BACKGROUND
genes are non-randomly ordered on chromosomes.
in eukaryotes, co-expressed genes tend to cluster across all kingdoms  <cit> , with cluster sizes ranging from kilobases in yeast to megabases in mammals  <cit> .
co-expression of neighbouring genes may result from use of common promoters or upstream activating sequences, while the transcriptional activity of larger chromosome domains is regulated by the structure of the chromatin and/or its spatial positioning within the nucleus  <cit> .
some of the observed grouping of co-expressed genes likely reflects clustering of functionally related genes  <cit> , while some may be attributed to transcriptional leakage  <cit> .
in bacteria and archaea, the most obvious case of gene clustering is the organisation of genes into co-transcribed cassettes, operons.
this facilitates tight co-regulation of genes encoding proteins involved in the same cellular pathway, or of subunits of the same protein complex  <cit> .
adjacent location of multi-gene functional entities also increases the chance for their co-transfer in lateral transfer events, likely to be important for efficient horizontal propagation  <cit> .
conserved bidirectionally transcribed gene pairs have also been observed, typically involving a transcriptional regulator that shares the promoter region with a target operon  <cit> .
non-random gene order is also evident at much larger scales  <cit> , such as a  <dig> -  <dig> kb periodic pattern of gene co-expression observed in escherichia coli and bacillus subtilis  <cit> , which likely reflects how the chromosome is spatially structured in the nucleoid.
in bacteria, other trends have also been observed, such as higher incidence of essential genes on the leading strand  <cit> , clustering of evolutionary persistent genes  <cit> , and clustering of genes involved in transcription and translation near the origin of replication in fast-growing bacteria  <cit> .
archaeal organisms exhibit both bacterial and eukaryotic-like features.
in particular, the information-processing systems  closely resemble their eukaryotic counterparts  <cit> .
sulfolobus species are thermoacidophilic crenarchaea, serving as model systems for the archaeal cell cycle  <cit> .
the sulfolobus cell cycle is characterised by a short pre-replicative phase, an s phase of about a third of the generation time, and a long post-replicative phase  <cit> .
global gene expression analysis has revealed that at least 10% of the sulfolobus acidocaldarius genes display cyclic expression during cell cycle progression  <cit> , including a unique cell division machinery, the cdv system, that recently was identified based on the expression data  <cit> .
in contrast to all studied bacteria and most archaea, sulfolobus chromosomes harbour multiple origins of replication.
marker frequency analysis has shown that replication is initiated in near synchrony at the three origins and, due to the uneven spacing of the origins, asynchronously terminated on the circular chromosome  <cit> .
the selective advantage of multiple origins  is not clear: the fact that the origins are unevenly distributed on the chromosome  is, for instance, not in agreement with models in which shortening of the replication time would be the main selective force.
here, we performed global gene expression analysis in exponentially and stationary phase cells, and investigated other properties of genome organisation in sulfolobus.
the results demonstrate that the sulfolobus chromosome is organised in a highly replication-biased manner, such that levels of gene expression, as well as genome sequence-derived parameters, are correlated with distance to nearest replication origin.
RESULTS
to monitor the distribution of gene expression over the sulfolobus chromosome, we harvested rna from sulfolobus solfataricus and s. acidocaldarius cell cultures in exponential and stationary phase.
the rna was reverse transcribed into cdna, labelled and hybridised onto spotted whole-genome dna microarrays  <cit> .
to compensate for differences in array probe concentrations and hybridisation efficiencies, the cdna was co-hybridised with differentially labelled genomic dna derived from stationary phase cultures.
as stationary phase cells exclusively contain fully replicated chromosomes  <cit>  and all genes, consequently, are present in equal copy number, the cdna/genomic dna ratios serve as estimates of relative transcript abundances.
gene expression was non-randomly distributed over the chromosomes in both species .
transcriptionally active regions coincided with replication origins, and gene expression was negatively correlated with distance to the nearest origin in both genomes .
the gene expression gradients were significantly more pronounced than what would be anticipated from gene dosage effects alone in growing populations.
thus, while the average gene copy number ratio between the earliest and latest replicating chromosome regions in a growing sulfolobus population is about  <dig>  fold  <cit> , the average expression ratio between genes located proximally and distantly relative to the nearest origin was > <dig> fold .
a negative, but weaker, correlation to distance from the nearest origin was observed also when cdna derived from s. acidocaldarius cells in stationary phase was hybridised , whereas in s. solfataricus there was no such correlation .
comparative genomics has revealed a core of  <dig> genes present in all archaea  <cit> .
