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Novel alkane hydroxylase (alkB) gene diversity in sediments associated with 1

hydrocarbon seeps in the Timor Sea, Australia. 2

3

Kenneth Wasmund1,2

, Kathryn A. Burns1, D. Ipek Kurtböke

2, David G. Bourne

1* 4

5

1Australian Institute of Marine Science, PMB 3 Townsville, Australia, 4810 6

2Faculty of Science, Health and Education, University of the Sunshine Coast, 7

Maroochydore DC, Australia, 4558. 8

Corresponding Author: D. G. Bourne 9

Email: [email protected] 10

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01370-09 AEM Accepts, published online ahead of print on 9 October 2009

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Abstract 11

Hydrocarbon seeps provide inputs of petroleum hydrocarbons to widespread 12

areas of the Timor Sea. Alkanes constitute the largest proportion of chemical 13

components found in crude oils and therefore genes involved in the biodegradation of 14

these compounds may act as bioindicators for this ecosystems response to seepage. To 15

assess alkane biodegradation potential, the diversity and distribution of alkane 16

hydroxylase (alkB) genes in sediments of the Timor Sea was studied. AlkB protein 17

sequences derived from clone libraries identified sequences only distantly related to 18

previously identified AlkB sequences, suggesting the Timor Sea maybe a rich 19

reservoir for novel alkane hydroxylase enzymes. Most sequences clustered with AlkB 20

sequences previously identified from marine Gammaproteobacteria, though protein 21

sequence identities only averaged 73% (with a range of 60%-94% sequence 22

identities). AlkB sequence diversity was lower in deep water (> 400 m) samples off 23

the continental slope compared to shallow water (< 100 m) samples on the 24

continental shelf, but not significantly different in response to levels of alkanes. Real-25

time PCR assays targeting Timor Sea alkB genes were designed and used to quantify 26

alkB gene targets. No correlation was found between gene copy numbers and levels of 27

hydrocarbons measured in sediments using sensitive GC-MS techniques, probably 28

due to the very low-levels of hydrocarbons found in most sediment samples. 29

Interestingly however, copy numbers of alkB genes increased substantially in 30

sediments exposed directly to active seepage, even though only low or undetectable 31

concentrations of hydrocarbons were measured in these sediments in complementary 32

geochemical analyses due to efficient biodegradation. 33

Key Words: Hydrocarbon seeps, Timor Sea, alkB, alkanes, real-time PCR and clone 34

libraries. 35


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Introduction 36

Alkanes are saturated hydrocarbons that are widespread in marine 37

environments due to a variety of anthropogenic and natural sources. They constitute 38

the major fraction of hydrocarbon components found in crude oils and refined 39

petroleum, and are also produced by various marine organisms (e.g., zooplankton) as 40

cellular components (2, 45). Alkanes are considered as pollutants, with short chained 41

alkanes acting as solvents towards cellular membranes and other lipid components 42

(35), while longer chained alkanes may contribute to the formation of oil films and 43

slicks that may limit nutrient and oxygen exchange (22). Importantly, alkanes also 44

serve as important carbon and energy sources for some microorganisms. In marine 45

environments, alkanes succumb to various removal and dispersal processes such as 46

dissolution, photochemical oxidation, evaporation, adsorption and sedimentation. 47

However, the greatest removal pathway for alkanes in marine sediments is via 48

biodegradation by bacteria (14). This mechanism also mediates the transfer of oil-49

derived carbon to higher trophic levels (29, 38) and therefore these bacteria have an 50

important role in carbon cycling in environments subject to long term inputs of 51

hydrocarbons such as marine seep associated ecosystems. Alkane biodegradation is 52

mediated by a diverse range of marine bacteria using various electron acceptors, 53

although degradation generally proceeds at greater rates under aerobic conditions in 54

comparison to anaerobic processes that proceed relatively slowly (8, 27). 55

In the presence of oxygen, well characterized alkane oxidation pathways are 56

initiated by an activation step whereby oxygen is introduced to the alkane substrate 57

before further catabolic steps can proceed. A number of oxygen-dependent alkane 58

hydroxylase enzyme systems have been discovered that catalyse this initial step 59

including the soluble di-iron methane monooxygenases and the membrane-bound 60


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copper-containing methane monooxygenases, both of which act upon short-chain 61

alkanes (i.e., C1 up to C8). Integral membrane non-heme iron alkane hydroxylases (the 62

‘alk’ system) that are related to the well characterised AlkB of Pseudomonas putida 63

