research article adaptation, ecology, and evolution of the...

Research ArticleAdaptation Ecology and Evolution of the HalophilicStromatolite Archaeon Halococcus hamelinensis Inferredthrough Genome Analyses

Reema K Gudhka1 Brett A Neilan12 and Brendan P Burns12

1School of Biotechnology and Biomolecular Sciences University of New South Wales Sydney NSW 2052 Australia2Australian Centre for Astrobiology University of New South Wales Sydney NSW 2052 Australia

Correspondence should be addressed to Brendan P Burns brendanburnsunsweduau

Received 29 October 2014 Revised 4 December 2014 Accepted 10 December 2014

Academic Editor William B Whitman

Copyright copy 2015 Reema K Gudhka et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Halococcus hamelinensis was the first archaeon isolated from stromatolites These geomicrobial ecosystems are thought to be someof the earliest known on Earth yet despite their evolutionary significance the role of Archaea in these systems is still not wellunderstood Detailed here is the genome sequencing and analysis of an archaeon isolated from stromatolites The genome ofH hamelinensis consisted of 3133046 base pairs with an average G+C content of 6008 and contained 3150 predicted codingsequences or ORFs 2196 (6867) of which were protein-coding genes with functional assignments and 954 (2983) of whichwere of unknown function Codon usage of the H hamelinensis genome was consistent with a highly acidic proteome a majoradaptive mechanism towards high salinity Amino acid transport andmetabolism inorganic ion transport andmetabolism energyproduction and conversion ribosomal structure and unknown function COG genes were overrepresented The genome of Hhamelinensis also revealed characteristics reflecting its survival in its extreme environment including putative genespathwaysinvolved in osmoprotection oxidative stress response and UV damage repair Finally genome analyses indicated the presence ofputative transposases as well as positive matches of genes of H hamelinensis against various genomes of Bacteria Archaea andviruses suggesting the potential for horizontal gene transfer

1 Introduction

Stromatolites are defined as organosedimentary structuresproduced by the trapping and precipitation of carbonates andsediments as a result ofmicrobial growth andmetabolic activ-ity [1] The presence of stromatolites dates back some 35ndash38billion years in Earthrsquos history a time period that witnessedthe appearance of the first forms of life [2] The microbialcommunities that constituted these complex structures alsoplayed a significant role in global biogeochemical cycles inc-luding early oxygenation of the atmosphere [3] Owing totheir importance stromatolites have formed the core of a gro-wing area of microbial ecology and evolution-based researchThe most extensive modern stromatolites to date reside inthe open marine waters in Exuma Sound in the Bahamas [4]

and the hypersaline marine environment of Shark Bay on thewestern coast of Australia [5 6]

Previous reports employing a combination of microbialisolation culture-independent nucleic acid based analysesand lipid profiling have shown that Shark Bay stromatolitessupport a significant range of metabolically and phylogeneti-cally diverse microorganisms [5ndash7] Major functional groupswere identified that are involved in cycling of key nutrientsincluding oxygenic and anoxygenic photosynthesisers aero-bic heterotrophs and organisms involved in sulphur cyclingFor the first time a diverse range of Archaea was also identi-fied in the stromatolites of Shark Bay [5ndash7]

Although most studies to date have focused on the func-tion of cyanobacteria and heterotrophic bacteria in stroma-tolites the physiological role and significance of Archaea in

Hindawi Publishing CorporationArchaeaVolume 2015 Article ID 241608 11 pageshttpdxdoiorg1011552015241608

2 Archaea

stromatolite systems still remains unexplored HalophilicArchaea in particular may be an important component of theShark Bay stromatolites vital to ecosystem function Halophi-lic Archaea are very well adapted to hypersaline environ-ments and thrive in many different areas including the DeadSea and solar salterns [8 9] Previously the first archaeon tobe isolated from stromatolites a Gram-negative nonmotilestrictly aerobic Halococcus isolate (Halococcus hamelinensis)was also characterised [10] This archaeon grows optimallyat 37∘C in medium containing 15 NaCl as opposed to 20ndash30 in other characterizedHalococcus strains It metabolizesmany simple and complex carbohydrates including glucosesucrose xylose maltose trehalose mannitol galactose andglycerol [10] It does not hydrolyze starch or produce indoleand is negative for sulfide reduction urease and gelatin lique-fication in contrast to otherHalococcus species Furthermoreit displays sensitivity to rifampicin novobiocin and baci-tracin and resistance to kanamycin tetracycline streptomy-cin neomycin and penicillin H hamelinensis was also oxi-dase-negative whereas all recognized Halococcus species areoxidase-positive and most aerobic halophilic Archaea arealso oxidase-positive [11] Whole-cell protein profiles enzy-me composition and carbon source utilization of this isolatewere distinct from other characterized Halococcus speciesrendering it a novel species of Halococcus [10] Prior to theisolation of this organism no archaeal species had been dis-covered at Shark Bay As no archaeal isolates were also identi-fied in the surrounding water despite samples being collectedseveral years apart [5 7] it may be that this organism has asignificant role to play in stromatolite ecosystem function

Recent studies have shown that H hamelinensis displaysa number of other novel characteristics including lackingpotassium accumulation as a primary osmoprotective strat-egy in contrast to most known haloarchaea [12] Insteadaccumulation of glycine betaine as well as other solutes suchas glutamate and trehalose was observed to be the primarymechanism of adaptation to salt stress in this organism [12]In addition a recent study examined the effects of UV stressdamage inH hamelinensis [13] revealing that this archaeon isable to survive high germicidalUVC radiation dosages aswe-ll as possessing several bacterial-like nucleotide excision rep-air genes [13] Changes in the expression levels of these genes(uvrA uvrB and uvrC) were investigated by qRT-PCR andall were upregulated during both light and dark repairs [13]

While these recent studies have provided useful insightsinto the physiological characteristics of this novel archaeonwhat is lacking is a comprehensive whole genomic profile andrelated computational analyses of this organismwhichwouldsignificantly enhance our understanding of its evolutionaryand adaptive traits Here we describe the sequence (by mas-sively parallel 454 pyrosequencing technology) annotationand analysis of the genome of H hamelinensis which pro-vides insights into the ecology evolution and adaptation ofthis novel microorganism

2 Materials and Methods

21 Genome Sequencing and Assembly Halococcus hameli-nensis was grown as previously described until logarithmic

phase [10] andDNAwas extracted using a xanthogenate pro-tocol previously optimised for this Archaea [14]The genomeof H hamelinensis was sequenced at The Clive and VeraRamaciotti Centre using 454 (Roche) sequencing technologySample quality and quantity were assessed prior to librarypreparation as described in the GS FLX Titanium GeneralLibrary Preparation Method Manual (Roche) Briefly 8 120583gof gDNA was fragmented by nebulization fragmented DNArun on a 08 agarose gel and fragments between sim500 and800 bp excised and purified using a QiaQuick Gel ExtractionKit (Qiagen) DNA sample quality assessment fragment endrepair and adaptor ligation small fragment removal libraryimmobilization and single-stranded DNA library isolationwere performed as per GS FLXTitanium SequencingMethodManual (Roche) The quality of the library was assessed on aBioanalyzer RNAPico 6000 Chip (Agilent Technologies) andquantified using the Quant-It RiboGreen assay (Invitrogen)Finally emulsion PCR and sequencing were performed asdescribed in the GS FLX Titanium SequencingMethodMan-ual A total of 272854 reads counting up to 118638438 bases(32-fold coverage of the genome) were assembled using 454Newbler GS de novo Assembler software which generated453 contigs ranging from 500 to 105929 bp with bases havingquality scores of 40 and above

22 Automated Sequence Annotation Sequencing output wasuploaded onto two different auto annotation servers theRapid Annotation using Subsystem Technology (RAST) [15]and Integrated Microbial GenomesExpert Review (IMGER) [16] RAST uses a strategy based on comparison withmanually curated subsystems and subsystem-based proteinfamilies guaranteeing a high degree of consistency and accu-racy Putative coding sequences were identified by Glimmersoftware [17] and peptides shorter than 30 amino acids wereeliminated tRNA genes and rRNA genes were predicted bytRNAScan-SE software and RNAmmer respectively [18 19]Functional annotation was performed by searching the NCBInonredundant protein database and the Kyoto Encyclopediaof Genes and Genomes (KEGG) protein database Data fromthe genomewerematched against data from external sourcesincluding RefSeq EBI Genome Reviews GenBankEMBLJGI microbial genome data LIGAND GO COG EnzymeInterPro KEGG KO Pfam TIGRFam and GOLD Pathwaysfor modes of nutrition and transport system were acquiredthrough the KEGG function analysis in IMG COG analysiswas carried out using COG functions and abundance profileanalysis within the IMGER

23 Bioinformatic Analyses Phylogenetic analyses were per-formed to elucidate evolutionary relationships The 16 SrRNA gene sequences were derived from various euryarcha-eal genomes from the NCBI database The sequences werethen aligned using ClustalX [20] A BLOSUM series proteinweight matrix was used with a Gap Opening of 10 and a GapExtension of 02 Using the ClustalXmultiple sequence align-ment neighbor joining phylograms were constructed boot-strapped and displayed using the MEGA version 4 program[21] Programs from the EMBOSS package COMPSEQ (cal-culates the composition of unique words in a sequence) and

