BACKGROUND
novosphingobium is a genus within the alpha subclass of proteobacteria that was separated from the general genus, sphingomonas a result of extensive classification on the basis of the  <dig> s rrna sequence, chemotaxonomic and physiological analyses  <cit> .
currently, the genus sphingomonas is divided into four genera namely; sphingomonas, sphingobium, novosphingobium and sphingopyxis.
bacteria belonging to the genus novosphingobium are often associated with the biodegradation of aromatic compounds such as phenol, aniline, nitrobenzene, 4-chlorobenzene, phenanthrene, pyrene, carbofuran, dibenzofuran and estrogen  <cit> .
given their extensive bioremediation properties these bacteria are frequently isolated from estuarine sediment, coastal sediment and marine aquatic environments that have been exposed to high level of anthropogenic activities.
the recent report of a plant-associated novosphingobium sp.
rr 2– <dig> isolated from the surface of a crown gall tumor on grapevine has provided an interesting insight regarding the putative role of this genus as a plant epiphyte  <cit> .
strain rr 2– <dig> was found to produce significant amounts of quorum sensing signals of the n-acyl-homoserine lactone  class.
a subset of these ahls has the potential to influence the onset of crown gall disease by pathogen tumefaciens in addition to other phytopathogical effects.
recently, novosphingobium sp.
ap <dig> has been isolated from the rhizosphere of populus deltoides as part of the initiatives to understand the metabolic interactions between plants and bacteria belonging to this genus  <cit> .
the common trend in the study of the culturable bacteria belonging to novosphingobium genus typically entails isolation, identification, carbon utilization test and the analysis of extracellular products  <cit> .
to a certain extent, mutagenesis experiments has also been employed to elucidate the function of certain genes involved in quorum sensing signal metabolism  <cit> .
given the well-established phenotypical characterization of various reported novosphingobium strains, it would be of great interest to the scientific community to compare phenotypical characteristics to the genetic make-up of these bacteria.
for example, traits associated with xenobiotic compound metabolism, cell-cell signaling and adaptation to marine osmotic condition are very much of interest to further the understanding of the genus pertaining to its lifestyle in certain environments and / or conditions.
in addition, the availability of this information has the potential to facilitate targeted gene-specific functional studies in novosphingobium species that are currently unknown.
the present study compares the genomes of six bacteria belonging to the genus novosphingobium.
the six bacteria are as follows; novosphingobium aromaticivorans dsm <dig> , novosphingobium pentaromaticivorans us6- <dig> , novosphingobium nitrogenifigens y <dig> , novosphingobium sp.
pp1y , novosphingobium sp rr 2– <dig>  and novosphingobium sp ap <dig>   <cit> .
the diverse isolation source and metabolic property of these strains provides an excellent opportunity to apply comparative genomics to identify the genetic features that differentiate each bacterium within this genus.
this study reveals that the quorum sensing system that utilizes the ahl class of signals is not universally present in the genus novosphingobium.
in addition, marine adaptation in novosphingobium strains is likely to be based on the organic osmolyte mechanism that is fundamentally different from those reported in common gram-negative marine bacteria that constantly export na+ ion via the sodium-pumping nadh dehydrogenase nqr  <cit> .
several dioxygenases putatively associated with the biodegradation of aromatic compounds were identified particularly in the known aromatic compound degrader strains based on both phylogenetic tree and similarity search approaches, providing valuable information for strain engineering in the field of bioremediation.
to our knowledge, this is the first comparative genomic analysis of the genus novosphingobium with a focus on the genes associated with quorum sensing metabolism, marine adaptation and bioremediation.
RESULTS
general features of the sequenced novosphingobium strains and their correlation with phylogenomic classification
tables  <dig> and  <dig> present the general and genomic features respectively of the strains used in this study.
the isolation source of strains pp1y and us6- <dig> indicates that both are osmotolerant and this property has been experimentally validated by previous studies  <cit> .
in addition, strains pp1y, us6- <dig> and dsm  <dig> were noted for their extensive ability in the biodegradation of aromatic pollutants.
although both strains rr 2– <dig> and ap <dig> are associated with plants, the difference in their isolation source, epiphytic for strain rr 2– <dig> and rhizospheric for strain ap <dig>  may suggest a different role in plant-bacteria interaction and ultimately may lead to significant difference in their genomic features.
abbreviations: go gene ontology, ec enzyme code, ipr interproscan, cds coding domain sequence, n.r not relevant, n.d not yet determined.
the size and g + c content of the genomes used in this study ranged from  <dig>  to  <dig>  mb and  <dig>  to  <dig> %, respectively.
a slightly lower number and percentage of cds with gene ontology annotation and assigned enzyme commission numbers were observed for the plant-associated strains rr 2– <dig> and ap <dig> .
the phylogenomic clustering of each strain was of high confidence  and generally correlated well with the isolation source of the novosphingobium strains .
however, when a 16s-rrna based phylogenetic tree was constructed using the same subjects, a slightly different topology with lower bootstrap support was observed instead .
in addition, a decrease in the overall resolution of the tree was observed when more 16s rrna sequences of additional novosphingobium species were included into the 16s rrna based phylogenetic tree analysis .
average amino acid identity, dinucleotide relative abundance values  and genome signature dissimilarity  support phylogenomic classification and provide insights into niche adaptation
the aai shared among different genome pairs ranged from  <dig> % to  <dig> % .
strain pp1y which is not classified to a species shared a strikingly high aai  with strain us6- <dig>  suggesting that it may belong to the species novosphingobium pentaromaticivorans.
strains y <dig> and dsm <dig> shared the highest aai  among themselves as compared to pairing with any other strains  thus providing additional support to the placement of strains y <dig> and dsm <dig> in a separate cluster in the phylogenomic tree.
the  values within each strain ranged from  <dig> to  <dig> .
strain pp1y shared the lowest genome dissimilarity value with strain us6- <dig>  again suggesting its identity at the species level as novosphingobium pentaromaticivorans.
the calculated ρ* values were in the normal range  for all six strains studied except for at  and ta .
