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Keywords:

  • Great Artesian Basin;
  • Thermophile;
  • Clone library

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

Culture-independent modes of analysis were chosen to investigate a microbial mat associated with the thermal waters of the Great Artesian Basin (GAB). 16S rDNA was amplified from total genomic mat DNA, and used to construct a clone library. Use of plasmid-specific primers to amplify the inserts from 92 selected recombinants proved to be an effective approach, and demonstrated that there were four size categories of insert: 1500 bp (62% of clones examined), 1400 bp (25% of clones examined), 500 bp (5% of clones examined) and 240 bp (8% of clones examined). Restriction enzyme analysis was evaluated for its ability to group the 1500 bp and 1400 bp size inserts into operational taxonomic units. Clone inserts were presumptively identified by analysis of partial sequence data, and each operational taxonomic unit was found to be phylogenetically cohesive. Phylogenetic analyses of the sequence data indicated the presence of a broad range of bacteria related to the cyanobacteria, Thermus species, thiobacilli, planctomycetes, thermophilic hydrogen oxidisers, thermotogales, clostridia, actinomycetes, and β and δ subclasses of the proteobacteria. Use of the restriction enzyme analysis protocol also enabled the extent of insert repetition within the library to be monitored, and would thus have eliminated 70% of sequencing expenses. This is the first time a community analysis has been performed on this extreme environment.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

The Great Artesian Basin (GAB) is a massive system of aquifers covering more than one-fifth of Australia [1]. Its naturally heated, non-volcanic waters underlie arid regions, and are vital for the maintenance of many rural establishments and a major pastoral industry [2]. Despite the economic value of this resource, little is known regarding its ecology. It has been established that the GAB waters are alkaline, very old [3] and differ chemically from those of volcanic hot springs [4]. Prolific microbial mats are associated with the bore pools and run-off drains. The main objective of this study was to investigate the bacteria associated with one such mat. Given the unique nature of this extreme environment, it is not unreasonable to suspect the presence of bacteria of biotechnological importance and evolutionary significance. To date, studies aimed at the isolation of thermophiles from the GAB have concentrated on anaerobes (e.g. [4–6]).

Difficulties associated with the isolation and growth of thermophiles have been documented [7, 8]. Failing to understand or accurately reproduce a microbe's original habitat means that culture-dependent approaches may meet with limited success [9]. Therefore, in an effort to accurately describe the microbial populations inhabiting the GAB, molecular methods were employed.

Numerous methods have been developed to retrieve ribosomal nucleic acids directly from environmental samples, and subsequent analysis of these sequences provides a non-culture dependent means of identifying community members. A common technique involves the selective amplification, cloning and sequencing of 16S rDNA (e.g. [10–14]), and this strategy was chosen to investigate a microbial mat associated with the GAB.

Restriction enzyme analysis (REA) has been used to screen 16S rDNA libraries prepared from a number of environments (e.g. [11, 13, 15]). The 16S rDNA lends itself to this kind of analysis as not only does the gene contain highly conserved regions, but also diagnostic variable regions that are unique to particular organisms or closely related groups of organisms [15]. The clonal types i.e. those with identical restriction patterns, can be grouped into operational taxonomic units (OTUs) [15]. The other method of analysis of clone libraries is partial or complete insert sequencing of clones. Representatives of the OTUs according to REA can be sequenced rather than sequencing all clones. Not only does REA reduce the sequencing load, it also provides one with a rapid preview of the biodiversity present in the library. Therefore REA was tested for its ability to screen the clone library prepared from the hot spring mat.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

2.1Collection of bacterial mat samples

Bore RN 1627 is an artesian well located in the Charleville district of western Queensland, Australia. Water leaves the outlet pipe at a temperature of 63°C and a pH of 8.1. Prolific microbial mats were visible in the bore pool and run-off drains. Mat samples were collected from the bore pool (62°C) in sterile containers which were sealed, placed on ice for 6 h, and then frozen.