GPo1 (also known as P. oleovorans TF4-1 I), act upon longer chain alkanes (i.e., C5 64

to C16) (41). Other systems including alkane-hydroxylating cytochrome P450 65

enzymes and other enzyme systems known to exist based purely on chemical analyses 66

of metabolites formed during alkane degradation experiments are known (23, 26, 30), 67

however, knowledge pertaining to the enzymes and genes involved, as well as their 68

importance in the environment is limited. Only recently have genes involved in the 69

degradation of long chain alkanes (e.g., C32 and C36) been identified in Acinetobacter 70

sp. strain DSM 17874 (40), though there is no information about the presence or 71

importance of such enzymes in the environment. 72

Although various chemical and microbiological aspects of petroleum oil and 73

alkane biodegradation in marine systems have been relatively well studied, there is a 74

general lack of knowledge concerning the diversity or abundance of functional genes 75

involved. The biochemical and molecular aspects of alkB genes and the enzymes they 76

encode have been relatively well studied and this has enabled the development of 77

molecular tools for the study of alkB genes in the environment (20). Elevated levels of 78

hydrocarbons or the introduction of hydrocarbons to environments has been shown to 79

increase gene copy numbers, indicating the potential use of alkB genes as 80

bioindicators of oil pollution and/or biodegradation (17, 34, 37, 44). However, to date 81

only one study has used culture-independent molecular methods to examine the 82

diversity of alkB genes in a marine environment (21) and no studies have examined 83

hydrocarbon degrading genes where natural hydrocarbon seepage occurs. 84


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In this study, the diversity and relative abundance of alkB genes was examined 85

in sediments of the Timor Sea, a region where natural seeps are sources of widespread 86

petroleum hydrocarbons. It was hypothesized that (i) novel alkB genes may exist in 87

this unique tropical marine environment, (ii) that variations in gene diversity would be 88

found in sediments with different hydrocarbon levels, and (iii) that the abundance of 89

certain alkB gene types may reflect the levels of measured hydrocarbons in sediments 90

and therefore this assay could be used as a complimentary tool for monitoring 91

petroleum inputs into sediments of the Timor Sea. 92

93

Methods 94

Sample collection and processing 95

Samples were collected from the Timor Sea, Northwestern Australia, during 96

the RV Southern Surveyor cruise SS05/06 of June 2005. A heavily weighted Smith-97

MacIntyre (0.25 m2 surface area) grab sampler was used to obtain sediment grabs 98

with undisturbed surface sediments. Grabs were then sub-sampled with a sterile core 99

device (5 cm inner diameter and 30 cm in length) shipboard. Locations of sediment 100

grabs are indicated in Table 1 and Figure 1. All cores were sectioned into 1 cm 101

intervals and stored at –20oC shipboard, in liquid nitrogen (–180

oC) during transport 102

(5 days) and at –80oC in the laboratory prior to molecular analysis. Details of 103

analytical methods for measuring hydrocarbon content are reported previously (4). 104

105

DNA extraction 106

The DNA extraction protocol was based on previously described procedures 107

for the extraction of total DNA from soil samples (10, 46). Briefly, the sediment 108

slurry (0.5 g wet weight) was placed in a 1.5 ml tube and suspended in 1.35 ml of 109


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extraction buffer [100 mM Tris-HCl (pH 8.0), 100 mM sodium EDTA (pH 8.0), 100 110

mM sodium phosphate (pH 8.0), 1.5 M NaCl, 1% (w/v) CTAB to which 5 µl of fresh 111

Proteinase K (20 mg ml−1

) was added. The sample was placed horizontally on a 200 112

rpm shaker at 37°C for 30 min. After shaking, 150 µl of SDS [20% (w/v)] was added 113

and the tube was placed at 65°C for 2 h and mixed by inversion every 15 min. The 114

supernatant was decanted into a clean tube after centrifugation for 10 min at 6000 x g 115

and 25°C. The sediment pellet was again suspended in 450 µl of extraction buffer and 116

50 µl of SDS [20% (w/v)] was added, incubated at 65°C, centrifuged as before and the 117

supernatant added to the first aliquot. The crude extract was gently extracted with an 118

equal volume of chloroform [containing 4% (v/v) isoamyl alcohol to minimize 119

foaming] and centrifuged at 16 000 x g for 10 min at 25°C. The aqueous phase (upper 120

layer) was transferred to a clean tube and the DNA was precipitated by adding 0.6 vol. 121

of 2-propanol. The DNA was left to precipitate for 1 h at 20°C. The DNA was 122

pelleted by centrifugation at 16 000 x g for 30 min at 20°C. The DNA pellet was 123

rinsed with 500 µl of 70% (v/v) ethanol and air-dried for 20 min. DNA was 124

resuspended in 50 µl of deionized water and diluted 1:10 to facilitate PCR 125

amplification. DNA concentrations were determined using a NanoDrop ND1000 126

(NanoDrop Technologies) in triplicate. All DNA extracts were stored at –20°C. 127