notably, strain y <dig> exhibited the highest ρ*aa, ρ*tt and ρ*ta and the lowest ρ*at in comparison to other novosphingobium strains .
pan-genome analysis reveals a high abundance of singletons among all strains
orthologous clustering for the pan-genome analysis was performed using panoct.
in comparison to other existing orthologous gene clusters determination tools, panoct utilizes a new parameter termed conservation of gene order in addition to blast score ratio to effectively reassign groups of paralogs into separate clusters of orthologs based on a weighted scoring scheme.
at a conservative percentage identity cut-off of 65%, a core genome of strains in the genus novosphingobium containing  <dig> orthologous groups was identified .
for strains dsm <dig> and pp1y that have complete genome sequences available, the origin of the singletons was determined.
in strain pp1y, 47% of the singletons are of plasmid origin.
however, in strain dsm <dig>  only 15% of the singletons were located on the plasmid .
the most closely related marine strains us6- <dig> and pp1y shared the largest unique core genes  that will serve as an initial gene pool for the identification of marine adaptation genes in the genus novosphingobium.
ectoine biosynthesis genes relevant to salt tolerance are conserved in the marine strains
two approaches were used to identify the genes that are associated with salt water adaptation in the marine novosphingobium strains .
first, the whole proteome was searched against a blast database consisting of sodium-pumping nadh dehydrogenase nqr  commonly associated with sodium dependence in gram-negative marine bacteria and clinical pathogens  <cit> .
however, no significant hit was observed .
second, given the identification of the unique core genome of strains pp1y and us6- <dig> , cdss in the unique core genome were subject to seed annotation that subsequently led to the identification cdss associated with ectoine biosynthesis in both marine strains .
a 20-kbp region of contig <dig> from strain us6- <dig> containing the ectoine biosynthesis gene cassette was compared against the whole genome sequences of novosphingobium strains.
genes related to ectoine synthesis  were conserved only in the marine strains us6- <dig> and pp1y .
ectoine is a type of highly water-soluble organic compound that can accumulate in the cell without significant effects on the cell’s metabolism even at high cytoplasmic concentrations  <cit> .
after the accumulation of ectoine, the diffusion of water through the cell membrane can be reduced thus enabling the survival of cells at high salt environment.
the synthesis of ectoine involves three main proteins namely  <dig> -diaminobutyric acid  transaminase , daba acetyltransferase  and ectoine synthase .
ectb transfers an amino group from glutamate to aspartate-semialdehyde to form daba.
subsequently, an acetyl group is transferred to daba from ecta.
the cyclic condensation of n-acetyl-l- <dig> -diaminobutyric acid by ectc will lead to the production of ectoine  <cit> .
the ecta homologs, pp1y_at <dig> and nsu_ <dig>  in strains pp1y and dsm <dig> respectively are 162-residue in length and slightly acidic .
this slightly acidic feature is also exhibited in the remaining protein components implicated in ectoine synthesis  .
interestingly, genomic region containing a gene coding for hypothetical protein  was only conserved in the marine strains.
this gene corresponded to nsu_ <dig> and pp1y_at <dig> of strains us6- <dig> and pp1y respectively.
protein signature recognition search using interproscan revealed the presence of a signature motif for the sodium alanine symporter in the hypothetical protein  that may be responsible for the passage of alanine molecules and sodium ions through the cytoplasmic membrane  <cit> .
luxri homologs are not universally present in the genus novosphingobium
in our previous study, novosphingobium sp.
rr 2– <dig> was found to produce an abundant amount of ahls that could activate the trar of agrobacterium tumefaciens <cit> .
it is therefore of interest to assess the prevalence of genes involved in ahl synthesis in the genus novosphingobium.
by performing blast searches against the curated luxi homologs, a total of five putative ahl synthases were identified in strains us6- <dig> , pp1y , rr 2– <dig>  and ap <dig> .
based on phylogenetic genetic tree analysis, the ahl synthases did not exhibit a tight clustering and were dispersed in the monophyletic group consisting of various ahl synthases from the family sphingomonadacaea .
alignment of the protein sequences with luxi homologs showed that similar to all curated luxi homologs, containing the three absolutely conserved amino acids, arg <dig>  phe <dig> and trp <dig> .
all identified luxi homologs in novosphingobium  genes are genetically linked to their cognate transcriptional regulator novr that encodes for a luxr-type transcriptional regulator .
it should be highlighted that phyh, that encodes for phytanoyl-coa dioxygenase, is located adjacent to novi in four out of five of the novi/novr pairs.
identification of a luxr solo homolog in novosphingobium sp.
pp1y and evidence for its acquisition through horizontal gene transfer
filtering based on the interproscan identifier ipr <dig>  in the proteome of all six novosphingobium strains indicated the presence of an additional novr  in strain pp1y which is closely related to a luxr homolog of sphingopyxis alaskensis rb <dig>  .
analysis of the gene neighborhood of the novr strain pp1y confirmed the absence of novi in its vicinity, suggesting its role as a novr solo.
additionally, a large abundance of mobile elements such as transposases and phage integrases were located upstream of novr  which was not a typical feature of various previously reported luxr homolog solo .
two transposase genes located immediately upstream of novr were found to code for different fragment of a transposase, suggesting that the genomic region encompassing both partial transposase genes may code for a functional transposase at one time.
identification of various aromatic ring hydroxylating dioxygenase  broadens the bioremediation potential of novosphingobium strains
ardo has always been implicated in the biotransormation of aromatic carbon into less toxic compound  <cit> .
through the action of aromatic ring hydroxylation, oxygen is incorporated into the ring structure that further destabilizes the structure and promotes ring cleavage in the ensuing enzymatic step.
a phylogenetic tree consisting of functionally validated aromatic ring hydroxylating dioxygenase  and the putative ardos from novosphingobium strain identified based on similarity search was constructed .
as expected, a higher abundance of ardos were found in strains us6- <dig>  pp1y and dsm  <dig> that are validated aromatic compound degraders  <cit> .
