BACKGROUND
the family sphingomonadaceae has been subdivided into five genera: sphingomonas, sphingobium, novosphingobium, sphingopyxis and sphingosinicella <cit> .
to date, the genomes of nearly  <dig> sphingomonads have been sequenced, which has revealed the genetic basis for the degradation of a broad range of polycyclic aromatic hydrocarbons  and polysaccharides  <cit> .
however, sphingobium spp.
are of particular interest due to their ability to degrade hexachlorocylcohexane .
the majority of hch isomers  are formed during the production of the insecticide lindane , and have been active pollutants since the 1950s  <cit> .
among all these isomers, only γ-hch has insecticidal properties.
purification of γ-hch  from the mixture leads to the formation of hch muck  having mainly α , β , δ , and ϵ  isomers  <cit> .
this has been generally discarded in the open by the side of industrial units creating a large number of hch dumpsites between the 1960s to the 1980s around the world  <cit> .
sphingobium spp.
are often enriched in hch dumpsites and have been shown to acquire and maintain genes associated with hch degradation .
the degradation potential for hch isomers has been attributed to the lin pathway , which has been studied in detail in both sphingobium japonicum ut26s  <cit>  and sphingobium indicum b90a  <cit> .
the lin pathway is subdivided into an upper degradation pathway consisting of hch dehydrochlorinase , haloalkane dehalogenase  and dehydrogenase , and a lower degradation pathway consisting of reductive dechlorinase , ring cleavage oxygenase , maleylacetate reductase , an acyl-coa transferase , a thiolase  and transcription factors .
the link, linl, linm and linn i.e., a permease, atpase, periplasmic protein and a lipoprotein respectively, together constitute a putative abc-type transporter  <cit> .figure  <dig>
degradation of α-, β-, γ-, δ- and ϵ-hch mediated by
lin
pathway in
sphingobium
spp.
each color that encircles a specific lin gene depicts that the pathway is blocked within these sphingobium strains.
the compounds that are formed during hch degradation are broadly known as pcch: pentachlorocyclohexane, pchl: pentachlorocyclohexanol, tcdn: tetrachloro- <dig> -cyclohexadiene, dnol: trichloro- <dig> -cyclohexadiene-1-ol, ddol: dichloro- <dig> -cyclohexadiene- <dig> -diol, tcdl: tetrachlorocyclohexanediol, dchq: dichlorohydroquinone, chq: chlorohydroquinone, hq: hydroquinone.
green dots represent spontaneous reaction.
there is evidence that indicates high levels of polymorphisms in the amino acid sequences of the lina and linb genes.
further studies have revealed that these differences contribute to the efficacy of hch degradation and substrate specificity  <cit> .
while there are several strains of sphingomonads isolated from hch dumpsites with demonstrated differences in hch degradation ability  <cit> , genome-wide comparative analyses to better understand the lin pathway, localization of lin genes in the genome and methods of recruitment have not yet been undertaken.
in order to understand the evolution of the hch-degradation pathway, the draft genomes of six sphingobium spp.
isolated from hch dumpsites and the complete genomes of three previously-sequenced, well-studied strains were analysed.
here, we characterize the genetic divergence between these strains in reference to the lin catabolic system and auxiliary characteristics associated with bioremediation potential.
we also present evidence for possible plasmid and is <dig> based horizontal gene transfer  as the method for spread of the lin system genes among sphingomonads.
additionally, variation in the lin gene sequences is a matter of further investigation for improved degradation ability of these strains.
RESULTS
genomic features of sphingobiumstrains
the genome sizes for the six newly sequenced sphingobium spp.
averaged  <dig>  mbp and ranged from  <dig>  to  <dig>  mbp, with s. chinhatense ip <dig> maintaining the largest genome .
these sizes are consistent with existing sphingobium spp.
<cit> .
the variation in genome size can be partially correlated to the presence of genomic islands; ip <dig> maintained the largest genome and the highest genomic island content, while ll <dig> had the least .
this potentially reflects differential degrees of hgt and mobile genetic element acquisition among these strains.
ut26s, b90a, ip <dig> and hdipo <dig> all shared high sequence identity , whereas ll <dig>  p <dig>  rl <dig> and ds <dig> have accumulated more sequence variation despite being under similar selection pressures  .table  <dig>
general characteristic features of the
sphingobium
genomes
project id
prjna208542
prjna201012
prjna208544
prjna201016
prjna201649
prjna202090
prjna50313
prjda19949
prjna73353
ncbi accession no.
auda00000000
atdo00000000
auwy00000000
atho00000000
atdp00000000
atib00000000
ajxq00000000
ap <dig> to ap010806
ap <dig>  ap012223
source of isolation
hch dumpsite, india
hch dumpsite, india
hch dumpsite, india
hch dumpsite, india
hch dumpsite, india
hch dumpsite, czech republic
rhizosphere soil, india
soil contaminated with γ-hch, japan
waste water of kraft mill pulp, japan
hch degarder
yes
yes
yes
no
no
no
yes
yes
no
genome size
<dig> ,129
<dig> ,576
<dig> ,053
<dig> ,546
<dig> ,246
<dig> ,216
<dig> ,196
<dig> ,878
<dig> ,133
g + c content
<dig>
<dig>
65
<dig>
<dig>
64
<dig>
<dig>
66
predicted cds
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
pseudogenes
10
46
2
70
45
39
-
-
-
average gene size
903
889
896
903
890
852
886
890
933
% of cds
<dig> %
<dig> %
<dig> %
<dig> %
<dig> %
<dig> %
<dig> %
<dig> %
<dig> %
is
6100
21
18
19
24
15
22
11
5
0
trna
66
54
56
49
59
56
54
55
50
rrna
11
13
12
9
15
12
3
9
6
genomic islands
1104688
705420
180116
201158
179141
48062
427792
395714
244403
*strain having crispr element.
comparative genome map of
sphingobium
spp.
mapped  over sphingobium japonicum ut26s as a reference genome in which all genetic elements of ut26s were concatenated in the order of chr <dig> , chr <dig> , pchq <dig> , put <dig>  and put <dig> .
genes for hch, phenol/toluene, chlorophenol, anthranilate and homogentisate degradation pathways are identified in the outermost region of the figure.
genetic breakpoints between ut26s and other sphingobium spp.
; from the outside in; outermost circle1: orthologous genes, circle 2: draft genome of s. lactosutens ds <dig>  circle 3: draft genome of s. baderi ll <dig>  circle 4: draft genome of s. quisquilarium p <dig>  circle 5: draft genome of s. ummariense rl <dig>  circle 6: draft genome of sphingobium sp.
hdipo <dig>  circle 7: draft genome of s. chinhatense ip <dig>  circle  <dig> : draft genome of s.indicum b90a.
.
crispr elements were only found associated with s. baderi ll <dig>  and s. lactosutens ds <dig> .
these spacer sequences are known bacterial defense mechanisms against viral and plasmid challenges acquired from foreign invading dna, with the number of new phage-derived spacers being correlated with phage resistance  <cit> .
however, their spacer sequences had no similarity to known viral phage sequences.
furthermore, ll <dig> maintained a type ii crispr element with the cas <dig> gene involved in target interference, whereas ds <dig> had type i crispr elements with the cas <dig> gene  <cit> .
strains ll <dig> and ds <dig> were isolated from hch dumpsites in the czech republic and india, respectively, and these strains had two different crispr/cas systems, that may correspond to their different geographical locations.
these data also reflected that ll <dig> should have the greatest phage resistance.
comparative phylogenetic analysis
four different phylogenetic methods ) were used to analyze the relationships of the nine strains under study .
the consensus tree topology obtained by these methods clustered s. indicum b90a, s. japonicum ut26s, s. chinhatense ip <dig>  and sphingobium sp.
hdipo <dig>  with the exception of the single copy gene approach.
notably, these four strains were the only ones with an entirely complete lin pathway, thus suggesting convergent evolution through hch selection pressure.
furthermore, ani topology supported the grouping of sphingobium sp.
hdipo <dig> and s. chinhatense ip <dig> as subspecies    <cit> .
the other five strains i.e., ll <dig>  ds <dig>  rl <dig>  p <dig> and syk <dig> did not produce a consensus phylogeny, with relationships differing between these approaches; in short, strains with the complete lin pathway formed a closed group whereas, the others have diverged.
in addition, 16s rrna and single copy gene approaches may be problematic for differentiation among highly related strains .
however, ani based pairwise comparison has clustered ll <dig> and rl <dig>  in a monophyletic clade with p <dig>  forming a close relationship.
moreover, ds <dig> and syk <dig>  were clustered together.
this suggests that ani based phylogeny is more appropriate and mirrors their relationship with respect to hch degradation.figure  <dig>
phylogenic constructions of
sphingobium
spp.
sphingobium baderi ll <dig>  s. lactosutens ds <dig>  s. chinhatense ip <dig>  s. quisquilarium p <dig>  s. ummariense rl <dig>  sphingobium sp.
hdipo <dig>  s. indicum b90a, s. japonicum ut26s and sphingobium sp.
syk <dig>   16s rrna gene sequence-based phylogeny was performed with the neighbor joining method using treeconw  <dig> b   the amino acid sequences of  <dig> single copy genes were concatenated for all ten genomes and phylogeny performed with the neighbor joining method using treeconw  <dig> b   & whole genome based tetranucleotide correlation and ani based pairwise comparisions between the genomes was carried out for all the genomes.
a pearson correlation matrix was constructed on the basis of their tetranucleotide correlation and ani values, followed by hierarchical clustering on the resultant matrix using mev <dig> .