128

PCR amplification of alkB genes 129

Partial alkB genes were amplified using the forward primer alkB-1f 130

(5′-AAYACNGCNCAYGARCTNGGNCAYAA-3’) and the reverse primer alkB-1r 131

(5′-GCRTGRTGRTCNGARTGNCGYTG-3’) (20). All PCR reactions (final volume 132

of 25 µl) contained 1 x Qiagen PCR Buffer (Qiagen), 1 U of HotStarTaq DNA 133

Polymerase (Qiagen), 200 uM of each dNTP, 25 pmoles of each primer, 0.5 µl of 134


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purified Bovine Serum Albumin (BSA) 10 ug µl-1

(New England Biolabs), 0.5 µl of 135

DNA template (~ 50 ng) and deionized water up to 25 µl. PCR cycling conditions 136

included an initial ‘enzyme activation’ step at 95°C for 15 min, followed by an 137

additional 39 cycles of 94°C for 1 min, annealing at 55°C for 1 min and extension at 138

72°C for 1 min. A final extension step of 72°C for 10 min was included to facilitate 139

‘A-tailing’ of PCR products for cloning. 140

141

Clone library construction, colony hybridization and DNA sequencing 142

PCR products from three replicate reactions for each sample were pooled and 143

subject to agarose gel electrophoresis. Bands of expected size were excised and 144

purified using a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturers 145

instructions. Purified DNA was then cloned using a TOPO-TA Cloning Kit 146

(Invitrogen) according to the manufacturers instructions. Individual colonies were 147

suspended in 100 µl of sterile deionized water using a sterile toothpick, vortexed 148

briefly, allowed to stand for 60 min, vortexed again and this cell suspension used as 149

template for PCR using the M13 primers to confirm inserts. Colony hybridisation to 150

identify positive alkB inserts in clones was performed as previously described (20). 151

Generated PCR products were dried and sent to Macrogen Inc. (Seoul, South Korea) 152

for purification and sequencing using an ABI3730 XL automatic DNA sequencer 153

using the M13F vector specific primer. 154

155

Phylogenetic analysis of alkB genes 156

Retrieved alkB gene nucleotide sequences were initially checked using 157

Chromas Lite software version 2.01 (Technelysium) before being truncated to exclude 158

primer and vector sequence. Nucleotide sequences were translated into protein 159


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sequences using the Translate tool in the ExPASy (Expert Protein Analysis System) 160

proteomics server of the Swiss Institute

of Bioinformatics 161

(http://us.expasy.org/tools/dna.html). Deduced protein sequences from each library 162

were grouped into ‘Operational Protein Units’ (OPUs) using DOTUR software (32) 163

with a distance threshold of 0.20 (80% sequence similarity). This cut-off value was 164

selected after initial examination of phylogenetic trees which included all nucleotide 165

sequences generated by neighbour-joining analysis. The phylogenetic comparison 166

identified a general clustering of AlkB sequences at this level, i.e., when sequences 167

from distinct clusters were aligned using the BLAST (bl2seq) they generally exhibited 168

more than 80% sequence identity. This approximate distance threshold was also used 169

in a similar study of AlkB sequences in Antarctic sediments (21) and therefore allows 170

comparisons between studies. Deduced protein sequences were aligned using 171

ClustalX version 1.83 (39) with related reference sequences identified from BLAST 172

(1) searches, in addition to other sequences of various representative AlkB sequences 173

identified from the literature. Distance matrices were calculated using the PROTDIST 174

program in PHYLIP (11, 12). Phylogenetic trees were generated from distance 175

matrices using the neighbor-joining method (31) and Kimura substitution algorithm 176

(19) using PHYLIP. Bootstrapping with 1000 replicates was performed using SeqBoot 177

as integrated in PHYLIP. 178

179

Statistical analysis of clone library data 180

Rarefaction analysis (16), Ace, Chao1 nonparametric richness estimates (7), 181

Simpson Index of diversity (24) and Shannon-Weaver Index of diversity (33) were 182

generated using DOTUR software (32). Statistical evaluations were obtained using the 183

80% amino acid sequence similarity. 184


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185

Real-time PCR amplification of alkB genes 186

Real-time PCR assays were carried out using a Rotor-Gene 3000 real-time 187

DNA amplification system (Corbett Research) using a Platinum SYBR Green qPCR 188

SuperMix-UDG kit (Invitrogen). PCR reactions (final volume of 25 µl) contained 189

12.5 µl of 2 x Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen), 4.0 mM 190