Archaea 3

CHIPS (calculatesNc codon usage statistics) were run to det-ermine the number and frequency of occurrence of codontriplets of the predicted proteins Over- or underrepresentedcodons were corrected based on the GC content (6608) ofthe H hamelinensis Predictions of amino acid usage werebased on the codon frequency output for each amino acidRadial graphs were created using IMGER Phylogenetic Dis-tribution of genes software The program Alien Hunter wasused to detect potential horizontal transfer regions within theH hamelinensis genome The Alien hunter output was readthrough theArtemis program and the data output was viewedusing the DNA PLOTTER program

3 Results

31 H hamelinensis GenomeOverview Whole genome sequ-encing by 454 pyrosequencing produced a greater than 32xcoverage of the H hamelinensis genome The genome sequ-ence ofH hamelinensis is accessible at GenBank under acces-sion number PRJNA80845 An overview of the genome fea-tures is given in Table 1The genome ofH hamelinensis cons-isted of 3133046 base pairs (bp)with an averageG+C contentof 6008Thegenome consisted of 223DNAscaffolds and 48RNA genes 45 of which were tRNA genes and the remainingthree were rRNA genes comprising of a single rRNA operon(16 S-23 S-5 S) Furthermore the genome contained 3150predicted coding sequences 2196 (6867) of which wereprotein-coding genes with functional assignments and 954(2983) of which were of unknown function Based on 16 SrDNA phylogenetic analyses H jeotgali was identified as theclosest sequenced genome to H hamelinensis with a BLASTidentity score of 91Other related organismswereH turkm-enica isolated in sulfate saline soil in Turkmenistan [22] NMagadii isolates from a Kenyan soda lake [23] andH xana-duensis isolated from a saline Lake Shangmatala in China[24]

The results for codon frequency indicated a bias towardshigh GC content in the third position (Figure 1) From thethree-letter codon prediction codons the following aminoacids were observed to have high frequency alanine (GCCGCG and GCA) arginine (CGA CGC CGG and CGT)aspartic acid (GAC) glutamic acid (GAA GAG) glycine(GGC GGG and GGT) histidine (CAC) leucine (CTC)proline (CCC CCG) serine (TCC TCG) threonine (ACCACG) and valine (GTC) Frank Wrightrsquos NC statistic for theeffective number of codons used was also calculated wherethe Nc output can take values from 20 in the case of extremebias where one codon is exclusively used for each aa to 61when the use of alternative synonymous codons is equallylikely The Nc output value for the H hamelinensis genomewas calculated as 478 indicating very little bias between indi-vidual codon usage Organisms high in GC tend to havecodons ending in C or G and this is also reflected in the Hhamelinensis genome

32 Carbon Assimilation Preliminary studies carried out onH hamelinensis whole cells indicated that the organism utili-zes glucose sucrose xylose maltose trehalose and glycerolas both single and complex carbon sources in addition to

Table 1 General features of the H hamelinensis genome

General features NumberSize (bp) 3133046GC1 6608RNA genes 48rRNA genes 35 S rRNA 116 S rRNA 123 S rRNA 1

tRNA genes 45DNA scaffolds 223DNA coding bases 2529448Protein coding genes (PCG) 3150PCG with known and putative function () 6867PCG with enzymes () 2239PCG with COGs () 6513PCG with Pfam () 6598PCG with TIGRfam () 1948PCG with InterPro () 8712PCG coding signal peptides () 1007PCG coding transmembrane proteins () 2248PCG in paralog clusters () 2004Fused Protein coding genes 219COG clusters 1177KOG clusters 635Pfam clusters 1207TIGRfam clusters 5391GC percentage shown as count of Grsquos and Crsquos divided by a total numberof Grsquos Crsquos Arsquos and Trsquos COGsmdashClusters of Orthologous Groups of proteinsPfammdashprotein family database TIGRFAMsmdashresource consisting of curatedmultiple sequence alignments HiddenMarkov Models for protein sequenceclassification and associated information designed to support automatedannotation of proteins KOGmdasheuKaryotic Orthologous Groups

mannitol galactose and ethanol as single carbon sources[10] Detailed genome analyses conducted in the presentstudy identified glucose catabolism to proceed via the Embd-en-Meyerhof pathway Genes encoding enzymes from theEntner-Doudoroff pathway were not present in theH hame-linensis genome

33 Nitrogen Assimilation Archaea can use ammonia as wellas a large variety of nitrogen compounds as sole sources of nit-rogen To assimilate ammonia only one enzyme is requiredglutamine synthetase that was identified here in theH hame-linensis genome (gene ID 2502298936) Once converted toL-glutamine the enzyme glutamate synthase (gene ID2502300824 2502301225 and 2502301417) converts it to glu-tamate In cases of low ammonia H hamelinensis can puta-tively convert nitrate to ammonia via nitrite using theenzymes nitrate reductase (catalytic subunit) (gene ID2502301552) and ferredoxin nitrite reductase (gene ID2502300324) The organism also has the genetic potentialto produce glycine and cyclic amidines using the enzymesaminomethyltransferaseglycine cleavage system T protein

4 Archaea

Codon frequencyThree-letterfrequency

Observed frequencyExpected frequency

AACAAG AATACAACC

ACGACT

AGAAGC

AGGAGT

ATAATC

ATG

ATT

CAA

CAC

CAG

CATCCA

CCCCCG

CCTCGA

CGCCGG

CGTCTACTCCTGCTTGAAGACGAGGAT

GCAGCC

GCGGCT

GGAGGC

GGGGGT

GTAGTC

GTG

GTT

TAA

TAC

TAG

TAT

TCATCC

TCGTCT

TGATGC

TGGTGT

TTATTCTTGTTT AAA

005

0045

004

0035

003

0025

002

0015

001

0005

0

Figure 1 Radial graph displaying the three-letter codon frequency prediction of H hamelinensis Expected codon frequencies are indicatedin the inner circle Squares in the outer circle are indications are overrepresented codons

(gene ID 2502301667) and NAD+ synthetase (Gene ID2502298992 2502299198 2502299746) In addition to theseenzymesH hamelinensis also possesses genes encoding threeABC transport proteins involving glutamine namely glu-tamine ABC transporter periplasmic glutamine-bindingprotein (gene ID 2502298730) ABC-type glutamineglutam-atepolar amino acids transport system permease protein(gene ID 2502298731) and ABC-type glutamineglutamatepolar amino acids transport system ATP-binding protein(gene ID 2502298732)

34 Energy Acquisition Analyses here confirmed the pres-ence of genes encoding 3Na+H+ antiporters Na+H+ antip-orter NhaC Na+H+ antiporter MnhB subunit-related pro-tein multisubunit Na+H+ antiporter MnhF subunit andtwo Na+ symporters namely Na+solute symporter and pan-tothenate Na+ symporter suggesting a functioning pathwaybetween H+ and osmotic metabolism Genome analysis alsoconfirmed the presence of an ATP synthase gene cluster (V-type ATP synthase subunit A B C E F H I and a ATP syn-thase subunit C) and putative regulatory ligand-bindingprotein related to C-terminal domains of K+ channels indi-cating complete pathways from H+ to K+ gradients andATP production respectively Respiratory chain enzymes in

halophilic organisms require high salt concentration to beactive [25] Genome analyses indicated that H hamelinensiscontained a putative electron-transport flavoprotein (alphaand beta subunit) and two cytochrome B coding genes

The halophilic Archaea possess light driven ion pumps[26] bacteriorhodopsin and halorhodopsin that pump pro-tons and chloride ions respectively across the membraneThe proton pump serves to create a proton potential that canbe used to drive ATP synthesis [25] Although this has beenthought to be exclusively an archaeal protein recently bacter-iorhodopsin-like proteins such as xanthorhodopsin and pro-teorhodopsin have been discovered in bacteria [27 28] Inte-restingly despite exhaustive genome analyses no homolo-gous genes encoding bacteriorhodopsin halorhodopsin orbacterioruberin were identified in the H hamelinensis geno-me in the present study

35 Osmoadaptation H hamelinensis is known to accumu-late compatible solutes such as glycine betaine trehalose glu-tamate and proline [12] which display a general stabilizingeffect by preventing the unfolding and denaturation of pro-teins caused by heating freezing and drying Genome anal-yses here revealed several putative genes involved in glycinebetaine accumulation processes The presence of three genes

Archaea 5

(designated betI betII and betIII) encoding for three putativeglycine betaine transport proteins supports the previous find-ings showing glycine betaine accumulation as an osmopro-tective strategy in this archeon [12] Two ABC-type glycinebetaine permease components and glycine betaine ABCtransporter ATP-binding protein identified here (Table 2)also indicate the presence of a glycine betaine ABC-transportsystem used to accumulate glycine betaine In addition thereare two further genes encoding a putative glycine betainetransporter (OpuD) present in the genome Phylogeneticanalysis carried out on the three Bet proteins fromH hameli-nensis that showed high similarity to putative glycine betainesecondary transporters of other halophilic Archaea as well asthose ofDesulfomicrobium baculatum ButA of the halophilicbacteriumTetragenococcus halophilus andOpuDof themar-ine gamma-proteobacterium Teredinibacter turnerae [29ndash31] Twelve transmembrane domains were present a sig-nature of the BCCT (betainecarnitinecholine transporter)family Trehalose is another compatible solute putativelysynthesized in H hamelinensis for osmoadaptation [12] Aputative trehalose biosynthesis pathway was found to becomplete in the H hamelinensis genome here where UPD-glucose is metabolised to trehalose-6-phosphate and thentrehalose via trehalose 6-phosphate