<dig>  and visualized on mega <dig>
common gene content and functional profiling of sphingobiumspp
core genome analysis identified  <dig> orthologs conserved between the nine genomes.
the majority of these genes were involved in housekeeping functions such as the synthesis of ribosomal proteins, dna replication, transcription & translation machinery, amino acid metabolism and membrane transporters.
core genome analysis for the eight strains that were either isolated from an hch dumpsite or showed hch degradation potential  predicted  <dig> orthologs , which suggests a significant increase in genomic conservation  resulting from the selective pressure of hch exposure.
this conservation is also seen in the degradation potential for other aromatic compounds such as benzoate,  <dig> -dichlorobenzene,  <dig> -methylnapthalene, caprolactam, toluene and xylene, trinitrotoluene, biphenyl and styrene degradation .
genes involved in the degradation of p-hydroxybenzoate, benzoate, quinate, gentisare, and catechol were also identified in the nine sphingobium genomes .
the presence of degradation pathways for phenol/toluene, chlorophenol, anthranilate, and homogentisate are identified in ut26s  <cit> .
these pathways were observed in at least two of the newly sequenced strains .
this suggests that these sphingobium spp.
possess broad aromatic compound degradation potential, although we did not observe the presence of these compounds at the hch dumpsite  <cit> .
the link between these aromatic degradation pathways and the hch degradation pathway requires further investigation.figure  <dig>
functional profiling of the
sphingobium
genomes.
heat map showing the normalized relative abundance of the top  <dig> subsystems enriched in the nine sphingobium genomes.
the strains and enriched pathways were clustered using pearson correlation with a  <dig> % minimum abundance.
the color scale represents the relative abundance of gene content for each pathway, normalized by sample mean.
functional profiling was used to analyze pathways that were differentially enriched in these strains.
for this, a dendogram was constructed based upon the top  <dig> subsystems at  <dig> % minimum abundance using pearson correlation distance.
the analysis revealed that the two-component system for gene expression was highly abundant in all of the sphingobium genomes .
this system is known to facilitate adaptation to extreme environmental conditions and likely contributes to the ability to survive in conditions of high hch pressure, salinity, and acidity that exist at the hch dumpsite  <cit> .
additionally, the nine strains collectively showed an abundance of abc transporters within their genomes.
the abundance of these transporters implies that these strains are highly engaged in transport of a wide variety of substrates across extra- and intracellular membranes  <cit> , which is consistent with the sphingobium proficiency for degradation of a wide range of xenobiotics .
interestingly, hdipo <dig> and ip <dig>  which had a close phylogenetic relationship, demonstrated differences in their functional repertoire, based on the top  <dig> subsystems.
this is primarily driven by an increased abundance of  <dig> -dichlorobenzene degradation, toluene and xylene degradation, caprolactam degradation, ppar signaling and atrazine degradation pathways in hdipo <dig>  which was clustered with functional profiling of p <dig> and ut26s as they shared these enrichments.
furthermore, lipopolysaccharide biosynthesis, tyrosine metabolism, glycan and glycosaminoglycan degradation pathways were found enriched in ip <dig> as compared to hdipo <dig>  this variation suggests that while these strains exhibit similar genomic content, they exhibit differential dominance in their metabolic preferences.
nitrogen assimilation and the presence of flagellar genes in non-motile sphingobium
the genomes of all the nine strains were found to contain an enrichment of the two component signal transduction system for nitrogen stress response  .
additionally, the large subunit of assimilatory nitrate reductase, a key regulator that potentially enables the utilization of nitrate as a nitrogen source, was found to be under diversifying natural selection , which suggests that these strains can tolerate low inorganic nitrogen concentrations and are evolving in response to this inorganic nitrogen stress.
at high nitrogen concentration, the transmembrane protein glnc  responds to nitrogen availability and phosphorylates glng , which in turn leads to the activation of glna   <cit> .
another key regulator of the pathway is glnb, which interacts and regulates the activity of glnc.
when the nitrogen availability is low, glnb is subjected to post transcriptional modification by uridylation .
this modification is reversed in n-sufficient conditions  <cit> .
thus, the presence of ntrc pathway and nitrate reductase genes explains the ongoing phenomena of nitrogen assimilation by these strains at hch dumpsites to acclimatize themselves in such nitrate concentrations.
increasing exposure to elevated hydrocarbon concentrations was found to be positively correlated with the relative abundance of genes associated with nitrogen metabolism  <cit> .
the ntrc pathway is also associated with genes regulating chemotactic response, such as chey, mota, motb, and flagellar biosynthesis proteins, such as flha, flio, flip, flir etc.
all these genes were also found in the core-genome.
chey modulates the cell’s ability to interact with the flagellum and controls swimming behavior  <cit> .
interestingly, while these sphingobium strains are considered non-motile , each genome housed more than half of the genes needed for flagellar assembly and functioning.
this raises the possibility that they are either in a process of acquiring or losing motility.
the abundance of chemotaxis and motility genes has already been demonstrated in the metagenome of the hch dumpsite  <cit>  from where hdipo <dig>  ip <dig>  p <dig>  ds <dig>  and rl <dig> were isolated.
however, further analysis is needed to probe the reason for retention or loss of flagellar genes in the sphingobium strains, and to investigate whether sphingobium have the potential to gain motility through acquisition of the remaining genes under the high selective pressure of hch in the stressed environments.
recruitment of linpathway through different routes
the genome analysis revealed a mosaic distribution of lin genes and is <dig> elements in hch-degrading sphingobium spp.
coupled with high polymorphism levels in the lin genes.
this indicates the recruitment of lin genes through different routes in sphingobium spp.
under hch stress, and further that the pathway has not yet stabilized in these strains but is instead subjected to further rearrangements and polymorphisms.
is6100-mediated recruitment based on mosaic distribution pattern of lingenes
the is <dig> elements, known for disseminating lin genes through hgt among sphingomonads , were found to be present in all of the newly sequenced strains associated with hch degradation, including strain ds <dig> which did not degrade hch.
the number, as determined from the genome sequence, varied from  <dig> copies in ut26s to  <dig> copies in p <dig> .
the presence of a large number of is <dig> elements reflects a high degree of genomic rearrangement, as the is <dig> elements have already been demonstrated to play an important role in the spread and reorganization of the lin pathway in sphingomonads .
to further explore the mechanism of hgt in the spread and diversification of the lin system, we examined the colocalization of lin genes with mobile elements such as the insertion sequence is <dig> and transposons, and their presence on plasmids.
in all of strains where lina gene was present in, it was found in nearly identical association with is elements as in ut26s i.e.
is <dig> was found within proximity of <5kbp.
however, in rl <dig>  two is <dig> copies lies in the same orientation within the above mentioned range.
hence, this suggests that among these strains the association of lina with is <dig> is consistent, but the reason and possible involvement of is <dig> in the mechanism of duplication of the lina gene in rl <dig> needs to be identified.
in ip <dig>  linb was found to be flanked on both sides by is <dig> .
additionally, resolvase genes were found at the flanking ends of both of these transposons.
strains ll <dig> and p <dig> did not contain the linb gene, as confirmed by pcr amplication.
thus, either these strains have yet to acquire the linb gene, or, given the flanking is <dig> elements, it is suggested that the loss of linb could have occurred via an intra-chromosomal single homologous recombination between two copies of is <dig> <cit> .figure  <dig>
lin
genes and their association with is
6100
in hch degrading
sphingobium
spp.
linb associated with is <dig> at both its end in strain ip <dig>  association of linder with homogentisate degradation pathway genes hmga and hmgb separated by is <dig> in strain rl <dig>
in hdipo <dig>  a truncated copy of linf along with complete set of linc and linb was found with an is <dig> element  .