evolutionary persistent genes like these are highly enriched in house-keeping functions essential to the organism  <cit> , and such genes have been found to cluster in both bacteria  <cit>  and eukaryotes  <cit> .
we found that genes representing clusters of orthologous groups  present in all archaea  clustered in the early replicating regions and were absent in large regions of the genomes , and that the density of archaeal core genes was significantly negatively correlated with distance to nearest origin in both organisms .
also ribosomal rna  genes and transfer rna  genes clustered in the highly expressed regions .
the genomic parameters were calculated in  <dig> kb, non-overlapping windows .
numbers indicate spearman rank-order correlation coefficients, ρ, with p values  within parenthesis.
compared to eukaryotes, prokaryotic genomes are highly compact, with short intergenic regions and generally few repeated elements.
nevertheless, elevated gene densities were observed in the early replicating  regions .
gene density was hence negatively correlated with distance to nearest origin, and positively correlated with gene expression, in both genomes .
as this could potentially be a consequence of that highly expressed genes relatively frequently belong to polycistronic transcripts, with short intergenic regions, we investigated distances between adjacent genes on opposite strands .
these distances increased with distance to origin , indicating that the increased gene density in early replicating regions could not be explained only by an increased operon incidence.
the s. solfataricus chromosome is one of the most transposon-dense of all sequenced genomes  <cit> .
also for this feature a non-random distribution could be observed, as also noted previously  <cit> , with transposon density being positively correlated with distance to nearest origin .
discussion
we observed a strongly replication-biased genome organization in the two sulfolobus species, despite that massive genomic rearrangements have occurred since the organisms diverged , which indicates that the trait is under selection.
replication-biased genome organisation has also been reported in bacteria .
the nature of the sulfolobus genome organisation is, however, different in several aspects.
fast-growing bacteria with multiple simultaneously ongoing rounds of replication  display increased densities of highly expressed genes  near origins.
however, the relationship only holds true for genes involved in transcription and translation and has been suggested to reflect selection for gene dosage effects, advantageous during rapid growth  <cit> .
in contrast, in sulfolobus the correlation between gene expression and distance to origin remains even if transcription and translation genes are excluded .
moreover, in slow-growing bacteria  only rrna genes are generally located close to origins  <cit> .
if the replication-biased genome organisation in sulfolobus does not correspond to selection for gene dosage effects, what does it reflect?
the clustering of archaeal core genes near the origins may indicate selection for early replication of essential genes, since evolutionary persistent genes often are essential  <cit> .
also, since essential genes often are highly expressed , the observed expression gradient could be a secondary effect of their biased distribution.
to evaluate if core genes cluster near origins independently of expression level, we binned the s. acidocaldarius genes based on expression levels into  <dig> equally sized bins .
the binning was sufficiently fine-grained to remove expression differences between core and non-core genes; within the  <dig> bins that included genes of both categories there were no significant differences in expression levels between the two groups .
however, the distance to nearest origin was significantly shorter  for core than non-core genes in  <dig> out of  <dig> bins, showing that essential genes cluster near origins independently of expression level, and may, thus, cause the correlation between expression and distance to origin.
if so, one would perhaps expect the expression gradient to disappear when analysing core and non-core genes separately.
this is however not the case; although weaker than for the whole dataset, the correlations remain for both groups of genes .
however, the extent to which individual genes contribute to fitness likely varies within the two groups, and a more fine-grained binning according to fitness contribution would be needed to determine its effect on the expression gradient.
the multiple origins may hence potentially serve to promote fast backup of essential genetic material.
this could also explain the shorter intergenic distances, lack of transposons, and clustering of rrna and trna genes in early replicating regions.
having two copies of a gene facilitates expression of a functional protein even if one copy is damaged, thereby preventing cell death.
the intact chromosomal copy could potentially also facilitate repair of the damaged dna by homologous recombination.
in support, sister chromatid junctions have been observed near replication origins in s. solfataricus  <cit> .
sulfolobus species inhabit geothermal environments where thermal dna damage, in particular deamination, depurination and oxidation, are prone to occur frequently  <cit> .
in addition, the aerobic metabolism requires surface growth with consequent uv exposure.
it has been demonstrated that the dna repair systems of sulfolobus are constitutively expressed in batch cultures  <cit> , reflecting this life style, and that expression is correlated to the replicative cell cycle stage  <cit> .
a need for backup of genetic material would also be in line with the organisation of the sulfolobus cell cycle, in which replication is initiated shortly after cell division, and two complete chromosomes thus are present during most of the cell cycle, as well as in all cells in stationary phase  <cit> .
this organisation of the cell cycle has been shown to be widely conserved among crenarchaea  <cit> .
