The Pathogen of the Great Barrier Reef Sponge ...

Mar Biotechnol DOI 10.1007/s10126-015-9627-y

ORIGINAL ARTICLE

The Pathogen of the Great Barrier Reef Sponge Rhopaloeides odorabile Is a New Strain of Pseudoalteromonas agarivorans Containing Abundant and Diverse Virulence-Related Genes Jayanta D. Choudhury 1 & Arnab Pramanik 1 & Nicole S. Webster 2 & Lyndon E. Llewellyn 2 & Ratan Gachhui 3 & Joydeep Mukherjee 1

Received: 17 September 2014 / Accepted: 11 March 2015 # Springer Science+Business Media New York 2015

Abstract Sponge diseases have increased dramatically, yet the causative agents of disease outbreaks have eluded identification. We undertook a polyphasic taxonomic analysis of the only confirmed sponge pathogen and identified it as a novel strain of Pseudoalteromonas agarivorans. 16S ribosomal RNA (rRNA) and gyraseB (gyrB) gene sequences along with phenotypic characteristics demonstrated that strain NW4327 was most closely related to P. agarivorans. DNA-DNA hybridization and in silico genome comparisons established NW4327 as a novel strain of P. agarivorans. Genes associated with type IV pili, mannose-sensitive hemagglutinin pili, and curli formation were identified in NW4327. One gene cluster encoding ATP-binding cassette (ABC) transporter, HlyD and TolC, and two clusters related to the general secretion pathway indicated the presence of type I secretion system (T1SS) and type II secretion system (T2SS), respectively. A contiguous gene cluster of at least 19 genes related to type VI secretion system (T6SS) which included all 13 core genes was found. The absence of T1SS and T6SS in nonpathogenic P. agarivorans S816 established NW4327 as the virulent

Electronic supplementary material The online version of this article (doi:10.1007/s10126-015-9627-y) contains supplementary material, which is available to authorized users. * Joydeep Mukherjee [email protected]; [email protected] 1

School of Environmental Studies, Jadavpur University, Kolkata 700 032, India

2

Australian Institute of Marine Science, PMB 3, Townsville MC, Townsville, Queensland 4810, Australia

3

Department of Life Science and Biotechnology, Jadavpur University, Kolkata 700 032, India

strain. Serine proteases and metalloproteases of the classes S8, S9, M4, M6, M48, and U32 were identified in NW4327, many of which can degrade collagen. Collagenase activity in NW4327 and its absence in the nonpathogenic P. agarivorans KMM 255T reinforced the invasiveness of NW4327. This is the first report unambiguously identifying a sponge pathogen and providing the first insights into the virulence genes present in any pathogenic Pseudoalteromonas genome. The investigation supports a theoretical study predicting high abundance of terrestrial virulence gene homologues in marine bacteria. Keywords Pseudoalteromonas . Sponge disease . Taxonomy . Genome analysis . Collagenase

Introduction Reports of sponge disease have been steadily increasing with associated population declines of ecologically and commercially important sponge species in the Mediterranean, Caribbean, Papua New Guinea (PNG), and the Great Barrier Reef (GBR) (Webster 2007; Maldonado et al. 2010; Stabili et al. 2012; Angermeier et al. 2012; Olson et al. 2014). The majority of these reports suspected a microbial origin for the diseases, although few studies have elucidated potential pathogens (Webster 2007). Investigations were aimed at identification of putative pathogen(s) suspected to cause sponge diseases, and infection experiments were attempted by some investigators. For instance, nearly 20 % of the Amphimedon compressa population in Florida were affected by a disease syndrome characterized by white patches along the sponge branches (Angermeier et al. 2012). Transmission electron microscopy of diseased A. compressa tissue revealed a spongin-boring bacterial morphotype consistent with previous observations

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of diseased specimens of the GBR sponge Rhopaloeides odorabile (Webster et al. 2002). However, tissue transplantation experiments could not demonstrate infectivity of the disease and no pathogen was identified (Angermeier et al. 2012). Sponge disease has also impacted populations of Ianthella basta throughout Papua New Guinea, Torres Strait, and the Great Barrier Reef (Cervino et al. 2006; Luter et al. 2010a), and cultivation-based techniques identified two Bacillus spp. and three Pseudomonas spp. that were present in diseased specimens but apparently absent from healthy sponges in PNG (Cervino et al. 2006). Other potential pathogens identified from sponges include a δ-Proteobacteria, a εProteobacteria, and a Cytophaga strain reported from lesions of diseased Aplysina aerophoba in Slovenia (Webster et al. 2008); a nonflagellated, twisted, rod-shaped bacterium from infected Ircinia sp. in the Western Mediterranean (Maldonado et al. 2010); Vibrio rotiferianus isolated from diseased Ircinia variabilis in the southern Adriatic and Ionian seas (Stabili et al. 2012); and Leptolyngbya sp. isolated from red band lesions of diseased Aplysina cauliformis sponges in the Caribbean (Olson et al. 2014). Other studies have indicated that microbes may not be playing a primary role in sponge pathogenesis. For example, infection assays and a thorough comparison of bacteria, viruses, fungi, and microeukaryotes in healthy and diseased I. basta revealed that microbes were not responsible for the brown spot lesions in this species (Luter et al. 2010b). Similarly, despite an identified shift from the Synechococcus/ Prochlorococcus clade of cyanobacteria in healthy Xestospongia muta toward several clades of undefined cyanobacteria in diseased specimens, underwater infection experiments were unable to establish the disease in healthy individuals (Angermeier et al. 2011). Although a substantial number of taxonomically diverse pathogens have been implicated, our understanding of sponge pathogenesis, such as taxonomic identification of the causative agents, mechanisms of pathogen transmission and virulence, is exceedingly inadequate. Strain NW4327 was confirmed as the primary pathogen of the GBR sponge R. odorabile (Webster et al. 2002) which, to date, remains the only study that has confirmed Koch’s postulates. Later, Mukherjee et al. (2009) purified a collagenolytic enzyme from NW4327 that was speculated to enhance the pathogenicity of this bacterial strain against the sponge. The authors considered the partially purified collagenase to be either a nonselective collagen degrading protease or tainted with nonspecific proteases. The highest enzyme activity was observed at pH 5, the internal pH of R. odorabile, and 30 °C, the mean seawater temperature of the GBR (Mukherjee et al. 2009). Very recently, the draft genome of this confirmed sponge pathogen (NW4327) was published (Choudhury et al. 2014). Coral pathogenesis has previously been studied using genomebased approaches (Reshef et al. 2008; de O Santos et al.