interestingly, saro_ <dig> , pp1y_at <dig> and pp1y_at <dig>  formed a separate cluster that shared a common ancestor with group  <dig> and group  <dig> ardos, suggesting potentially a new group of ardos .
the alignment of all ardos used in this analysis revealed an interesting feature in four ardos namely nsu_pla <dig> , saro_ <dig> , pp1y_at <dig> and pp1y_at <dig> .
instead of having  <dig> amino acids between the first histidine and the second cystiene residues in the rieske-type  cluster binding site sequence typical of group ii ardo, there are  <dig> amino acids separating these residues .
three ardos, two belonging to group i ardo, and one belonging to the group ii ardo, were identified in strain rr 2– <dig> that was not known for its biodegradation ability.
the identified group i ardos of strain rr 2– <dig> which interesting were the only group i ardos of novosphingobium origin in this study formed a monophyletic group with the oxygenase component of 4-aminobenzenesulfonate  <dig> -dioxygenase .
similar to the 4-aminobenzenesulfonate  <dig> -dioxygenase operon of hydrogenophaga sp.
pbc, a gene coding for glutamine synthase that may be involved in the amino group transfer of aromatic amines was located directly next to both of the ardo genes  <cit>  .
discussion
the elucidation of evolutionary relationship via phylogenomic approaches provides a very high confidence clustering when compared to the more facile 16s rrna based approaches.
furthermore, the decrease in the resolution of 16s rrna based phylogenetic tree upon the inclusion of the more novosphingobium 16s rrna sequences suggests that single locus-based phylogenetic tree construction may not provide enough information to accurately distinguish evolutionary relationship among member of the novosphingobium genus  <cit> .
aai and karlin’s genomic signature dissimilarity generally agreed with the phylogenomic clustering and provided support to the classification of novosphingobium sp.
pp1y as novosphingobium pentaromaticivorans pp1y.
future whole genome sequencing of additional novosphingobium strains will greatly aid in the determination of the cut-off values necessary to enable a wgs-based species definition as demonstrated by thompson et al.
<cit> .
it is worth noting that strain y <dig> which was isolated from a bioreactor treating pulp and paper-mill effluent has the highest aa + tt dinucleotide frequency among the strains studied.
in pasteurella multocida, the continuous culturing at higher temperatures led to increased aa, ta, and tt frequencies in comparison with the virulent strain  <cit> .
since the conformation of aa/tt dinucleotides is not heavily altered by its adjacent nucleotide, these dinucleotides are more stable when exposed to high temperature  <cit> .
the genomic feature exhibited by strain y <dig> may therefore correspond to an adaptation to the continuous exposure to mill effluent which is generally above ambient temperatures  <cit> .
pan-genome analysis based on a conservative percent identity cut-off of 65% revealed a large abundance of singletons in the genus novosphingobium and a rather small core genome .
it should be noted that the pan-genome analysis was performed using strains of the same genus and not strains of the same species thus explaining the small core genome.
a further significant reduction in the core genome and number of singletons in each strain is very likely with the addition of more newly sequenced strains in the future.
for example, a core genome of up to  <dig> genes has been reported for strains of the species vibrio cholerae and the inclusion of more genomes from different vibrio species  further reduced the core genome down to  <dig> genes.
nevertheless, combining the prior knowledge of the wet-lab validated phenotypical trait of the bacterial strains and the identification of unique core genome from such an unorthodox pan-genome analysis, we were able to systematically determine the putative proteins that are involved in marine adaptation.
such approach reinforced the idea that unique core genome should reflect the phenotypical traits that are specific to a group of bacteria  <cit> .
an additional similarity search directly against the nucleotide sequences consisting of the bacterial whole genome sequences eliminated the possible presence of homologous genes that were not included in the orthologous grouping either due to gene prediction failure or partial sequence length.
due to the lack of active transport system for water in many microorganisms, the biosynthesis of compatible solutes such as ectoine in the marine novosphingobium strains is advantageous in regulating osmotic balance across the cell membrane.
based on molecular dynamic simulations, ectoine has been shown to be a durable water structure-forming solute across a broad temperature range  <cit> .
the effectiveness of ectoine has been demonstrated in the halo tolerant brevibacterium sp.
jcm  <dig>  <cit> .
upon osmotic shift towards higher salinity, the supplementation of ectoine has been shown to provide the most remarkable growth stimulation independent of the range of osmotic shifts compared to other compatible solutes,.
sphingopyxis alaskensis was the first member in the family of sphingomonadaceae reported to possess the complete gene set for synthesis of ectoine.
<cit> .
subsequently, based on blastp query, it appears that at least one member in each of the genus sphingomonas, sphingobium and novosphingobium possesses such gene set , suggesting that ectoine-based organic osmolyte mechanism could be a fairly common strategy employed by marine sphingomonads to persist in high-salt environments.
the gene coding for hypothetical protein containing the sodium/alanine symporter protein motif, which is present only in the marine novosphingobium strains us6- <dig> and pp1y, may also be involved in osmoregulation.
in desulfovibrio vulgaris, the gene coding for sodium/alanine symporter was overexpressed upon exposure to salt shock  <cit> .
in addition, the accumulation of alanine in cells was concurrent with the alleviation of salt-related growth inhibition.
alanine has also been reported to play a role in the relief of salt mediated inhibition on the enzymes involved in nitrogen fixation, photosynthesis and respiration of nitrogen-fixing cyanobacterium  <cit> .the slightly acidic proteome  and the presence of compatible solute synthesis in both marine sphingomonads closely mimic the salt adaptation strategy employed by halomonas elongata <cit>  thus supporting the notion that highly acidic proteome   <cit>  is not required for salt adaptation provided that there is a mechanism to accumulate and/or synthesize osmolyte in the cell.
the presence of conserved amino acids hypothesized to be essential for conformational change during substrate binding  <cit> , provides significant bioinformatics support that the five luxi homologs identified in this study are potentially capable of ahl signal production.
it would be important to further genetically and biochemically validate the function of the putative ahl synthase and determine the identity of their ahl signals.