2.2Extraction and purification of nucleic acids from the microbial mat

A 10-g (wet weight) section of the microbial mat and 40 ml of saline EDTA were ground with a cold (4°C) sterile mortar and pestle. The resultant slurry was transferred to two sterile 40-ml centrifuge tubes, 20 μl lysozyme (1 mg ml−1) was added to each sample, the tubes were incubated at 37°C for 30 min; then 1 ml of SDS (25%) and 1 ml proteinase K (1%) were added to each sample and incubated at 60°C for 30 min. The tubes were placed on iced water in a sonicating waterbath and sonicated for 30 s. The samples were centrifuged at 10 000×g for 10 min, and the supernatants extracted with equal volumes of phenol-chloroform, chloroform-isoamyl alcohol. Aliquots (200 μl) of the supernatant were purified using the Prep-a-Gene DNA purification kit (Bio-Rad Laboratories) as per manufacturer's instructions, with the following modification: DNA binder was increased to 20 μl and the binding buffer increased to 650 μl.

2.3Amplification of 16S rDNA

The mixed 16S ribosomal genes were selectively amplified from the purified community genomic DNA by using the heat-soaked PCR [16]. Each 100-μl reaction contained 0.05 μg purified extracted DNA; dATP, dCTP, dGTP, and dTTP at 0.2 mM each; 50 mM KCl; 10 mM Tris-HCl (pH 8.3); 1.8 mM MgCl2, 0.1% gelatin; 2.5 U of Taq polymerase (Boehringer Mannheim) and 0.4 μM of the following modified amplification primers (numbering based on the Escherichia coli 16S rDNA): 27F, 5′-GCGGGATCC/GAGTTTGATCCTGGCTCAG-3′ and 1492R, 5′-GGCCGTCGAC/GGTTACCTTGTTACGACTT-3′. The underlined regions represent overhangs with a BamHI and a SalI restriction site, respectively. These restriction enzymes were chosen as they rarely cut the 16S rRNA gene [14]. Amplification was performed with a Perkin Elmer Cetus 480 DNA thermal cycler. A total of 30 cycles of the following thermal profile were employed: denaturation at 94°C for 1 min, annealing at 49°C for 1 min, and extension at 72°C for 2 min. A final extension was performed at 72°C for 10 min.

2.4Construction of bacterial 16S rDNA clone library

Products from triplicate PCRs were pooled, ethanol precipitated, resuspended in 10 μl sterile HPLC water, and analysed on a 0.7% agarose gel against DNA reference standards. Bands of the appropriate size were excised from the gel using a sterile scalpel blade, and purified using the Prep-a-Gene kit (Bio-Rad) according to the manufacturer's instructions.

The purified PCR product and the pBS+ vector (Stratagene) were each restricted with 10 U of BamHI and 10 U SalI (Boehringer Mannheim). The ligation reaction mixture contained 150 ng of restricted PCR product, 30 ng of cut vector, 1 μl of 10× ligase buffer (500 mM Tris-HCl pH 7.5, 70 mM MgCl2, 10 mM DTT), 1 μl of 10 mM dATP, and 2 U of T4 DNA ligase (Stratagene) in a final volume of 10 μl. The reaction was allowed to proceed overnight at 4°C and then 50 ng DNA transformed into E. coli strain XL1-Blue (Stratagene) according to the manufacturer's protocol. Clones were screened for α-complementation using X-Gal as the substrate [17] on LB agar (1% Bacto tryptone, 1% yeast extract, 0.5% NaCl, 1.5% agar) supplemented with ampicillin (100 μg ml−1).

2.516S rDNA REA

Plasmids were extracted by an alkaline lysis/PEG precipitation method (Applied Biosystems) and the 16S rDNA inserts were amplified by PCR using the following vector-specific primers: T3 (5′-ATTAACCCTCACTAAAGGGA-3′) and T7 (5′-TAATACGACTCACTATAGGG-3′). 10 μl of an unpurified 100-μl PCR product was digested with 2 U of HinfI (Boehringer Mannheim) for 2.5 h at 37°C, or 2 U TaqI (Boehringer Mannheim) for 2.5 h at 65°C, in a final volume of 15 μl. Enzymes with 4-bp recognition sites were chosen due to the frequency with which they restrict DNA [11, 15]. The resulting REA patterns were visualised on 2% agarose gels stained with ethidium bromide.