Archaea are also known to maintain a constant K+ con-centration intracellularly for essential functions such as mai-ntenance of cell turgor and homeostasis adaptation of cellsto osmotic stress and activation of cell cytoplasmic enzymesThere are various mechanisms to accumulate K+ in the celland the three best characterized systems are the Trk Kdp andKup K+ transport systems [32] Previous analyses screeningfor putative K+ uptake genes by degenerative PCR failed todetect two putative potassium transport proteins namelyTrkH and KdpB in H hamelensis [12] While no homologsto the kdpB system were identified in the present study othergenes coding for K+ uptake proteins were identified whichincluded two putative K+ uptake proteins TrkH and TrkA(Table 2) These form a part of the Trk system which is alow affinity rapid transport system mainly used at neutral oralkaline pHThe presence of homologs to TrkH in the presentstudy is in contrast to PCR results reported recently [12]and illustrates the advantage of whole genome sequencingto complement targeted PCR approaches Two Kef-type K+transport proteins andmultiple sodium-potassiumhydrogenantiporter subunits were also identified here which comprisetheK+ efflux system [33] Interestingly genes encoding for thesynthesis of archaeatidylglyercolmethyl phosphate a typicallipid identified previously inH hamelinensis [12] and knownto contribute to osmotolerance were not found here

36 Strategies of DNA Repair and Heavy Metal Resistance Arecent study revealed the presence of a UvrABC DNA repairsystem in H hamelinensis possessing three bacterial-likenucleotide repair genes namely excinucleaseABC subunit AB and C [13] Three other putative DNA repair systems wereidentified in the present study These were a bacterial MutL-MutS system consisting of mismatch repair proteins DNArepair base excision system consisting endonucleases and

Table 2 Putative osmoadaptive genes identified in H hamelinensisgenome

Gene name Gene IDProbable glycine betaine transporterprotein (Bet III) 2502299791

Glycine betaine transporters OpuD 2502299823Glycine betaine transporter 2502299930ABC-type prolineglycine betainepermease component 2502300305

Glycine betaine ABC transporterATP-binding protein 2502300306

ABC-type prolineglycine betainepermease component 2502300307

Probable glycine betaine transportprotein 2502301633

Potassium uptake protein TrkH fig|3321683peg1799TrkA system potassium uptake protein ffig|3321683peg105Potassiumchannel protein 2502299500Kef-type K+ transport systems fig|3321683peg334

ligases and the 2-phosphoglycolate salvage system consist-ing of phosphoglycolate phosphatase In addition there werealso several DNA repair and recombination proteins detectedwithin the H hamelinensis genome

Various Archaea are also known to possess mechanismsfor heavy metal resistance Analyses were conducted here toverify the presence of any toxicheavymetal resistance codinggenes in H hamelinensis Several putative genes were identi-fied here and are shown in Table 3 Two copper-translocatingATPases were detected and there was also the presenceof genes encoding arsenate reductase enzymes and copper-zinc-cadmium resistance proteins within theH hamelinensisgenome There were four permease enzymes belonging tothe drugmetabolite transporter super family which arepresumed to transport toxic metabolites out of the cell

37 Cluster of Orthologous Group (COG) Categories The Hhamelinensis genome has a total of 2296 genes within COGcategories A searchwas carried out to determine the overrep-resented COG categories withinH hamelinensis when com-pared to other sequenced archaeal genomes There were 8overrepresented categories the general prediction category(1690) followed by the amino acid transport and metabol-ism (980) unknown function (932) inorganic ion trans-port andmetabolism (762) energy production and conver-sion (714) and translation and ribosomal structure and bio-genesis (693) Following these analyses a search for bothhomolog and nonhomolog genes between H hamelinensisand Archaea was conducted and then for the EuryarchaeotaResults indicate a higher number of homolog genes betweenboth Archaea and Euryarchaeota and fewer nonhomologgenes Genome analysis reveals 165 genes fromH hamelinen-sis that have homologs in the 86 genomes of other Euryarcha-eota whereas there are only 95 genes fromH hamelinensis in

6 Archaea

Table 3 Putative genes coding for heavy metal resistance in Hhamelinensis

Gene name Gene IDArsenical pump-driving ATPase fig|3321683peg1469Arsenate reductase (EC 12041) fig|3321683peg2242Arsenate reductase (EC 12041) fig|3321683peg3119Arsenical-resistance protein ACR3 fig|3321683peg3118DNA gyrase subunit A (EC 59913) fig|3321683peg722DNA gyrase subunit B (EC 59913) fig|3321683peg721Aminoglycoside N61015840-acetyltransferase(EC 23182) fig|3321683peg687

Copper-translocating P-type ATPase(EC 3634) fig|3321683peg2377


Cobalt-zinc-cadmium resistanceprotein CzcD fig|3321683peg2193

Cobalt-zinc-cadmium resistanceprotein fig|3321683peg471

Permease of the drugmetabolitetransporter (DMT) superfamily fig|3321683peg1282




total that have homologs after comparison with all other 120Archaea genomes

38 Potential Horizontal Gene Transfer Nine putative trans-posase coding genes were detected in the H hamelinensisgenomeThesewere distributed amongst six different contigsTwo transposase DDE domains were found close to eachother on contig 00141 Neighbouring these transposase geneswere three genes two ABC transport protein coding genesand one putative cobalamin protein coding gene from theinorganic ion transport and metabolism COG category Twoputative transposase coding genes are found in close proxim-ity to a glycine betaine transporter and a glycine cleavage T-protein Other genes neighbouring the transposases are genesbelonging to the general function prediction COG categoryas well as the inorganic ion transport and metabolism COGcategory A third putative transposase DDE domain was fou-nd isolated on contig 00142with a hypothetical protein neigh-bouring it A phylogenetic distribution of genes was assessedby carrying out individual BLAST searches between eachprotein coding gene of H hamelinensis and genes across allother phyla This was conducted to analyze potential hori-zontally transferred genes It was found that H hamelinensishas 2639 positive matches within the archaeal lineage outof which 9689 fall within the Euryarchaeota phylum and117 hits within the bacterial lineage Under the bacteriallineage the highest numbers of genes with positive hits were

against the following the Actinobacteria phylum (22)Bacilli (15) Gammaproteobacteria (11) Clostridia (9)Cyanobacteria (8) and Bacteroidetes (7)

A radial phylogenetic tree comparison betweenH hame-linensis and other bacteria and Archaea (Figure 2) also indi-cate potential evolutionary relationships andor potentialHGT that may have occurred To further examine the impor-tance of potential horizontal gene transfer to theH hameline-nsis genome a gene-independent analysis of potential HGTusing interpolated variable motif scores calculated fromthe program Alien Hunter was performed Several putativeregions were considered likely to have been acquired by HGT(data not shown)

39 Screening for RestrictionModification and CRISPR Sys-tems in H hamelinensis Microbial defence systems againstmobile elementsforeign DNA include restriction modifica-tion (RM) systems [34] and clustered regularly interspacedshort palindromic repeat (CRISPR) systems [35] CRISPRsystems have been recently identified as an adaptivemicrobialimmune system that provides acquired immunity againstviruses and plasmidsThis system represents a family of DNArepeats found in approximately 40 of bacterial genomesand most archaeal (sim90) genomes thus far screened [35]Interestingly after analysis of the H hamelinensis genomeno putative CRISPRCas elements could be identified RMsystems were also examined by screening theH hamelinensisannotated genome for genes involved in RM systems nohomologs to known host restriction modification systemswere identified and only one homolog to a methylase puta-tively involved in methylating (and thus protecting) hostDNA from host restriction enzymes (CTAG modificationmethylase) was identified

4 Discussion

Thepresent study for the first time presents detailed bioinfor-matic analyses of the Halococcus hamelinensis genome Thismicroorganism was originally isolated from modern stro-matolites in the hypersaline reaches of Shark Bay Australiaand is known to exhibit several novel characteristics [10 12]However few genomes of stromatolite organisms have beenfully sequenced and are available for comparative genomicsFurthermore to date little is known in regards to the adaptivemechanisms employed by Archaea that reside in such ecosys-tems and the exact role of Archaea in stromatolites rema-ins clouded To better understand the basis of survival andpotentially any role H hamelinensis may play in its uniqueenvironment the genome was sequenced annotated anddetailed bioinformatics analyses conducted

The environment where H hamelinensis was isolatedShark Bay is prone to high salinity exposed ultraviolet radi-ation and high temperatures leading to desiccation [36]The-refore it is likely that H hamelinensis would need to possessvarious strategies to cope with such stresses and recent stud-ies have revealed several mechanisms this archaeon utilizesto cope with both high saline and UV stress [12 13] In termsof saline stress most halophilic Archaea are known to import

