BACKGROUND
species in the genus rhodobacter are purple nonsulfur bacteria found mostly in freshwater environments  <cit> .
a hallmark of purple nonsulfur bacteria is that they display tremendous physiological diversity  <cit> .
genome sequences are available from two rhodobacter species, r. capsulatus
<cit>  and r. sphaeroides
<cit> , and transcriptional profiling studies have been performed with both species .
these two species have been widely studied as model organisms for anoxygenic photosynthesis, carbon and nitrogen fixation, chemotaxis and flagellar motility, and various regulatory systems including quorum sensing, two-component phosphorelays and those responsible for regulation in response to o <dig> and light
.
r. capsulatus is also a model organism for study of a gene transfer agent, rcgta, which is a virus-like particle that packages small segments of the genome of a gta-producing cell that can then be transferred to recipient cells  <cit> .
weighted gene co-expression network analysis  has been widely used to analyze transcriptional profiles since its introduction in  <dig>  <cit> , and has proved to be a useful approach for the functional annotation of uncharacterized genes  <cit> .
in a recent critical assessment of methods for constructing gene networks  <cit>  wgcna was found to be one of the methods that performed the best for constructing global co-expression networks.
after network construction, functional analysis focuses on groups of tightly connected genes  instead of single genes.
because genes within the same modules tend to maintain a consistent, correlated expression relationship independent of phenotype or experimental condition, such genes are assumed to be functionally associated, and shared regulatory and/or functional pathways may be inferred.
in addition, wgcna offers functionality to assess whether gene modules are preserved in other networks  <cit> .
preserved gene modules indicate biological processes that are conserved between species and may be candidates for functional information transfer between species.
non-preserved gene modules reflect species-specific modules, which may provide insight into biological processes that have diverged between species.
recently, a metric for expression divergence called local network similarity  was proposed to assess expression conservation of a pair of orthologs  <cit> .
lns is the correlation between the correlations of the pair of orthologs’ expression and the expression patterns of all other identified orthologs.
this metric differs from the module preservation statistics obtained by wgcna in that it is applied to a pair of genes instead of to a gene module.
lns and wgcna may also be applied to diverse datasets such as mrna expression and protein abundance data.
observations of low to moderate correlations between mrna expression and protein abundance data are recurrent in the literature  <cit> , indicating that network-based metrics of similarity may be more suitable to compare these two types of data.
in this study, we constructed an r. capsulatus gene co-expression network, and took advantage of the module preservation functionality in wgcna to identify r. capsulatus gene modules preserved in a collection of published r. sphaeroides mrna expression data, and in a r. capsulatus proteomics dataset.
in addition, we calculated lns for all  <dig> pairs of orthologs between the two rhodobacter species, and we also applied this metric to assess whether r. capsulatus genes and proteins have similar co-expression relationships in the protein abundance and mrna expression data.
we also related lns to wgcna module preservation statistics and investigated the effect of the size of the datasets in lns.
in sum, we produced comparative transcriptomics resources to guide further functional studies of r. capsulatus, and, to the best of our knowledge, performed the first application of network-based expression preservation metrics between transcriptomics and proteomics data.
RESULTS
r. capsulatusco-expression network
we used  <dig> gene expression experiments encompassing  <dig> different conditions and/or mutant strains for the  <dig> genes on the r. capsulatus microarrays to construct a gene co-expression network using wgcna.
a total of  <dig> gene co-expression modules were identified.
to assess the stability of modules, we performed a resampling analysis of cluster robustness as described in  <cit> .
the results of cluster stability analysis indicated that module assignments were reasonably stable with many of the modules being identified in most resampled data sets .
the modules varied in size from  <dig> to  <dig> genes with an average size of  <dig> genes.
a total of  <dig>  genes out of the  <dig>  genes represented on the microarrays were assigned to modules.
thirty-seven modules had enrichment of at least one type of biological gene set , and  <dig> modules were related to at least one biological pathway, which indicated that the modules were biologically meaningful.
some modules of interest are discussed below to illustrate the validity of this analysis.
one gene co-expression module containing  <dig> genes  was associated with the production of rcgta.
this module was enriched  with the rcgta gene cluster   <cit> .
it also contained the endolysin and holin genes  required for rcgta release  <cit> , and genes predicted to be involved in dna uptake and recombination, with two genes annotated as related to competence  and three genes associated with dna repair and protection and incorporation of dna received from rcgta particles   <cit> .
