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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Alkane monooxygenases (Alk) are the key enzymes for alkane degradation. In order to understand the dispersion and diversity of alk genes in Antarctic marine environments, this study analysed by clone libraries the presence and diversity of alk genes (alkB and alkM) in sediments from Admiralty Bay, King George Island, Peninsula Antarctica. The results show a differential distribution of alk genes between the sites, and the predominant presence of new alk genes, mainly in the pristine site. Sequences presented 53.10–69.60% nucleotide identity and 50.90–73.40% amino acid identity to alkB genes described in Silicibacter pomeroyi, Gordonia sp., Prauserella rugosa, Nocardioides sp., Rhodococcus sp., Nocardia farcinica, Pseudomonas putida, Acidisphaera sp., Alcanivorax borkumensis, and alkM described in Acinetobacter sp. This is the first time that the gene alkM was detected and described in Antarctic marine environments. The presence of a range of previously undescribed alk genes indicates the need for further studies in this environment.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

The Antarctic marine ecosystem contains low concentrations of a range of hydrocarbons, being mostly biogenic in origin. N-alkanes originating from petroleum and other types of fossil fuel have increased in Antarctic ecosystems, especially around settlements, including scientific bases (Aislabie et al., 2006), and in sites where accidental spills occurred (Cripps, 1992). Being hydrophobic, organic compounds such as hydrocarbons tend to be incorporated into the particulate material and deposited in the subsurface marine sediment (Volkman et al., 1992). Alkanes deposited in the sediment can be used as carbon source by microbial communities that bear alkane monooxygenases (Alk), a membrane-bound enzyme that catalyses the initial oxidation of the alkane substrate to a 1-alkanol (Wyatt, 1984).

Bacterial oxidation of n-alkanes has been reported worldwide in nature (Watkinson and Morgan, 1990; Sotsky et al., 1994; Whyte et al., 1996; 2002; van Beilen et al., 2003; Head et al., 2006; van Beilen and Funhoff, 2007). More than 60 Alk homologues are presently known, showing high sequence diversity (van Beilen et al., 2003; van Beilen and Funhoff, 2007). Despite the studies reporting the detection of alk genes by hybridization analysis, isolation and phylogenetic identification of indigenous cold-adapted degrading microorganisms and the characterization of communities with hydrocarbon degradation capacity (Sotsky et al., 1994; Delille et al., 1997; MacCormack and Fraile, 1997; Whyte et al., 1999; 2002; Bej et al., 2000; Delille and Delille, 2000; Powell et al., 2003; 2007; Luz et al., 2004; Aislabie et al., 2006), information on the DNA code of these alk genes in polar environments remains scarce.

In order to understand the dispersion and the diversity of alk genes in Antarctic marine environments, this study analysed the presence and diversity of alkane monooxygenases genes (alkB and alkM) in sediments from Admiralty Bay, King George Island, Peninsula Antarctica. The results show a range of alk genes not previously described, highlighting the need for more studies in this environment.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Seventy-six alk gene sequences from sediment samples from the Comandante Ferraz Station (CF) and 101 from Botany Point (BP) were sequenced and analysed. We defined the cut-off of 18% difference between sequences to form the Operational Protein Family (OPF) (Schloss and Handelsman, 2008). This decision was primarily based on a previous phylogenetic analysis of all clones and an identification of clusters in a phylogenetic tree and secondarily based on the results of blast and tblastx with sequences from GenBank. Sequences considered belonging to the same cluster in the phylogenetic tree matched the same sequences from GenBank with low difference identity values and e-values (data not shown). These phylogenetic clusters were 100% identical to the OPF generated in dotur and FastGroup II.

In both libraries, rarefaction curves expressed an asymptotic behaviour, showing that the sampling effort was satisfactory (data not shown). Coverage estimators suggest that 100% of the diversity present in CF and 94% in BP were covered (Table 1). Libraries were large enough to produce stable coverage estimates by using a program available at http://www.aslo.org/lomethods/free/2004/0114a.html (Kemp and Aller, 2004). Shannon–Weiner index (H′), SACE and SCHAO1 scores indicated that the BP alk gene library is more diverse than the CF library (Table 1).

Table 1.  Diversity and richness estimators of the alk gene clone libraries.
SiteShannon–Weiner (H′)Richness observedaSACESCHAO1% Coverageb
  • a.

    Numbers in parentheses indicate the number of clones used in analysis. Richness is the number of OPF observed.

  • b.

    Per cent coverage is the ratio of the number of OPF observed over the average number of OPF estimated (based on the average of estimates from SACE and SCHAO1).

