible. Dokl Earth Sci 2009, 429(8):1333-1336.View ArticleGoogle Scholar
- Kloos K, Munch JC, Schloter M: A new method for the detection of alkane-monooxygenase homologous genes ( alkB ) in soils based on PCR-hybridization. J Microbiol Methods 2006, 66: 486-496. 10.1016/j.mimet.2006.01.014View ArticleGoogle Scholar
- Larkin MJ, De Mot R, Kulakov LA, Nagy I: Applied aspects of Rhodococcus genetics. Antonie Leeuwenhoek 1998, 74: 133-153. 10.1023/A:1001776500413View ArticleGoogle Scholar
- Likhoshvay A, Khanaeva T, Gorshkov A, Zemskaya T, Grachev M: Do oil-degrading Rhodococci contribute to the genesis of deep water bitumen mounds in Lake Baikal? Geomicrobiol 2013, 30(3):209-213. 10.1080/01490451.2012.665149View ArticleGoogle Scholar
- Saitou N, Nei M: The neighbor-joining methods: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987, 4: 406-425.Google Scholar
- Sambrook J, First EF, Maniatis T: Molecular Cloning. A Laboratory Manual Cold. Spring Harbor Laboratory Press, New York; 1989.Google Scholar
- Sekine M, Tanikawa S, Omata S, Saito M, Fujisawa T, Tsukatani N, Tajima T, Sekigawa T, Kosugi H, Matsuo Y, Nishiko R, Imamura K, Ito M, Narita H, Tago S, Fujita N, Harayama S: Sequence analysis of three plasmids harboured in Rhodococcus erythropolis strain PR4. Environ Microbiol 2006, 8(2):334-346. 10.1111/j.1462-2920.2005.00899.xView ArticleGoogle Scholar
- Tamura K, Dudley J, Nei M, Kumar S: MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007, 24: 1596-1599. 10.1093/molbev/msm092View ArticleGoogle Scholar
- Turova TP, Nazina TN, Mikhailova EM, Rodionova TA, Ekimov AN, Mashukova AV, Poltaraus AB: AlkB Homologues in thermophilic bacteria of the genus Geobacillus . Mol Boil 2008, 42(2):247-257.Google Scholar
- van Beilen JB, Funhoff EG: Alkane hydroxylase homologues involved in microbial alkane degradation. Appl Microbiol Biotechnol 2007, 1: 13-21.View ArticleGoogle Scholar
- van Beilen JB, Panke S, Lucchini S, Franchini AG, Röthlisberger M, Witholt B: Analysis of Pseudomonas putida alkane degradation gene clusters and flanking insertion sequences: Evolution and regulation of the alk -genes. Microbiol 2001, 147: 1621-1630.View ArticleGoogle Scholar
- van Beilen JB, Li Z, Duetz WA, Smits THM, Witholt B: Diversity of alkane hydroxylase systems in the environment. Oil Gas Scien Technol 2003, 58(4):427-440. 10.2516/ogst:2003026View ArticleGoogle Scholar
- van Beilen JB, Smits THM, Roos FF, Brunner T, Balada SB, Rothlisberger M, Witholt B: Identification of an amino acid position that determines the substrate range of integral membrane alkane hydroxylases. J Bacteriol 2005, 187(1):85-91. 10.1128/JB.187.1.85-91.2005View ArticleGoogle Scholar
- Warhurst AW, Fewson CA: Biotransformations catalyzed by the genus Rhodococcus . Crit Rev Biotechnol 1994, 14: 29-73. 10.3109/07388559409079833View ArticleGoogle Scholar
- Whyte LG, Hawari J, Zhou E, Bourbonnière L, Inniss WE, Greer CW: Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp. Appl Environ Microbiol 1998, 64: 2578-2584.Google Scholar
- Whyte LG, Slagman SJ, Pietrantonio F, Bourbonnière L, Koval SF, Lawrence JR, Inniss WE, Greer CW: Physiological adaptations involved in alkane assimilation at a low temperature by Rhodococcus sp. strain Q15. Appl Environ Microbiol 1999, 65(7):2961-2968.Google Scholar
- Whyte LG, Smits THM, Labbe’ D, Witholt B, Greer CW, van Beilen JB: Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531. Appl Environ Microbiol 2002, 68: 5933-5942. 10.1128/AEM.68.12.5933-5942.2002View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.