2011; Kimes et al. 2012), yet no similar analysis on marine sponges has been reported. To further investigate the only primary sponge pathogen described to date, we present a complete polyphasic taxonomic identification of strain NW4327 and describe the virulence-related genes present in the draft genome. Comparisons with nonpathogenic strains were performed to highlight specific genotypic and phenotypic differences that likely underpin the virulence of the sponge pathogen.

Materials and Methods Microorganism, Isolation, and Growth Conditions Strain NW4327 was originally isolated from a heavily fouled and necrotic sample of R. odorabile collected at a water depth of 8 m from Davies Reef, GBR, Australia (Webster et al. 2002). The strain was revived from −80 °C glycerol stock for the present study. NW4327 is publicly available (MTCC 11073 and DSM 25418). For the morphological, biochemical, and molecular studies, bacterium NW4327 was maintained on solid BD Marine Agar 2216 at 0–4 °C with monthly subculturing. Biomass for molecular systematic studies was obtained by growing the isolate in liquid medium (BD Marine Broth 2216, pH 7.5 to 8.0) prepared in purified, deionized water and incubating at 28 °C and 100 rpm in a rotary shaker for 48 h. Electron Microscopy For observing cell morphology and flagellation, NW4327 was grown overnight on BD Marine Agar 2216 and suspended in physiological saline solution. A drop of the suspension was negatively stained with a drop of 1 % phosphotungstic acid and placed on a carbon-coated copper grid. Observation was carried out in a FEI-Tecnai G 2 Spirit BioTWIN n (FP 5018/40) transmission electron microscope at an acceleration voltage of 80 kV. Chemotaxonomic Studies Fatty acid analyses were performed by the Identification Service of the DSMZ, Braunschweig, Germany. Fatty acid methyl esters (FAMEs) were acquired from 40 mg cells by saponification, methylation, and extraction as described previously (Miller 1982; Kuykendall et al. 1988). The FAME mixtures were separated by applying the Sherlock Microbial Identification System (MIS) (MIDI, Microbial ID, Newark, DE 19711, USA). Peaks were automatically integrated and fatty acid names as well as percentages determined by the MIS Standard Software (Microbial ID). A detailed method is available at http://www.dsmz.de/services/services-microorganisms/

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identification/analysis-of-cellular-fatty-acids.html. Isoprenoid quinones were extracted from the early stationary phase cells of strain NW4327 and analyzed by thin layer chromatography (TLC) following Hiraishi et al. (1992). Amplification and Sequencing of the 16S Ribosomal RNA Gene The 16S ribosomal RNA (rRNA) gene of strain NW4327 was amplified and sequenced by Identification Service of the DSMZ, Braunschweig, Germany, through direct sequencing of PCR-amplified 16S rDNA. Genomic DNA extraction, PCR amplification, and sequencing of the 16S rRNA gene of strain NW4327 were done following Rainey et al. (1996) and det a i l e d a t h t t p : / / w w w. d s m z . d e / s e r v i c e s / s e r v i c e s microorganisms/identification/full-phylogenetic-study-bycomplete-16s-rdna-sequence-analysis.html. Phylogenetic Analysis of the 16S rRNA Gene Pairwise similarities of the nearly complete 16S rRNA gene sequence of strain NW4327 were determined on the EzTaxone server (http://eztaxon-e.ezbiocloud.net/) (last accessed on 10th September, 2014) (Kim et al. 2012) applying identity analysis. Reference strains required for phylogenetic and phenotypic analyses were chosen from the top hits of this analysis. The 16S rRNA gene sequence of strain NW4327 was aligned with sequences of the type strains of related species of the genus Pseudoalteromonas. Basic phylogenetic methods described in our earlier publication (Arumugam et al. 2011) were followed. A consensus tree was constructed with PAUP* version 4b10 applying the majority rule option. Bootstrap values of the final tree were obtained from 50 % majority rule consensus of 100 trees. Bootstrap analysis was performed with 1000 resamplings to confirm the support for each clade. GyraseB (gyrB) Gene Amplification and Sequencing The nucleotide sequences of the gyrB gene of strain NW4327 and Pseudoalteromonas agarivorans KMM 255T were amplified through PCR with universal primer sets UP1 and UP2r applying the PCR conditions described (Yamamoto and Harayama 1995). PCR-amplified DNAs were analyzed by gel electrophoresis on 1 % agarose, and the correct band (approx. 1.2 kb) was cut and eluted with Qiagen Gel Extraction Kit (Cat. No. 28704). The purified product was ligated into vector pTZ57R/T using Fermentas InsTAclone PCR Cloning Kit (Cat. No. K1213) and transformed into competent Escherichia coli DH5α cells following standard protocols. The cloned gyrB gene was sequenced with M13 primers using an ABI Prism 3730 DNA analyzer (PE Applied Biosystems, Foster City, USA).

Phylogenetic Analysis of the gyrB Gene Sequence The gyrB gene sequence of NW4327 was contrasted with other gyrB sequences available from the NCBI database (last accessed on 10th September, 2014) and that of P. agarivorans KMM 255T obtained in this study. The nearly complete gyrB gene sequence of strain NW4327 was aligned with sequences of the type strains of related species of the genus Pseudoalteromonas. Phylogenetic analyses were performed by applying the neighbor-joining (NJ) method using PAUP* version 4b10. The unrooted tree was visualized through the TREEVIEW program, version 1.6.6 (http://taxonomy. zoology.gla.ac.uk/rod/treeview.html). Morphological, Physiological, and Biochemical Characteristics of Strain NW4327 Reference strains as listed in the supplementary table (Online Resource 3) were selected based on the results obtained from molecular phylogenetic analyses. Morphological characteristics included cell morphology, Gram staining, and motility (Baumann et al. 1971). Biochemical and physiological tests (as described in Online Resource 3) were carried out according to established procedures (Baumann et al. 1971, 1972; Baumann and Baumann 1981; Smibert and Krieg 1993; Barrow and Feltham 1993) unless stated otherwise. The following physiological and biochemical properties were examined: acid production from sugars; arginine dihydrolase activity; sodium requirement; oxidase, catalase, and urease activities; indole and H2S production; and ability to hydrolyze gelatin, starch, esculin, agar (Akagawa and Yamasato 1989), alginate (Sawabe et al. 2000), DNA, Tween 80, Tween 20, and casein. The requirement of Na+ ions was determined on medium B (Ivanova et al. 1996). Soluble pigment production was studied by using medium BT (medium B supplemented with 0.1 % tyrosine). The temperature and pH ranges of growth were examined on BD Marine agar 2216 and broth, respectively. Utilization/nonutilization of various organic substrates as sole carbon sources was tested following Baumann et al. (1971). Accumulation of poly-β-hydroxybutyrate was tested according to Burdon (1946). Nitrate and nitrite reductions were ascertained following Barrow and Feltham (1993). Susceptibility/resistance to antibiotics was determined through the routine diffusion plate technique. Antibacterial activity of strain NW4327 was assessed by the agar diffusion assay as described by Barry (1980) against Staphylococcus aureus MTCC 96, Bacillus subtilis MTCC 441, Micrococcus luteus MTCC 106, Bacillus cereus MTCC 430, Pseudomonas aeruginosa MTCC 2453, Proteus mirabilis MTCC 425, E. coli MTCC 739, and Shigella flexneri MTCC 1457. Bovine Achilles tendon collagen was used as the sole nutrient source in the production medium BD Marine Broth 2216. Collagenase activity was determined using azocoll following