2.6DNA sequencing and phylogenetic analysis

The alkaline lysis/PEG purified plasmids were sequenced using the Prism™ Dyedeoxy™ Terminator Ready Reaction cycle sequencing kit (Applied Biosystems) with 1 μg of template DNA and T7 as the sequencing primer. Extension products were purified by phenol/chloroform and analysed on an ABI model 373A automated sequencer (Applied Biosystems). The clone sequences were compared against sequence databases, such as GenBank, available on the Australian National Genomic Information Service (ANGIS) using BLAST [18] to determine their general taxonomic affiliation.

Phylogenetic analysis was as per the method of Bond et al. [10]. When clone inserts with greater than 98% sequence similarity occurred, representatives were selected in order to reduce the total number of sequences to be analysed.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

3.1Library construction and amplification of clone inserts

Total genomic DNA was extracted from a 62°C artesian bore microbial mat, and the 16S rRNA genes amplified and cloned. Use of plasmid-specific primers to amplify the inserts from the 92 selected recombinants avoided any contamination from E. coli host cell 16S rDNA [11], and demonstrated that there were four size categories of insert. Group I inserts were ca. 1500 bp in length and constituted 62% of clones examined, group II inserts were ca. 1400 bp in length and constituted 25% of clones examined, group III inserts were ca. 500 bp in length and constituted 5% of clones examined, and group IV inserts were ca. 240 bp in length and constituted 8% of clones examined.

3.2REA typing

Amplified inserts were to be typed according to their restriction profiles. It was deemed logical to compare only those clones with the same size inserts, and not to subject the two smallest groups to REA. As these group III and IV inserts contained less than 50% of the information content of entire intact 16S rDNA, REA may be of little value, and the inserts were directly sequenced instead (Table 1). All clone inserts examined, with one exception (which may have lacked the appropriate restriction sites), were cut by the enzymes used in this study. The restricted inserts were divided into OTUs based on the various banding patterns observed by gel electrophoresis after digestion with HinfI (example given in Fig. 1), i.e. digested inserts with the same banding pattern were assigned to the same OTU. Eighty clones were found to be distributed amongst 20 OTUs (Table 2). Of these, 11 OTUs had only one member.

Table 1.  Classification of group III and IV clones based on BLAST analysis of sequence data
Group% ClonesPresumptive clone source identification% nt similarityAccession no. of presumed sourcePhylogenetic affiliation of presumed source
  1. aThese clone sequences remain phylogenetically unaffiliated.

IV3.2Thermus sp. str. W28A.1100L10068Deinococcus/Thermus
IV2.2Aureobacterium liquefaciens77X77444Actinomycetes (high mol% G+C Gram-positives)
III4.3Atopobium parvulum82X67150Actinomycetes (high mol% G+C Gram-positives)
IV1.1Clostridium termitidis80X71854Low mol% G+C Gram-positive
IV2.2SBR1064 (unknown activated sludge bacterium)88X84498a
image

Figure 1. Ethidium bromide stained agarose gel (2%) showing amplified clone inserts digested with HinfI. Clone insert numbers are marked above their corresponding restriction patterns. ‘M’ denotes the molecular weight marker SPP-1 DNA/EcoRI. ‘1627b’ indicates where the amplified 16S rDNA insert from bore RN 1627 isolate Bacillus flavothermus was restricted with HinfI.

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Table 2.  Classification and frequency of group I and II clones based on REA of amplified inserts with HinfI, and BLAST and phylogenetic analysis of sequence data
Insert sizeOTU% of totalAffiliation by BLAST Analysis% nt similarityAccession no. of BLAST matchAffiliation by phylogenetic analysisBootstrap value
  1. aThese clone sequences remain phylogenetically unaffiliated.

  2. bnd=not determined.

  3. cClone insert remained uncut by restriction enzymes.