in contrast, in the case of the reference ut26s, these elements were dispersed, with linf present on chromosome  <dig> and linb and linc on chromosome  <dig>  the association of these three elements suggests that they may have been brought together by is6100-mediated transposition, a hypothesis supported by the fact that hdipo <dig> contains a high number of is <dig> comparable to ut26s , and that they may be in the process of forming an operon.
of the three copies of linder present in rl <dig>  one was closely associated with the hmgb and hmga genes of the homogentisate degradation pathway, separated by a copy of is <dig> .
in contrast, in ut26s, linder was housed on a plasmid , while hmgb and hmga were found on chromosome  <dig>  <cit> .
therefore, in rl <dig>  it is possible that these two different aromatic compound degradation pathways were brought into close proximity by is <dig> mediated transposition.
thus, is <dig>  apart from the spread of lin gene system, might be effective in the spread of homogentisate pathways despite the absence of homogentisate selective pressure at the hch dumpsite, consistent with the fact that already sphingomonads that degrade aromatic hydrocarbons were found to contain catabolic genes associated with is <dig> <cit> .
in strain ll <dig>  isolated from the czech republic, linghij genes were associated with is <dig>  whereas in the ut26s genome, isolated from japan, is <dig> was absent from the region proximal to these lower pathway genes.
as is <dig> is reported to be a key driver in the recruitement of the lin system  <cit> , differential organization of the is <dig> element with respect to lin genes for strains from geographically-disparate locations reflects an ongoing is6100–driven evolution of the lin system, including the lower degradation pathway components such as linghij.
is <dig> elements have also been found in the genome of ds <dig>  which did not degrade hch isomers .
however, in ds <dig>  the regions flanking the is <dig> elements comprised a variety of xenobiotic tolerance and degradation genes , which further supports the role of is <dig> in distributing genes for a broad-range of such functions in sphingobium spp.
the fact that ds <dig>  a non-hch degrader, maintained  <dig> copies of is <dig> elements clearly suggests the potential of this strain to acquire lin genes through is <dig> mechanisms in the future.
plasmid mediated recruitment
in investigating the presence and spread of the lin genes, the recently sequenced genome of an hch-degrader sphingomonas sp.
mm- <dig> is of interest as it was found to have five plasmids housing the genes of the lin pathway  <cit> .
in the mm- <dig> genome, the linf was found on pisp0; lina, linc, and a truncated linf on pisp <dig>  linder on pisp <dig>  and linb, linc, and another truncated linf on pisp <dig>  <cit>  and linghij was found on pisp <dig>  genes for an abc transporter were found on the chromosome, but these did not share at least 80% identity to the linklmn genes of ut26s.
in addition to this, in strain ut26s, hch-specific genes of the lin pathway were found to be housed on regions unique to the ut26s genome  <cit> ; with lina, linb, linc genes in chromosome  <dig>  linf on chromosome  <dig>  and linder on the plasmid pchq <dig>  <cit> .
the lower pathway genes, including linghij and linklmn were found on chromosomes  <dig> and  <dig>  respectively, in regions that were conserved among sphingomonads  <cit> .
genome recruitment plots were created to map the raw reads of the six novel-sequenced strains to sphingobium plasmid sequences to investigate the possibility of these plasmids playing a role in transfer of the lin genes.
mm- <dig> plasmids pisp <dig> and pisp <dig> in particular were found to have a high percentage of coverage which was maximum with s. ummariense rl <dig> .
as pisp <dig> houses linder, it is highly probable that plasmid uptake and duplication may explain the triplication of linder in rl <dig>  the recent metagenomics analysis of the hch dumpsite also reflected the enrichment of pisp <dig>  suggesting its availability for other sphingomonads strains present at the hch dumpsite  <cit> .
furthermore, pisp <dig> encodes linb, linc, and linf, and similarly shows a high degree of coverage by rl <dig>  consistent with the absence of linc from the rl <dig> draft genome, which was confirmed by pcr amplification by using the primer 5′-gcggatccgcatgtctgatttgagcggc-3′ and 3′-gcctcgagtcagatcgcggtaaagccgccgtc-5′, there is a gap in the coverage seen in the plasmid region containing linc , which is a region flanked by two is <dig> elements in mm- <dig> .
this points to the possibility that the plasmid has undergone either acquisition in mm- <dig> or looping out from rl <dig> of the linc gene during the course of evolution, mediated directly by is <dig>  mapping the raw reads of the six newly-sequenced sphingobium strains to the plasmid sequences for mm- <dig> and ut26s, several of the mm- <dig> plasmids, but none of the ut26s plasmids demonstrated a high degree of coverage.
additionally, the proportionally higher presence of lin genes on plasmids in mm- <dig> than in ut26s suggests that strain mm- <dig> acts as a reservoir for plasmids allowing for the effective spread of the lin system, and thus may be an important strain to include in the consortium development as a potential disseminator of the lin system.
also, strains sharing similar arrangement profile of lin genes with mm- <dig> i.e., rl <dig>  ip <dig> and hdipo <dig>  should be included into designing a consortium.figure  <dig>
genome recruitment plot mapping on
sphingomonas
sp mm- <dig> plasmid a) pisp <dig> and b) pisp4: the raw reads of the six novely sequenced strains were mapped on plasmids of
sphingomonas
sp.
mm <dig>  the orange bars are depicting is <dig> while the lin genes were marked with grey and yellow bars showing their position in reference.
strains in transition to acquire linpathway
of the nine sphingomonads under study, seven possessed components of the upper hch degradation pathway to varying degrees of completion, and two, syk <dig> and ds <dig>  were completely devoid of them .
syk <dig> did not contain any components of the lin system and the ds <dig> genome contained only genes of the lower lin pathway- linklmn an abc transporter.
of the hch-degraders, not every strain was found to house the complete array of lin genes characterized in ut26s or b90a.
for instance, the p <dig> genome lacked linb, linc, linder, lingh, lini and linj genes while, strains rl <dig> and ll <dig> both lacked linc and ll <dig> lacked linb, as confirmed by pcr amplificiation .
the differential composition of the lin system between these strains may be indicative of different steps in the evolution of the lin pathway, with ip <dig> in the stage of probable homologous recombination and looping out of linb, while ll <dig> shows potential gain of linghij through is6100-mediated hgt.
strain ds <dig> possesses abc transporters and shows potential for acquisition of the lin genes, as it holds  <dig> copies of is <dig>  while p <dig>  in addition to the abc transporter, has lina and linf but is yet to acquire the other lin genes.
linsystem sequence diversity and its effect on metabolic efficiency
upper linpathway
the upper pathway genes lina, linb and linc degrade γ-hch and α-hch, and additionally linb acts on β-hch, leading to the formation of β- <dig> , <dig> ,6-pentachlorocyclohexanol  .
as α- and β-hch form the major components of contamination at the hch dumpsite , both lina and linb are extremely important enzymes encoding hch dehydrochlorinase and haloalkane dehalogenase, respectively .
to gain deeper insights into the lin gene sequence diversity and its impact on hch degradation, the genetic divergence of the lin system components was analyzed with respect to the copy number and nucleotide sequence divergence of the lin genes in both upper and lower degradation pathways, using b90a as a reference.
the highest level of divergence in the upper hch degradation pathway lin genes was reported for the lina gene encoding for hch dehydrochlorinase in b90a  <cit> .
the two previously characterized lina variants observed in b90a differed by 10% of their amino acid sequence, and were named lina <dig> and lina <dig>  the functional aspects of these variants have been well characterized, as they show enantioselectivity in  α-hch degradation, with lina <dig> selective for the  and lina <dig> for the  α hch  <cit> .
also, the degradation ability of lina <dig> was found to be lower than that of lina <dig>  <cit> .
among all the lin genes of the upper pathway, lina in the present study was found to be most diverged in hdipo <dig>  in which it appeared to be a hybrid of the two variants  with  <dig> % sequence similarity to lina <dig> and  <dig>  to lina <dig>  near to the catalytic dyad d <dig> and h <dig> critical for its enzymatic activity, the hdipo <dig> lina was found to be identical with lina <dig> <cit> .
however, the c-terminal region corresponded to lina <dig> .
this hybrid copy, now marked as lina <dig>  requires further experimentation, but might be responsible for the comparatively better dehydrochlorination activity of hdipo <dig> against α- and γ-hch, as reported earlier  <cit> .figure  <dig>
a) multiple sequence alignments of
lina
b)
linb
gene sequences in the strains showing variation.
amino acid sequences of lina and linb genes from the strains were aligned using bioedit to depict the substitutions at the respective sites.
apart from this divergence of the lina sequence in hdipo <dig>  not such changes to the lina gene sequences were observed; all strains showed 100% sequence similarity to that of the lina <dig> gene  <cit>  with the exception that lina <dig> of rl <dig> showed a single substitution of l78q.