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Mukherjee et al. (2009). Agarase activity was detected according to Akagawa and Yamasato (1989). All experiments were performed thrice in duplicate sets.

Determination of DNA G+C Content of Strain NW4327 The DNA G+C content of strain NW4327 was measured by applying the thermal denaturation method of Marmur and Doty (1962). The Tm value was ascertained by UV spectroscopy (Lambda 25 UV/visible spectrophotometer; PerkinElmer, USA).

DNA-DNA Hybridization Dot blot hybridization experiments were carried out with digoxigenin-labeled DNA using the DIG High Prime DNA Labelling and Detection Starter Kit (Cat. No. 11745832910, Roche Applied Sciences, Germany) following the manufacturer’s instructions. Four type strains showing the highest 16S rRNA and gyrB gene sequence similarities to NW4327 were selected for DNA-DNA hybridization (DDH). The genomic DNA probe was prepared from strain NW4327 by labeling it with digoxigenin-11-dUTP (DIG). Total DNA from strain NW4327 and reference strains was extracted, dissolved in saline sodium citrate (SSC) buffer, and boiled for 10 min. Equal amounts of DNA from different strains were spotted on a nylon membrane and treated with denaturation solution (1.5 M NaCl/0.5 M NaOH) and neutralization solution (1 M NaCl/0.5 M Tris Cl, pH 7.0). Hybridization temperature was determined applying the formulas: Tm =49.82+0.41 (% G+C) −(600/l) (l=length of hybrid in base pairs) and Topt =Tm −20 to 25 °C. Following overnight hybridization, the membrane was washed by applying high stringency conditions (twice with 2× SSC/0.1 % SDS at room temperature for 5 min each, twice with 0.5× SSC/0.1 % SDS at 65 °C for 15 min each). Colorimetric detection was performed by using NBT/BCIP. Dot intensities were quantified using ImageJ (http://rsb.info.nih.gov/ij/index. html) by determining relative intensities in circles of equal size, considering the self-hybridization value as 100 %. Experiments were performed in triplicate.

Genome Annotation and Analyses Detailed annotation and genome analyses of the previously sequenced and assembled draft genome of strain NW4327 (Choudhury et al. 2014) were undertaken. The genome was annotated using the RAST server (Aziz et al. 2008). Circular representation of the genome was performed using the GeneWiz browser 0.94 server (Hallin et al. 2009).

Determination of Species Rank Using In Silico Methods The predicted open reading frames (ORFs) of Pseudoalteromonas sp. NW4327 were compared with the NCBI nonredundant protein database using the BLASTX program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (last accessed on 17th August, 2014). The top BLASTX hit for each ORF was selected and identified by the species name to determine the strain closest to NW4327 (Jakobsen et al. 2013). Only genomes with definite species designation were considered for this comparison to unambiguously identify strain NW4327. In instances where the top hit came from a Pseudoalteromonas genome without any species name, the second best hit was considered. To confirm the species identity of NW4327, genome-to-genome distance and average nucleotide identity (ANI) were calculated. In silico DNADNA hybridization was performed with genome-to-genome distance calculator (GGDC) version 2 (http://ggdc.dsmz.de) (last accessed on 6th September, 2014), which was claimed to mimic DNA-DNA hybridization carried out in the laboratory (Auch et al. 2010). The draft genome sequence of Pseudoalteromonas sp. NW4327 was compared with the draft genome sequence of P. agarivorans S816 (accession no. APME00000000) (Vynne et al. 2011). Average nucleotide identity (Goris et al. 2007) (parameters: identity (%) ≥30, alignment (%) ≥70, and length=1020 nucleotides) and correlation coefficiency using tetranucleotide distribution pattern (TETRA) (Teeling et al. 2004) of NW4327 and P. agarivorans S816 genomes were calculated using the JSpecies software (Richter and Rosselló-Móra 2009). For determination of ANI using the BLAST algorithm (ANIb), the following parameters were used: −x=150; −q=−1; F=F; −e=1e−15 and − a=2. To identify the genomic rearrangements between the genomes of Pseudoalteromonas strain NW4327 and P. agarivorans strain S816, the draft genomes were aligned and compared using Mauve version 2.3.1 (Darling et al. 2004). Identification of Putative Virulence Factors Putative virulence-associated genes were assigned to three broad categories: (1) adhesion and colonization, (2) secretion systems, and (3) proteases and toxins. The genes coding for the putative virulence factors classified under the RAST subsystem (Aziz et al. 2008) categories Bmembrane transport^ and Bprotein degradation^ were identified in the Pseudoalteromonas sp. NW4327 genome. The presumed virulence factors that were not allocated to any specific subsystem were manually categorized. The ascertained proteins of interest were verified by the BLASTP program (last accessed on 17th August, 2014) using an E-value cutoff (1e-6). The presence of signal peptide was searched using the default settings of Gram-negative bacteria on the SignalP Server 4.1