IA5.4MC65 (unknown actinomycete)78X68457Gram-positive high mol% G+C82
IB3.2MC65 (unknown actinomycete)81X68457Gram-positive high mol% G+C82
IIC1.1MC65 (unknown actinomycete)80X68457Gram-positive high mol% G+C82
ID8.7Phormidium minutum84–96X62685Cyanobacteria71
IE2.2Phormidium minutum89X62685Cyanobacteria71
IF21.0Anabaena sp.90–98X59559Cyanobacteria71
IIG18.5Thiobacillus hydrothermalis85–94M90662γ Subclass, Proteobacteria92
IIH2.2Thermus ruber100L09672Deinococcus/Thermus100
II1.1Thermus ruber100L09672Deinococcus/Thermus100
IJ11.0Thermus sp. str. NMX2.A198–100L09661Deinococcus/Thermus96
IIK1.1Clostridium lituseburense95M59107Clostridia and relatives100
IL1.1Dictyoglomus thermophilum78L39875Clostridia and relatives80
IIM2.2Gemmata obscuriglobis90X56305Planctomycetales100
IN1.1Hydrogenobacter thermophilus99Z30189Thermophilic oxygen reducers100
IO1.1Fervidobacterium icelandicum94M59176Thermotogales100
IP1.1Desulfovibrio sp.75L42995δ Subclass, Proteobacteria7
IQ1.1Pelobacter propionicus80X70954δ Subclass, Proteobacteria59
IR1.1Bordetella sp.84X57026β Subclass, Proteobacteria41
IS1.1SBR1064 (unknown activated sludge bacterium90X84498a100
IT1.1MC87 (unknown actinomycete)80X68455ndbnd
c1.1SBR1064 (unknown activated sludge bacterium)88X84498a45

Subsequent use of a second restriction enzyme, TaqI, revealed two further examples of diversity (in OTU J and G), although it failed to identify a HinfI OTU. The OTUs D, E and F had very similar profiles. Both TaqI and HinfI could distinguish OTU E, but only HinfI could differentiate between OTUs D and F. No REA pattern matched that of Bacillus flavothermus, a low mol% G+C Gram-positive bacterium, which had been previously isolated from the microbial mat investigated in this study (data unpublished).

3.3Sequencing and BLAST analysis

Forty-six clones were selected from the 20 OTUs for partial sequencing. Each OTU was represented, and multiple clones were taken from those OTUs with more than one member. A portion of each clone was sequenced with the T7 primer, yielding approximately 330 bases from the equivalent of E. coli position 27 onwards in the case of the group I and II inserts. BLAST analysis [18] was performed to determine which GenBank sequences most closely matched the insert sequences (Table 2). Partial insert sequences of clones from groups III and IV were also analysed in this manner (Table 1). These smaller inserts were often found to correspond to the 3′ end of the 16S rDNA, and therefore could not be used in the subsequent phylogenetic analysis (which was based on nucleotide positions located at the 5′ end of the 16S rDNA). Overall, BLAST matches ranged between 80–100% sequence similarity. The bacterial sources of the ‘SBR’ and ‘MC’ sequences (Tables 1 and 2) are still unknown.

No insert designated to a particular OTU by REA was re-assigned to a different OTU after sequencing. Members of each OTU grouped together phylogenetically. Three groups of OTUs – D and E; H and I; and A, B and C – were differentiated by REA, but not by partial sequencing followed by BLAST analysis. Full sequencing of the gene would likely reveal areas of nucleotide variation amongst these inserts, but would be slower and more expensive than REA.

3.4Phylogenetic analysis

A sequence similarity matrix based on the 46 sequenced group I and II inserts was prepared, but is not included here due to the large size of the data set. The number of clone sequences used in the analysis was reduced to 28 by including only single representatives of those clones with >99% sequence similarity. Phylogenetic analysis was based upon 200 nucleotide positions, and an evolutionary distance tree constructed (Fig. 2). The tree demonstrated the wide diversity of group I and II 16S rDNA sequences isolated from the artesian bore microbial mat, and their relationships with representatives of the domain Bacteria. Bootstrap values from 100 analyses are quoted (Table 2) and when these values fell below 75%, tree topology alone was used to infer relationships.

image

Figure 2. Distribution of clones throughout the domain Bacteria phylogenetic tree, based on analysis of masked, partial 16S rDNA nucleotide sequence data (corresponding to E. coli nucleotide positions 30–60, 101–180, 220–300). The bar represents one nucleotide substitution per 100 nucleotides (Knuc=0.10).