MgCl2, 5 pmoles of each primer, 0.5 µl of purified BSA (10 µg ul-1

; New England 191

Biolabs), 2.0 µl of DNA template (DNA extracts from 1:10 dilutions of crude extracts 192

were used and exhibited no PCR inhibition) and deionized water up to 25 µl. Primers 193

were designed to target specific clusters of abundant alkB genes detected in clone 194

libraries to avoid problems associated with aberrant PCR efficiencies associated with 195

the use of the highly degenerate primers used for the amplification of diverse alkB 196

genes. Two sets of primers targeting Clusters A and C (Figure 3) were designed after 197

examination of alignments from Clustal X. The design of primers suitable for real-198

time PCR application was aided by using the program NetPrimer 199

(http://www.premierbiosoft.com/netprimer/index.html). AlkB genes from 200

Cluster A were amplified using the forward primer 201

A-f (5′-TACGGGCACTTCGCGATTGA-3’) and the reverse primer A-r 202

(5′-CGCCCAGTTCGAMACGATGTG-3’). AlkB genes from Cluster C were 203

amplified using the forward primer C-f (5′-TCGTACTTGCCGTGCCTGTGTA-3’) 204

and the reverse primer C-r (5′-CGATCAGCGTCAGTTGAATCAC-3’). Real-time 205

PCR cycling conditions included an initial ‘UDG incubation’ step at 50°C for 2 min, 206

an ‘enzyme activation’ step at 95°C for 2 min, followed by 40 cycles of 95°C for 207

20 sec, annealing at 59°C for 20 sec and extension at 72°C for 40 sec. Acquisition of 208

fluorescence signal was performed during the 72°C extension step of each cycle. This 209


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was followed by a melt curve from 45°C to 95°C. In addition to melt curve analysis, 210

PCR products were checked on a 1% agarose gel to ensure they were the expected 211

size. 212

The DNA standards used in the real-time PCR assays consisted of serial 213

dilutions of purified PCR product derived from cloned alkB genes. Clones F44 and 214

G17 (see Figure 3) were used as standards for Cluster A and C, respectively. 215

Standards were PCR amplified directly from a colony using M13 vector specific 216

primers, checked using standard agarose gel electrophoresis, gel purified using a 217

QIAquick Gel Extraction Kit (Qiagen) according to the manufacturers instructions, 218

and DNA concentrations were determined using a Quant-iT PicoGreen dsDNA 219

quantitation kit (Molecular Probes) using fluorimetry. Measured concentrations of 220

purified PCR product were then converted to copies per microliter and the 221

concentration was adjusted to 1 x 109

copies µl-1

prior to performing serial dilutions. 222

A five point standard curve (1 x 105 to 1.0 x 10

1 copies per reaction) was run in 223

duplicate with each run, and each run was performed twice (totaling four replicates 224

per sample). Environmental samples and negative controls (no template DNA) were 225

included in each run and were also performed in duplicate. Data and copy numbers of 226

alkB targets in environmental samples were analysed using the Rotor-Gene software 227

version 6.1.71 (Corbett Research) following manufacturers guidelines. Final copy 228

numbers of alkB genes in environmental samples were calculated assuming 100% 229

DNA extraction efficiency and were expressed as copy numbers per gram of 230

sediment. 231

232

Genbank accession numbers 233


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Nucleic acid sequences determined in this study have been deposited in the 234

GenBank/EMBL/DDBJ databases. The accession numbers for sequences of the genes 235

are: GQ184383-GQ184421, GQ184423-GQ184432, GQ184434. 236

237

Results 238

Sample description and hydrocarbon chemistry 239

Samples were collected from shallow continental shelf waters associated with 240

the active Cornea seep area (Grabs D, E, and H), from waters north of the Cornea seep 241

area in a ‘paleo-riverbed’ (Grab G and F), and from deeper waters off the continental 242

slop (Grabs B, J, K L, and M) (Figure 1). Details of sample coordinates and 243

hydrocarbon concentrations are presented in Table 1. Concentrations of measured 244

total hydrocarbons (THC) were below detection limits in Cornea seep associated 245

sediments (Grabs D, E and H), while only 11.5 ng g-1

of total n-alkanes were 246

measured in sediments of Grab H that are presumed to be directly influenced by 247

hydrocarbons actively seeping from underlying sediments to the water column, as 248

detected by ship-board echo-sounder and towed video camera surveys. Concentrations 249

of polycyclic aromatic hydrocarbons (oil PAHs) at the Cornea seep area were among 250

the lowest in the Timor Sea, ranging from 3.2-62 ng g-1

. Relatively low to mid ranged 251

concentrations of THC (0.19-0.66 mg g-1

), total n-alkanes (40.5-57.8 ng g-1

) and oil 252

PAHs (5.2-5.4 ng g-1

) were measured in Grabs G and F taken from the paleo-riverbed 253

sediments to the north of the Cornea seep area. The highest concentrations of 254

petroleum hydrocarbons were measured in sediments (Grab B) to the west of the 255