the alignment of gene context / neighborhoods of and around the luxi homologs show conserved topology where novr is proximal to novi and where transcriptional orientation is convergent with the receptor  upstream , a genomic context feature based on proximity commonly found in complete qs regulatory circuits  in the alpha subclass of proteobacteria.
in addition, this feature is also generally found in the genomes of proteobacteria  <cit> .
interestingly, downstream of the novi genes in four of the five aligned contents showed a gene coding for phytanoyl-coa dioxygenase  whose expression is convergent with the qs regulatory circuits.
such topology has also been observed in a fosmid metagenomic library clone and in other genera within sphingomonadaceae including, sphingomonas and sphingopyxis <cit> .
based on transposon insertion mutagenesis and the short intergenic distance between luxi homolog and phyh, it was hypothesized that phyh is a gene in an operon that is qs regulated  <cit> .
phyh belongs to the family of iron-dependent oxygenases and is responsible for the alpha-hydroxylation of phytanoyl-coa, a derivative of phytanic acid.
the grapevine crown gall tumor epiphyte strain rr 2– <dig> possess a single ahl synthase homolog although it has been previously demonstrated that strain rr 2– <dig> could produce ahls of various chain lengths  <cit> .
three possible reasons could lead to this observation.
first, this ahl synthase may contain a rather broad specificity for its acyl-carrier counterpart as previously reported for some ahl synthases  <cit> .
second, additional genes coding for ahl synthases were not identified due to sequence divergence.
third, it is also possible that one or more of the ahl synthase genes were lost in the gaps of the draft genome.
further study entailing targeted disruption of the ahl synthase gene in strain rr 2– <dig> and assessment of ahl production in the mutant strain will be necessary to validate these hypotheses.
the successful construction of the ahl negative mutant will also be beneficial for future work involving whole transcriptome sequencing since this will directly provide insights into the genes that are regulated by quorum sensing in the genus novosphingobium.
the identification of a putative novr solo which was flanked upstream by various mobile elements suggests that its acquisition is via horizontal gene transfer.
two possible mechanisms were proposed for the acquisition of novr solo.
first, the novr solo may be part of the gene cassettes incorporated into an integron as evident by the presence of various genes coding for recombinase upstream of novr solo.
second, given the presence of two fragmented transposase genes upstream of novr solo, it is also plausible that novr solo was acquired via transposition and the subsequent transposase inactivation possibly due to a combination of nonsense and frame shift mutations prevented further movement of the gene.
unlike the novr solo, transposase elements were absent in the close vicinity of various well-studied luxr homolog solo  <cit> .
the association of mobile element with the complete luxir-type quorum sensing system has been previously reported.
for example, spnir has been reported to be localized in a mobile transposon and was found to regulate the transposition frequency  <cit> .
in addition, luxi and luxr homologues which were separated by transposase genes have also been reported previously in a metagenomic library clone  <cit> .
the solo novr  in strain pp1y could function in eavesdropping on ahl production within microbial communities and / or enable additional ahl regulated gene expression in itself.
this identification of a putative luxr homolog solo in strain pp1y is similar to bisr of rhizobium leguminosarum and expr of sinorhizobium meliloti.
it is worth noting that these two bacterial species produce multiple ahl signals and have multiple complete qs regulatory circuits  <cit> .
similarly, two complete qs regulatory circuits, one on the chromosome and one on plasmid, have also been identified in the italian marine strain, pp1y .
the identification of the putative ardos and the prediction of their function via phylogenetic analysis correlated particularly well with the remarkable bioremediation capability of strains us6- <dig>  pp1y and dsm  <dig> .
high abundance of ardos in plasmid pnl <dig> of strain dsm  <dig> is consistent with previous report on the sequencing and analysis of pnl <dig>  <cit> .
targeted mutagenesis of the putative ardos followed by the assessment of biodegradation ability will be imperative to provide functional analysis data.
in addition, assuming that the transcription of ardo gene sets is tightly regulated as observed in several dioxygenase systems  <cit> , it is also possible to potentially identify the key ardo for a specific aromatic compound via differential gene expression analysis.
the presence of gene coding for glutamine synthase directly next to the ardos of strain rr 2– <dig> is particularly intriguing.
in the 4-aminobenzenesulfonate  <dig> -dioxygenase system, sadb, a glutamine synthase, was hypothesized to catalyze the removal of amine group from the hydroxylated 4-aminobenzenesulfonate to form 4-sulfocatechol based on targeted gene deletion and random transposon mutagenesis studies  <cit> .
given the fact that 4-aminobenzenesulfonate is a xenobiotic, the identification of the original or natural substrate for this group of ardos may be further examined using strain rr 2– <dig> that has never been exposed to, to this compound according to the current literature  <cit> .
CONCLUSIONS
the facile sequencing of whole bacterial genomes as a result of next generation sequencing technologies has brought about a paradigm shift in the field of microbiology.
the genome analysis of six novosphingobium strains resulted in the identification of various genes putatively associated with the reported phenotype of novosphingobium strains pertaining to salt-tolerance, biosynthesis and perception of cell-cell signaling molecules and aromatic compound biodegradation.
of special mention is the identification of a luxr solo that was flanked by various mobile elements, providing new insights into the potential origin of this luxr solo.
the results from this study have the potential to provide information to facilitate future studies relating to the cloning and functional analysis of genes in novosphingobium species.