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

4.1Construction of the PCR clone library

A PCR-based clone library was prepared from genomic DNA extracted from a hot spring bacterial community. One of the criticisms of using such clone libraries to describe natural communities is the potential to ‘skew’ the population. A number of biases can be introduced throughout the procedure [11, 19, 15, 20, 21], resulting in a library that is not truly representative of the mixed population from which it was constructed [10, 22]. In an effort to minimise such biases, both chemical and physical means of lysis were employed in attempts to maximise DNA recovery. Only purified high molecular mass DNA was used as template in the PCR, as there is evidence that sheared DNA can give rise to PCR chimeras [23]. PCR reactions were pooled prior to cloning and gel purified [15]. Despite these precautions, there is still the risk that some community members will amplify either disproportionately, or not amplify at all. There is also the possibility that DNA from dead cells could be amplified, giving a false impression of the abundance of certain bacteria. Due to these potential biases, the distribution of the clonal types found within the library was not assumed to reflect the actual numerical abundance of their bacterial sources in the original environment [13].

It should also be noted that Archaea present in the community will not be detected within the clone library, as the 27F primer is specific for members of the domain Bacteria only.

4.2Clone insert lengths

The group I inserts appear to represent the near complete 16S rRNA gene, whilst the shorter sequences may be due to the fragmentation of the original PCR product during cloning [12]. Some of the amplified 16S rDNAs probably contained internal BamHI and/or SauI restriction sites, and therefore were restricted during the construction of the library. A further possibility is that some of the inserts are the result of chimeric rearrangement of 16S rDNA. Further sequencing of the cloned 16S rDNA sequence domains is recommended in order to determine if this is the case [24]. Use of the T7 and T3 vector-specific primers enabled amplification of the cleaved inserts.

4.3REA typing

In this study, the use of a restriction enzyme (HinfI) to digest the inserts was found to be a rapid and reproducible means of typing clones. The clones were distributed amongst 20 OTUs, 11 of which had only one member. The fact that so many unique members exist suggests that the full diversity of 16S rDNA types in this library has not been fully uncovered and ideally, significantly more than 100 clones should be examined. Another possibility is that some of the unique clones are actually PCR chimeras, as has been found in another study [25]. REA would then give a false impression of biodiversity within the library [23]. Further analysis of complete insert sequences – especially from those OTUs with only one member – would be required to screen for PCR chimeras.

Each restriction enzyme used in this study distinguished a unique OTU which the other enzyme failed to reveal. Employing more than one enzyme in the REA is therefore recommended.

No REA pattern of clone inserts matched that of the mat isolate Bacillus flavothermus. Other researchers have also reported the failure to find isolates using PCR and clone based protocols (e.g. [26]). This could be due to technical limitations such as PCR biases. Also, if a particular organism is present in the microbial community at less than 1%, at least 100 recombinants must be examined to have any chance of finding inserts derived from this organism.

The choice of tetrameric restriction enzymes used in this study was somewhat arbitrary, being influenced by practical factors such as availability and price. However, a systematic evaluation of 10 tetrameric restriction enzymes aimed at optimising REA of cloned 16S rDNAs has been reported and determined a number of enzymes considered to be the most efficacious for REA [27]. Although HinfI and TaqI were not listed amongst the optimal group of enzymes, they appear to have been adequate for the purposes of the work presented here. Use of these two enzymes to group clones into OTUs followed by partial sequencing of OTU representatives would have reduced the insert sequencing load by 70%, representing a significant saving both in expenses and time.

4.4BLAST analysis of partial sequences

BLAST analysis of sequence data derived from 16S rDNA clone libraries can be a useful aid in determining the range of bacteria present in an environment [28]. In this study, BLAST matches ranged between 75–100% sequence similarity, and indicated that the artesian bore recombinants were related to 16S rDNA sequences from a wide variety of known thermophilic bacteria (Table 2).