however, despite the fact that pronounced clustering of archaeal core genes, coinciding with elevated coding densities, is apparent in all sulfolobales genomes , this does not appear to be a general feature of crenarchaea .
it is possible that the anaerobic lifestyle characteristic of most other genome-sequenced crenarchaea may reduce the mutation frequency and, consequently, the selective advantage of this higher-order genome structure.
our data indicate that genome organisation reflects selection for early backup of essential genetic material, but we cannot rule out that other selective forces may affect gene localisation.
in higher eukaryotes, chromosome structure is tightly linked to gene regulation and to replication timing  <cit> .
although a suite of architectural proteins have been identified and characterised in archaea  <cit> , relatively little is known about archaeal chromosome structure and its potential role in gene regulation  <cit> .
however, the identification of dna-binding proteins that can undergo methylation  <cit>  and acetylation  <cit> , as well as expression of chromatin-organizing proteins that is dependent on growth phase  <cit>  and cell cycle progression  <cit> , indicate dynamic chromosome structures in archaea which may have implications for transcriptional regulation.
thus, the organisation of highly expressed genes near replication origins in sulfolobus might, in addition to selective forces related to dna repair and genetic back-up, reflect a higher order chromosome structure centred at replication origins.
a recent comparative genomics study of seven s. islandicus isolates revealed a large genomic region that was enriched in gene insertion and deletions  <cit> .
our analysis shows that this region coincides with a region that is very low in archaeal core genes and has low gene density .
whether this reflects selection against insertions and deletions in regions dense in essential genetic material, or that the chromosome structure of these regions physically prevents recombination events, is an interesting topic for further investigation.
CONCLUSIONS
our study reports a strong replication-biased structuring of the sulfolobus chromosome which implies that the multiple replication origins serve purposes other than only shortening the time required for replication.
the higher-level chromosomal organisation may be of importance for minimizing the impact of dna damage during growth in extreme environments and is possibly related to chromosome structure.
the findings provide a basis for further investigation of chromosome organisation, transcription patterns and gene regulation in archaea, as well as of the evolutionary forces that promote different levels of transcriptional and chromatin organisation.

 genes are non-randomly ordered on chromosomes. in eukaryotes, co-expressed genes tend to cluster across all kingdoms, with cluster sizes ranging from kilobases in yeast to megabases in mammals. co-expression of neighbouring genes may result from use of common promoters or upstream activating sequences, while the transcriptional activity of larger chromosome domains is regulated by the structure of the chromatin and/or its spatial positioning within the nucleus. some of the observed grouping of co-expressed genes likely reflects clustering of functionally related genes, while some may be attributed to transcriptional leakage. in bacteria and archaea, the most obvious case of gene clustering is the organisation of genes into co-transcribed cassettes, operons. this facilitates tight co-regulation of genes encoding proteins involved in the same cellular pathway, or of subunits of the same protein complex. adjacent location of multi-gene functional entities also increases the chance for their co-transfer in lateral transfer events, likely to be important for efficient horizontal propagation. conserved bidirectionally transcribed gene pairs have also been observed, typically involving a transcriptional regulator that shares the promoter region with a target operon. non-random gene order is also evident at much larger scales, such as a - kb periodic pattern of gene co-expression observed in escherichia coli and bacillus subtilis, which likely reflects how the chromosome is spatially structured in the nucleoid. in bacteria, other trends have also been observed, such as higher incidence of essential genes on the leading strand, clustering of evolutionary persistent genes, and clustering of genes involved in transcription and translation near the origin of replication in fast-growing bacteria. archaeal organisms exhibit both bacterial and eukaryotic-like features. in particular, the information-processing systems closely resemble their eukaryotic counterparts. sulfolobus species are thermoacidophilic crenarchaea, serving as model systems for the archaeal cell cycle. the sulfolobus cell cycle is characterised by a short pre-replicative phase, an s phase of about a third of the generation time, and a long post-replicative phase. global gene expression analysis has revealed that at least 10% of the sulfolobus acidocaldarius genes display cyclic expression during cell cycle progression, including a unique cell division machinery, the cdv system, that recently was identified based on the expression data. in contrast to all studied bacteria and most archaea, sulfolobus chromosomes harbour multiple origins of replication. marker frequency analysis has shown that replication is initiated in near synchrony at the three origins and, due to the uneven spacing of the origins, asynchronously terminated on the circular chromosome. the selective advantage of multiple origins is not clear: the fact that the origins are unevenly distributed on the chromosome is, for instance, not in agreement with models in which shortening of the replication time would be the main selective force. here, we performed global gene expression analysis in exponentially and stationary phase cells, and investigated other properties of genome organisation in sulfolobus. the results demonstrate that the sulfolobus chromosome is organised in a highly replication-biased manner, such that levels of gene expression, as well as genome sequence-derived parameters, are correlated with distance to nearest replication origin.  