 novosphingobium is a genus within the alpha subclass of proteobacteria that was separated from the general genus, sphingomonas a result of extensive classification on the basis of the s rrna sequence, chemotaxonomic and physiological analyses. currently, the genus sphingomonas is divided into four genera namely; sphingomonas, sphingobium, novosphingobium and sphingopyxis. bacteria belonging to the genus novosphingobium are often associated with the biodegradation of aromatic compounds such as phenol, aniline, nitrobenzene, 4-chlorobenzene, phenanthrene, pyrene, carbofuran, dibenzofuran and estrogen. given their extensive bioremediation properties these bacteria are frequently isolated from estuarine sediment, coastal sediment and marine aquatic environments that have been exposed to high level of anthropogenic activities. the recent report of a plant-associated novosphingobium sp. rr 2– isolated from the surface of a crown gall tumor on grapevine has provided an interesting insight regarding the putative role of this genus as a plant epiphyte. strain rr 2– was found to produce significant amounts of quorum sensing signals of the n-acyl-homoserine lactone class. a subset of these ahls has the potential to influence the onset of crown gall disease by pathogen tumefaciens in addition to other phytopathogical effects. recently, novosphingobium sp. ap has been isolated from the rhizosphere of populus deltoides as part of the initiatives to understand the metabolic interactions between plants and bacteria belonging to this genus. the common trend in the study of the culturable bacteria belonging to novosphingobium genus typically entails isolation, identification, carbon utilization test and the analysis of extracellular products. to a certain extent, mutagenesis experiments has also been employed to elucidate the function of certain genes involved in quorum sensing signal metabolism. given the well-established phenotypical characterization of various reported novosphingobium strains, it would be of great interest to the scientific community to compare phenotypical characteristics to the genetic make-up of these bacteria. for example, traits associated with xenobiotic compound metabolism, cell-cell signaling and adaptation to marine osmotic condition are very much of interest to further the understanding of the genus pertaining to its lifestyle in certain environments and / or conditions. in addition, the availability of this information has the potential to facilitate targeted gene-specific functional studies in novosphingobium species that are currently unknown. the present study compares the genomes of six bacteria belonging to the genus novosphingobium. the six bacteria are as follows; novosphingobium aromaticivorans dsm, novosphingobium pentaromaticivorans us6-, novosphingobium nitrogenifigens y, novosphingobium sp. pp1y, novosphingobium sp rr 2– and novosphingobium sp ap. the diverse isolation source and metabolic property of these strains provides an excellent opportunity to apply comparative genomics to identify the genetic features that differentiate each bacterium within this genus. this study reveals that the quorum sensing system that utilizes the ahl class of signals is not universally present in the genus novosphingobium. in addition, marine adaptation in novosphingobium strains is likely to be based on the organic osmolyte mechanism that is fundamentally different from those reported in common gram-negative marine bacteria that constantly export na+ ion via the sodium-pumping nadh dehydrogenase nqr. several dioxygenases putatively associated with the biodegradation of aromatic compounds were identified particularly in the known aromatic compound degrader strains based on both phylogenetic tree and similarity search approaches, providing valuable information for strain engineering in the field of bioremediation. to our knowledge, this is the first comparative genomic analysis of the genus novosphingobium with a focus on the genes associated with quorum sensing metabolism, marine adaptation and bioremediation.  general features of the sequenced novosphingobium strains and their correlation with phylogenomic classification tables  and present the general and genomic features respectively of the strains used in this study. the isolation source of strains pp1y and us6- indicates that both are osmotolerant and this property has been experimentally validated by previous studies. in addition, strains pp1y, us6- and dsm were noted for their extensive ability in the biodegradation of aromatic pollutants. although both strains rr 2– and ap are associated with plants, the difference in their isolation source, epiphytic for strain rr 2– and rhizospheric for strain ap may suggest a different role in plant-bacteria interaction and ultimately may lead to significant difference in their genomic features. abbreviations: go gene ontology, ec enzyme code, ipr interproscan, cds coding domain sequence, n.r not relevant, n.d not yet determined. the size and g + c content of the genomes used in this study ranged from to  mb and to %, respectively. a slightly lower number and percentage of cds with gene ontology annotation and assigned enzyme commission numbers were observed for the plant-associated strains rr 2– and ap. the phylogenomic clustering of each strain was of high confidence and generally correlated well with the isolation source of the novosphingobium strains. however, when a 16s-rrna based phylogenetic tree was constructed using the same subjects, a slightly different topology with lower bootstrap support was observed instead. in addition, a decrease in the overall resolution of the tree was observed when more 16s rrna sequences of additional novosphingobium species were included into the 16s rrna based phylogenetic tree analysis. average amino acid identity, dinucleotide relative abundance values and genome signature dissimilarity support phylogenomic classification and provide insights into niche adaptation the aai shared among different genome pairs ranged from % to %. strain pp1y which is not classified to a species shared a strikingly high aai with strain us6- suggesting that it may belong to the species novosphingobium pentaromaticivorans. strains y and dsm shared the highest aai among themselves as compared to pairing with any other strains thus providing additional support to the placement of strains y and dsm in a separate cluster in the phylogenomic tree. the values within each strain ranged from to. strain pp1y shared the lowest genome dissimilarity value with strain us6- again suggesting its identity at the species level as novosphingobium pentaromaticivorans. the calculated ρ* values were in the normal range for all six strains studied except for at and ta. notably, strain y exhibited the highest ρ*aa, ρ*tt and ρ*ta and the lowest ρ*at in comparison to other novosphingobium strains. pan-genome analysis reveals a high abundance of singletons among all strains orthologous clustering for the pan-genome analysis was performed using panoct. in comparison to other existing orthologous gene clusters determination tools, panoct utilizes a new parameter termed conservation of gene order in addition to blast score ratio to effectively reassign groups of paralogs into separate clusters of orthologs based on a weighted scoring scheme. at a conservative percentage identity cut-off of 65%, a core genome of strains in the genus novosphingobium containing orthologous groups was identified. for strains dsm and pp1y that have complete genome sequences available, the origin of the singletons was determined. in strain pp1y, 47% of the singletons are of plasmid origin. however, in strain dsm only 15% of