The 16S rDNA sequences of known bacteria, identified by BLAST analysis as possible sources of the group II, III, and IV inserts, were screened with the MAPSORT program (GCG package, available through ANGIS) to check for BamHI and SauI restriction sites. Thiobacillus hydrothermalis contained a single SauI site within the 16S rDNA, yielding the nucleotide fragments 130 bp/1374 bp. In this case, an internal restriction site is probably the cause of the shorter length of the insert.

As for the other truncated inserts, it must be remembered that the closest matches highlighted by BLAST may be only distantly related to organisms from which the clone inserts came [29]. Thus the clone sequences may still possess BamHI and/or SauI restriction sites. It should also be stated that only partial sequences were employed in the BLAST analysis and this factor can influence which sequence the insert is matched with. Additional sequence data could result in a different BLAST match, possibly one which does possess the relevant restriction site. Cleavage of the insert can be a problem when constructing clone libraries using restriction enzymes, but as can be seen from this study, meaningful data can still be obtained.

4.5Phylogenetic analysis

As the primary aim of this study was to characterise the molecular diversity associated with a unique thermal environment, our approach involved the analysis of many relatively short sequences [12]. Phylogenetic trees constructed from the analysis of short sections of the 16S rDNA are reported to be similar to those generated from full gene sequence data [10, 30], although 200 bases are not considered sufficient data for resolving deep phylogenetic relationships [12]. Despite this, the tree topology was generally the same as that based on near complete sequences, although bootstrap values were sometimes low as others have previously reported [31]. Bootstrap values are considered significant when >75%[10, 32], and values on branches involving clones were often in excess of 80% (Table 2).

Overall, the results generated by REA, BLAST analysis of partial sequences, and phylogenetic analysis were in agreement (Table 2). REA indicated a great variety of 16S rDNAs had been amplified from the environmental sample. BLAST analysis of partial sequence data supported this finding, and correlated well with subsequent phylogenetic analysis – indicating that data obtained in this study from the partial sequences are reliable. Clones were shown to be distributed throughout many different branches of the domain Bacteria, indicating a high level of biodiversity within the hot spring mat.

4.6Bacteria present in the GAB

Many of the bacteria indicated by this study to be present in the GAB are related to thermophiles found in alkaline hot springs, e.g. the Octopus Spring mat (50–55°C) in Yellowstone National Park (e.g. [8, 33]). This no doubt reflects the nature of the ecosystem - a number of major bacterial groups such as cyanobacteria, Thermus species and sulfate-reducing bacteria (SRB) are well known to be associated with alkaline hot spring systems (e.g. [33, 34]).

The cyanobacteria are aerobic photosynthetic microorganisms and are usually the major primary producer in a hot spring mat [34]. Cyanobacterial-like sequences dominated the 16S rDNA library (Table 3) and could reflect the prevalence of these photosynthetic primary producers in the bore RN 1627 mat. Based on REA, there appear to be three cyanobacterial sequence types while BLAST analysis of partial sequence data indicated two cyanobacterial sequence types. This parallels the Octopus Spring studies, where a number of cyanobacterial sequence types were revealed [8, 33]. The authors of the latter study [8] concluded that cyanobacterial diversity within the hot spring community had been underestimated due to its morphological simplicity, although it was possible that each sequence type did not necessarily represent a unique cyanobacterium. Similarly, the bore RN 1627 mat may support a population of cyanobacteria. Photosynthates produced would fuel the associated bacterial community.

Table 3.  List of additional partial 16S rDNA sequences from reference Bacterial strains used in the phylogenetic analysis
AbbreviationOrganismPhylogenetic affiliation
  1. and=not determined.