to monitor the distribution of gene expression over the sulfolobus chromosome, we harvested rna from sulfolobus solfataricus and s. acidocaldarius cell cultures in exponential and stationary phase. the rna was reverse transcribed into cdna, labelled and hybridised onto spotted whole-genome dna microarrays. to compensate for differences in array probe concentrations and hybridisation efficiencies, the cdna was co-hybridised with differentially labelled genomic dna derived from stationary phase cultures. as stationary phase cells exclusively contain fully replicated chromosomes and all genes, consequently, are present in equal copy number, the cdna/genomic dna ratios serve as estimates of relative transcript abundances. gene expression was non-randomly distributed over the chromosomes in both species. transcriptionally active regions coincided with replication origins, and gene expression was negatively correlated with distance to the nearest origin in both genomes. the gene expression gradients were significantly more pronounced than what would be anticipated from gene dosage effects alone in growing populations. thus, while the average gene copy number ratio between the earliest and latest replicating chromosome regions in a growing sulfolobus population is about fold, the average expression ratio between genes located proximally and distantly relative to the nearest origin was > fold. a negative, but weaker, correlation to distance from the nearest origin was observed also when cdna derived from s. acidocaldarius cells in stationary phase was hybridised, whereas in s. solfataricus there was no such correlation. comparative genomics has revealed a core of genes present in all archaea. evolutionary persistent genes like these are highly enriched in house-keeping functions essential to the organism, and such genes have been found to cluster in both bacteria and eukaryotes. we found that genes representing clusters of orthologous groups present in all archaea clustered in the early replicating regions and were absent in large regions of the genomes, and that the density of archaeal core genes was significantly negatively correlated with distance to nearest origin in both organisms. also ribosomal rna genes and transfer rna genes clustered in the highly expressed regions. the genomic parameters were calculated in kb, non-overlapping windows. numbers indicate spearman rank-order correlation coefficients, ρ, with p values within parenthesis. compared to eukaryotes, prokaryotic genomes are highly compact, with short intergenic regions and generally few repeated elements. nevertheless, elevated gene densities were observed in the early replicating regions. gene density was hence negatively correlated with distance to nearest origin, and positively correlated with gene expression, in both genomes. as this could potentially be a consequence of that highly expressed genes relatively frequently belong to polycistronic transcripts, with short intergenic regions, we investigated distances between adjacent genes on opposite strands. these distances increased with distance to origin, indicating that the increased gene density in early replicating regions could not be explained only by an increased operon incidence. the s. solfataricus chromosome is one of the most transposon-dense of all sequenced genomes. also for this feature a non-random distribution could be observed, as also noted previously, with transposon density being positively correlated with distance to nearest origin. discussion we observed a strongly replication-biased genome organization in the two sulfolobus species, despite that massive genomic rearrangements have occurred since the organisms diverged, which indicates that the trait is under selection. replication-biased genome organisation has also been reported in bacteria. the nature of the sulfolobus genome organisation is, however, different in several aspects. fast-growing bacteria with multiple simultaneously ongoing rounds of replication display increased densities of highly expressed genes near origins. however, the relationship only holds true for genes involved in transcription and translation and has been suggested to reflect selection for gene dosage effects, advantageous during rapid growth. in contrast, in sulfolobus the correlation between gene expression and distance to origin remains even if transcription and translation genes are excluded. moreover, in slow-growing bacteria only rrna genes are generally located close to origins. if the replication-biased genome organisation in sulfolobus does not correspond to selection for gene dosage effects, what does it reflect? the clustering of archaeal core genes near the origins may indicate selection for early replication of essential genes, since evolutionary persistent genes often are essential. also, since essential genes often are highly expressed, the observed expression gradient could be a secondary effect of their biased distribution. to evaluate if core genes cluster near origins independently of expression level, we binned the s. acidocaldarius genes based on expression levels into equally sized bins. the binning was sufficiently fine-grained to remove expression differences between core and non-core genes; within the bins that included genes of both categories there were no significant differences in expression levels between the two groups. however, the distance to nearest origin was significantly shorter for core than non-core genes in out of bins, showing that essential genes cluster near origins independently of expression level, and may, thus, cause the correlation between expression and distance to origin. if so, one would perhaps expect the expression gradient to disappear when analysing core and non-core genes separately. this is however not the case; although weaker than for the whole dataset, the correlations remain for both groups of genes. however, the extent to which individual genes contribute to fitness likely varies within the