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(Petersen et al. 2011) to predict if the protein of interest was secreted. Additionally, nonclassically secreted proteins were predicted using the SecretomeP 2.0 Server applying default settings for Gram-negative bacteria (Bendtsen et al. 2005). The presence of a complete type VI secretion system (T6SS) was verified by looking for all the core proteins defined by Bingle et al. (2008) and Boyer et al. (2009) using BLASTP and applying E-value (1e-6). The conserved domains were confirmed by carrying out a search in Pfam (http://pfam.sanger.ac.uk/) and InterPro (https://www.ebi.ac. uk/interpro) (last accessed on 17th August, 2014) protein databases. Homologues of type VI secretion system components found in terrestrial bacteria Rhizobium leguminosarum bv. Trifolii (Bladergroen et al. 2003) and Edwardsiella tarda strain PPD130/91 (Zheng and Leung 2007) were identified through BLASTP with E-value (1e-6). Nucleotide Sequence Accession Numbers The 16S rRNA and gyrB gene sequences of strain NW4327 were deposited in GenBank with accession numbers FR839670 and KC198083, respectively. The gyrB gene sequence of P. agarivorans KMM 255T was deposited in GenBank with accession number KF793928. The draft genome sequence of NW4327 was deposited in DDBJ/EMBL/ GenBank under accession number AZIO00000000.

Results and Discussion General Description The bacterial strain NW4327 (MTCC 11073 = DSM 25418) possessed all morphological, biochemical, and physiological features characteristic of the genus Pseudoalteromonas. On BD 2216 Marine agar incubated at 28 °C, young colonies were smooth, translucent, circular, and convex with regular edges having a diameter of 1–2 mm. Cells were 0.5 to 1.15 μm in length and 0.4 to 0.5 μm in diameter. The strain was motile by means of a single, polar flagellum. Detailed description of the strain is provided as supplemental text (Online Resource 1).

12:0 3OH was detected in NW4327 in moderate amount (7.50 %), which was not generally identified in Pseudoalteromonas species. Ubiquinone 8 was found to be the major respiratory quinone. Molecular Phylogenetic Analyses The similarity analysis performed on the 1524-bp 16S rRNA gene sequence of strain NW4327 established that strain NW4327 belonged to the genus Pseudoalteromonas, showing greater than 99 % 16S rRNA gene sequence similarity to many members of this genus. From the consensus tree shown in Fig. 1, it was concluded that strain NW4327 formed a robust clade with P. agarivorans KMM 255T with very high confidence (94 %). Besides, Pseudoalteromonas atlantica IAM 12927T and Pseudoalteromonas espejiana NCIMB 2127T were also found to be close relatives of NW4327 in the consensus tree. Sequence similarity data were consistent with the phylogenetic analysis (Fig. 1) and supported by phenotypic tests described later. The more rapidly evolving gyrB gene was utilized as a high-resolution molecular identification marker for discriminating strains of Pseudoalteromonas (Venkateswaran and Dohmoto 2000; Zeng and Zheng 2011). Sequence similarity analysis of the 1130-bp gyrB gene sequence of strain NW4327 further confirmed that the strain belonged to the genus Pseudoalteromonas, showing high gyrB gene sequence similarities with P. atlantica IAM 12927T, P. agarivorans KMM 255T, and Pseudoalteromonas tetraodonis IAM 14160T. The highest gyrB sequence similarity (98.39 %) occurred with P. agarivorans. The relative phylogenetic positions occupied by each of the Pseudoalteromonas type strains, based on gyrB nucleotide sequences, are shown in Fig. 2. The branching order of the gyrB-based tree resembled that of the tree derived from 16S rRNA nucleotide sequences (Fig. 1), where strain NW4327 was closely related to P. agarivorans KMM 255T, P. atlantica IAM 12927T, and P. espejiana NCIMB 2127T. Additionally, strain NW4327 appeared to be closely related to P. tetraodonis IAM 14160T in the gyrB-based tree. For this reason, P. tetraodonis was included in the determination of phenotypic characteristics. Determination of Phenotypic Characteristics

Chemotaxonomic Studies The predominant fatty acids of NW4327 were 16:1ω7c (34.11 %), 18:1ω7c (19.74 %), and 16:0 (17.7 %) as represented in the supplemental table (Online Resource 2). These values were in accordance with those generally found in Pseudoalteromonas species (Ivanova et al. 2000). The content of 18:1ω7c was, however, slightly higher than that reported for the genus Pseudoalteromonas. Interestingly, 18:1ω7c was not found in P. agarivorans KMM 255T (Online Resource 2).

Although strain NW4327 was highly similar to P. atlantica, P. espejiana, P. agarivorans, and P. tetraodonis based on 16S rRNA and gyrB sequencing, several differences in the phenotypic characteristics were noted. NW4327 differed from P. atlantica by 11, P. espejiana by 7, P. tetraodonis by 8, and P. agarivorans by only 2 (production of alginase and agarase) of the 19 biochemical and physiological characters examined as evident from the table (Online Resource 3 marked with asterisks). Strain NW4327 differed from

Mar Biotechnol Fig. 1 Consensus phylogenetic tree based on 16S rRNA gene sequences obtained by the majority rule option showing the position of Pseudoalteromonas agarivorans strain NW4327 among its phylogenetic neighbors. Numbers at nodes indicate levels of bootstrap support (%) based on analysis of 1000 resampled datasets; only values greater than 50 % are shown. The consensus tree provides the branching order among the genus Pseudoalteromonas, but not indicative of the evolutionary distances between the branches or nodes. Sulfitobacter pontiacus ChLG-10T was selected as outgroup. GenBank accession numbers are given in parentheses. Bar, 0.1 nt substitutions per site

P. atlantica by 18, P. espejiana by 12, P. agarivorans by 10, and P. tetraodonis by 9 of the 29 carbon source utilization characters examined (Online Resource 3 marked with asterisks) and differed from P. atlantica by 4, P. espejiana by 3, P. tetraodonis by 5, and P. agarivorans by none of the 11 antibiotic sensitivity characters examined (Online Resource 3 marked with asterisks). Overall, 59 differential characteristics were examined (Online Resource 3), of which strain NW4327 differed from type strains of P. atlantica by 33, from P. espejiana by 22, from P. tetraodonis by 22, and from P. agarivorans by 12 characteristics. Strain NW4327 was most closely related to P. agarivorans as determined by phenotypic characteristics. The cell-free supernatant of strain

NW4327 did not show antibacterial activity against any of the test strains. DNA-DNA Hybridization The highest reassociation values between strain NW4327 and P. agarivorans KMM 255T (93.12±6.9) as evident from the table (Online Resource 4) indicated NW4327 to be a strain of P. agarivorans, following the recommended threshold of 70 % DNA-DNA relatedness for differentiating species (Wayne et al. 1987). Strain NW4327 was originally isolated from a diseased specimen of the GBR sponge R. odorabile and previously