the singletons were located on the plasmid. the most closely related marine strains us6- and pp1y shared the largest unique core genes that will serve as an initial gene pool for the identification of marine adaptation genes in the genus novosphingobium. ectoine biosynthesis genes relevant to salt tolerance are conserved in the marine strains two approaches were used to identify the genes that are associated with salt water adaptation in the marine novosphingobium strains. first, the whole proteome was searched against a blast database consisting of sodium-pumping nadh dehydrogenase nqr commonly associated with sodium dependence in gram-negative marine bacteria and clinical pathogens. however, no significant hit was observed. second, given the identification of the unique core genome of strains pp1y and us6-, cdss in the unique core genome were subject to seed annotation that subsequently led to the identification cdss associated with ectoine biosynthesis in both marine strains. a 20-kbp region of contig from strain us6- containing the ectoine biosynthesis gene cassette was compared against the whole genome sequences of novosphingobium strains. genes related to ectoine synthesis were conserved only in the marine strains us6- and pp1y. ectoine is a type of highly water-soluble organic compound that can accumulate in the cell without significant effects on the cell’s metabolism even at high cytoplasmic concentrations. after the accumulation of ectoine, the diffusion of water through the cell membrane can be reduced thus enabling the survival of cells at high salt environment. the synthesis of ectoine involves three main proteins namely -diaminobutyric acid transaminase, daba acetyltransferase and ectoine synthase. ectb transfers an amino group from glutamate to aspartate-semialdehyde to form daba. subsequently, an acetyl group is transferred to daba from ecta. the cyclic condensation of n-acetyl-l- -diaminobutyric acid by ectc will lead to the production of ectoine. the ecta homologs, pp1y_at and nsu_ in strains pp1y and dsm respectively are 162-residue in length and slightly acidic. this slightly acidic feature is also exhibited in the remaining protein components implicated in ectoine synthesis. interestingly, genomic region containing a gene coding for hypothetical protein was only conserved in the marine strains. this gene corresponded to nsu_ and pp1y_at of strains us6- and pp1y respectively. protein signature recognition search using interproscan revealed the presence of a signature motif for the sodium alanine symporter in the hypothetical protein that may be responsible for the passage of alanine molecules and sodium ions through the cytoplasmic membrane. luxri homologs are not universally present in the genus novosphingobium in our previous study, novosphingobium sp. rr 2– was found to produce an abundant amount of ahls that could activate the trar of agrobacterium tumefaciens. it is therefore of interest to assess the prevalence of genes involved in ahl synthesis in the genus novosphingobium. by performing blast searches against the curated luxi homologs, a total of five putative ahl synthases were identified in strains us6-, pp1y, rr 2– and ap. based on phylogenetic genetic tree analysis, the ahl synthases did not exhibit a tight clustering and were dispersed in the monophyletic group consisting of various ahl synthases from the family sphingomonadacaea. alignment of the protein sequences with luxi homologs showed that similar to all curated luxi homologs, containing the three absolutely conserved amino acids, arg phe and trp. all identified luxi homologs in novosphingobium genes are genetically linked to their cognate transcriptional regulator novr that encodes for a luxr-type transcriptional regulator. it should be highlighted that phyh, that encodes for phytanoyl-coa dioxygenase, is located adjacent to novi in four out of five of the novi/novr pairs. identification of a luxr solo homolog in novosphingobium sp. pp1y and evidence for its acquisition through horizontal gene transfer filtering based on the interproscan identifier ipr in the proteome of all six novosphingobium strains indicated the presence of an additional novr in strain pp1y which is closely related to a luxr homolog of sphingopyxis alaskensis rb. analysis of the gene neighborhood of the novr strain pp1y confirmed the absence of novi in its vicinity, suggesting its role as a novr solo. additionally, a large abundance of mobile elements such as transposases and phage integrases were located upstream of novr which was not a typical feature of various previously reported luxr homolog solo. two transposase genes located immediately upstream of novr were found to code for different fragment of a transposase, suggesting that the genomic region encompassing both partial transposase genes may code for a functional transposase at one time. identification of various aromatic ring hydroxylating dioxygenase broadens the bioremediation potential of novosphingobium strains ardo has always been implicated in the biotransormation of aromatic carbon into less toxic compound. through the action of aromatic ring hydroxylation, oxygen is incorporated into the ring structure that further destabilizes the structure and promotes ring cleavage in the ensuing enzymatic step. a phylogenetic tree consisting of functionally validated aromatic ring hydroxylating dioxygenase and the putative ardos from novosphingobium strain identified based on similarity search was constructed. as expected, a higher abundance of ardos were found in strains us6- pp1y and dsm that are validated aromatic compound degraders. interestingly, saro_, pp1y_at and pp1y_at formed a separate cluster that shared a common ancestor with group and group ardos, suggesting potentially a new group of ardos. the alignment of all ardos used in this analysis revealed an interesting feature in four ardos namely nsu_pla, saro_, pp1y_at and pp1y_at. instead of having amino acids between the first histidine and the second cystiene residues in the rieske-type cluster binding site sequence typical of group ii ardo, there are amino acids separating these residues. three ardos, two belonging to group i ardo, and one belonging to the group ii ardo, were identified in strain rr 2– that was not known for its biodegradation ability. the identified group i ardos of strain rr 2– which interesting were the only group i ardos of novosphingobium origin in this study formed a monophyletic group with the oxygenase component of 4-aminobenzenesulfonate -dioxygenase. similar to the 4-aminobenzenesulfonate -dioxygenase operon of hydrogenophaga sp. pbc, a gene coding for glutamine synthase that may be involved in the amino group transfer of aromatic amines was located directly next to both of the ardo genes. discussion the elucidation of evolutionary relationship via phylogenomic approaches provides a very high confidence clustering when compared to the more facile 16s rrna based approaches. furthermore, the decrease in the resolution of 16s rrna based phylogenetic tree upon the inclusion of the more novosphingobium 16s rrna sequences suggests that single locus-based phylogenetic tree construction may not provide enough information to accurately distinguish evolutionary relationship among member of the novosphingobium genus. aai and karlin’s genomic signature dissimilarity generally agreed with the phylogenomic clustering and provided support to the classification of novosphingobium sp. pp1y as novosphingobium pentaromaticivorans pp1y. future whole genome sequencing of additional novosphingobium strains will greatly aid in the determination of the cut-off values necessary to enable a wgs-based species