two groups, and a more fine-grained binning according to fitness contribution would be needed to determine its effect on the expression gradient. the multiple origins may hence potentially serve to promote fast backup of essential genetic material. this could also explain the shorter intergenic distances, lack of transposons, and clustering of rrna and trna genes in early replicating regions. having two copies of a gene facilitates expression of a functional protein even if one copy is damaged, thereby preventing cell death. the intact chromosomal copy could potentially also facilitate repair of the damaged dna by homologous recombination. in support, sister chromatid junctions have been observed near replication origins in s. solfataricus. sulfolobus species inhabit geothermal environments where thermal dna damage, in particular deamination, depurination and oxidation, are prone to occur frequently. in addition, the aerobic metabolism requires surface growth with consequent uv exposure. it has been demonstrated that the dna repair systems of sulfolobus are constitutively expressed in batch cultures, reflecting this life style, and that expression is correlated to the replicative cell cycle stage. a need for backup of genetic material would also be in line with the organisation of the sulfolobus cell cycle, in which replication is initiated shortly after cell division, and two complete chromosomes thus are present during most of the cell cycle, as well as in all cells in stationary phase. this organisation of the cell cycle has been shown to be widely conserved among crenarchaea. however, despite the fact that pronounced clustering of archaeal core genes, coinciding with elevated coding densities, is apparent in all sulfolobales genomes, this does not appear to be a general feature of crenarchaea. it is possible that the anaerobic lifestyle characteristic of most other genome-sequenced crenarchaea may reduce the mutation frequency and, consequently, the selective advantage of this higher-order genome structure. our data indicate that genome organisation reflects selection for early backup of essential genetic material, but we cannot rule out that other selective forces may affect gene localisation. in higher eukaryotes, chromosome structure is tightly linked to gene regulation and to replication timing. although a suite of architectural proteins have been identified and characterised in archaea, relatively little is known about archaeal chromosome structure and its potential role in gene regulation. however, the identification of dna-binding proteins that can undergo methylation and acetylation, as well as expression of chromatin-organizing proteins that is dependent on growth phase and cell cycle progression, indicate dynamic chromosome structures in archaea which may have implications for transcriptional regulation. thus, the organisation of highly expressed genes near replication origins in sulfolobus might, in addition to selective forces related to dna repair and genetic back-up, reflect a higher order chromosome structure centred at replication origins. a recent comparative genomics study of seven s. islandicus isolates revealed a large genomic region that was enriched in gene insertion and deletions. our analysis shows that this region coincides with a region that is very low in archaeal core genes and has low gene density. whether this reflects selection against insertions and deletions in regions dense in essential genetic material, or that the chromosome structure of these regions physically prevents recombination events, is an interesting topic for further investigation.  our study reports a strong replication-biased structuring of the sulfolobus chromosome which implies that the multiple replication origins serve purposes other than only shortening the time required for replication. the higher-level chromosomal organisation may be of importance for minimizing the impact of dna damage during growth in extreme environments and is possibly related to chromosome structure. the findings provide a basis for further investigation of chromosome organisation, transcription patterns and gene regulation in archaea, as well as of the evolutionary forces that promote different levels of transcriptional and chromatin organisation. 
the higher-level chromosomal organisation could be of importance for minimizing the impact of dna damage, and may also be linked to transcriptional regulation. we demonstrate that global gene expression in two sulfolobus species is highly biased, such that early replicating genome regions are more highly expressed at all three origins. in addition, early replicating regions are denser in archaeal core genes, display lower intergenic distances, and are devoid of mobile genetic elements. the strong replication-biased structuring of the sulfolobus chromosome implies that the multiple replication origins serve purposes other than simply shortening the time required for replication. species of the crenarchaeon sulfolobus harbour three replication origins in their single circular chromosome that are synchronously initiated during replication. the bias by far exceeds what would be anticipated by gene dosage effects alone.
 species of the crenarchaeon sulfolobus harbour three replication origins in their single circular chromosome that are synchronously initiated during replication.  we demonstrate that global gene expression in two sulfolobus species is highly biased, such that early replicating genome regions are more highly expressed at all three origins. the bias by far exceeds what would be anticipated by gene dosage effects alone. in addition, early replicating regions are denser in archaeal core genes, display lower intergenic distances, and are devoid of mobile genetic elements.  the strong replication-biased structuring of the sulfolobus chromosome implies that the multiple replication origins serve purposes other than simply shortening the time required for replication. the higher-level chromosomal organisation could be of importance for minimizing the impact of dna damage, and may also be linked to transcriptional regulation. 