Mar Biotechnol Fig. 2 Radial cladogram showing the position of Pseudoalteromonas agarivorans strain NW4327 in relation to type strains of various species of Pseudoalteromonas based on gyrB nucleotide sequences. Bar, 0.1 nt substitutions per site

designated as a novel member of the α-Proteobacteria with 94 % 16S rRNA gene sequence similarity to Sulfitobacter pontiacus (Webster et al. 2002). S. pontiacus is a heterotrophic marine member of the Sulfitobacter genus, which specializes in sulfite oxidation (Sorokin 1995). The predominant fatty acid in Sulfitobacter genus is 18:1ω7c, ranging from 59.9 to 85.6 % of the total fatty acids (Park et al. 2007), whereas in this study, strain NW4327 was found to contain fatty acids that are characteristic of the genus Pseudoalteromonas. Similarly, the DNA G+C content of strain NW4327 was 41.2– 43.6 % which is characteristic of the Pseudoalteromonas genus (range 37–50 %), whereas the DNA G+C content of various Sulfitobacter species ranges from 55 to 64 % (Yoon et al. 2007). Phylogenetic analysis of strain NW4327 based on almost full-length 16S rRNA gene and gyrB gene sequences also placed strain NW4327 unequivocally within the genus Pseudoalteromonas. In light of these new findings, we now revise the generic assignment of strain NW4327 from Sulfitobacter to Pseudoalteromonas. Two strains of Pseudoalteromonas with 92 % gyrB gene sequence similarity were recently classified as two distinct species based on a species cutoff value of 70 % DNA-DNA

relatedness (Zeng and Zheng 2011). On this basis, it was proposed that the generally accepted cutoff value of 90 % (Venkateswaran et al. 1999) for gyrB gene sequence similarity may be too low to determine interspecies relationships within the genus Pseudoalteromonas. Tindall et al. (2010) stipulated DNA-DNA hybridization as a mandatory requirement for the description of a new species within a taxon when strains shared greater than 97 % 16S rRNA similarity although this criterion is still actively debated in the literature (Stackebrandt and Ebers 2006; Meier-Kolthoff et al. 2013). For the genus Pseudoalteromonas, it was recently suggested that the cutoff value for 16S rRNA sequence similarity to perform DNADNA hybridization should be 99.0 % (Oh et al. 2011). By searching the EzTaxon-e server, it was found that type strains of some of the established distinct species, clearly distinguishable by DNA-DNA homology and/or phenotypical properties, shared more than 99 % similarity in their 16S rRNA sequences. This finding was consistent with Mikhailov et al. (2002) in that most of the nonpigmented species of the genus Pseudoalteromonas were closely related to each other (99.4– 99.9 % 16S rRNA sequence similarity) constituting the major cluster of the genus. Additionally, Pseudoalteromonas

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aurantia and Pseudoalteromonas citrea were shown to have DNA-DNA hybridization values of only 10 % despite sharing 99.7 % similarity in their 16S rRNA sequence (Venkateswaran and Dohmoto 2000). On the basis of these observations, we included only the closest type strains for DNA-DNA hybridization studies. General Features of the Draft Genome Details of the draft genome assembly of strain NW4327 genome was previously described in Choudhury et al. (2014). The G+C content determined by the RAST server was 40.9 % (Choudhury et al. 2014) which was comparable to the value determined by the thermal denaturation method (41.2– 43.6 %) in this study. About 47 % of all genes matched with the subsystem categories of RAST (1712 nonhypothetical and 147 hypothetical proteins), and the remaining 53 % genes did not align with any subsystem categories (1071 nonhypothetical and 1060 hypothetical proteins). Circular representation of the NW4327 genome and its comparison with P. agarivorans strain S816 genome is shown in Fig. 3. Determination of Species Rank Comparison of the ORFs of strain NW4327 with the five closest bacterial genomes by BLASTX confirmed that P. agarivorans S816 (Vynne et al. 2011) alone was the best match for approximately 81 % (total ORFs=3970, Choudhury et al. 2014) of the ORFs of strain NW4327 (Fig. 4). GGDC calculations applying BLAST+ generated DDH values of 81.90±3.60 % (formula 1), 82.80±2.66 % (formula 2), and 85.00±2.95 % (formula 3). All three formulas returned DDH values well above the proposed cutoff value of 70 % for species differentiation. Using the JSpecies software, ANIb value between strains NW4327 and S816 genomes was 97.93 % (reciprocal value 97.82 %), which was, again, higher than the recommended cutoff value of 95 % for species differentiation (Goris et al. 2007). Calculated TETRA value between the two genomes was 0.99934 which was above the threshold of 0.99. The plotted values represented a straight line (Fig. 5). TETRA values greater than 0.99 and ANI values greater than 95–96 % indicate alike species (Richter and Rosselló-Móra 2009). The Mauve analysis (Fig. 6) revealed that the two genomes shared substantial homologous regions between them although some specific sequence elements unique to each organism were also present as evident from the presence of white areas inside the colored blocks in both genomes. DNA-DNA hybridization has long been considered a cornerstone for bacterial identification. However, with the advent of next generation sequencing, several in silico methods have also been proposed to replace and/or complement the conventional DDH. For example, it was shown (Goris et al. 2007) that 95 % ANI of common genes present in two strains