Archaea 7

Unclassified D

ictyosteliida

Euphorbiaceae

Ustilaginaceae

Schizophyllaceae

Unclassified Saccharomycetales

Saccharomycetaceae

Magnaporthaceae

Cryptosporidiidae

Thermaceae

Trueperaceae

Deinococcaceae

SynergistaceaeNitrospiraceae

Unclassified gammaproteob

Unclassified Deltaproteobacteria

ThiotrichaceaeHalomonadaceaeEnterobacteriaceaeEctothiorhodospiraceae

Chromatiaceae

Colwelliaceae

Alteromonadaceae

Myxococcaceae

Haliangiaceae

Cystobacteraceae

Geobacteraceae

Desulfovibrionaceae

Oscillatoriaceae

Nostocac

eae

Hapalo

siphon

aceae

Microcys

taceae

Therm

oana

eroba

cterac

eae

Synt

roph

omon

adac

eae

Lach

nosp

irace

ae

Clos

tridi

ales f

amily

XVI

I in

cCl

ostri

diac

eae

Ther

mob

acul

acea

eKt

edon

obac

tera

ceae

Her

peto

sipho

nace

aeCh

loro

flexa

ceae

Ther

mofi

lace

aeA

rcha

eogl

obac

eae

Hal

oarc

ulac

eae

Halo

bact

eriac

eae

Uncla

ssifi

ed H

aloba

cter

iales

Meth

anoc

ellac

eae

Meth

anoc

orpu

scul

acea

e

Meth

anoc

ullac

eae

Meth

anos

arcina

ceae

Therm

ococca

ceae

Nitrosopumilac

eae

Unclassifi

ed Archaea

Acidobacteriaceae

Frankiaceae

Mycobacteriaceae

Nocardiaceae

Pseudonocardiaceae

Streptomycetaceae

Streptosporangiaceae

ThermomonosporaceaeRubrobacteraceaeConexibacteraceae

BradyrhizobiaceaeMethylobacteriaceaeXanthobacteraceae

Rhodobacteraceae

Rhodospirillaceae

Alicyclobacillaceae

BacillaceaeUnclassified Bacillales

Enterococcaceae

Lactobacillaceae

Cyclobacteriaceae

Cytophagaceae

Rhodothermaceae

Saprospiraceae

Burkholderiaceae

Comam

onadaceae

Anaerolineaceae

Paenibacillaceae

Figure 2 Radial phylogenetic tree comparison between H hamelinensis and the bacterial and archaeal lineage within IMGER databaseGenomes of Archaea Bacteria and Eukaryota are displayed clockwise on the circular plot The bar charts in between the circular plot andphylogenetic dendrograms are percentage representations of positive hits of genes between H hamelinensis and respective genomes

potassium ions as the primary osmoadaptive strategy [12 37]Interestingly recent studies indicated that no potassium wastaken up by H hamelinensis either after growth at bothlow and high salt concentrations [12] Upon analysis of thegenome of H hamelinensis in the present study however itwas found that a putative potassium uptake protein TrkHwas present however no homologs to KdpB were identifiedOther putative potassium uptake proteins such as TrkA and

several others from the potassium efflux system were identi-fied (Table 2) although no complete pathway for potassiumuptake was detected within the H hamelinensis genome It ishypothesized that these putative proteinsmay be inactive dueto the lack of K+ uptake seen previously [12] however it is alsopossible they may be expressed under different conditionsOther research based on multidrug susceptibility suggestedthat resistance to antibiotics in microorganisms is associated

8 Archaea

with several factors including efflux pumps [38] Specificallyone study found that the disruption of the putative potassiumuptake regulator TrkA leads to rifampicin resistance [39]as well as conferring resistance to other antibiotics such asnovobiocin [40 41] Interestingly H hamelinensis displayssensitivity to both rifampicin and novobiocin [10] however itremains to be determined whether the putative TrkA presentin this organism plays any role in its observed antibiotic sen-sitivities

Early studies have also shown that halophilic enzymesexhibit highly polar surfaces in order to remain in solution[41] and comparative studies have shown that in particularribosomal proteins from moderate halophiles often have ahigher content of acidic amino acids than did the comparableproteins from E coli and other nonhalophiles [42]Thereforecytoplasmic proteins of halophiles often reveal high ratios ofacidic amino acids particularly glutamate and aspartate res-idues A presence of high ratios of acidic amino acids wasalso observed in the genome ofH hamelinensis in the presentstudy through codon prediction analyses suggesting thisfeature may correlate with its ability to tolerate a high salineenvironment

Glycine betaine accumulation has been shown to be themajor adaptive response to osmotic stress in H hamelinensis[12] and the presence of several glycine betaine uptake geneswere confirmed in the present study In addition other solutesinvolved in osmoadaptive strategies in this archaeon includetrehalose glutamate and proline [12] Levels of trehalosewere found to oscillate depending on the levels of glycinebetaine present in the cell [12] therefore the organism is likelyable to synthesize and degrade this sugar depending on thelevel of other compatible solutes present in the cell andormicroenvironment The high frequency of abundance dis-played for codons that code for proline glycine and glutamicacid discovered in the present study in theH hamelinensis inthe genome further supports osmotoleranceH hamelinensiscan potentially use glycine betaine not only as an osmoticstabilizer but also as a carbon and energy source An earlystudy was carried out on a number of haloalkaliphilic isolatesfrom Mono Lake California which grew on glycine betaineby sequential demethylation via dimethylglycine to sarcosinelater excreting glycine betaine into the medium [43] At highsalinities glycine betaine catabolism was suppressed and thecompound was taken up solely to serve as an osmotic solutethereby allowing extension of the salt range enabling growthin 25ndash35M NaCl [12 43] Another study carried out on theShark Bay stromatolites reported the synthesis of glycinebetaine as a compatible solute in cyanobacteria [44] and ithas been suggested that Archaea and cyanobacteria coexistin these systems through aspects of metabolic cooperationinvolving compatible solutes [12] However further work isneeded to clarify the exact role of glycine betaine inH hame-linensis physiology

Bacteriorhodopsin halorhodopsin and bacterioruberinare unique energy transducers involved in energy acquisition[25 45]These energy transducers are signature tomost halo-philes however there were no homologs to these systemsdetected here in H hamelinensis This was intriguing sincecultures of this organism display a bright red-pink color [12]

characteristic of these pigments Furthermore earlier studiesemploying Raman spectroscopy confirmed the presence ofbacterioruberin in H hamelinensis [46] indicating furtherwork is needed to deduce whether genes involved in the syn-thesis of these pigments are less conserved than expected orare present as genes encoding unknown or hypothetical pro-teins

To further distinguish the H hamelinensis from otherorganisms analyses were conducted here to review the com-position and representation of its COG categories H hame-linensis COG categories with the largest number of PCGswere surprisingly also the overrepresented COG categorieswithinH hamelinensis The overrepresented COG categoriesconsisted of amino acids transport and metabolism car-bohydrate transport and metabolism and general functionprediction only These mostly consisted of PCGs relating tonutrient metabolism Other overrepresented COG categorieswere energy productionconversion and inorganic transportand metabolism These categories consisted of PCGs of var-ious enzymes such as ATP-synthases dehydrogenases andmultiple ABC-transporter genes These COG categories sug-gest potential adaptation of H hamelinensis towards highnutrient uptake and synthesis which would be beneficialas this archaeon is prone to a low nutrient environment[47] The secondary metabolites biosynthesis transport andcatabolism and signal transduction mechanism COG cate-gories were also overrepresented in H hamelinensis whencompared to other archaeal genomes These categories con-sisted of putative genes for synthesis of coenzyme A antibi-otics such as novobiocin and streptomycin and presence ofstress response proteins respectively These suggest putativedefence mechanism that may be present in H hamelinensis

H hamelinensis is associated witha highly diverse micro-bial community [7] and this organism displays potentiallyunique genome characteristics not present in many ArchaeaTo test the importance of horizontal gene transfer onH ham-elinensis various detailed bioinformatics analyses were con-ducted Interestingly there was significant similarity betweenH hamelinensis and the bacterial lineage There was 2222similarity between theH hamelinensis genome and the phylaof Actinobacteria which consists of many marine bacte-ria [48] Additionally there was 769 similarity betweencyanobacteria andH hamelinensis which alludes to potentialhorizontal gene transfer between these organisms Of furthersignificance four putative genes hadgt30 similarity betweenH hamelinensis and dsDNA viruses genomes elucidatinga possible function of viral DNA being involved in genetransfer Most of the bacterial phyla indicated lt1 gene hitswith theH hamelinensis genes therefore it is difficult to con-clude based on this evidence that HGT was prevalent in thegenomeDespite this the apparent lack of CRISPR-associatedgenes in H hamelinensis is consistent with a recent studyindicating these predicted genes do not seem to be presentin all haloarchaea [49] Although it is possible other genescould be encoded for in some of the unknownhypotheticalgenes of the H hamelinensis genome the apparent lack ofthese systems suggests this organism may be thus amenableto HGT a trait which may be of critical importance in itsspecialized niche in the Shark Bay stromatolites