it is important to mention here that the lina gene has already been reported to be under continuous selection pressure and a large number of variants of this gene exist  <cit>  and better variants of lina may be used for developing enzymatic bioremediation system for hch.
in contrast to lina, there were less variations in linb sequences among strains under study.
the sequence differences among lina and linb genes among different sphingobium spp.
are particularly interesting in light of findings that marginal differences in the amino acid sequences of linb in ut26s  <cit> , sp+ <cit> , b90a  <cit> , bhc-a  <cit>  and m <dig>  <cit>  can alter the efficacy and substrate range, with the former group degrading β-hch to β-pchl and the latter group taking the pathway beyond pchl to tchl.
hdipo <dig> housed two identical linb copies with a t81a substitution and overall  <dig> % similarity to b90a while rl <dig> linb gene had  <dig> % identity, with three substitutions  as compared to linb of b90a .
here, the copy number difference is suspected to have a more impact, as the two copies of linb might explain the high β-hch degradation efficacy of hdipo <dig>  <cit> .
apart from these two strains, no such diversity was observed, thus demonstrating the stability of linb gene in the population.
linc, which encodes for hch dehydrogenase was most conserved among the genes of the upper degradation pathway and demonstrated only a single substitution: y172c in case of ip <dig>  in any case these studies reflect that lina genes are more prone to evolutionary changes under hch stress and have not stablized yet.
lower linpathway
the lower pathway of γ-hch degradation begins from  <dig> -dichlorohydroquinone  an intermediate of γ-hch , which is mineralized by the lower pathway lin genes   <cit> .
in contrast to the upper degradation pathway, very less is known about the divergence and polymorphisms of the genes of the lower degradation pathway.
among all the lin genes of the lower pathway, linf was the most highly conserved, as its amino acid sequence was 100% identical in all genomes .
in the linder operon, the set of lind genes similarly showed minimal divergence, with the ip <dig>  rl <dig>  and ll <dig> genes sharing a substitution of n82s, and additionally ip <dig> having a substitution q30p.
further, linr and line had very little divergence; linr diverged only in one substitution in hdipo <dig>  and line was 100% identical in all strains.
this highlights the fact that the linder operon, which makes up the backbone of the downstream hch degradation pathway, remained highly stable during the course of evolution.
a greater degree of the variation of this operon was found, however, in copy number, as rl <dig> and hdipo <dig> housed three and two copies, respectively .figure  <dig>
genetic sequence and copy number variation of
lin
genes: the genetic divergence, as quantified by percentage nucleotide identity to the archetypal strain ut <dig>  and copy number variation in
lin
genes across the nine
sphingobium
strains under study.
in particular lingh, lini and linj, which mediate the later stages of the lower degradation pathway, i.e., conversion of β-ketoadipate to succinyl coa and acetyl coa , showed variation in the sequences of linh and lini, whereas ling and linj sequences were 100% conserved among all these strains.
here, linh of hdipo <dig> and ip <dig> were similar to each other, and both diverged from b90a with  <dig> % identity.
they held two substitutions  while ll <dig> shared the i31v substitution and additionally had a n131d substitution.
lini was found to be identical in hdipo <dig> and ip <dig>  with a single substitution  and  <dig> % identity to b90a, while ll <dig> had two substitutions  and  <dig> % identity.
however, the significance of sequence divergence in ling, linh, lini, and linj genes among these strains is yet to be investigated.
another important lin gene system of the lower pathway is the abc transporter system i.e., link, linl, linm, and linn, which encode a permease, atpase, periplasmic protein, and lipoprotein, respectively.
this abc transpoter system is very important as it allows for the transport of hch isomers and clearance of dead-end metabolites of hch from the cell  <cit> .
out of the entire lin system these genes have shown the highest level of of divergence with link at  <dig> % in rl <dig>  linl at  <dig> % in ds <dig>  linm at  <dig> % in ds <dig> and linn at  <dig> % in p <dig>  based on the prevalence of similar but non lin-specific abc type transporters which are found by sequence identity searches across a variety of microbial species, it is hypothesized that the linklmn operon derived from convergent evolution in response to environment changes.
with the introduction of the hch to the environment, pre-existing abc-type transporters were likely recruited to the hch degradation pathway, and thus several genetic variants might have undergone convergent evolution to select for transporters with increased efficiency for hch-metabolite efflux, and these later generation genes were the one that subsequently underwent hgt.
this is in contrast to the likely origin of the linder operon, which encodes more highly hch-specific enzymatic functions and is almost perfectly conserved, and thus was likely generated once, and spread through hgt from a single genetic ancestor.
the lin pathway shows a characteristic pattern in which the upper hch degradation pathway was diverged along a gradient from the most, lina  to the least, linc .
while in the lower degradation pathway linklmn was the most highly diverged, followed by linghij, linder and linf respectively.
in literature, the degradation ability of these strains under consideration is in the order: hdipo4>ip26>rl <dig>  in order to further substantiate the relationship between lin system diversity and degradation efficiency, principle component analysis  was performed on the copy number and sequence divergence for these strains, with hch degradation plotted as a supplementary variable to visualize correlation.
the analysis demonstrated a close grouping of the four lin-deficient strains, i.e.
syk <dig>  ds <dig>  ll <dig>  and p <dig> under pca  <dig> and  <dig>  .
hdipo <dig> and ip <dig> were colocalized in quadrant  <dig>  opposite from the non-degrader cluster in terms of both dimension  <dig> and dimension  <dig>  which is appropriate given that these two strains have degradation rates documented to be faster than archetypal strains ut26s and b90a  <cit>  .
while copy number variation for linf, linder, and linb were mapped most closely to the hch degradation vector , the copy numbers for linc, linghij and again linb played a more important role in differentiating these strains, as they correlated significantly to pca <dig> .
in terms of sequence, lina and linr showed high correlation to degradation ability , while linl, linm, and link were significantly correlated to pca <dig>  and lini, linh, linc, linb, and linf were the most significant contributors to pca <dig> .
this suggests that while variation can be seen throughout the lin pathway and the copy number of linf, linder and linb sequence for lina and r might have the most impact in optimizing the efficacy of an hch enzymatic bioremediation system.figure  <dig>
principal component analysis of genes involved in hch degradation pathway.
pca individual factor plot showing the grouping of the nine sphingobium strains based upon the sequence divergence and copy number of the set of lin genes.
principle components  <dig>  and  <dig>  were chosen as the separation of the strains by these pcs demonstrated the highest fidelity to known hch degradation ability.
pca variable factor plot using principle component  <dig> and  <dig>  showing the contribution of the lin genetic sequences  and copy number  to the variation of the nine sphingobium strains.
hch degradation ability was plotted as a supplementary categorical variable , with non-hch degraders coded as  <dig>  partial degraders as  <dig>  and complete degraders as  <dig>
genes under diversifying natural selection
to identify the substitutions that have fixed along the independent lineages and their direction of evolution, dn/ds  analysis was performed for sets of orthologous genes, and in particular those responsible for the degradation of hch, phenol/toluene, homogentisate, chlorophenol and transposons/integrases .
the genes of interest found to be under diversifying selection  includes those for fe dependent oxygenase, abc transporters, assimilatory nitrate reductase, and general secretory pathway protein.
these genes are largely associated with stress tolerance.
as mentioned earlier, abc transporters are involved in uptake of high molecular weight pharmacological agents including xenobiotic compounds  <cit> .
hence, these transporters and their activity are crucial for their capability of hch degradation and other aromatic compounds and likewise, their positive selection indicates the importance of their function in the hch-stressed environments from which these bacterial strains were isolated.
additionally, nitrate reductase catalyzes the conversion of nitrate into free nitrogen and likely would enable the sphingobium spp.
to more effectively enact a nitrogen stress response.
finally, the diversification of genes such as oxygenases, secretory pathway proteins and translocase components adds to the sphingomonads skill of degradation of a wide range of aromatic compounds.
notably, only linh has shown the dn/ds >  <dig>  while the other lin genes did not.
this clearly indicates that under strong hch pressure, the whole lin pathway is likely to get stabilized in the population with more synonomous substitutions compared to non-synonomous ones and tends to be retained in the population.figure  <dig>
dn/ds v/s ds plot of orthologs, xenobiotic degradation pathway genes and is elements/integrases.
sequences of orthologs, xenobiotic degradation pathway genes and is elements/integrases were used for calculating their dn/ds.
genes having dn/ds value > <dig> are under positive natural selection.