Sahul Shoals, with relatively high THC (8.42 mg g-1

), total n-alkanes (344 ng g-1

) and 256

oil PAHs (144.4 ng g-1

). Relatively high concentrations of hydrocarbons in grabs from 257

the other deep water locations within the Cartier Trough (Grabs J, K, and L) were also 258


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found, with concentrations of THC (0.05-0.40 mg g-1

), total n-alkanes (45.8-210.3 ng 259

g-1

) and oil PAHs (19.9-57.1 ng g-1

) being measured. 260

261

Diversity of AlkB sequences 262

Rarefaction analysis (Figure 2) of a clone library constructed from deep-water 263

samples that possessed the highest measured hydrocarbon content (Grab B) displayed 264

similar AlkB deduced protein diversity when compared directly against a low 265

concentration site (Grab M). The highest AlkB diversity of all samples was found 266

within Grab G from the paleo-river to the north of the Cornea seep area and was only 267

slightly higher than diversity predicted in sediments closely associated with the largest 268

seep plume identified in the Timor Sea (Grab H) and those from another paleo-river 269

sample (Grab F). Rarefaction curves did not reach an asymptote for any for the 270

libraries (Figure 2), suggesting a greater diversity of AlkB sequences were present in 271

the samples than revealed by the sequencing effort. Ace and Chao estimators also 272

suggested more OPUs were present in the samples than were detected in the clone 273

libraries (Table 2). Rarefaction curves for libraries F, G and H (on the continental 274

shelf) indicated higher diversity than for libraries B and M (off the continental shelf) 275

(Figure 2). Both Shannon-Weaver and Simpson indices of diversity were consistent 276

with rarefaction analysis showing greater diversity of AlkB sequences in libraries G, 277

F, and H than in libraries B and M (Table 2). 278

279

Phylogenetic analysis of AlkB sequences 280

Phylogenetic analysis of AlkB deduced protein sequences revealed that all 281

Timor Sea sequences were not closely related to any previously identified AlkB 282

sequences present in the public databases (Figure 3). Overall, sequence similarity of 283


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Timor Sea AlkB sequences to sequences present in the public databases ranged from 284

60%-94%, with an average sequence similarity of only 73%. Most Timor Sea AlkB 285

sequences grouped in a large cluster of diverse sequences that were most closely 286

related to marine and mostly gammaproteobacterial derived AlkB sequences, although 287

a few soil derived sequences also clustered within this group. Two sequences 288

representing OPUs B4 and M61 affiliated with AlkB sequences derived from Gram-289

positive actinobacterial Nocardia sp. strain CF8 and Prauserella rugosa strains. Other 290

sequences belonging to OPUs G69, H12 and M60 also affiliated most closely with 291

other marine derived AlkB sequences. No trends in the clustering of sequences from 292

different sites were apparent (e.g., high hydrocarbon concentrations versus low 293

hydrocarbon concentrations, or deep water versus shallow water), and was most 294

evident by the fact that sequences from each site were well represented in Clusters A 295

and C, which were comprised of the most abundant sequences in each library. 296

Importantly, all deduced AlkB protein sequences included in this analysis revealed the 297

presence of two highly conserved regions (motif B, EHXXGHH and motif C, 298

NYXEHYG) identified as important for alkane hydroxylase activity (results not 299

shown) (36, 43). 300

301

Quantification of alkB genes using real-time PCR 302

Previously designed primers targeting alkB genes belonging to marine alkane 303

degrading bacteria Thalassolituus sp. and Alcanivorax borkumesus (5, 25) were 304

unable to produce positive amplification products when applied to Timor Sea 305

sediment samples. Therefore quantitative real-time PCR primers were designed 306

targeting the most abundant alkB gene sequences identified in Timor Sea clone 307

libraries. Two sets of primers targeting Clusters A and C (Figure 3) proved to be 308


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functional under real-time PCR conditions, i.e., produced acceptable amplification 309

efficiencies across all samples and standards, and were specific for the alkB genes 310

from which they were designed for, as tested by PCR using plasmid DNA from target 311

and non-target clones as template (results not shown). 312

When these primers were used for the evaluation of the relative abundance of 313

these gene groups in Timor Sea sediments, gene copy numbers were found to be 314

highest in sediments of Grab H sampled from the area of the largest active seep plume 315

in the Timor Sea. Approximately 2.9 x 105 and 1.1 x 10

5 copies of the alkB gene per g 316

of sediment were recovered for primer sets targeting Clusters A and C, respectively 317