Anabaena_spAnabaena sp.Cyanobacteria and chloroplasts
Asta.lon_CAstasia longa str. CCAPCyanobactera and chloroplasts
Aqu.pyrophAquifex pyrophilus str. Kol5aThermophilic oxygen reducers
Bac.fragilBacteroides fragilisFlexibacter/Cytophaga/Bacteroides
Cfx.aurantChloroflexus aurantiacus str. J-10-flGreen non-sulfur and relatives
Clos.litusClostridium lituseburenseClostridia and relatives
Chl.vibriChlorobium vibrioforme str. 6BCGreen sulfur
Clm.pneumoChlamydia pneumoniaePlanctomycetales/Chlamydia group
Clm.psittaChlamydia psittaciPlanctomycetales/Chlamydia group
D._radiodurDeinococcus radioduransDeinococcus/Thermus
Dsv.desulfDesulfovibrio desulfuricansδ Subclass, Proteobacteria
E.coliEscherichia coliγ Subdivision, Proteobacteria
Fervido.icFervidobacterium icelandicum str. H-21Thermotogales
Fls.sinusaFlexistipes sinusarabici str. Mas 10Flexistipes
Fus.simiaeFusobacterium simiaeFusobacteria
Gmt.obscurGemmata obscuriglobusPlanctomycetales/Chlamydia group
Got.subterGeotoga subterranea str cc-1Thermotogales
Hal.hydrosHaliscomenobacter hydrossisFlexibacter/Cytophaga/Bacteroides
Hdg.thermoHydrogenobacter thermophilusThermophilic oxygen reducers
Her.aurantHerpetosiphon aurantiacusGreen non-sulfur and relatives
Lpp. spLeptospirillum sp.Leptospirillum
Mc.vannieMethanococcus vannielii str. EY33Archaea
MC18Mount Cootha 16S rDNA clonenda
MC65Mount Cootha 16S rDNA cloneGram-positive high G+C
MC66Mount Cootha 16S rDNA cloneGram-positive high G+C
Myx.xanthuMyxococcus xanthusδ Subclass, Proteobacteria
N.moscovieNitrospira moscoviensisβ Subdivision, Proteobacteria
P.minutumPhormidium minutumCyanobacteria and chloroplasts
P.propioniPelobacter propionicusδ Subdivision, Proteobacteria
Pln.stayeliPirellula stayeliPlanctomycetales/Chlamydia group
Prg.modestPropionigenium modestumFusobacteria
Ps.testostComamonas testosteroni str. RH1104β Subdivision, Proteobacteria
Rcy.purpurRhodocyclus purpureusβ Subdivision, Proteobacteria
SBR1064Unidentified activated sludge bacteriumnd
SBR1040Unidentified activated sludge bacteriumnd
Spi.aurantSpirochaeta aurantia str. J1Spirochetes and relatives
Sng.jonesiiSynergistes jonesii str. 78-1Paraphyletic assemblage
Sul.solfatSulfolobus solfataricusArchaea
Syn6301Synechococcus sp. (PCC 6301)Cyanobacteria and chloroplasts
Thb.hydrotThiobacillus hydrothermalisγ Subdivision, Proteobacteria
Tmc.roseumThermomicrobium roseumGreen non-sulfur and relatives
T.nmx2Thermus sp. str. NMX2 A.1Deinococcus/Thermus
T.ruberThermus ruberDeinococcus/Thermus
T.thermopThermus thermophilusDeinococcus/Thermus
Tt.maritimThermotoga maritima str. MSB8Thermotogales

The Gram-negative SRB are members of the δ subclass of the proteobacteria, and are represented in our library by OTU P. The SRB often occur in association with cyanobacterial mats [34], and a novel species of SRB has been previously isolated from the GAB [6]. The hydrogen sulfide present at some bores, such as RN 1627, is believed to result from the microbial reduction of sulfate [2]. Yet only a sole representative of the SRB was recovered from the clones examined. The paucity of these sequences within the clone library could be the result of biases associated with PCR, or sampling and storage regimes. OTU Q also contains a member of the delta subclass proteobacteria, possibly Pelobacter propionicus, an organism capable of both iron and sulfur reduction [35].

Other non-photosynthetic organisms commonly found in hot springs include members of the β and γ subclasses of the proteobacteria. Sequences from members of the γ subclass of the proteobacteria were found within the clone library, in the form of close relatives of Thiobacillus hydrothermalis (94% nt similarity), an obligate chemolithotroph. T. hydrothermalis, originally isolated from a 35°C marine hydrothermal vent in the North Fiji Basin, was described as a mesophile with negligible growth occurring at 50°C [36]. The GAB clone insert sequence could represent a thermophilic relative of T. hydrothermalis, capable of growing lithoautotrophically on sulfide of biogenic origin present in the mat community. These organisms could also play a ‘protective’ role with respect to the cyanobacteria, preventing their exposure to toxic levels of sulfide [34].