CONCLUSIONS
in sequencing the genomes of six novel sphingobium species and comparing these to the known genomes of three other sphingobium species, this study has begun to probe the natural variation in the lin pathway for hch degradation.
analysis of the variation in the lin system, as well as in the phylogenetic relationships, core genomes, and functional profiles of these bacterial strains demonstrated unique characteristics of b90a, hdipo <dig> and ip <dig> which could explain their higher efficacy as the degraders of hch isomers.
the information thus obtained can now be used to select these better-performing strains for the development of a bacterial consortium for on-site bioremediation of the hch dumpsites.
focusing on the lin system, analysis of the similarities in the lin genes sequences and varying copy numbers between these strains has identified variations in the specific genes as key differentiators and these key components will be of critical interest as the most effective targets for optimization of an enzymatic bioremediation system.
the analysis so far made reflect that better lina and linb variants can eventually be the ideal candidates for developing an enzymatic bioremediation system.
moreover, this study has uncovered evidence for genus-level hgt of plasmids housing components of the lin system, specifically between sphingomonas sp.
mm- <dig> and rl <dig>  the additional lin-deficient strains are of further importance as they demonstrate varying degrees of acquisition of the lin system and will be useful in future homologous recombination studies to work with manipulated pathway completion through introduction of synthetic lin genes.

 the family sphingomonadaceae has been subdivided into five genera: sphingomonas, sphingobium, novosphingobium, sphingopyxis and sphingosinicella. to date, the genomes of nearly sphingomonads have been sequenced, which has revealed the genetic basis for the degradation of a broad range of polycyclic aromatic hydrocarbons and polysaccharides. however, sphingobium spp. are of particular interest due to their ability to degrade hexachlorocylcohexane. the majority of hch isomers are formed during the production of the insecticide lindane, and have been active pollutants since the 1950s. among all these isomers, only γ-hch has insecticidal properties. purification of γ-hch from the mixture leads to the formation of hch muck having mainly α, β, δ, and ϵ isomers. this has been generally discarded in the open by the side of industrial units creating a large number of hch dumpsites between the 1960s to the 1980s around the world. sphingobium spp. are often enriched in hch dumpsites and have been shown to acquire and maintain genes associated with hch degradation. the degradation potential for hch isomers has been attributed to the lin pathway, which has been studied in detail in both sphingobium japonicum ut26s and sphingobium indicum b90a. the lin pathway is subdivided into an upper degradation pathway consisting of hch dehydrochlorinase, haloalkane dehalogenase and dehydrogenase, and a lower degradation pathway consisting of reductive dechlorinase, ring cleavage oxygenase, maleylacetate reductase, an acyl-coa transferase, a thiolase and transcription factors. the link, linl, linm and linn i.e., a permease, atpase, periplasmic protein and a lipoprotein respectively, together constitute a putative abc-type transporter.figure degradation of α-, β-, γ-, δ- and ϵ-hch mediated by lin pathway in sphingobium spp. each color that encircles a specific lin gene depicts that the pathway is blocked within these sphingobium strains. the compounds that are formed during hch degradation are broadly known as pcch: pentachlorocyclohexane, pchl: pentachlorocyclohexanol, tcdn: tetrachloro- -cyclohexadiene, dnol: trichloro- -cyclohexadiene-1-ol, ddol: dichloro- -cyclohexadiene- -diol, tcdl: tetrachlorocyclohexanediol, dchq: dichlorohydroquinone, chq: chlorohydroquinone, hq: hydroquinone. green dots represent spontaneous reaction. there is evidence that indicates high levels of polymorphisms in the amino acid sequences of the lina and linb genes. further studies have revealed that these differences contribute to the efficacy of hch degradation and substrate specificity. while there are several strains of sphingomonads isolated from hch dumpsites with demonstrated differences in hch degradation ability, genome-wide comparative analyses to better understand the lin pathway, localization of lin genes in the genome and methods of recruitment have not yet been undertaken. in order to understand the evolution of the hch-degradation pathway, the draft genomes of six sphingobium spp. isolated from hch dumpsites and the complete genomes of three previously-sequenced, well-studied strains were analysed. here, we characterize the genetic divergence between these strains in reference to the lin catabolic system and auxiliary characteristics associated with bioremediation potential. we also present evidence for possible plasmid and is based horizontal gene transfer as the method for spread of the lin system genes among sphingomonads. additionally, variation in the lin gene sequences is a matter of further investigation for improved degradation ability of these strains.  genomic features of sphingobiumstrains the genome sizes for the six newly sequenced sphingobium spp. averaged mbp and ranged from to mbp, with s. chinhatense ip maintaining the largest genome. these sizes are consistent with existing sphingobium spp.. the variation in genome size can be partially correlated to the presence of genomic islands; ip maintained the largest genome and the highest genomic island content, while ll had the least. this potentially reflects differential degrees of hgt and mobile genetic element acquisition among these strains. ut26s, b90a, ip and hdipo all shared high sequence identity, whereas ll p rl and ds have accumulated more sequence variation despite being under similar selection pressures.table general characteristic features of the sphingobium genomes project id prjna208542 prjna201012 prjna208544 prjna201016 prjna201649 prjna202090 prjna50313 prjda19949 prjna73353 ncbi accession no. auda00000000 atdo00000000 auwy00000000 atho00000000 atdp00000000 atib00000000 ajxq00000000 ap to ap010806 ap ap012223 source of isolation hch dumpsite, india hch dumpsite, india hch dumpsite, india hch dumpsite, india hch dumpsite, india hch dumpsite, czech republic rhizosphere soil, india soil contaminated with γ-hch, japan waste water of kraft mill pulp, japan hch degarder yes yes yes no no no yes yes no genome size,129,576,053,546,246,216,196,878,133 g + c content 65 64 66 predicted cds pseudogenes 10 46 2 70 45 39 - - - average gene size 903 889 896 903 890 852 886 890 933 % of cds % % % % % % % % % is 6100 21 18 19 24 15 22 11 5 0 trna 66 54 56 49 59 56 54 55 50 rrna 11 13 12 9 15 12 3 9 6 genomic islands 1104688 705420 180116 201158 179141 48062 427792 395714 244403 *strain having crispr element. comparative genome map of sphingobium spp. mapped over sphingobium japonicum ut26s as a reference genome in which all genetic elements of ut26s were concatenated in the order of chr, chr, pchq, put and put. genes for hch, phenol/toluene, chlorophenol, anthranilate and homogentisate degradation pathways are identified in the outermost region of the figure. genetic breakpoints between ut26s and other sphingobium spp.; from the outside in; outermost circle1: orthologous genes, circle 2: draft genome of s. lactosutens ds circle 3: draft genome of s. baderi ll circle 4: draft genome of s. quisquilarium p circle 5: draft genome of s. ummariense rl circle 6: draft genome of sphingobium sp. hdipo circle 7: draft genome of s. chinhatense ip circle: draft genome of s.indicum b90a.. crispr elements were only found associated with s. baderi ll and s. lactosutens ds. these spacer sequences are known bacterial defense mechanisms against viral and plasmid challenges acquired from foreign invading dna, with the number of new phage-derived spacers being correlated with phage resistance. however, their spacer sequences had no similarity to known viral phage sequences. furthermore, ll maintained a type ii crispr element with the cas gene involved in target interference, whereas ds had type i crispr elements with the cas gene. strains ll and ds were isolated from hch dumpsites in the czech republic and india, respectively, and these strains had two different crispr/cas systems, that may correspond to their different geographical locations. these data also reflected that ll should have the greatest phage resistance. comparative phylogenetic analysis four different phylogenetic methods ) were used to analyze the relationships of the nine strains under study. the consensus tree topology obtained by these methods clustered s. indicum b90a, s. japonicum ut26s, s. chinhatense ip and sphingobium sp. hdipo with the exception of the single copy gene approach. notably, these four strains were the only ones with an entirely complete lin pathway, thus suggesting convergent evolution through hch selection pressure. furthermore, ani topology supported the grouping of sphingobium sp. hdipo and s. chinhatense ip as subspecies. the other five strains i.e., ll ds rl p and syk did not produce a consensus phylogeny, with relationships differing between these approaches; in short, strains with the complete lin pathway formed a closed group whereas, the others have diverged. in addition, 16s rrna and single copy gene approaches may be problematic for differentiation among highly related strains. however, ani based pairwise comparison has clustered ll and rl in a monophyletic clade with p forming a close relationship. moreover, ds and syk were clustered together. this suggests that ani based phylogeny is more appropriate and mirrors their relationship with respect to hch degradation.figure phylogenic constructions of sphingobium spp. sphingobium baderi ll s. lactosutens ds s. chinhatense ip s. quisquilarium p s. ummariense rl sphingobium sp. hdipo s. indicum b90a, s. japonicum ut26s and sphingobium sp. syk 16s rrna gene sequence-based phylogeny was performed with the neighbor joining method using treeconw b the amino acid sequences of single copy genes were concatenated for all ten genomes and phylogeny performed with the neighbor joining method using treeconw b & whole genome based tetranucleotide correlation and ani based pairwise comparisions between the genomes was carried out for all the genomes. a pearson correlation matrix was constructed on the basis of their tetranucleotide correlation and ani values, followed by hierarchical clustering on the resultant matrix using mev. and visualized on mega common gene content and functional profiling of sphingobiumspp core genome analysis identified orthologs conserved between the nine genomes. the majority of these genes