(Figure 4). These gene copy numbers in sediments of Grab H were higher than gene 318

copy numbers identified in all other sediment samples. For example, other sediments 319

from the Cornea seep area (Grabs D and E) and sediments from the paleo-river to the 320

north of the Cornea seep area (Grabs G and F) displayed lower levels of alkB genes, 321

revealing no more than 2.3-3.3 x 104 alkB gene copies per g of sediment for both 322

Clusters A and C (Figure 4). Low or no copies of alkB genes were detected in all 323

deep water sediments with primers targeting Cluster A or were below detection limits 324

with primers targeting Cluster C (Figure 4). 325

326

Discussion 327

In this study, sediment samples were taken from various geographically 328

separated sites in the Timor Sea to explore and compare the diversity and abundance 329

of the alkB functional gene in relation to levels of hydrocarbons measured in 330

sediments. Phylogenetic analysis of Timor Sea AlkB sequences revealed a novel array 331

of AlkB sequence types. Essentially all AlkB OPUs were divergent to previously 332

characterized AlkB sequences with average amino acid sequence identities of only 333


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73% (range 60%-94%) to sequences available in public databases. These results 334

suggest that the Timor Sea harbors a unique suite of AlkB sequences probably with a 335

range of substrate specificities and/or induction patterns enabling the degradation of 336

various n-alkanes. Most sequences were related to AlkB sequences derived from 337

marine Proteobacteria (mostly gammaproteobacterial). Effectively all marine bacteria 338

so far implicated in the degradation of alkanes (elucidated predominately through 339

culture-dependent studies) belong to the Gammaproteobacteria (15), and therefore 340

these results independently support the notion that members of the 341

Gammaproteobacteria are responsible for the aerobic degradation of alkanes in 342

marine environments. Results from this study are in contrast to analysis of alkB genes 343

from soils which identified mostly Gram-positive derived genes (20) and suggest 344

markedly different alkane degrading bacteria exist in the marine environment in 345

comparison to terrestrial environments. 346

Interestingly, AlkB sequence diversity did not appear to be influenced by 347

levels of measured alkanes since gene diversity was not substantially different in 348

sediments with high concentrations of hydrocarbons versus sediments with low 349

concentrations, or in sediments in direct association with active seepage that are 350

presumably exposed to high concentrations of hydrocarbons. For instance, in libraries 351

from Grab B (the highest hydrocarbon levels measured in the Timor Sea) and Grab M 352

(very low hydrocarbon levels) that were taken from comparable depths and away 353

from any active seepage, differences in AlkB sequence diversity as assessed by 354

rarefaction analysis and diversity statistics of clone libraries, were minimal. In a study 355

of AlkB sequence diversity in samples influenced by anthropogenic activities 356

polluting Antarctic marine sediments, much lower AlkB sequence diversity was 357

identified in heavily contaminated in comparison to less-contaminated sediments (21), 358


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suggesting an enrichment of specific gene types with increasing alkane concentrations 359

and resulting lower diversity indices. Levels of alkanes in contaminated Antarctic 360

sediments were an order of magnitude higher in contaminated sediments (e.g., 5 µg g-

361

1 total n-alkanes) versus ‘control’ (e.g., 0.29 µg g

-1 total n-alkanes) sediments. These 362

Antarctic ‘control’ sediments had alkane concentrations comparable to the highest 363

levels measured in the Timor Sea, suggesting concentrations measured in Timor Sea 364

sediments may not have been sufficient to cause significant shifts in gene diversity as 365

was observed in the Antarctic study. 366

In addition, Ace and Chao indices predicted over three times higher diversity 367

in Timor Sea sediments than in Antarctic sediments. It is possible that since Timor 368

Sea sediments have been exposed to naturally seeping hydrocarbons since the 369

Pliocene era (28) that a greater diversification of AlkB sequences has occurred in this 370

ecosystem, in comparison to the historically pristine Antarctic environments. The high 371

diversity in the Timor Sea may be ‘constitutively’ present through out the region and 372

therefore the detectable diversity may be relatively unaltered when exposed to alkane 373

inputs. In contrast, Antarctic sediments which have been pristine in the past, have 374

apparently less diversity and therefore become dominated by certain AlkB sequence 375

types when exposed to anthropogenic sources. 376

The only notable differences in AlkB sequence diversity identified in this 377

study were between shallow water sites (i.e., Grabs F, G and H, on the continental 378

shelf, in < 100 m water) and deep water (i.e., Grabs B and M, off the continental 379

shelf, in> 400 m water). All shallow water samples generally displayed greater AlkB 380

sequences diversity as assessed by rarefaction analysis and statistical analyses of 381

clone libraries. Interestingly, no studies have investigated bacterial diversity in 382

sediments over depth gradients in the ocean. It is possible that bacterial diversity 383