CF0.92674 (76)4.004.00100
BP1.774915 (101)16.3415.6694

All 177 sequences from both libraries showed the presence of the two internal conserved regions (motif B, EHXXGHH and motif C, NYXEHYG) (Smits et al., 1999; van Beilen et al., 2005) of Alks and matched with Alks from GenBank using the tblastx program. Four OPF in CF (S1 to S4) and 15 OPF in BP (S1 to S15) were found (Fig. 1). Two of these OPF (S2 and S15) matched with the alkM gene from Acinetobacter sp. (lineages ADP-1 and M1 respectively). This is the first time that the gene alkM was detected and described in Antarctic environments. Two Acinetobacter spp. Hydrocarbon-degrading bacteria were isolated from Antarctic soil chronically exposed to oil (MacCormack and Fraile, 1997), but the specific homologue gene alkM has not been described yet. Luz and colleagues (2004), searching for alkB and alkM genes in contaminated and pristine soils from the vicinity of the Brazilian Station, detected no alkM genes. Furthermore, a study of alpine soils from Austria, using an alkM universal primer, reports that alkM OPF were detected in 50% of the contaminated soils but were not found in any of the eight pristine soils analysed (Margesin et al., 2003).

image

Figure 1. Alkane monooxygenase gene frequency and OPF identification in marine sediments from Comandante Ferraz Station (CF) and Botany Point (BP). The table shows alk genes previously described in isolated microorganisms that have the highest identity and identity matches to the environmental sequences. On the right, the results of the identity average from the environmental sequences compared with GenBank database sequences: (nt) percentage of identity by nucleotide level; (aa) percentage of identity by amino acid level.

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All the other OPF matched with alkB genes from different genera from α-, β- and γ-Proteobacteria and from Actinobacteria, and alkB genes from environmental clones (S7, S11 and S14) (Figs 1 and 2). With the exception of OPF S2, blastn searches showed neither significance nor identity with nucleotide database sequences. In general, the sequences show a range from only 53.10% (S11) to 69.60% (S15) identity to reference sequences by nucleotide level, and 50.90% (S11) to 73.40% (S15) identity to reference sequences by amino acid level (Fig. 1). Smits and colleagues (1999) had similar results screening alk genes in isolates. These results indicate the presence of novel putative alkane monooxygenase genes in Antarctic marine sediments not previously identified in any other environment or isolate.

image

Figure 2. Phylogenetic tree based on the alignment of amino acid sequences of membrane-bound alkane monooxygenases from clones obtained in this study and reference strains [comprising 162–169 amino acids, corresponding to position 143–310 of alkB from Pseudomonas putida GPo1 (van Beilen et al., 2005)]. The tree was constructed by using the neighbour-joining (NJ) method in mega3 program with the p-distance model and pairwise deletion of gaps/missing data, substitution model PAM Matrix (Dayhoff), and bootstrap of 1000 repetition. The scale bar denotes divergence percentage between sequences. Symbols: clones from Comandante Ferraz (●); clones from Botany Point (▴). Access numbers of alk genes from GenBank database reference strains are in parentheses.

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Considering the distribution of OPF in the libraries, it was noted that CF library had two predominant OPF (S1 and S2) in a total of four OPF, and BP had two predominant OPF also (S1 and S5), but in a total of 15 OPF. OPF S1 represented 59.2% of the library from CF and 52.4% of the library in BP (Fig. 1). This OPF had 64.34% identity with the alkB gene from Silicibacter pomeroyi DSS-3. The second predominant group was the OPF S2, corresponding to 32.9% of the library from CF, being the sole OPF that exhibit significant nucleotide identity to any alkane monooxygenase sequences currently in the GenBank database (99.88%).

The sequences from the Antarctic samples were more closely related to each other than to reference sequences. Only BP presented sequences related to alkB genes previously described in β- and γ-Proteobacteria. Alkane monooxygenases alkB described in α-Proteobacteria (102 clones) were the most abundant alk gene in the libraries. Despite reports that there is no clear linkage between the diversity of the alk genes and phylogenetic lines (Smits et al., 1999; van Beilen et al., 2003; Rojo, 2005), in this study, it was possible to see four distinct clusters in the phylogenetic tree detached from the cluster related to alkB from α-Proteobacteria, and alkM separate from alkB in γ-Proteobacteria (Fig. 2).

The results clearly showed the difference between CF and BP libraries and the high diversity indices found in BP. Preliminary studies on the presence and concentrations of aliphatic hydrocarbons in sediment samples from Admiralty Bay show differences of total n-alkanes (TNA) between these same sites compared with this study in three different years: 1990 [CF: 5.48 μg g−1 (dry weight); BP: 0.23 μg g−1], 1992 (CF: 3.27 μg g−1; BP: 0.14 μg g−1) and 1993 (CF: 2.89 μg g−1; BP: 0.29 μg g−1) (Bícego et al., 1998). A more recent investigation (Martins et al., 2004) found concentrations of TNA ranging from 0.10 to 9.63 μg g−1 (dry weight) in different sites in the Admiralty Bay. Despite detection of low concentrations of aliphatic hydrocarbons in the bay, the authors identify hydrocarbons in the CF area originating from human activities, as logistic activities and from sewage effluent. The authors also confirm that the sewage can reach no farther than 1 km away from the sewage outfall, implying that BP, located more than 3 km away, is not affected by the station effluent (Martins et al., 2004). These data may suggest that the difference in the composition of alk gene community may result from anthropogenic activities near the Brazilian scientific station, and also that the gene library from BP could better represent the presence and distribution of alk genes in Antarctic marine environment since it is from a pristine area with no anthropogenic influences.