corresponded to the recommended cutoff DDH value (70 %). Later, Auch et al. (2010) claimed that replacing DDH by genome-to-genome distances (GGD) would be a promising alternative for taxonomists. Therefore, to unambiguously confirm the species level identification of strain NW4327, in silico DDH was performed in parallel to the conventional laboratory-based DDH. To the best of our knowledge, this study is the first to apply an array of in silico methods for taxonomic analysis of the genus Pseudoalteromonas. As strain NW4327 was found to be a strain of P. agarivorans by conventional DDH, the genome sequence of P. agarivorans S816 was chosen for comparison. Genome sequence of the type strain of P. agarivorans (KMM 255T) is in an unfinished state and, thus, could not be included in this study. P. agarivorans S816 was collected during a global research cruise for finding an antibiotic against Vibrio anguillarum (Vynne et al. 2011). The authors claimed to accurately identify the strains by detailed 16S rRNA gene sequence analysis. A high genomic similarity (as determined by ANI, GGD, and TETRA) of strain NW4327 with P. agarivorans S816 was in agreement with the conventional DDH, confirming NW4327 as a strain of P. agarivorans. A bacterium previously isolated from diseased specimens of the sponge A. compressa (strain HA007) (Angermeier et al. 2012) was found to be a close relative of NW4327 with 99.4 % sequence identity covering 1375 bp (GenBank accession number JQ582943). However, consequential to the revision of the taxonomic assignment of NW4327 described in this communication, α-Proteobacterium strain HA007 would not be related to NW4327 in terms of the full-length 16S rRNA gene sequence. For a definitive taxonomic assignment of HA007, further analysis of the fatty acid profile, gyrB gene sequence, phenotypic characteristics, DNA-DNA reassociation, and genome analysis will be required. Identification of Putative Virulence Factors Comparative analyses of virulence-associated genes (under three subsections: coding for adhesion and colonization, secretion systems, and proteases and toxins) in P. agarivorans strain NW4327 with other confirmed or potential pathogens of marine origin revealed interesting similarities as presented in the following three subsections. Homologues in terrestrial pathogens are described in the fourth subsection. Adhesion and Colonization The genome of NW4327 possessed several components coding for adherence and attachment to the host surface that would be important in the first stage of infection. At least 24 genes associated with the formation of type IV pili were identified in NW4327 and were arranged in several clusters (Online Resource 5). The major pilin subunit pilA gene was

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Fig. 3 Circular representation of Pseudoalteromonas agarivorans strain NW4327 genome. The genome atlas was constructed using GeneWiz Browser 0.94 server. The reference genome was Pseudoalteromonas agarivorans strain NW4327 and the outermost ring represents the genome of P. agarivorans strain S816 (query genome). The protein sequences of NW4327 were aligned with P. agarivorans strain S816 proteins using BLASTP algorithm. Color intensities of various regions of the outermost ring are proportional to similarities (as percent) of S816 protein sequences with NW4327 sequences. The dark red areas in the outermost ring signify regions in the S816 genome that bear very high similarity with NW4327 genome. The white or light colored areas denote regions of S816 genome that bear no or very little similarity with

NW4327 genome. Moving from outside to inside, coding sequences CDS+ and CDS− in the next two rings represent the open reading frames (ORFs) present on the positive and negative strands of NW4327 genome, respectively. The next two rings refer to global direct repeats and global inverted repeats, respectively, which denote sequences that are present in at least two copies on the same or opposite strands, correspondingly. Colored regions represent repeats. The next ring shows the GC skew of the NW4327 genome which is calculated as (G −C)/(G+C). The next inner ring shows the percent AT which indicates the percent of A’s and T’s in the NW4327 genome. The small ring in the upper left corner indicates the size (4,482,415 bp) of the NW4327 genome

identified clustering with pilB, pilC, and pilD (prepilin peptidase). Another cluster was composed of genes encoding type IV pili components FimT, FimU, PilE, PilY1, PilX, PilW, and

PilV. Genes homologous to the two component response regulator system algZ/algR were found in proximity to this cluster. Similar gene clusters for type IV pili biosynthesis were

Fig. 4 The five bacterial species most closely related to Pseudoalteromonas agarivorans strain NW4327 as determined by the BLASTX search. The numbers displayed on the top of each bar refer to the number of ORFs having closest match with strain NW4327 proteins

Mar Biotechnol Fig. 5 Scatterplot of tetranucleotide usage patterns (TETRA) between Pseudoalteromonas agarivorans strain NW4327 and Pseudoalteromonas agarivorans strain S816 as calculated by the JSpecies software. Correlation coefficient was calculated as 0.99934

also reported in the potential pathogen Pseudoalteromonas tunicata (Thomas et al. 2008). The absence of the complete alginate biosynthesis gene cluster in NW4327 as well as in P. tunicata and the presence of the isolated alginate biosynthesis regulator (AlgZ/AlgR) indicated that this regulatory system may be required for biogenesis or function of the pili cluster in both bacteria (Thomas et al. 2008). There were 17 genes in a cluster coding for a mannose-sensitive hemagglutinin (MSHA) biogenesis locus (Online Resource 5) in NW4327. The MSHA pili operon structure of NW4327 closely resembled that of P. tunicata (Dalisay et al. 2006) in which

the MSHA pilus played a role in the attachment of the bacterium to abiotic and living surfaces (Dalisay et al. 2006). While NW4327 lacked the mshF gene found in P. tunicata, both strains contained the two mshI genes. The genome of NW4327 was shown to harbor several genes related to curli formation (Online Resource 5). csgAEFG genes were present in a single cluster forming an operon along with a transcriptional regulator (csgD) present in reverse orientation. The apparent absence of the curli-associated csgB gene in P. agarivorans strain NW4327 was consistent with the csg operon structure found in other Pseudoalteromonas (Dueholm

Fig. 6 Mauve alignment of the genomes: Pseudoalteromonas sp. NW4327 (reference genome) and Pseudoalteromonas agarivorans S816 (query genome). The Mauve software aligns homologous regions shared by two genomes and identifies genome rearrangements. The colored blocks represent apparently conserved regions between two genomes which are free from any genomic rearrangement. Regions outside the blocks do not possess obvious homology between query and reference genomes. When a block lies below the center line (shown

as CL in the figure) in relation to the reference genome sequence, it indicates regions aligned in inverse orientation. The lines connect the colored blocks of one genome with similarly colored blocks of the other genome (i.e., homologous regions). White areas inside the colored blocks represent unique sequence elements which are specific to a particular genome. Scales represent nucleotide sequence coordinates of the genomes