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decreases with increasing depth, driven by factors such as decreasing amounts of 384

labile organic matter and therefore available substrates for nutrition, in addition to 385

increasing barometric pressures and decreasing temperatures. If such changes in 386

bacterial diversity do occur through depth, these may account for the reduction in the 387

AlkB sequence diversity at depth observed in this study. 388

Quantification of alkB genes belonging to Clusters A and C (Figure 3) using 389

real-time PCR identified significantly higher copy numbers in sediments associated 390

with an actively venting seep (Grab H) where hydrocarbons are seeping from the 391

underlying sediments and into the water column. Interestingly, hydrocarbon 392

concentrations in sediments exposed to active seepage (and in other sediments from 393

the Cornea seep area, i.e., Grabs D and E) were relatively low or even undetectable. 394

This suggests that rapid microbial degradation of hydrocarbons is occurring and that 395

this rapid degradation removes detectable quantities of hydrocarbons before they can 396

be measured by geochemical analyses. The quantification of alkB gene copy numbers 397

in sediments therefore provides an insight into the microbial response to the seepage 398

of hydrocarbons and acts as a useful complementary tool for understanding this 399

ecosystems response to hydrocarbons in addition to geochemical measurements that 400

suggest little or no exposure to hydrocarbons if used solely without other 401

complementary observations or data. Interestingly, no considerable increases in gene 402

copy numbers were detected in sediments that had high measured hydrocarbon 403

concentrations (i.e., Grab B which had the highest hydrocarbon concentrations 404

measured in the Timor Sea), in comparison to other samples from comparable water 405

depths that had low or undetectable hydrocarbon concentrations (e.g., Grab M). 406

Quantitative molecular methods targeting hydrocarbon degrading genes in 407

environmental samples have generally identified some correlations between 408


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hydrocarbon degrading gene copy numbers and hydrocarbon concentrations (6, 9), 409

although other studies have found only weak correlations (18). Within this study it is 410

likely that hydrocarbon concentrations in the Timor Sea (with the exception of 411

sediments exposed directly to active seepage, e.g., Grab H) may not be high enough to 412

invoke dramatic shifts on gene copy numbers such as occurs in study sites where such 413

correlations have been made. Quantitative mRNA-based approaches may be useful as 414

a more sensitive tool for monitoring subtle changes in gene copy numbers in marine 415

sediments exposed to low levels of hydrocarbons. The assay developed here however, 416

may be sensitive enough to be applied as a tool for monitoring, for example, the 417

effects of oil released from petroleum oil extraction operations in the Timor Sea, such 418

as those studied by Burns and Codi which monitored the spatial effects of oil 419

discharge from oil rigs in the Timor Sea (3). 420

Since the actual hydrocarbon concentrations measured in sediments did not 421

appear to have a significant influence on gene copy numbers, other factors appear 422

important in determining gene copy numbers in this oceanic environment. Water 423

depth in combination with fluxes of hydrocarbons from the water column to 424

sediments may be important in determining numbers of alkB genes in this ecosystem, 425

at least in sediments not exposed directly to active seepage (e.g., all grabs excluding 426

Grab H). For example, alkB gene copy numbers were very low or undetectable in 427

deep water (> 400 m) sediment samples (i.e., Grabs B, K, L, and M) off the 428

continental shelf and well away from any known active seepage, but easily detectable 429

in all samples (Grabs D, E, F, G and H) from shallow water sediments (c. 100 m) on 430

the continental shelf. Complementary research that measured fluxes of hydrocarbons 431

from the water column to sediments using a layered sediment trap approach revealed 432

that only a small flux of alkanes actually reach deeper waters of more than 400 m, 433


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e.g., up to 85% of n-alkanes are degraded while settling from 100 m to 400 m water 434

depth (unpublished results). Therefore it seems fluxes of alkanes to deep water 435

sediments are probably not high enough to sustain detectable numbers of alkB genes 436

using the real-time PCR assays applied here. 437

This study provides new insights into the diversity of alkane degrading 438

enzymes in the marine environment. The identification of novel AlkB sequence 439

diversity points to the existence of a diverse range of what are most likely 440

gammaproteobacterial species/gene-types capable of degrading alkanes in the Timor 441