Apparently, all genes identified here could be functional in the environment since their enzyme activity sites are extremely conserved and observed in all sequences. The region analysed here, around the two histidine clusters, was previously shown to be essential for the function of the structurally related desaturases (Shanklin et al., 1994). The activity of these genes in an Antarctic marine environment will be confirmed by the analysis of the alk genes carried by Antarctic isolates and after the development of experiments that corroborate the association of the alk gene expression and the degradation of the specific substrate. Moreover, the results show a range of alk genes not previously described, highlighting the need for more studies in this environment.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Sediment samples

Marine sediment samples were collected approximately at 20 m depth, in the summer of 2003/2004, using a van Veen grab. Sediment was sampled in two different sites in the Admiralty Bay. One site was located in front of the Brazilian scientific station Comandante Ferraz (CF) (62°05.12′S, 58°23.07′W), and is affected by human activity (Martins et al., 2004). The second one is a pristine site located across the Admiralty Bay, 3.2 km away, identified as Botany Point (BP) (62°05.97′S, 58°20.49′W).

Total DNA extraction and PCR amplification of alk genes

DNA of CF and BP marine sediment samples were extracted using the protocol of Piza and colleagues (2004). To detect the alk genes, degenerate primers were designed based on the first and third histidine-containing sequence motifs of the alkane monooxygenases, which contain four amino acid conserved sequences: alkF (5′-GCI CAI GAR ITI RKI CAY AA-3′) and alkR (5′-GCI TGI TGI TCI SWR TGI CGY TG-3′). The PCR was performed in a final volume of 25 μl containing 1× PCR buffer, supplemented with 1.5 mM MgCl2, 200 μM of each dNTPs, 1.2 μM of each primer, 1 U de Platinum Taq DNA Polimerase and 50 ng from the DNA template. The PCR consisted of an initial denaturation step at 97°C for 3 min, followed by 28 cycles of 1 min at 94°C, 1 min at 45°C and 1 min at 72°C, and a final elongation step of 5 min at 72°C. Four separate reactions were run for each sample and then pooled and purified with GFXTM PCR-DNA Band Purification kit (GE Healthcare).

Clone library construction and sequencing

Cloning of alk gene fragments (524 bp) was performed using the pGEM-T Vector (Promega), according manufacturer's guidelines, and transformed into Escherichia coli DH10b cells. M13 primers of the vector were used to amplify alk gene products from the vector of individual clone colonies to remove the amplicon from the vector. These PCR products were purified and then sequenced using the forward primer T7 of the vector pGEM and the DYEnamic ET Dye Terminator Cycle Sequencing kit (GE Healthcare) for MegaBace DNA Analysis Systems.

Phylogenetic analysis

The sequences from CF and BP clones were analysed using the BioEdit software package. All nucleotide sequences were assembled and the primer sequences were removed, maintaining the reading frame for translation to visualize the protein and the two internal motifs (motif B, EHXXGHH and motif C, NYXEHYG) of alkane monooxygenases (Smits et al., 1999; van Beilen et al., 2005). Reference sequences were obtained from GenBank/NCBI using the programs blastn and tblastx. Nucleotide sequences were translated in silico and phylogenetic trees were constructed by neighbour-joining (NJ) analysis using mega3 software with 1000 bootstrap replicates.

Diversity indices and statistics

Diversity was analysed using the software dotur (Schloss and Handelsman, 2005) and FastGroup II (Seguritan and Rohwer, 2001). For each clone library the Shannon–Weiner index (H′), the collect curve, CHAO and ACE non-parametric estimates of richness, and rarefaction curves were calculated. Sequences with > 82% identity (cut-off value) were assigned to the same OPF.

Nucleotide alk gene sequences accession numbers

The nucleotide sequences reported in this study were deposited in the GenBank database with the following accession numbers: EF446020 to EF446095 (CF clone library) and EF467066 to EF467166 (BP clone library).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

The authors wish to thank Dr Charles Greer (NRC-BRI) for helping with primer design. This research was supported by the Brazilian Antarctic Program (PROANTAR) by grants from the National Council for Scientific and Technological Development (CNPq). E. Kuhn was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
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