Members of the genus Thermus have previously been isolated from a number of GAB bores [37], and appear to be present in the GAB mat. These aerobic heterotrophs utilise organic matter derived from autotrophic bacteria. More than one species was found to occur in the same sample by this study, according to sequence analysis. This is consistent with other reports regarding the distribution of this genus [38]. This study also extends the known range of T. ruber and Thermus sp. strain NMX.A2, further evidence that Thermus species are not restricted to particular geographic locations [30].

Representatives of the two deepest branching lineages of the domain Bacteria, the thermophilic oxygen reducers and the thermotogales, were present in the GAB microbial mat clone library. Microaerophilic thermophilic oxygen reducers have been previously associated with hydrothermal vents [39] and Octopus Spring [7]. New members of the order Thermotogales, including the anaerobe Fervidobacterium gondwanense sp. nov., have been recently isolated from the GAB [5].

The high mol% G+C Gram-positive representatives (actinomycetales) found in this library are, by 16S rDNA analysis, most similar to clones first found in an Australian soil sample [40]. Other groups represented include the low mol% G+C Gram-positive clostridia and their relatives, and the ‘SRB’ activated sludge-like clones [10]. Planctomycete-like sequences were also retrieved. These organisms have been associated with other hot spring systems [8].

4.7Limitations of the molecular investigation

It is important to remember that phenotype cannot be inferred with certainty from sequence data, or from the phylogenetic analysis. This is especially so in those lineages that contain diverse physiological types, such as the β subclass of the proteobacteria [41]. At most, we can propose various metabolic roles for the sources of the sequences obtained from the GAB mat – further work involving culturing, physiological studies, and in situ probing would be required to confirm these hypotheses.

From the sequence data, the cyanobacteria, thiobacilli, Thermus species and unknown actinomycetes are the most numerous members of the library (Table 3). Care must be taken when attempting to extrapolate these data in a quantitative manner to the source environment. One way to test if predominance in the library equates to dominance in the ecosystem would be the use of whole cell probes to enumerate the target organism in situ. However, clone libraries still have value as a means identifying uncultured organisms and of compiling new sequence data. The development of databases based on such information paves the way for the design of probes to detect these ‘marker’ sequences in the environment.

5Conclusions

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

Molecular techniques were successfully applied to a thermophilic environment, and indicated that a broad spectrum of bacteria are associated with the microbial mat sample taken from the GAB. REA provided an efficient means of screening the clone inserts, and provided the first major indication of the biodiversity present. From the phylogenetic analysis, it can be seen that the clones are spread throughout the phylogenetic tree. The results obtained from the different modes of molecular analysis were generally in agreement. Ten distinct bacterial phylogenetic lineages are present in the clone library, based on partial sequence analysis. This compares to seven lineages present in a 16S rDNA clone library prepared from a hydrothermal vent [25], and seven lineages each for both a 16S rcDNA library [8], and a DGGE-based study of an Octopus Spring mat [42].

Now that some of the mat inhabitants, or at least their known relatives, have been presumptively identified by molecular means, the next step would be to modify cultural conditions to suit these bacteria and attempt their isolation. Also whole cell probing should be employed, to demonstrate the location and prevalence of the different mat members.

This is the first report describing the diversity associated with a thermal environment in Australia. As the GAB appears to be isolated from other thermal waters, it would be interesting to check full sequence data of the inserts, and compare these to the 16S rDNA of thermophiles isolated from other geographic locations, in order to determine the degree of speciation that has occurred in isolation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

We thank Dr Fred Rainey and Naomi Ward-Rainey for advice on preparation of clone libraries and Drs Mark Fegan and Michael Poidinger for useful discussions regarding the phylogenetic analysis.

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  2. Abstract
  3. 1Introduction
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  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References
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