were involved in housekeeping functions such as the synthesis of ribosomal proteins, dna replication, transcription & translation machinery, amino acid metabolism and membrane transporters. core genome analysis for the eight strains that were either isolated from an hch dumpsite or showed hch degradation potential predicted orthologs, which suggests a significant increase in genomic conservation resulting from the selective pressure of hch exposure. this conservation is also seen in the degradation potential for other aromatic compounds such as benzoate, -dichlorobenzene, -methylnapthalene, caprolactam, toluene and xylene, trinitrotoluene, biphenyl and styrene degradation. genes involved in the degradation of p-hydroxybenzoate, benzoate, quinate, gentisare, and catechol were also identified in the nine sphingobium genomes. the presence of degradation pathways for phenol/toluene, chlorophenol, anthranilate, and homogentisate are identified in ut26s. these pathways were observed in at least two of the newly sequenced strains. this suggests that these sphingobium spp. possess broad aromatic compound degradation potential, although we did not observe the presence of these compounds at the hch dumpsite. the link between these aromatic degradation pathways and the hch degradation pathway requires further investigation.figure functional profiling of the sphingobium genomes. heat map showing the normalized relative abundance of the top subsystems enriched in the nine sphingobium genomes. the strains and enriched pathways were clustered using pearson correlation with a % minimum abundance. the color scale represents the relative abundance of gene content for each pathway, normalized by sample mean. functional profiling was used to analyze pathways that were differentially enriched in these strains. for this, a dendogram was constructed based upon the top subsystems at % minimum abundance using pearson correlation distance. the analysis revealed that the two-component system for gene expression was highly abundant in all of the sphingobium genomes. this system is known to facilitate adaptation to extreme environmental conditions and likely contributes to the ability to survive in conditions of high hch pressure, salinity, and acidity that exist at the hch dumpsite. additionally, the nine strains collectively showed an abundance of abc transporters within their genomes. the abundance of these transporters implies that these strains are highly engaged in transport of a wide variety of substrates across extra- and intracellular membranes, which is consistent with the sphingobium proficiency for degradation of a wide range of xenobiotics. interestingly, hdipo and ip which had a close phylogenetic relationship, demonstrated differences in their functional repertoire, based on the top subsystems. this is primarily driven by an increased abundance of -dichlorobenzene degradation, toluene and xylene degradation, caprolactam degradation, ppar signaling and atrazine degradation pathways in hdipo which was clustered with functional profiling of p and ut26s as they shared these enrichments. furthermore, lipopolysaccharide biosynthesis, tyrosine metabolism, glycan and glycosaminoglycan degradation pathways were found enriched in ip as compared to hdipo this variation suggests that while these strains exhibit similar genomic content, they exhibit differential dominance in their metabolic preferences. nitrogen assimilation and the presence of flagellar genes in non-motile sphingobium the genomes of all the nine strains were found to contain an enrichment of the two component signal transduction system for nitrogen stress response. additionally, the large subunit of assimilatory nitrate reductase, a key regulator that potentially enables the utilization of nitrate as a nitrogen source, was found to be under diversifying natural selection, which suggests that these strains can tolerate low inorganic nitrogen concentrations and are evolving in response to this inorganic nitrogen stress. at high nitrogen concentration, the transmembrane protein glnc responds to nitrogen availability and phosphorylates glng, which in turn leads to the activation of glna. another key regulator of the pathway is glnb, which interacts and regulates the activity of glnc. when the nitrogen availability is low, glnb is subjected to post transcriptional modification by uridylation. this modification is reversed in n-sufficient conditions. thus, the presence of ntrc pathway and nitrate reductase genes explains the ongoing phenomena of nitrogen assimilation by these strains at hch dumpsites to acclimatize themselves in such nitrate concentrations. increasing exposure to elevated hydrocarbon concentrations was found to be positively correlated with the relative abundance of genes associated with nitrogen metabolism. the ntrc pathway is also associated with genes regulating chemotactic response, such as chey, mota, motb, and flagellar biosynthesis proteins, such as flha, flio, flip, flir etc. all these genes were also found in the core-genome. chey modulates the cell’s ability to interact with the flagellum and controls swimming behavior. interestingly, while these sphingobium strains are considered non-motile, each genome housed more than half of the genes needed for flagellar assembly and functioning. this raises the possibility that they are either in a process of acquiring or losing motility. the abundance of chemotaxis and motility genes has already been demonstrated in the metagenome of the hch dumpsite from where hdipo ip p ds and rl were isolated. however, further analysis is needed to probe the reason for retention or loss of flagellar genes in the sphingobium strains, and to investigate whether sphingobium have the potential to gain motility through acquisition of the remaining genes under the high selective pressure of hch in the stressed environments. recruitment of linpathway through different routes the genome analysis revealed a mosaic distribution of lin genes and is elements in hch-degrading sphingobium spp. coupled with high polymorphism levels in the lin genes. this indicates the recruitment of lin genes through different routes in sphingobium spp. under hch stress, and further that the pathway has not yet stabilized in these strains but is instead subjected to further rearrangements and polymorphisms. is6100-mediated recruitment based on mosaic distribution pattern of lingenes the is elements, known for disseminating lin genes through hgt among sphingomonads, were found to be present in all of the newly sequenced strains associated with hch degradation, including strain ds which did not degrade hch. the number, as determined from the genome sequence, varied from copies in ut26s to copies in p. the presence of a large number of is elements reflects a high degree of genomic rearrangement, as the is elements have already been demonstrated to play an important role in the spread and reorganization of the lin pathway in sphingomonads. to further explore the mechanism of hgt in the spread and diversification of the lin system, we examined the colocalization of lin genes with mobile elements such as the insertion sequence is and transposons, and their presence on plasmids. in all of strains where lina gene was present in, it was found in nearly identical association with is elements as in ut26s i.e. is was found within proximity of <5kbp. however, in rl two is copies lies in the same orientation within the above mentioned range. hence, this suggests that among these strains the association of lina with is is consistent, but the reason and possible involvement of is in the mechanism of duplication of the lina gene in rl needs to be identified. in ip linb was found to be flanked on both sides by is. additionally, resolvase genes were found at the flanking ends of both of these transposons. strains ll and p did not contain the linb gene, as confirmed by pcr amplication. thus, either these strains have yet to acquire the linb gene, or, given the flanking is elements, it is suggested that the loss of linb could have occurred via an intra-chromosomal single homologous recombination between two copies of is.figure lin genes and their association with is 6100 in hch degrading sphingobium spp. linb associated with is at both its end in strain ip association of linder with homogentisate degradation pathway genes hmga and hmgb separated by is in strain rl in hdipo a truncated copy of linf along with complete set of linc and linb was found with an is element. in contrast, in the case of the reference ut26s, these elements were dispersed, with linf present on chromosome and linb and linc on chromosome the association of these three elements suggests that they may have been brought together by is6100-mediated transposition, a hypothesis supported by the fact that hdipo contains a high number of is comparable to ut26s, and that they may be in the process of forming an operon. of the three copies of linder present in rl one was closely associated with the hmgb and hmga genes of the homogentisate degradation pathway, separated by a copy of is. in contrast, in ut26s, linder was housed on a plasmid, while hmgb and hmga were found on chromosome. therefore, in rl it is possible that these two different aromatic compound degradation pathways were brought into close proximity by is mediated transposition. thus, is apart from the spread of lin gene system, might be effective in the spread of homogentisate pathways despite the absence of homogentisate selective pressure at the hch dumpsite, consistent with the fact that already sphingomonads that degrade aromatic hydrocarbons were found to contain catabolic genes associated with is. in strain ll isolated from the czech republic, linghij genes were associated with is whereas in the ut26s genome, isolated from japan, is was absent from the region proximal to these lower pathway genes. as is is reported to be a key driver in the recruitement of the lin system, differential organization of the is element with respect to lin genes for strains from geographically-disparate locations reflects an ongoing is6100–driven evolution of the lin system, including the lower degradation pathway components such as linghij. is elements have also been found in the genome of ds which did not degrade hch isomers. however, in ds the regions flanking the is elements comprised a variety of xenobiotic tolerance and degradation genes, which further supports the role of is in distributing genes for a broad-range of such functions in sphingobium spp. the fact that ds a non-hch degrader, maintained copies of is elements clearly suggests the potential of this strain to acquire lin genes through is mechanisms in the future. plasmid mediated recruitment in investigating the presence and spread of the lin genes, the recently sequenced genome of an hch-degrader