definition as demonstrated by thompson et al.. it is worth noting that strain y which was isolated from a bioreactor treating pulp and paper-mill effluent has the highest aa + tt dinucleotide frequency among the strains studied. in pasteurella multocida, the continuous culturing at higher temperatures led to increased aa, ta, and tt frequencies in comparison with the virulent strain. since the conformation of aa/tt dinucleotides is not heavily altered by its adjacent nucleotide, these dinucleotides are more stable when exposed to high temperature. the genomic feature exhibited by strain y may therefore correspond to an adaptation to the continuous exposure to mill effluent which is generally above ambient temperatures. pan-genome analysis based on a conservative percent identity cut-off of 65% revealed a large abundance of singletons in the genus novosphingobium and a rather small core genome. it should be noted that the pan-genome analysis was performed using strains of the same genus and not strains of the same species thus explaining the small core genome. a further significant reduction in the core genome and number of singletons in each strain is very likely with the addition of more newly sequenced strains in the future. for example, a core genome of up to genes has been reported for strains of the species vibrio cholerae and the inclusion of more genomes from different vibrio species further reduced the core genome down to genes. nevertheless, combining the prior knowledge of the wet-lab validated phenotypical trait of the bacterial strains and the identification of unique core genome from such an unorthodox pan-genome analysis, we were able to systematically determine the putative proteins that are involved in marine adaptation. such approach reinforced the idea that unique core genome should reflect the phenotypical traits that are specific to a group of bacteria. an additional similarity search directly against the nucleotide sequences consisting of the bacterial whole genome sequences eliminated the possible presence of homologous genes that were not included in the orthologous grouping either due to gene prediction failure or partial sequence length. due to the lack of active transport system for water in many microorganisms, the biosynthesis of compatible solutes such as ectoine in the marine novosphingobium strains is advantageous in regulating osmotic balance across the cell membrane. based on molecular dynamic simulations, ectoine has been shown to be a durable water structure-forming solute across a broad temperature range. the effectiveness of ectoine has been demonstrated in the halo tolerant brevibacterium sp. jcm. upon osmotic shift towards higher salinity, the supplementation of ectoine has been shown to provide the most remarkable growth stimulation independent of the range of osmotic shifts compared to other compatible solutes,. sphingopyxis alaskensis was the first member in the family of sphingomonadaceae reported to possess the complete gene set for synthesis of ectoine.. subsequently, based on blastp query, it appears that at least one member in each of the genus sphingomonas, sphingobium and novosphingobium possesses such gene set, suggesting that ectoine-based organic osmolyte mechanism could be a fairly common strategy employed by marine sphingomonads to persist in high-salt environments. the gene coding for hypothetical protein containing the sodium/alanine symporter protein motif, which is present only in the marine novosphingobium strains us6- and pp1y, may also be involved in osmoregulation. in desulfovibrio vulgaris, the gene coding for sodium/alanine symporter was overexpressed upon exposure to salt shock. in addition, the accumulation of alanine in cells was concurrent with the alleviation of salt-related growth inhibition. alanine has also been reported to play a role in the relief of salt mediated inhibition on the enzymes involved in nitrogen fixation, photosynthesis and respiration of nitrogen-fixing cyanobacterium.the slightly acidic proteome and the presence of compatible solute synthesis in both marine sphingomonads closely mimic the salt adaptation strategy employed by halomonas elongata thus supporting the notion that highly acidic proteome is not required for salt adaptation provided that there is a mechanism to accumulate and/or synthesize osmolyte in the cell. the presence of conserved amino acids hypothesized to be essential for conformational change during substrate binding, provides significant bioinformatics support that the five luxi homologs identified in this study are potentially capable of ahl signal production. it would be important to further genetically and biochemically validate the function of the putative ahl synthase and determine the identity of their ahl signals. the alignment of gene context / neighborhoods of and around the luxi homologs show conserved topology where novr is proximal to novi and where transcriptional orientation is convergent with the receptor upstream, a genomic context feature based on proximity commonly found in complete qs regulatory circuits in the alpha subclass of proteobacteria. in addition, this feature is also generally found in the genomes of proteobacteria. interestingly, downstream of the novi genes in four of the five aligned contents showed a gene coding for phytanoyl-coa dioxygenase whose expression is convergent with the qs regulatory circuits. such topology has also been observed in a fosmid metagenomic library clone and in other genera within sphingomonadaceae including, sphingomonas and sphingopyxis. based on transposon insertion mutagenesis and the short intergenic distance between luxi homolog and phyh, it was hypothesized that phyh is a gene in an operon that is qs regulated. phyh belongs to the family of iron-dependent oxygenases and is responsible for the alpha-hydroxylation of phytanoyl-coa, a derivative of phytanic acid. the grapevine crown gall tumor epiphyte strain rr 2– possess a single ahl synthase homolog although it has been previously demonstrated that strain rr 2– could produce ahls of various chain lengths. three possible reasons could lead to this observation. first, this ahl synthase may contain a rather broad specificity for its acyl-carrier counterpart as previously reported for some ahl synthases. second, additional genes coding for ahl synthases were not identified due to sequence divergence. third, it is also possible that one or more of the ahl synthase genes were lost in the gaps of the draft genome. further study entailing targeted disruption of the ahl synthase gene in strain rr 2– and assessment of ahl production in the mutant strain will be necessary to validate these hypotheses. the successful construction of the ahl negative mutant will also be beneficial for future work involving whole transcriptome sequencing since this will directly provide insights into the genes that are regulated by quorum sensing in the genus novosphingobium. the identification of a putative novr solo which was flanked upstream by various mobile elements suggests that its acquisition is via horizontal gene transfer. two possible mechanisms were proposed for the acquisition of novr solo. first, the novr solo may be part of the gene cassettes incorporated into an integron as evident by the presence of various genes coding for recombinase upstream of novr solo. second, given the presence of two fragmented transposase genes upstream of novr solo, it is also plausible that novr solo was acquired via transposition and the subsequent transposase inactivation possibly due to a combination of nonsense and frame shift mutations prevented further movement of the gene. unlike the novr solo, transposase