there were also two genes encoding predicted signal transduction proteins, rcc <dig> encoding a sensor domain protein and rcc <dig> encoding a diguanylate cyclase/phosphodiesterase, which had previously been identified as affected by the loss of the response regulator ctra similar to the rcgta gene cluster  <cit> .
the trends for genes in this module were increased expression in the stationary phase relative to logarithmic phase, reduced expression in the ctra and gtai mutants but not in the ccka mutant, and greatly increased expression in the rcgta overproducer strain, de <dig> .figure  <dig>
expression profiles of genes in selected co-expression modules across all conditions and/or mutant strains.
heatmap colours indicate robust z-scores.
colours on top of the columns refer to clusters of conditions/mutant strains highlighted in figure  <dig>  numbers below the columns correspond to the indices of the conditions mutant strains described in table  <dig>   orange co-expression module;  pink co-expression module;  darkturquoise co-expression module.
signal transduction and transcriptional regulation proteins affected by the loss of the response regulator protein ctra  <cit>  are significantly over-represented  among the  <dig> genes forming the pink module, with  <dig> out of the  <dig> previously identified proteins in this module.
the pink module showed a significant enrichment of genes involved in chemotaxis , two-component systems , and flagellar assembly .
this module also contains all  <dig> r. capsulatus proteins containing a methyl-accepting chemotaxis protein  signalling domain .
genes in the pink module showed significantly lower expression in both the ccka and ctra strains .
this corresponds to previous work that demonstrated that ctra and ccka are required for expression of flagellar and chemotaxis genes  <cit> .
genes within this module have also been shown to be involved in control of motility  <cit>  and expression of the rcgta genes .
the darkturquoise module also contained an over-representation of flagellar genes , and visually showed a very similar expression profile  as the pink module.
the one exception was that expression of genes in the darkturquoise module was elevated in the de <dig> strain in the transition and stationary growth phases while expression of genes in the pink module was not.
the median expression profiles of the pink and darkturquoise modules reciprocally correlate the most with each other .
correlations between module median expression profiles are shown in additional file  <dig>  in addition to flagellar genes, the darkturquoise module contained an over-representation of gas vesicle genes.
in total, the orange, pink and darkturquoise modules represent 84% of what was previously identified as the ctra “regulon”  <cit> .
several modules showed patterns of expression that were most affected by the culture growth medium .
this included the darkred and orangered <dig> modules that showed a relative decrease in expression in rcv medium and the cyan, greenyellow and paleturquoise modules that showed increased expression in rcv medium.
not surprisingly, these modules contained many genes involved in transport and various aspects of metabolism such as sugar and vitamin biochemistry.
two modules, midnightblue and salmon <dig>  which showed high relative expression across all strains and conditions contained many of the genes required for phototrophic growth.
this included genes encoding the photosynthetic reaction centre, the light-harvesting complexes i and ii, and bacetriochlorophyll and carotenoid pigment biosynthesis proteins.
high expression of these genes is expected because all rna samples used in the microarray experiments came from cultures grown photoheterotrophically, and these genes are well characterized for their global regulation by several key regulators  <cit> .
the darkgreen module contained genes responsible for synthesis of the capsule, rcc0181- <dig> and rcc01958- <dig>  required as an rcgta receptor  <cit> .
this module showed increased expression in strain de <dig> and decreased gene expression in the gtai mutant in the logarithmic phase when grown in rcv medium.
this module also contains one of the r. capsulatus crispr-associated  gene clusters  <cit> .
the skyblue <dig> module could also be implicated as affected by quorum sensing because of decreased expression levels in the gtai mutant relative to wild type .
this module included another one of the genes required for capsule synthesis, rcc01932
<cit> , and rcc01955- <dig>  which are located adjacent to the genes in the darkgreen module mentioned above that are involved in quorum sensing-dependent capsule production.
three modules, skyblue, turquoise and violet, showed lower relative expression across all strains and conditions.
these modules obviously represent genes with low or no expression under the conditions of these experiments, and the turquoise module was the largest of all  <dig> modules, with  <dig> genes.
of note in the turquoise module is a large number of prophage genes, representing  <dig> distinct uncharacterized prophage regions as well as the majority of the genes of rcapmu  <cit> .
this module also includes genes for nitrogen fixation and several alternative sigma factors.
as a result of this gene co-expression network analysis, 99% of the  <dig> r. capsulatus genes described as “hypothetical protein” were assigned to modules.
these uncharacterized genes might now be putatively implicated in specific biological processes to guide functional characterization.