sphingomonas sp. mm- is of interest as it was found to have five plasmids housing the genes of the lin pathway. in the mm- genome, the linf was found on pisp0; lina, linc, and a truncated linf on pisp linder on pisp and linb, linc, and another truncated linf on pisp and linghij was found on pisp genes for an abc transporter were found on the chromosome, but these did not share at least 80% identity to the linklmn genes of ut26s. in addition to this, in strain ut26s, hch-specific genes of the lin pathway were found to be housed on regions unique to the ut26s genome; with lina, linb, linc genes in chromosome linf on chromosome and linder on the plasmid pchq. the lower pathway genes, including linghij and linklmn were found on chromosomes and respectively, in regions that were conserved among sphingomonads. genome recruitment plots were created to map the raw reads of the six novel-sequenced strains to sphingobium plasmid sequences to investigate the possibility of these plasmids playing a role in transfer of the lin genes. mm- plasmids pisp and pisp in particular were found to have a high percentage of coverage which was maximum with s. ummariense rl. as pisp houses linder, it is highly probable that plasmid uptake and duplication may explain the triplication of linder in rl the recent metagenomics analysis of the hch dumpsite also reflected the enrichment of pisp suggesting its availability for other sphingomonads strains present at the hch dumpsite. furthermore, pisp encodes linb, linc, and linf, and similarly shows a high degree of coverage by rl consistent with the absence of linc from the rl draft genome, which was confirmed by pcr amplification by using the primer 5′-gcggatccgcatgtctgatttgagcggc-3′ and 3′-gcctcgagtcagatcgcggtaaagccgccgtc-5′, there is a gap in the coverage seen in the plasmid region containing linc, which is a region flanked by two is elements in mm-. this points to the possibility that the plasmid has undergone either acquisition in mm- or looping out from rl of the linc gene during the course of evolution, mediated directly by is mapping the raw reads of the six newly-sequenced sphingobium strains to the plasmid sequences for mm- and ut26s, several of the mm- plasmids, but none of the ut26s plasmids demonstrated a high degree of coverage. additionally, the proportionally higher presence of lin genes on plasmids in mm- than in ut26s suggests that strain mm- acts as a reservoir for plasmids allowing for the effective spread of the lin system, and thus may be an important strain to include in the consortium development as a potential disseminator of the lin system. also, strains sharing similar arrangement profile of lin genes with mm- i.e., rl ip and hdipo should be included into designing a consortium.figure genome recruitment plot mapping on sphingomonas sp mm- plasmid a) pisp and b) pisp4: the raw reads of the six novely sequenced strains were mapped on plasmids of sphingomonas sp. mm the orange bars are depicting is while the lin genes were marked with grey and yellow bars showing their position in reference. strains in transition to acquire linpathway of the nine sphingomonads under study, seven possessed components of the upper hch degradation pathway to varying degrees of completion, and two, syk and ds were completely devoid of them. syk did not contain any components of the lin system and the ds genome contained only genes of the lower lin pathway- linklmn an abc transporter. of the hch-degraders, not every strain was found to house the complete array of lin genes characterized in ut26s or b90a. for instance, the p genome lacked linb, linc, linder, lingh, lini and linj genes while, strains rl and ll both lacked linc and ll lacked linb, as confirmed by pcr amplificiation. the differential composition of the lin system between these strains may be indicative of different steps in the evolution of the lin pathway, with ip in the stage of probable homologous recombination and looping out of linb, while ll shows potential gain of linghij through is6100-mediated hgt. strain ds possesses abc transporters and shows potential for acquisition of the lin genes, as it holds copies of is while p in addition to the abc transporter, has lina and linf but is yet to acquire the other lin genes. linsystem sequence diversity and its effect on metabolic efficiency upper linpathway the upper pathway genes lina, linb and linc degrade γ-hch and α-hch, and additionally linb acts on β-hch, leading to the formation of β-,,6-pentachlorocyclohexanol. as α- and β-hch form the major components of contamination at the hch dumpsite, both lina and linb are extremely important enzymes encoding hch dehydrochlorinase and haloalkane dehalogenase, respectively. to gain deeper insights into the lin gene sequence diversity and its impact on hch degradation, the genetic divergence of the lin system components was analyzed with respect to the copy number and nucleotide sequence divergence of the lin genes in both upper and lower degradation pathways, using b90a as a reference. the highest level of divergence in the upper hch degradation pathway lin genes was reported for the lina gene encoding for hch dehydrochlorinase in b90a. the two previously characterized lina variants observed in b90a differed by 10% of their amino acid sequence, and were named lina and lina the functional aspects of these variants have been well characterized, as they show enantioselectivity in α-hch degradation, with lina selective for the and lina for the α hch. also, the degradation ability of lina was found to be lower than that of lina. among all the lin genes of the upper pathway, lina in the present study was found to be most diverged in hdipo in which it appeared to be a hybrid of the two variants with % sequence similarity to lina and to lina near to the catalytic dyad d and h critical for its enzymatic activity, the hdipo lina was found to be identical with lina. however, the c-terminal region corresponded to lina. this hybrid copy, now marked as lina requires further experimentation, but might be responsible for the comparatively better dehydrochlorination activity of hdipo against α- and γ-hch, as reported earlier.figure a) multiple sequence alignments of lina b) linb gene sequences in the strains showing variation. amino acid sequences of lina and linb genes from the strains were aligned using bioedit to depict the substitutions at the respective sites. apart from this divergence of the lina sequence in hdipo not such changes to the lina gene sequences were observed; all strains showed 100% sequence similarity to that of the lina gene with the exception that lina of rl showed a single substitution of l78q. it is important to mention here that the lina gene has already been reported to be under continuous selection pressure and a large number of variants of this gene exist and better variants of lina may be used for developing enzymatic bioremediation system for hch. in contrast to lina, there were less variations in linb sequences among strains under study. the sequence differences among lina and linb genes among different sphingobium spp. are particularly interesting in light of findings that marginal differences in the amino acid sequences of linb in ut26s, sp+, b90a, bhc-a and m can alter the efficacy and substrate range, with the former group degrading β-hch to β-pchl and the latter group taking the pathway beyond pchl to tchl. hdipo housed two identical linb copies with a t81a substitution and overall % similarity to b90a while rl linb gene had % identity, with three substitutions as compared to linb of b90a. here, the copy number difference is suspected to have a more impact, as the two copies of linb might explain the high β-hch degradation efficacy of hdipo. apart from these two strains, no such diversity was observed, thus demonstrating the stability of linb gene in the population. linc, which encodes for hch dehydrogenase was most conserved among the genes of the upper degradation pathway and demonstrated only a single substitution: y172c in case of ip in any case these studies reflect that lina genes are more prone to evolutionary changes under hch stress and have not stablized yet. lower linpathway the lower pathway of γ-hch degradation begins from -dichlorohydroquinone an intermediate of γ-hch, which is mineralized by the lower pathway lin genes. in contrast to the upper degradation pathway, very less is known about the divergence and polymorphisms of the genes of the lower degradation pathway. among all the lin genes of the lower pathway, linf was the most highly conserved, as its amino acid sequence was 100% identical in all genomes. in the linder operon, the set of lind genes similarly showed minimal divergence, with the ip rl and ll genes sharing a substitution of n82s, and additionally ip having a substitution q30p. further, linr and line had very little divergence; linr diverged only in one substitution in hdipo and line was 100% identical in all strains. this highlights the fact that the linder operon, which makes up the backbone of the downstream hch degradation pathway, remained highly stable during the course of evolution. a greater degree of the variation of this operon was found, however, in copy number, as rl and hdipo housed three and two copies, respectively.figure genetic sequence and copy number variation of lin genes: the genetic divergence, as quantified by percentage nucleotide identity to the archetypal strain ut and copy number variation in lin genes across the nine sphingobium strains under study. in particular lingh, lini and linj, which mediate the later stages of the lower degradation pathway, i.e., conversion of β-ketoadipate to succinyl coa and acetyl coa, showed variation in the sequences of linh and lini, whereas ling and linj sequences were 100% conserved among all these strains. here, linh of hdipo and ip were similar to each other, and both diverged from b90a with % identity. they held two substitutions while ll shared the i31v substitution and additionally had a n131d substitution. lini was found to be identical in hdipo and ip with a single substitution and % identity to b90a, while ll had two substitutions and % identity. however, the significance of sequence divergence in ling, linh, lini, and linj genes among these strains is yet to be investigated. another important lin gene system of the lower pathway is the abc transporter system i.e., link, linl, linm, and linn, which encode a permease, atpase, periplasmic protein, and lipoprotein, respectively. this abc transpoter system is very important as it allows for the transport of hch isomers and clearance of dead-end metabolites of hch from the cell. out of the entire lin system these genes have shown the highest level of of divergence with link at % in rl linl at % in ds linm at % in ds and linn at % in p based on the prevalence of similar but non