Archaea 9

Pregenome Postgenome

BetI

BetII

BetIII

UvrA

UvrB

UvrC

phr2

TrkATrkH

Additional GB transporters

OpuD

Trehalose biosynthesis

Proline transport

Putative transposases

MnhF

Glutamate transport

Oxidative stress response

GloA

Rth

MgshpII

Putative aquaporins

Novobiocin biosynthesis

Beta lactamase family

Heavy metal resistance

As Cu Co Zn

DNA repair

EndIII

RadAMutL

MutS

ABC-type

UVHeavy metals

toxins

Foreign DNA

K+ transportNa+H+ antiporter

Na+

H2O

Figure 3 Schematic representation of some of the putative adaptive traits present in H hamelinensis The traits identified pregenome isindicated on the left and those elucidated postgenome in the present study are indicated on the right

5 Conclusions

This study reports the detailed genome analyses ofH hameli-nensis a novel archaeon first isolated frommodern stromato-lites Early studies carried out indicated the putative presenceof three distinct and unique glycine-betaine transportersBetI BetII and BetIII (unpublished data) and the presence ofa bacterial-like nucleotide excision repair (NER) system con-taining UvrA UvrB and UvrC genes as well as a photolyase[12 13] in the H hamelinensis genome Postgenome analysispresented here revealed a range of other potential stressresponse mechanisms and Figure 3 summarises some of thekey adaptive responses identified through analyses in thecurrent study It also highlights the advantages of high-thr-oughput sequencing and comparative genomics to build onpregenome sequencingwork In addition toDNA repairmec-hanisms and osmotic response there were selective putativegenes for heavy metal resistance such as arsenic A recentstudy has suggested there may be a link between salinity andheavy metal uptake in some haloarchaea [50] and furtherwork at the transcript and growth level may help elucidatethe importance of these mechanisms in H hamelinensis

It is important to note that the genome analyses presentedhere describe the potential for a given function and futuredetailed analyses at the gene andor protein expression levelsare required to definitively attribute the traits delineatedin the this investigation Such studies may ultimately helpelucidate the role of this domain of life in these evolutionarilysignificant ecosystems

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was funded by the Australian Research Council

References

[1] M R Walter R Buick and J S R Dunlop ldquoStromatolites3400ndash3500 Myr old from the North Pole area Western Aus-traliardquo Nature vol 284 no 5755 pp 443ndash445 1980

[2] J W Schopf ldquoFossil evidence of Archaean liferdquo PhilosophicalTransactions of the Royal Society B Biological Sciences vol 361no 1470 pp 869ndash885 2006

[3] C Dupraz and P T Visscher ldquoMicrobial lithification in marinestromatolites and hypersalinematsrdquoTrends inMicrobiology vol13 no 9 pp 429ndash438 2005

[4] R P Reid P T Visscher A W Decho et al ldquoThe role of mic-robes in accretion lamination and early lithification of modernmarine stromatolitesrdquo Nature vol 406 no 6799 pp 989ndash9922000

[5] F Goh M A Allen S Leuko et al ldquoDetermining the specificmicrobial populations and their spatial distribution within thestromatolite ecosystem of Shark BayrdquoThe ISME Journal vol 3no 4 pp 383ndash396 2009

[6] M A Allen F Goh B P Burns and B A Neilan ldquoBacterialarchaeal and eukaryotic diversity of smooth and pustularmicrobial mat communities in the hypersaline lagoon of SharkBayrdquo Geobiology vol 7 no 1 pp 82ndash96 2009

[7] B P Burns F Goh M Allen and B A Neilan ldquoMicrobialdiversity of extant stromatolites in the hypersaline marine envi-ronment of Shark Bay Australiardquo Environmental Microbiologyvol 6 no 10 pp 1096ndash1101 2004

[8] A Oren ldquoPopulation dynamics of halobacteria in the Dead Seawater columnrdquo Limnology and Oceanography vol 28 no 6 pp1094ndash1103 1983

10 Archaea

[9] S Benlloch S G Acinas J Anton S P Luz and F Rodrıguez-Valera ldquoArchaeal biodiversity in crystallizer ponds from a solarsaltern culture versus PCRrdquoMicrobial Ecology vol 41 no 1 pp12ndash19 2001

[10] F Goh S Leuko M A Allen et al ldquoHalococcus hamelinensissp nov a novel halophilic archaeon isolated from stromatolitesin Shark Bay Australiardquo International Journal of Systematic andEvolutionary Microbiology vol 56 no 6 pp 1323ndash1329 2006

[11] W D Grant M Kamekura T J McGenity and A VentosaldquoThe Archaea and the deeply branching and phototrophicBacteriardquo inClass III Halobacteria class nov In Bergeyrsquos Manualof Systematic Bacteriology D R Boone R W Castenholz andG M Garrity Eds vol 1 Springer New York NY USA 2ndedition 2001

[12] F Goh Y J Jeon K Barrow B A Neilan and B P BurnsldquoOsmoadaptive strategies of the archaeon Halococcus hameli-nensis isolated from a hypersaline stromatolite environmentrdquoAstrobiology vol 11 no 6 pp 529ndash536 2011

[13] S Leuko B A Neilan B P Burns M R Walter and L JRothschild ldquoMolecular assessment of UVC radiation-inducedDNA damage repair in the stromatolitic halophilic archaeonHalococcus hamelinensisrdquo Journal of Photochemistry and Photo-biology B Biology vol 102 no 2 pp 140ndash145 2011

[14] S Leuko F Goh R Ibanez-Peral B P Burns M R Walterand B A Neilan ldquoLysis efficiency of standard DNA extractionmethods for Halococcus spp in an organic rich environmentrdquoExtremophiles vol 12 no 2 pp 301ndash308 2008

[15] R K Aziz D Bartels A Best et al ldquoThe RAST Server rapidannotations using subsystems technologyrdquo BMCGenomics vol9 article 75 2008

[16] V M Markowitz F Korzeniewski K Palaniappan et al ldquoTheintegrated microbial genomes (IMG) systemrdquo Nucleic acidsresearch vol 34 pp D344ndashD348 2006

[17] A L Delcher K A Bratke E C Powers and S L SalzbergldquoIdentifying bacterial genes and endosymbiont DNA withGlimmerrdquo Bioinformatics vol 23 no 6 pp 673ndash679 2007

[18] K Lagesen P Hallin E A Roslashdland H-H Staeligrfeldt T Rognesand DW Ussery ldquoRNAmmer consistent and rapid annotationof ribosomal RNA genesrdquo Nucleic Acids Research vol 35 no 9pp 3100ndash3108 2007

[19] T M Lowe and S R Eddy ldquotRNAscan-SE a program forimproved detection of transfer RNA genes in genomic sequ-encerdquo Nucleic Acids Research vol 25 no 5 pp 955ndash964 1997

[20] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[21] K Tamura J Dudley M Nei and S Kumar ldquoMEGA4 Molec-ular Evolutionary Genetics Analysis (MEGA) software version40rdquo Molecular Biology and Evolution vol 24 no 8 pp 1596ndash1599 2007

[22] E Saunders B J Tindall R Fahnrich et al ldquoComplete genomesequence of Haloterrigena turkmenica type strain (4k119879)rdquo Stan-dards in Genomic Sciences vol 2 no 1 pp 107ndash116 2010

[23] A Witte U Baranyi R Klein et al ldquoCharacterization ofNatronobacterium magadii phage 120601Ch1 a unique archaealphage containing DNA and RNArdquoMolecular Microbiology vol23 no 3 pp 603ndash616 1997

[24] M C Gutierrez A M Castillo M Kamekura et al ldquoHalopigerxanaduensis gen nov sp nov an extremely halophilic arch-aeon isolated from saline Lake Shangmatala in Inner MongoliaChinardquo International Journal of Systematic and EvolutionaryMicrobiology vol 57 no 7 pp 1402ndash1407 2007

[25] A Oren ldquoMolecular ecology of extremely halophilic Archaeaand Bacteriardquo FEMSMicrobiology Ecology vol 39 no 1 pp 1ndash72002

[26] R T Papke C J Douady W F Doolittle and F Rodrıguez-Valera ldquoDiversity of bacteriorhodopsins in different hypersalinewaters from a single Spanish salternrdquo Environmental Microbiol-ogy vol 5 no 11 pp 1039ndash1045 2003

[27] S P Balashov E S Imasheva V A Boichenko J Anton J MWang and J K Lanyi ldquoXanthorhodopsin a proton pumpwith alight-harvesting carotenoid antennardquo Science vol 309 no 5743pp 2061ndash2064 2005

[28] E F DeLong and O Beja ldquoThe light-driven proton pump pro-teorhodopsin enhances bacterial survival during tough timesrdquoPLoS Biology vol 8 no 4 Article ID e1000359 2010

[29] A Copeland S Spring M Goker et al ldquoComplete genomesequence of Desulfomicrobium baculatum type strain (X119879)rdquoStandards in Genomic Sciences vol 1 no 1 pp 29ndash37 2009

[30] A Juste B Lievens I Frans et al ldquoGenetic and physiologicaldiversity of Tetragenococcus halophilus strains isolated fromsugar- and salt-rich environmentsrdquo Microbiology vol 154 no9 pp 2600ndash2610 2008