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et al. 2012). Additionally, a putative tad locus (tadABC) was identified in the NW4327 genome (Online Resource 5). A response regulator found adjacent to this tad locus was annotated as histidine kinase (gi: 566569636), which might be responsible for transcriptional regulation of this cluster. Therefore, the presence of the genes for biogenesis and function of pili and curli was indicative of host attachment and colonization by strain NW4327 and likely played a role in disease initiation. Secretion Systems Type I secretion system (T1SS) is the simplest bacterial secretion system that requires only three component proteins: an outer membrane protein (OMP), an ATP-binding cassette (ABC), and a membrane fusion protein (MFP) (Delepelaire 2004). One set of genes encoding an ABC transporter, a MFP (HlyD) and an OMP (TolC), was found in a cluster (Online Resource 5), indicating the presence of T1SS in the NW4327 genome. Interestingly, no cluster with similar domain structure could be identified in the genome of P. agarivorans S816. Two clusters related to the general secretion pathway (components of type II secretion system or T2SS) were present (Online Resource 5) in the NW4327 genome. A contiguous cluster was identified possessing gspC-N genes. pilD gene (prepilin peptidase), a variant of gspO, was found in NW4327 genome, clustering with other components of type IV pili formation and supposed to play a crucial role in T2SS. In Pseudoalteromonas haloplanktis TAC125 genome, an ORF was found downstream to gspN, which was annotated as PSHAa0243 gene and encoded a hypothetical protein of 195 aa (termed as GspXX) related to T2SS (Parrilli et al. 2008). The T2SS gene cluster of NW4327 also contained an ORF downstream of gspN, which was shown to have high homology with gspXX of P. haloplanktis TAC125 (Parrilli et al. 2008). Another cluster possessing gspA and gspB was identified. The presence of a complete gene cluster for T2SS in NW4327 implicated its role in transporting different effector molecules to cause host pathogenicity. T2SS have previously been demonstrated to be responsible for the extracellular export of a large number of proteases in P. haloplanktis TAC125 (Parrilli et al. 2008). The type VI secretion system is a recently identified secretion system, the primary function of which is to facilitate extracellular transport of virulence factors into eukaryotic cells (Pukatzki et al. 2006). Though the composition of the T6SS gene cluster varies among different species, a core of 13 proteins, which is highly conserved among all T6SS, was identified (Boyer et al. 2009). T6SSs are regulated in a manner specific to different bacteria, which allows the coupling of the explicit role of the T6SS with the particular bacterium’s lifestyle and associated regulatory networks (Coulthurst 2013). In NW4327, a contiguous gene cluster was found

containing at least 19 genes related to T6SS (Online Resource 5 and 6), including all of the core genes reported previously (Bingle et al. 2008; Boyer et al. 2009). In addition to the core genes of T6SS loci, a serine/threonine protein phosphatase gene and a serine/threonine protein kinase gene were located in close vicinity of the T6SS cluster. These genes were believed to be involved in posttranslational regulation of the T6SS (Mougous et al. 2007). Interestingly, no such gene cluster for T6SS was observed in P. agarivorans S816 genome. The presence of a complete gene cluster for T6SS in NW4327, as shown in Online Resource 7, reinforced its role in pathogenicity. Genes ascribed to the T6SS have also been detected in the Vibrio coralliilyticus strain P1 (LMG23696) obtained from diseased Montipora aequituberculata coral colonies of the GBR (de O Santos et al. 2011). Components of type III (T3SS), IV (T4SS), and V (T5SS) secretion systems were not identified in NW4327. A wide-ranging in silico analysis carried out by Persson et al. (2009) recently verified the occurrence of virulence gene homologues of known terrestrial animal and plant bacterial pathogens in genomes of marine bacteria. Interestingly, genes of the virulence-associated T3SS, T4SS, T5SS, and T6SS were found in 60 of the 119 genomes of marine bacteria that were not known to be associated with any infectious disease. T6SS was primarily found among γ-Proteobacteria, the class to which NW4327 belongs. Proteases and Toxins Many bacterial proteases interact with host cells during a pathogenic infection (Hoge et al. 2010) and can therefore be considered as potential virulence factors. At least 39 serine proteases and metalloproteases were identified in the genome of NW4327 (Online Resource 5), 30 of which were predicted to be secreted. Diverse bacterial proteases of the classes M4, M6, M48, S8, S9, and U32 and others were found in the genome of NW4327. SDS-PAGE analysis previously carried out with the active fractions of P. agarivorans strain NW4327 collected during the purification of collagenase (Mukherjee et al. 2009) also indicated the presence of multiple proteases. Among the proteases, two metalloproteases belonging to the M4 peptidase family were found. One M4 protease of NW4327 (gi: 566571703) shared 57 % amino acid sequence identity to the MvP1 protease of the fish pathogen Moritella viscosa which was able to degrade casein, gelatin, and lumpfish collagen (Bjornsdottir et al. 2009). Three M4 metalloprotease genes were identified in the coral pathogen V. coralliilyticus strain P1 (de O Santos et al. 2011). The genome of NW4327 also contained a protease of the M6 family. A M6 metalloprotease gene was previously detected in the coral pathogen V. coralliilyticus (de O Santos et al. 2011). Among other metalloproteases, a Zn-dependent protease with chaperone function belonging to the M48 family was found.

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Three M48 metalloprotease genes were detected in V. coralliilyticus (de O Santos et al. 2011). Strain NW4327 possessed multiple serine proteases of the S8 family. A protease (gi: 566570228) was predicted to contain the same domain architecture as the collagenolytic serine protease Deseasin MCP-01 found in Pseudoalteromonas sp. SM9913 (Zhao et al. 2008) and bore high homology with it (79 % sequence identity and 87 % sequence similarity). The C-terminal of this protease was shown to possess a polycystic kidney disease (PKD) domain, which can bind insoluble collagen. Deseasin MCP-01 was suggested to have an immense capacity for degrading various types of collagen in deep-sea sediments and implicated to play an important role in marine nitrogen recycling (Zhao et al. 2008). However, this particular species of Pseudoalteromonas was not reported to be pathogenic. A dipeptidyl peptidase IV protease, which was predicted to be secreted, was also identified in NW4327 genome. Yet another virulence-related protease of peptidase U32 family was identified in NW4327 genome. Though the protease U32 in NW4327 was not predicted to be secreted through in silico prediction, it may be hypothesized to be secreted via any nonclassical pathways as suggested by Kavermann et al. (2003). It was suggested that the U32 collagenase degraded type I collagens that helped the coral pathogen V. coralliilyticus in the invasion of the host (de O Santos et al. 2011). These presumed virulence factors may be responsible for the invasiveness of strain NW4327. Besides proteases, at least two putative hemolysins (Online Resource 5) were found in the NW4327 genome. A hemagglutinin encoding gene (Online Resource 5) was also found in NW4327 genome. At least two putative phospholipase genes (Online Resource 5) were identified in the NW4327 genome. Furthermore, a toxin/antitoxin (TA) system (Online Resource 5) was identified in NW4327 genome. Although hemolysins are well-known cytotoxic proteins in vertebrate pathogenicity as they cause destruction of erythrocytes, genes coding for hemolysins have also been found in plant pathogens (Van Sluys et al. 2002) and a coral pathogen (de O Santos et al. 2011). Notably, some hemolysins (HlyA, a pore-forming toxin) and proteins belonging to the hemolysin family (the hemolysin-related protein RbmC) as well as a putative RTX toxin and RTX transporters were found in the genome of the coral pathogen V. coralliilyticus (de O Santos et al. 2011). Toxin-antitoxin systems probably played an important role in the survival of NW4327 against host defenses (Nieto et al. 2007). Homologues in Terrestrial Pathogens Homologues of all predicted virulence-associated genes of NW4327 (adhesion and colonization, secretion systems, proteases and toxins) were also present in numerous terrestrial pathogens (Online Resource 6 and 8). Persson et al. (2009)