Sea. Such AlkB enzymes are worthy of further investigation for biotechnological 442

applications, since these enzymes have various applications in the synthetic 443

production of various compounds such as secondary metabolites, pharmaceuticals and 444

agrochemical intermediates (42). High copy numbers of alkB genes were identified in 445

association with an actively venting seep, demonstrating enhanced alkane degrading 446

capacity at this site. However, despite high measured gene copy numbers this did not 447

equate to distinct changes in observed alkB gene diversity when compared to other 448

sampled sediments. Chronic exposure of sediments to hydrocarbon inputs appeared to 449

not alter alkB gene copy numbers within the sediments, though efficient and rapid 450

biodegradation of hydrocarbons was occurring, mediated by microbial communities 451

that are well adapted to the readily available carbon source. It is anticipated that future 452

examination of alkB gene transcripts could be used as a more sensitive tool for 453

assessing the response of alkB gene diversity and copy numbers to hydrocarbons in 454

this environment. 455

456

Acknowledgements 457


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We thank the crew and scientific support staff of the RV Southern Surveyor 458

during cruise SS05/06 to the Timor Sea for collection of samples during June of 2005. 459

A special thanks to Gregg Brunskill and Irena Zagorskis in aiding in sampling and 460

geochemical analyses. Tim Simmonds is thanked for help in preparation of the 461

manuscript figures. The author thanks the Australian Biological Resources Study 462

(ABRS) and Commonwealth Scientific and Industrial Research Organization 463

(CSIRO) for stipend and financial support. 464

465

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614


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Figure 1. Locations of sediment grab samples in the Timor Sea. Sediment grab 615

locations are indicated by open circles. Sediment grabs D, E and H are in close 616

proximity to each other and are represented by one open circle at the Cornea seep 617

area. 618

619

Figure 2. Rarefaction analysis of AlkB deduced protein sequences from the Timor 620

Sea. OPUs were defined using a 20% sequence similarity cut-off. 621

622

Figure 3. Phylogenetic tree based on deduced AlkB amino acid sequences retrieved 623

from this study relative to reference sequences from cultured bacteria and sequences 624

retrieved from other studies. Unique sequences from each library are presented in bold 625

type. Numbers presented in brackets after each sequence retrieved in this study 626

indicate the number of sequences from each OPU retrieved from each clone library. 627

Bootstrap values of ≥50% and ≥90% from 1000 resamplings are presented at nodes as 628

filled (•) and open circles (o), respectively. The tree was rooted with a xylene 629

monooxygenase subunit-1 amino acid sequence from Pseudomonas putida mt-2 (13). 630

The scale bar represents 10% sequence divergence. 631

632

Figure 4. Quantitative real-time PCR analysis of Timor Sea sediments targeting alkB 633

genes recovered in clone libraries. Error bars indicate standard deviations in gene 634

copy number measured from replicate PCRs. 635

636


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Table 1. Details of sediment grab locations and hydrocarbon content. 637

638

Sample

code Coordinates Depth

Geographical

description

Total

hydrocarbons

(mg g-1

)

Total n-alkanes

C11-C38 (ng g-1

)

Sum oil

PAHs

(ng/g)

B 11:26.61S

123:59.67E 482m

West of Sahul

shoals 8.42 344 144.4

D 13:39.31S

124:42.69E 89.2m

Cornea seep

area < D.L.

a < D.L. 6.2

E 13:39.15S

124:42.69E 88.4m

Cornea seep

area < D.L. < D.L. 3.2

F 13:24.71S

124:41.80E 107.2m North of seep 0.66 57.8 5.4

G 13:30.62S

124:42.48E 108.4m North of seep 0.19 40.5 5.2

H 13:39.27S

124:42.70E 90m

Cornea active

seep < D.L. 11.5 4.3

J 11:42.32S

125:01.61E 269m Cartier trough 0.05 45.8 19.9

K 11:30.65S


L 11:22.00S


M 11:12.50S

125:12.51E 430m Cartier trough < D.L. < D.L. 7.9

N 11:22.65S

126:52.60E

106.8m Darwin Shelf b 0.10 71.2 15.0

639

a Detection Levels (D.L.) were based on individual peak detection within a complex mixture. For these 640

summaries the estimate was < ~0.05 mg g-1

total hydrocarbons (4). 641

b Darwin Shelf was a station with no known active oil seepage nearby and was used as a ‘control’ site. 642

643

644


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Table 2. Diversity analysis of alkB deduced protein sequences. 645

Library No. of clones No. of OPUs ACE a Chao1

b 1 – D

c H′

d

B 57 8 31.5 27 0.77 1.82

F 40 10 33.6 28 0.84 2.11

G 41 13 51.8 50 0.86 2.28

H 56 14 49.8 36 0.84 2.23

M 52 8 37 48 0.75 1.67

646

a ACE richness estimate 647

b Chao1 richness estimate 648

c Simpson Index of diversity 649

d Shannon-Weaver Index of diversity 650


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