[31] J C Yang R Madupu A S Durkin et al ldquoThe complete geno-me of Teredinibacter turnerae T7901 an intracellular endosym-biont of marine wood-boring bivalves (shipworms)rdquo PLoSONE vol 4 no 7 Article ID e6085 2009

[32] A Ventosa J J Nieto and A Oren ldquoBiology of moderatelyhalophilic aerobic bacteriardquoMicrobiology andMolecular BiologyReviews vol 62 no 2 pp 504ndash544 1998

[33] L Lyngberg J Healy W Bartlett et al ldquoKefF the regulatorysubunit of the potassium efflux system KefC shows quinoneoxidoreductase activityrdquo Journal of Bacteriology vol 193 no 18pp 4925ndash4932 2011

[34] T Allers and M Mevarech ldquoArchaeal geneticsmdashthe third wayrdquoNature Reviews Genetics vol 6 no 1 pp 58ndash73 2005

[35] P Horvath and R Barrangou ldquoCRISPRCas the immunesystem of bacteria and Archaeardquo Science vol 327 no 5962 pp167ndash170 2010

[36] B P Burns R Anitori P Butterworth et al ldquoModern analoguesand the early history of microbial liferdquo Precambrian Researchvol 173 no 1ndash4 pp 10ndash18 2009

[37] D D Martin R A Ciulla andM F Roberts ldquoOsmoadaptationin Archaeardquo Applied and Environmental Microbiology vol 65no 5 pp 1815ndash1825 1999

[38] M A Webber and L J V Piddock ldquoThe importance of effluxpumps in bacterial antibiotic resistancerdquo Journal of Antimicro-bial Chemotherapy vol 51 no 1 pp 9ndash11 2003

[39] A Castaneda-Garcıa T T Do and J Blazquez ldquoThe K+ uptakeregulator trka controls membrane potential pH homeostasisand multidrug susceptibility in Mycobacterium smegmatisrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1489ndash1498 2011

[40] J Su H Gong J Lai A Main and S Lu ldquoThe potassiumtransporter Trk and external potassium modulate Salmonellaenterica protein secretion and virulencerdquo Infection and Immu-nity vol 77 no 2 pp 667ndash675 2009

[41] S P Kennedy W V Ng S L Salzberg L Hood and SDasSarma ldquoUnderstanding the adaptation of Halobacteriumspecies NRC-1 to its extreme environment through computa-tional analysis of its genome sequencerdquo Genome Research vol11 no 10 pp 1641ndash1650 2001

Archaea 11

[42] G Storz and R Hengge-Aronis Bacterial Stress Responses ASMPress Washington DC USA 2000

[43] M R Diaz and B F Taylor ldquoMetabolism of methylatedosmolytes by aerobic bacteria from Mono Lake a moderatelyhypersaline alkaline environmentrdquo FEMS Microbiology Ecol-ogy vol 19 no 4 pp 239ndash247 1996

[44] F Goh K D Barrow B P Burns and B A Neilan ldquoIden-tification and regulation of novel compatible solutes fromhypersaline stromatolite-associated cyanobacteriardquo Archives ofMicrobiology vol 192 no 12 pp 1031ndash1038 2010

[45] M Kolbe H Besir L-O Essen and D Oesterhelt ldquoStructureof the light-driven chloride pump halorhodopsin at 18 AResolutionrdquo Science vol 288 no 5470 pp 1390ndash1396 2000

[46] C P Marshall S Leuko C M Coyle M R Walter B P Burnsand B A Neilan ldquoCarotenoid analysis of halophilic archaea byresonance Raman spectroscopyrdquo Astrobiology vol 7 no 4 pp631ndash643 2007

[47] B Morton ldquoThe biology and functional morphology of Fragumerugatum (Bivalvia Cardiidae) from Shark Bay Western Aus-tralia the significance of its relationship with entrained zoox-anthellaerdquo Journal of Zoology vol 251 no 1 pp 39ndash52 2000

[48] H-P Fiedler C Bruntner A T Bull et al ldquoMarine actinomyce-tes as a source of novel secondary metabolitesrdquo Antonie vanLeeuwenhoek vol 87 no 1 pp 37ndash42 2005

[49] E A Lynch G I Langille A Darling et al ldquoSequencing ofseven haloarchael genomes reveals patterns of genomci fluxrdquoPlos ONE vol 7 pp 1ndash13 2012

[50] G Popescu and L Dumitru ldquoBiosorption of some heavy met-als from media with high salt concentrations by halophilicArchaeardquo Biotechnology amp Biotechnological Equipment vol 23supplement 1 pp 791ndash795 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology

2 Archaea

stromatolite systems still remains unexplored HalophilicArchaea in particular may be an important component of theShark Bay stromatolites vital to ecosystem function Halophi-lic Archaea are very well adapted to hypersaline environ-ments and thrive in many different areas including the DeadSea and solar salterns [8 9] Previously the first archaeon tobe isolated from stromatolites a Gram-negative nonmotilestrictly aerobic Halococcus isolate (Halococcus hamelinensis)was also characterised [10] This archaeon grows optimallyat 37∘C in medium containing 15 NaCl as opposed to 20ndash30 in other characterizedHalococcus strains It metabolizesmany simple and complex carbohydrates including glucosesucrose xylose maltose trehalose mannitol galactose andglycerol [10] It does not hydrolyze starch or produce indoleand is negative for sulfide reduction urease and gelatin lique-fication in contrast to otherHalococcus species Furthermoreit displays sensitivity to rifampicin novobiocin and baci-tracin and resistance to kanamycin tetracycline streptomy-cin neomycin and penicillin H hamelinensis was also oxi-dase-negative whereas all recognized Halococcus species areoxidase-positive and most aerobic halophilic Archaea arealso oxidase-positive [11] Whole-cell protein profiles enzy-me composition and carbon source utilization of this isolatewere distinct from other characterized Halococcus speciesrendering it a novel species of Halococcus [10] Prior to theisolation of this organism no archaeal species had been dis-covered at Shark Bay As no archaeal isolates were also identi-fied in the surrounding water despite samples being collectedseveral years apart [5 7] it may be that this organism has asignificant role to play in stromatolite ecosystem function

Recent studies have shown that H hamelinensis displaysa number of other novel characteristics including lackingpotassium accumulation as a primary osmoprotective strat-egy in contrast to most known haloarchaea [12] Insteadaccumulation of glycine betaine as well as other solutes suchas glutamate and trehalose was observed to be the primarymechanism of adaptation to salt stress in this organism [12]In addition a recent study examined the effects of UV stressdamage inH hamelinensis [13] revealing that this archaeon isable to survive high germicidalUVC radiation dosages aswe-ll as possessing several bacterial-like nucleotide excision rep-air genes [13] Changes in the expression levels of these genes(uvrA uvrB and uvrC) were investigated by qRT-PCR andall were upregulated during both light and dark repairs [13]

While these recent studies have provided useful insightsinto the physiological characteristics of this novel archaeonwhat is lacking is a comprehensive whole genomic profile andrelated computational analyses of this organismwhichwouldsignificantly enhance our understanding of its evolutionaryand adaptive traits Here we describe the sequence (by mas-sively parallel 454 pyrosequencing technology) annotationand analysis of the genome of H hamelinensis which pro-vides insights into the ecology evolution and adaptation ofthis novel microorganism

2 Materials and Methods

21 Genome Sequencing and Assembly Halococcus hameli-nensis was grown as previously described until logarithmic

phase [10] andDNAwas extracted using a xanthogenate pro-tocol previously optimised for this Archaea [14]The genomeof H hamelinensis was sequenced at The Clive and VeraRamaciotti Centre using 454 (Roche) sequencing technologySample quality and quantity were assessed prior to librarypreparation as described in the GS FLX Titanium GeneralLibrary Preparation Method Manual (Roche) Briefly 8 120583gof gDNA was fragmented by nebulization fragmented DNArun on a 08 agarose gel and fragments between sim500 and800 bp excised and purified using a QiaQuick Gel ExtractionKit (Qiagen) DNA sample quality assessment fragment endrepair and adaptor ligation small fragment removal libraryimmobilization and single-stranded DNA library isolationwere performed as per GS FLXTitanium SequencingMethodManual (Roche) The quality of the library was assessed on aBioanalyzer RNAPico 6000 Chip (Agilent Technologies) andquantified using the Quant-It RiboGreen assay (Invitrogen)Finally emulsion PCR and sequencing were performed asdescribed in the GS FLX Titanium SequencingMethodMan-ual A total of 272854 reads counting up to 118638438 bases(32-fold coverage of the genome) were assembled using 454Newbler GS de novo Assembler software which generated453 contigs ranging from 500 to 105929 bp with bases havingquality scores of 40 and above