lin-specific abc type transporters which are found by sequence identity searches across a variety of microbial species, it is hypothesized that the linklmn operon derived from convergent evolution in response to environment changes. with the introduction of the hch to the environment, pre-existing abc-type transporters were likely recruited to the hch degradation pathway, and thus several genetic variants might have undergone convergent evolution to select for transporters with increased efficiency for hch-metabolite efflux, and these later generation genes were the one that subsequently underwent hgt. this is in contrast to the likely origin of the linder operon, which encodes more highly hch-specific enzymatic functions and is almost perfectly conserved, and thus was likely generated once, and spread through hgt from a single genetic ancestor. the lin pathway shows a characteristic pattern in which the upper hch degradation pathway was diverged along a gradient from the most, lina to the least, linc. while in the lower degradation pathway linklmn was the most highly diverged, followed by linghij, linder and linf respectively. in literature, the degradation ability of these strains under consideration is in the order: hdipo4>ip26>rl in order to further substantiate the relationship between lin system diversity and degradation efficiency, principle component analysis was performed on the copy number and sequence divergence for these strains, with hch degradation plotted as a supplementary variable to visualize correlation. the analysis demonstrated a close grouping of the four lin-deficient strains, i.e. syk ds ll and p under pca and. hdipo and ip were colocalized in quadrant opposite from the non-degrader cluster in terms of both dimension and dimension which is appropriate given that these two strains have degradation rates documented to be faster than archetypal strains ut26s and b90a. while copy number variation for linf, linder, and linb were mapped most closely to the hch degradation vector, the copy numbers for linc, linghij and again linb played a more important role in differentiating these strains, as they correlated significantly to pca. in terms of sequence, lina and linr showed high correlation to degradation ability, while linl, linm, and link were significantly correlated to pca and lini, linh, linc, linb, and linf were the most significant contributors to pca. this suggests that while variation can be seen throughout the lin pathway and the copy number of linf, linder and linb sequence for lina and r might have the most impact in optimizing the efficacy of an hch enzymatic bioremediation system.figure principal component analysis of genes involved in hch degradation pathway. pca individual factor plot showing the grouping of the nine sphingobium strains based upon the sequence divergence and copy number of the set of lin genes. principle components and were chosen as the separation of the strains by these pcs demonstrated the highest fidelity to known hch degradation ability. pca variable factor plot using principle component and showing the contribution of the lin genetic sequences and copy number to the variation of the nine sphingobium strains. hch degradation ability was plotted as a supplementary categorical variable, with non-hch degraders coded as partial degraders as and complete degraders as genes under diversifying natural selection to identify the substitutions that have fixed along the independent lineages and their direction of evolution, dn/ds analysis was performed for sets of orthologous genes, and in particular those responsible for the degradation of hch, phenol/toluene, homogentisate, chlorophenol and transposons/integrases. the genes of interest found to be under diversifying selection includes those for fe dependent oxygenase, abc transporters, assimilatory nitrate reductase, and general secretory pathway protein. these genes are largely associated with stress tolerance. as mentioned earlier, abc transporters are involved in uptake of high molecular weight pharmacological agents including xenobiotic compounds. hence, these transporters and their activity are crucial for their capability of hch degradation and other aromatic compounds and likewise, their positive selection indicates the importance of their function in the hch-stressed environments from which these bacterial strains were isolated. additionally, nitrate reductase catalyzes the conversion of nitrate into free nitrogen and likely would enable the sphingobium spp. to more effectively enact a nitrogen stress response. finally, the diversification of genes such as oxygenases, secretory pathway proteins and translocase components adds to the sphingomonads skill of degradation of a wide range of aromatic compounds. notably, only linh has shown the dn/ds >  while the other lin genes did not. this clearly indicates that under strong hch pressure, the whole lin pathway is likely to get stabilized in the population with more synonomous substitutions compared to non-synonomous ones and tends to be retained in the population.figure dn/ds v/s ds plot of orthologs, xenobiotic degradation pathway genes and is elements/integrases. sequences of orthologs, xenobiotic degradation pathway genes and is elements/integrases were used for calculating their dn/ds. genes having dn/ds value > are under positive natural selection.  in sequencing the genomes of six novel sphingobium species and comparing these to the known genomes of three other sphingobium species, this study has begun to probe the natural variation in the lin pathway for hch degradation. analysis of the variation in the lin system, as well as in the phylogenetic relationships, core genomes, and functional profiles of these bacterial strains demonstrated unique characteristics of b90a, hdipo and ip which could explain their higher efficacy as the degraders of hch isomers. the information thus obtained can now be used to select these better-performing strains for the development of a bacterial consortium for on-site bioremediation of the hch dumpsites. focusing on the lin system, analysis of the similarities in the lin genes sequences and varying copy numbers between these strains has identified variations in the specific genes as key differentiators and these key components will be of critical interest as the most effective targets for optimization of an enzymatic bioremediation system. the analysis so far made reflect that better lina and linb variants can eventually be the ideal candidates for developing an enzymatic bioremediation system. moreover, this study has uncovered evidence for genus-level hgt of plasmids housing components of the lin system, specifically between sphingomonas sp. mm- and rl the additional lin-deficient strains are of further importance as they demonstrate varying degrees of acquisition of the lin system and will be useful in future homologous recombination studies to work with manipulated pathway completion through introduction of synthetic lin genes. 
further, the diversity in the lin gene sequences and copy number, their arrangement with respect to is and evidence for potential plasmid content elucidate possible evolutionary acquisition mechanisms for this pathway. are efficient degraders of a wide range of chlorinated and aromatic hydrocarbons. isolated from hch dumpsites, with three existing genomes. the bacteria isolated from hch dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy. in particular, strains which harbour the lin pathway genes mediating the degradation of hexachlorocyclohexane isomers are of interest due to the widespread persistence of this contaminant. here, we examined the evolution and diversification of the lin pathway under the selective pressure of hch, by comparing the draft genomes of six newly-sequenced sphingobium spp. while strains hdipo and ip contained the complete set of lin genes, ds was entirely devoid of lin genes whereas, ll p and rl were identified as lin deficient strains, as they housed incomplete lin pathways. genes associated with nitrogen stress response and two-component systems were found to be enriched. this study further opens the horizon for selection of bacterial strains for inclusion in an hch bioremediation consortium and suggests that hdipo ip and b90a would be appropriate candidates for inclusion. hexachlorocyclohexane sphingobiumlin genesxenobiotic compoundsissue-copyright-statement© the author 2014 less than 10% of the total gene content was shared among all nine strains, but among the eight hch-associated strains, that is all except syk the shared gene content jumped to nearly 25%.
 sphingobium spp. are efficient degraders of a wide range of chlorinated and aromatic hydrocarbons. in particular, strains which harbour the lin pathway genes mediating the degradation of hexachlorocyclohexane isomers are of interest due to the widespread persistence of this contaminant. here, we examined the evolution and diversification of the lin pathway under the selective pressure of hch, by comparing the draft genomes of six newly-sequenced sphingobium spp. isolated from hch dumpsites, with three existing genomes.  efficient hch degraders phylogenetically clustered in a closely related group comprising of ut26s, b90a, hdipo and ip where hdipo and ip were classified as subspecies with ani value >98%. less than 10% of the total gene content was shared among all nine strains, but among the eight hch-associated strains, that is all except syk the shared gene content jumped to nearly 25%. genes associated with nitrogen stress response and two-component systems were found to be enriched. the strains also housed many xenobiotic degradation pathways other than hch, despite the absence of these xenobiotics from isolation sources. additionally, these strains, although non-motile, but posses flagellar assembly genes. while strains hdipo and ip contained the complete set of lin genes, ds was entirely devoid of lin genes whereas, ll p and rl were identified as lin deficient strains, as they housed incomplete lin pathways. further, in hdipo lina was found as a hybrid of two natural variants i.e., lina and lina known for their different enantioselectivity.  the bacteria isolated from hch dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy. further, the diversity in the lin gene sequences and copy number, their arrangement with respect to is and evidence for potential plasmid content elucidate possible evolutionary acquisition mechanisms for this pathway. this study further opens the horizon for selection of bacterial strains for inclusion in an hch bioremediation consortium and suggests that hdipo ip and b90a would be appropriate candidates for inclusion. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords hexachlorocyclohexane sphingobiumlin genesxenobiotic compoundsissue-copyright-statement© the author 2014 