elements were absent in the close vicinity of various well-studied luxr homolog solo. the association of mobile element with the complete luxir-type quorum sensing system has been previously reported. for example, spnir has been reported to be localized in a mobile transposon and was found to regulate the transposition frequency. in addition, luxi and luxr homologues which were separated by transposase genes have also been reported previously in a metagenomic library clone. the solo novr in strain pp1y could function in eavesdropping on ahl production within microbial communities and / or enable additional ahl regulated gene expression in itself. this identification of a putative luxr homolog solo in strain pp1y is similar to bisr of rhizobium leguminosarum and expr of sinorhizobium meliloti. it is worth noting that these two bacterial species produce multiple ahl signals and have multiple complete qs regulatory circuits. similarly, two complete qs regulatory circuits, one on the chromosome and one on plasmid, have also been identified in the italian marine strain, pp1y. the identification of the putative ardos and the prediction of their function via phylogenetic analysis correlated particularly well with the remarkable bioremediation capability of strains us6- pp1y and dsm. high abundance of ardos in plasmid pnl of strain dsm is consistent with previous report on the sequencing and analysis of pnl. targeted mutagenesis of the putative ardos followed by the assessment of biodegradation ability will be imperative to provide functional analysis data. in addition, assuming that the transcription of ardo gene sets is tightly regulated as observed in several dioxygenase systems, it is also possible to potentially identify the key ardo for a specific aromatic compound via differential gene expression analysis. the presence of gene coding for glutamine synthase directly next to the ardos of strain rr 2– is particularly intriguing. in the 4-aminobenzenesulfonate -dioxygenase system, sadb, a glutamine synthase, was hypothesized to catalyze the removal of amine group from the hydroxylated 4-aminobenzenesulfonate to form 4-sulfocatechol based on targeted gene deletion and random transposon mutagenesis studies. given the fact that 4-aminobenzenesulfonate is a xenobiotic, the identification of the original or natural substrate for this group of ardos may be further examined using strain rr 2– that has never been exposed to, to this compound according to the current literature.  the facile sequencing of whole bacterial genomes as a result of next generation sequencing technologies has brought about a paradigm shift in the field of microbiology. the genome analysis of six novosphingobium strains resulted in the identification of various genes putatively associated with the reported phenotype of novosphingobium strains pertaining to salt-tolerance, biosynthesis and perception of cell-cell signaling molecules and aromatic compound biodegradation. of special mention is the identification of a luxr solo that was flanked by various mobile elements, providing new insights into the potential origin of this luxr solo. the results from this study have the potential to provide information to facilitate future studies relating to the cloning and functional analysis of genes in novosphingobium species. 
notably, a solo luxr homolog was found in strain pp1y that may have been recently acquired via horizontal gene transfer as evident by the presence of multiple mobile elements upstream of the gene. the taxonomic clustering of novosphingobium strains is generally influenced by their isolation source. the combination of prior knowledge on the distinctive phenotypes of novosphingobium strains and meta-analysis of their whole genomes enables the identification of several genes that are relevant in industrial applications and bioremediation. additionally, we show the taxonomic relationship between the sequenced bacteria based on phylogenomic analysis, average amino acid identity and genomic signatures. bacteria belonging to the genus novosphingobium are known to be metabolically versatile and occupy different ecological niches. the results from such targeted but comprehensive comparative genomics analysis have the potential to contribute to the understanding of adaptation, cell-cell communication and bioremediation properties of bacteria belonging to the genus novosphingobium. additionally, phylogenetic tree analysis and sequence comparison with functionally validated aromatic ring hydroxylating dioxygenases revealed the presence of several ardos in novosphingobium bacteria with the majority of them belonging to the groups ii and iii of the enzyme. novosphingobiummarine adaptation genesquorum sensingaromatic ring hydroxylating dioxygenase aai and genomic signature provide strong support the classification of novosphingobium sp. in this study, we analyzed the whole genome sequencing data of six bacteria in the novosphingobium genus and provide evidence to show the presence of genes that are associated with salt tolerance, cell-cell signaling and aromatic compound biodegradation phenotypes. the identification and subsequent functional annotation of the unique core genome in the marine novosphingobium bacteria show that ectoine synthesis may be the main contributing factor in salt water adaptation.
 bacteria belonging to the genus novosphingobium are known to be metabolically versatile and occupy different ecological niches. in the absence of genomic data and/or analysis, knowledge of the bacteria that belong to this genus is currently limited to biochemical characteristics. in this study, we analyzed the whole genome sequencing data of six bacteria in the novosphingobium genus and provide evidence to show the presence of genes that are associated with salt tolerance, cell-cell signaling and aromatic compound biodegradation phenotypes. additionally, we show the taxonomic relationship between the sequenced bacteria based on phylogenomic analysis, average amino acid identity and genomic signatures.  the taxonomic clustering of novosphingobium strains is generally influenced by their isolation source. aai and genomic signature provide strong support the classification of novosphingobium sp. pp1y as novosphingobium pentaromaticivorans pp1y. the identification and subsequent functional annotation of the unique core genome in the marine novosphingobium bacteria show that ectoine synthesis may be the main contributing factor in salt water adaptation. genes coding for the synthesis and receptor of the cell-cell signaling molecules, of the n-acyl-homoserine lactones class are identified. notably, a solo luxr homolog was found in strain pp1y that may have been recently acquired via horizontal gene transfer as evident by the presence of multiple mobile elements upstream of the gene. additionally, phylogenetic tree analysis and sequence comparison with functionally validated aromatic ring hydroxylating dioxygenases revealed the presence of several ardos in novosphingobium bacteria with the majority of them belonging to the groups ii and iii of the enzyme.  the combination of prior knowledge on the distinctive phenotypes of novosphingobium strains and meta-analysis of their whole genomes enables the identification of several genes that are relevant in industrial applications and bioremediation. the results from such targeted but comprehensive comparative genomics analysis have the potential to contribute to the understanding of adaptation, cell-cell communication and bioremediation properties of bacteria belonging to the genus novosphingobium. novosphingobiummarine adaptation genesquorum sensingaromatic ring hydroxylating dioxygenase 