22 Automated Sequence Annotation Sequencing output wasuploaded onto two different auto annotation servers theRapid Annotation using Subsystem Technology (RAST) [15]and Integrated Microbial GenomesExpert Review (IMGER) [16] RAST uses a strategy based on comparison withmanually curated subsystems and subsystem-based proteinfamilies guaranteeing a high degree of consistency and accu-racy Putative coding sequences were identified by Glimmersoftware [17] and peptides shorter than 30 amino acids wereeliminated tRNA genes and rRNA genes were predicted bytRNAScan-SE software and RNAmmer respectively [18 19]Functional annotation was performed by searching the NCBInonredundant protein database and the Kyoto Encyclopediaof Genes and Genomes (KEGG) protein database Data fromthe genomewerematched against data from external sourcesincluding RefSeq EBI Genome Reviews GenBankEMBLJGI microbial genome data LIGAND GO COG EnzymeInterPro KEGG KO Pfam TIGRFam and GOLD Pathwaysfor modes of nutrition and transport system were acquiredthrough the KEGG function analysis in IMG COG analysiswas carried out using COG functions and abundance profileanalysis within the IMGER

23 Bioinformatic Analyses Phylogenetic analyses were per-formed to elucidate evolutionary relationships The 16 SrRNA gene sequences were derived from various euryarcha-eal genomes from the NCBI database The sequences werethen aligned using ClustalX [20] A BLOSUM series proteinweight matrix was used with a Gap Opening of 10 and a GapExtension of 02 Using the ClustalXmultiple sequence align-ment neighbor joining phylograms were constructed boot-strapped and displayed using the MEGA version 4 program[21] Programs from the EMBOSS package COMPSEQ (cal-culates the composition of unique words in a sequence) and