carried out a widespread in silico analysis to assess the presence of virulence gene homologues of confirmed terrestrial animal and plant pathogenic bacteria in genome-sequenced marine bacteria. The authors noted that, first, a number of marine bacteria harbored virulence gene homologues that could possibly be employed for attachment to and, thus, compromise eukaryotic cells in an invasive manner to obtain organic matter and nutrients, a way similar to that used by known terrestrial pathogens. Second, the authors observed that selective advantage for opportunistic marine bacteria could be gained through the expression of virulence genes as a process to obtain resources. Third, employing the specific protein secretion systems as well as other virulence gene homologues, that may even be considered as essential functionalities, a successful transformation from a free-living lifestyle to a life associated with other living components of the marine ecosystem can be attained by the marine bacteria. The identification of virulence-associated genes (including protein secretion genes) in the genome of NW4327 that attacked and killed the sponge, R. odorabile, in a manner probably similar to several known terrestrial pathogens (as apparent through comparative genome analysis) is supportive of the three premises put forward on the basis of theoretical considerations by Persson et al. (2009). Ecophysiology of P. agarivorans Strain NW4327 Collagenase activity was measured by azocoll degradation, and the intensity of color of the liberated purple-colored azo dye was estimated by measuring its absorbance at 520 nm. After a 2-h incubation at 37 °C, the cell-free supernatant of strain NW4327 showed a significant increase in absorbance520 (0.600±0.01) due to the liberation of the azo dye. The cell-free supernatant of the nonpathogenic type strain showed marginal increase in absorbance520 (0.028±0.004) after incubation under identical conditions. In contrast, appearance of a groove around colonies of P. agarivorans KMM 255T growing on BD MA 2216 indicated degradation of agar, a property not observed in NW4327. Though NW4327 was found to be a strain of P. agarivorans, it differed from the type strain in several taxonomic features, most importantly in the inability to produce agarase which is one of the most distinguishing characteristics of P. agarivorans KMM 255T (Romanenko et al. 2003). Additionally, there are no previous reports of any P. agarivorans strains producing collagenase, which is the most ecologically significant property of NW4327 (Mukherjee et al. 2009). Interestingly, no genes coding for agar-degrading enzymes were found in the genome of NW4327. Thomas et al. (2008) also reported that enzymes for the hydrolysis of agaropectin and agarose were absent in P. tunicata genome. Ivanova et al. (2003) demonstrated that the enzyme profiles of different Pseudoalteromonas strains often correlate with their

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phenotypic response to environmental conditions. Moreover, occurrence of certain enzymes was shown to be strain specific and dependent on the ecological habitat from where the organism was sourced (Ivanova et al. 2003). Therefore, it would seem reasonable to consider that strain NW4327, possessing a unique ecophysiological niche (being a resident of sponge tissue), may have evolved differently to the type strain and expressed collagenolytic enzyme(s) to acquire nutrients from its surroundings (spongin fibers). Yung et al. (2011) hypothesized that occurrence of some low-abundance bacteria with collagenase activity might be indicative of the probable role of collagenase in sponge tissue degradation. There are numerous reports of the members of Pseudoalteromonas genus being opportunistically pathogenic to marine animals including lobsters (Ridgway et al. 2008), damselfish (Nelson and Ghiorse 1999), sea cucumbers (Liu et al. 2010), sea urchins (Wang et al 2013), and tunicates (Song et al. 2012). These previous descriptions of Pseudoalteromonas affecting marine species have classified the putative pathogens mainly based on 16S rRNA sequences that are generally insufficient to resolve species within the Pseudoalteromonas genus. This study, to the best of our knowledge, is the first to unambiguously identify not only a confirmed sponge pathogen, but also any Pseudoalteromonas pathogen affecting marine animals through complete polyphasic taxonomy and genome analysis. Furthermore, no previous studies (Ridgway et al. 2008; Nelson and Ghiorse 1999; Liu et al. 2010; Wang et al 2013; Song et al. 2012) applied genome analysis of the pathogens to presume the probable mode for pathogenicity.

Conclusions Significant concerns over the increasing prevalence of sponge disease have been raised in recent years. Here we confirmed the identity of the primary pathogen of the GBR sponge, R. odorabile through polyphasic taxonomy, unambiguously assigning it as a novel strain of P. agarivorans. Within the genome sequence, we identified a large number of genes coding for adhesins (pili, curli, etc.), secretion systems, collagenases, toxins, and phospholipases, which may be used by the pathogen for attaching, colonizing, invading, and killing its host, R. odorabile. Although there were expected similarities between the genomes of the nonpathogenic (S816) and pathogenic (NW4327) strains, the confirmed presence of secretion systems T1SS and T6SS in NW4327 and their absence in the nonpathogenic S816 established NW4327 as the virulent strain. Production of collagenase by NW4327 and the absence of this invasive property in the nonpathogenic type strain of P. agarivorans further reiterated the unique pathogenic property of strain NW4327. In the future, we intend to reinfect R. odorabile with NW4327 and study the expression of the

putative virulence genes identified in this study. We also plan to examine if NW4327 is able to infect other sponge species. Identification of a primary sponge pathogen and description of the underpinning molecular mechanisms likely contributing to disease causation in R. odorabile provides an ideal platform for future transcriptomic and proteomic work to validate expression of these pathways and for establishing the ubiquity of this virulence process in other sponge species. Acknowledgements Financial support through sanction no. SR/SO/ BB-0114/2010 to JM and RG and INSPIRE fellowship no. IF110045 to JDC from the Department of Science and Technology (http://www.dst. gov.in), Ministry of Science and Technology, Government of India is thankfully acknowledged. NSW was funded through an Australian Research Council Future Fellowship (FT120100480). Conflict of Interest The authors declare that they have no conflict of interest.

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