Archaea 3


3 Results









4 Archaea



AACAAG AATACAACC

ACGACT

AGAAGC

AGGAGT

ATAATC

ATG

ATT

CAA

CAC

CAG

CATCCA

CCCCCG

CCTCGA

CGCCGG


GCAGCC

GCGGCT

GGAGGC

GGGGGT

GTAGTC

GTG

GTT

TAA

TAC

TAG

TAT

TCATCC

TCGTCT

TGATGC

TGGTGT

TTATTCTTGTTT AAA

005

0045

004

0035

003

0025

002

0015

001

0005

0







Archaea 5














6 Archaea
















4 Discussion



Archaea 7

Unclassified D

ictyosteliida

Euphorbiaceae

Ustilaginaceae

Schizophyllaceae


Saccharomycetaceae

Magnaporthaceae

Cryptosporidiidae

Thermaceae

Trueperaceae

Deinococcaceae





Chromatiaceae

Colwelliaceae

Alteromonadaceae

Myxococcaceae

Haliangiaceae

Cystobacteraceae

Geobacteraceae

Desulfovibrionaceae

Oscillatoriaceae

Nostocac

eae

Hapalo

siphon

aceae

Microcys

taceae

Therm

oana

eroba

cterac

eae

Synt

roph

omon

adac

eae

Lach

nosp

irace

ae

Clos

tridi

ales f

amily

XVI

I in

cCl

ostri

diac

eae

Ther

mob

acul

acea

eKt

edon

obac

tera

ceae

Her

peto

sipho

nace

aeCh

loro

flexa

ceae

Ther

mofi

lace

aeA

rcha

eogl

obac

eae

Hal

oarc

ulac

eae

Halo

bact

eriac

eae

Uncla

ssifi

ed H

aloba

cter

iales

Meth

anoc

ellac

eae

Meth

anoc

orpu

scul

acea

e

Meth

anoc

ullac

eae

Meth

anos

arcina

ceae

Therm

ococca

ceae

Nitrosopumilac

eae

Unclassifi

ed Archaea

Acidobacteriaceae

Frankiaceae

Mycobacteriaceae

Nocardiaceae

Pseudonocardiaceae

Streptomycetaceae




Rhodobacteraceae

Rhodospirillaceae

Alicyclobacillaceae


Enterococcaceae

Lactobacillaceae

Cyclobacteriaceae

Cytophagaceae

Rhodothermaceae

Saprospiraceae

Burkholderiaceae

Comam

onadaceae

Anaerolineaceae

Paenibacillaceae




8 Archaea








Archaea 9


BetI

BetII

BetIII

UvrA

UvrB

UvrC

phr2

TrkATrkH


OpuD


Proline transport


MnhF

Glutamate transport


GloA

Rth

MgshpII

Putative aquaporins




As Cu Co Zn

DNA repair

EndIII

RadAMutL

MutS

ABC-type

UVHeavy metals

toxins

Foreign DNA


Na+

H2O


5 Conclusions





Acknowledgment


References









10 Archaea


































Archaea 11

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 3


3 Results









4 Archaea



AACAAG AATACAACC

ACGACT

AGAAGC

AGGAGT

ATAATC

ATG

ATT

CAA

CAC

CAG

CATCCA

CCCCCG

CCTCGA

CGCCGG


GCAGCC

GCGGCT

GGAGGC

GGGGGT

GTAGTC

GTG

GTT

TAA

TAC

TAG

TAT

TCATCC

TCGTCT

TGATGC

TGGTGT

TTATTCTTGTTT AAA

005

0045

004

0035

003

0025

002

0015

001

0005

0







Archaea 5














6 Archaea
















4 Discussion



Archaea 7

Unclassified D

ictyosteliida

Euphorbiaceae

Ustilaginaceae

Schizophyllaceae


Saccharomycetaceae

Magnaporthaceae

Cryptosporidiidae

Thermaceae

Trueperaceae

Deinococcaceae





Chromatiaceae

Colwelliaceae

Alteromonadaceae

Myxococcaceae

Haliangiaceae

Cystobacteraceae

Geobacteraceae

Desulfovibrionaceae

Oscillatoriaceae

Nostocac

eae

Hapalo

siphon

aceae

Microcys

taceae

Therm

oana

eroba

cterac

eae

Synt

roph

omon

adac

eae

Lach

nosp

irace

ae

Clos

tridi

ales f

amily

XVI

I in

cCl

ostri

diac

eae

Ther

mob

acul

acea

eKt

edon

obac

tera

ceae

Her

peto

sipho

nace

aeCh

loro

flexa

ceae

Ther

mofi

lace

aeA

rcha

eogl

obac

eae

Hal

oarc

ulac

eae

Halo

bact

eriac

eae

Uncla

ssifi

ed H

aloba

cter

iales

Meth

anoc

ellac

eae

Meth

anoc

orpu

scul

acea

e

Meth

anoc

ullac

eae

Meth

anos

arcina

ceae

Therm

ococca

ceae

Nitrosopumilac

eae

Unclassifi

ed Archaea

Acidobacteriaceae

Frankiaceae

Mycobacteriaceae

Nocardiaceae

Pseudonocardiaceae

Streptomycetaceae




Rhodobacteraceae

Rhodospirillaceae

Alicyclobacillaceae


Enterococcaceae

Lactobacillaceae

Cyclobacteriaceae

Cytophagaceae

Rhodothermaceae

Saprospiraceae

Burkholderiaceae

Comam

onadaceae

Anaerolineaceae

Paenibacillaceae




8 Archaea








Archaea 9


BetI

BetII

BetIII

UvrA

UvrB

UvrC

phr2

TrkATrkH


OpuD


Proline transport


MnhF

Glutamate transport


GloA

Rth

MgshpII

Putative aquaporins




As Cu Co Zn

DNA repair

EndIII

RadAMutL

MutS

ABC-type

UVHeavy metals

toxins

Foreign DNA


Na+

H2O


5 Conclusions





Acknowledgment


References









10 Archaea


































Archaea 11

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Archaea



AACAAG AATACAACC

ACGACT

AGAAGC

AGGAGT

ATAATC

ATG

ATT

CAA

CAC

CAG

CATCCA

CCCCCG

CCTCGA

CGCCGG


GCAGCC

GCGGCT

GGAGGC

GGGGGT

GTAGTC

GTG

GTT

TAA

TAC

TAG

TAT

TCATCC

TCGTCT

TGATGC

TGGTGT

TTATTCTTGTTT AAA

005

0045

004

0035

003

0025

002

0015

001

0005

0







Archaea 5














6 Archaea
















4 Discussion



Archaea 7

Unclassified D

ictyosteliida

Euphorbiaceae

Ustilaginaceae

Schizophyllaceae


Saccharomycetaceae

Magnaporthaceae

Cryptosporidiidae

Thermaceae

Trueperaceae

Deinococcaceae





Chromatiaceae

Colwelliaceae

Alteromonadaceae

Myxococcaceae

Haliangiaceae

Cystobacteraceae

Geobacteraceae

Desulfovibrionaceae

Oscillatoriaceae

Nostocac

eae

Hapalo

siphon

aceae

Microcys

taceae

Therm

oana

eroba

cterac

eae

Synt

roph

omon

adac

eae

Lach

nosp

irace

ae

Clos

tridi

ales f

amily

XVI

I in

cCl

ostri

diac

eae

Ther

mob

acul

acea

eKt

edon

obac

tera

ceae

Her

peto

sipho

nace

aeCh

loro

flexa

ceae

Ther

mofi

lace

aeA

rcha

eogl

obac

eae

Hal

oarc

ulac

eae

Halo

bact

eriac

eae

Uncla

ssifi

ed H

aloba

cter

iales

Meth

anoc

ellac

eae

Meth

anoc

orpu

scul

acea

e

Meth

anoc

ullac

eae

Meth

anos

arcina

ceae

Therm

ococca

ceae

Nitrosopumilac

eae

Unclassifi

ed Archaea

Acidobacteriaceae

Frankiaceae

Mycobacteriaceae

Nocardiaceae

Pseudonocardiaceae

Streptomycetaceae




Rhodobacteraceae

Rhodospirillaceae

Alicyclobacillaceae


Enterococcaceae

Lactobacillaceae

Cyclobacteriaceae

Cytophagaceae

Rhodothermaceae

Saprospiraceae

Burkholderiaceae

Comam

onadaceae

Anaerolineaceae

Paenibacillaceae




8 Archaea








Archaea 9


BetI

BetII

BetIII

UvrA

UvrB

UvrC

phr2

TrkATrkH


OpuD


Proline transport


MnhF

Glutamate transport


GloA

Rth

MgshpII

Putative aquaporins




As Cu Co Zn

DNA repair

EndIII

RadAMutL

MutS

ABC-type

UVHeavy metals

toxins

Foreign DNA


Na+

H2O


5 Conclusions





Acknowledgment


References









10 Archaea


































Archaea 11

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 5














6 Archaea
















4 Discussion



Archaea 7

Unclassified D

ictyosteliida

Euphorbiaceae

Ustilaginaceae

Schizophyllaceae


Saccharomycetaceae

Magnaporthaceae

Cryptosporidiidae

Thermaceae

Trueperaceae

Deinococcaceae





Chromatiaceae

Colwelliaceae

Alteromonadaceae

Myxococcaceae

Haliangiaceae

Cystobacteraceae

Geobacteraceae

Desulfovibrionaceae

Oscillatoriaceae

Nostocac

eae

Hapalo

siphon

aceae

Microcys

taceae

Therm

oana

eroba

cterac

eae

Synt

roph

omon

adac

eae

Lach

nosp

irace

ae

Clos

tridi

ales f

amily

XVI

I in

cCl

ostri

diac

eae

Ther

mob

acul

acea

eKt

edon

obac

tera

ceae

Her

peto

sipho

nace

aeCh

loro

flexa

ceae

Ther

mofi

lace

aeA

rcha

eogl

obac

eae

Hal

oarc

ulac

eae

Halo

bact

eriac

eae

Uncla

ssifi

ed H

aloba

cter

iales

Meth

anoc

ellac

eae

Meth

anoc

orpu

scul

acea

e

Meth

anoc

ullac

eae

Meth

anos

arcina

ceae

Therm

ococca

ceae

Nitrosopumilac

eae

Unclassifi

ed Archaea

Acidobacteriaceae

Frankiaceae

Mycobacteriaceae

Nocardiaceae

Pseudonocardiaceae

Streptomycetaceae




Rhodobacteraceae

Rhodospirillaceae

Alicyclobacillaceae


Enterococcaceae

Lactobacillaceae

Cyclobacteriaceae

Cytophagaceae

Rhodothermaceae

Saprospiraceae

Burkholderiaceae

Comam

onadaceae

Anaerolineaceae

Paenibacillaceae




8 Archaea








Archaea 9


BetI

BetII

BetIII

UvrA

UvrB

UvrC

phr2

TrkATrkH


OpuD


Proline transport


MnhF

Glutamate transport


GloA

Rth

MgshpII

Putative aquaporins




As Cu Co Zn

DNA repair

EndIII

RadAMutL

MutS

ABC-type

UVHeavy metals

toxins

Foreign DNA


Na+

H2O


5 Conclusions





Acknowledgment


References









10 Archaea


































Archaea 11

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 Archaea
















4 Discussion



Archaea 7

Unclassified D

ictyosteliida

Euphorbiaceae

Ustilaginaceae

Schizophyllaceae


Saccharomycetaceae

Magnaporthaceae

Cryptosporidiidae

Thermaceae

Trueperaceae

Deinococcaceae





Chromatiaceae

Colwelliaceae

Alteromonadaceae

Myxococcaceae

Haliangiaceae

Cystobacteraceae

Geobacteraceae

Desulfovibrionaceae

Oscillatoriaceae

Nostocac

eae

Hapalo

siphon

aceae

Microcys

taceae

Therm

oana

eroba

cterac

eae

Synt

roph

omon

adac

eae

Lach

nosp

irace

ae

Clos

tridi

ales f

amily

XVI

I in

cCl

ostri

diac

eae

Ther

mob

acul

acea

eKt

edon

obac

tera

ceae

Her

peto

sipho

nace

aeCh

loro

flexa

ceae

Ther

mofi

lace

aeA

rcha

eogl

obac

eae

Hal

oarc

ulac

eae

Halo

bact

eriac

eae

Uncla

ssifi

ed H

aloba

cter

iales

Meth

anoc

ellac

eae

Meth

anoc

orpu

scul

acea

e

Meth

anoc

ullac

eae

Meth

anos

arcina

ceae

Therm

ococca

ceae

Nitrosopumilac

eae

Unclassifi

ed Archaea

Acidobacteriaceae

Frankiaceae

Mycobacteriaceae

Nocardiaceae

Pseudonocardiaceae

Streptomycetaceae




Rhodobacteraceae

Rhodospirillaceae

Alicyclobacillaceae


Enterococcaceae

Lactobacillaceae

Cyclobacteriaceae

Cytophagaceae

Rhodothermaceae

Saprospiraceae

Burkholderiaceae

Comam

onadaceae

Anaerolineaceae

Paenibacillaceae




8 Archaea








Archaea 9


BetI

BetII

BetIII

UvrA

UvrB

UvrC

phr2

TrkATrkH


OpuD


Proline transport


MnhF

Glutamate transport


GloA

Rth

MgshpII

Putative aquaporins




As Cu Co Zn

DNA repair

EndIII

RadAMutL

MutS

ABC-type

UVHeavy metals

toxins

Foreign DNA


Na+

H2O


5 Conclusions





Acknowledgment


References









10 Archaea


































Archaea 11

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 7

Unclassified D

ictyosteliida

Euphorbiaceae

Ustilaginaceae

Schizophyllaceae


Saccharomycetaceae

Magnaporthaceae

Cryptosporidiidae

Thermaceae

Trueperaceae

Deinococcaceae





Chromatiaceae

Colwelliaceae

Alteromonadaceae

Myxococcaceae

Haliangiaceae

Cystobacteraceae

Geobacteraceae

Desulfovibrionaceae

Oscillatoriaceae

Nostocac

eae

Hapalo

siphon

aceae

Microcys

taceae

Therm

oana

eroba

cterac

eae

Synt

roph

omon

adac

eae

Lach

nosp

irace

ae

Clos

tridi

ales f

amily

XVI

I in

cCl

ostri

diac

eae

Ther

mob

acul

acea

eKt

edon

obac

tera

ceae

Her

peto

sipho

nace

aeCh

loro

flexa

ceae

Ther

mofi

lace

aeA

rcha

eogl

obac

eae

Hal

oarc

ulac

eae

Halo

bact

eriac

eae

Uncla

ssifi

ed H

aloba

cter

iales

Meth

anoc

ellac

eae

Meth

anoc

orpu

scul

acea

e

Meth

anoc

ullac

eae

Meth

anos

arcina

ceae

Therm

ococca

ceae

Nitrosopumilac

eae

Unclassifi

ed Archaea

Acidobacteriaceae

Frankiaceae

Mycobacteriaceae

Nocardiaceae

Pseudonocardiaceae

Streptomycetaceae




Rhodobacteraceae

Rhodospirillaceae

Alicyclobacillaceae


Enterococcaceae

Lactobacillaceae

Cyclobacteriaceae

Cytophagaceae

Rhodothermaceae

Saprospiraceae

Burkholderiaceae

Comam

onadaceae

Anaerolineaceae

Paenibacillaceae




8 Archaea








Archaea 9


BetI

BetII

BetIII

UvrA

UvrB

UvrC

phr2

TrkATrkH


OpuD


Proline transport


MnhF

Glutamate transport


GloA

Rth

MgshpII

Putative aquaporins




As Cu Co Zn

DNA repair

EndIII

RadAMutL

MutS

ABC-type

UVHeavy metals

toxins

Foreign DNA


Na+

H2O


5 Conclusions





Acknowledgment


References









10 Archaea


































Archaea 11

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

8 Archaea








Archaea 9


BetI

BetII

BetIII

UvrA

UvrB

UvrC

phr2

TrkATrkH


OpuD


Proline transport


MnhF

Glutamate transport


GloA

Rth

MgshpII

Putative aquaporins




As Cu Co Zn

DNA repair

EndIII

RadAMutL

MutS

ABC-type

UVHeavy metals

toxins

Foreign DNA


Na+

H2O


5 Conclusions





Acknowledgment


References









10 Archaea


































Archaea 11

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 9


BetI

BetII

BetIII

UvrA

UvrB

UvrC

phr2

TrkATrkH


OpuD


Proline transport


MnhF

Glutamate transport


GloA

Rth

MgshpII

Putative aquaporins




As Cu Co Zn

DNA repair

EndIII

RadAMutL

MutS

ABC-type

UVHeavy metals

toxins

Foreign DNA


Na+

H2O


5 Conclusions





Acknowledgment


References









10 Archaea


































Archaea 11

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

10 Archaea


































Archaea 11

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 11

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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