gene module assignments are provided in additional file  <dig>
we also tested whether genes in certain modules were preferentially packaged in the rcgta particles using the available rcgta packaging microarray data  <cit> .
no modules were found to be over-represented in the rcgta-packed dna but we observed a strong inverse correlation  between the content of plasmid genes in a module and the intensity measurements detected in the rcgta dna.
this is expected as the rcgta dna was isolated from de <dig>  which lacks the ~100-kb plasmid present in the genome-sequenced strain, sb <dig>  <cit> .
although the plasmid genes were distributed amongst  <dig> different modules, the darkmagenta module showed the largest proportional plasmid gene content  of all co-expression modules, at 60% plasmid-borne genes.
the darkmagenta and three other modules that contained > <dig> plasmid genes, purple , royalblue  and turquoise , combined to contain 64% of all plasmid genes on the arrays, indicating widespread co-regulation of the plasmid-borne genes.
to confirm that the modules with a large number of plasmid genes were still identifiable in the absence of the samples from the plasmid-lacking de <dig> strain, we assessed  whether such modules were reproducible in the data subset without the de <dig> data.
indeed, there was moderate to strong evidence  that all  <dig> modules were present in the subset of data without the de <dig> samples.
thus, modules are robust to the lack of signal for the plasmid genes in the de <dig> data.
to explore similarity of expression profiles based on conditions and/or mutant strains, we performed a hierarchical cluster  analysis with multiscale bootstrap resampling   <cit>  using the pvclust r package   <cit> .
the dendrogram obtained  indicated that gene expression profiles form groups based on rcv growth medium , the de <dig> strain , and culture growth phase .figure  <dig>
dendrogram showing hierarchical clustering of conditions and/or mutant strains based on gene expression profiles.
values at branches are approximately unbiased  percentage values  and bootstrap  percentage values .
bp values indicate the percentage of bootstrap replicates in which a given cluster appears.
leaf numbers correspond to the indices of the conditions/mutant strains described in table  <dig>  coloured boxes indicate branches in the dendrogram of conditions/mutant strains having in common rcv growth medium , the de <dig> strain , and culture growth phase .
network based comparison of transcript levels and protein abundance
we explored the preservation of co-expression between r. capsulatus mrnas and proteins by applying the modulepreservation function from wgcna.
this function, using a permutation test, assessed whether the module nodes identified in r. capsulatus gene co-expression network remained connected in the protein co-expression network and whether the connectivity pattern between nodes in both networks was similar.
a composite preservation statistic, zsummary, can be used to evaluate whether modules are preserved.
a zsummary >  <dig> indicates that there is weak to moderate evidence of preservation and zsummary >  <dig> indicates that there is strong evidence that the module is preserved  <cit> .
note that module preservation can be assessed using the protein co-expression network  without cluster detection.
we realized that the small sample size of the proteomics dataset  might reduce the statistical power to pinpoint preserved modules; however, it seemed possible that strongly conserved biological signals could be identified.
indeed, we found evidence of preservation of two gene modules in the protein abundance data: the blue module , which was enriched with a number of housekeeping functions , and especially with ribosomal proteins , and the brown module , which was enriched in genes related to iron transport  .
additional file  <dig> shows preservation statistics of r. capsulatus mrna modules in the protein co-expression network.
this suggests that network-based analysis may be suitable for identifying preservation of global expression between transcriptomics and proteomics data.
comparison of these two data types has frequently yielded mixed results with reports of low to moderate correlations  <cit> .
a network-based analysis with a larger sample size of protein abundance data is needed to corroborate and further extend our finding.
comparative transcriptomics in rhodobacterspecies
we investigated the preservation of global co-expression between r. capsulatus and r. sphaeroides using the network-based statistics calculated by the modulepreservation function from wgcna.
there are two main network-based statistics found to accurately distinguish preserved from unpreserved modules: zsummary and medianrank  <cit> .
these statistics are calculated twice: once to assess whether modules are reproducible in the reference data subset consisting only of genes in common with the test dataset , and the second time to evaluate the conservation of the modules in the test data subset .
the quality statistics are a complementary approach to the cluster stability analysis to assess the robustness of the identified modules.
<dig> one-to-one orthologs between the species have been identified by reciprocal best match  <cit> , and we calculated the module preservation statistics in the data subsets containing these  <dig> orthologous genes between the r. capsulatus  and r. sphaeroides  co-expression networks.
the quality and preservation of the  <dig> r. capsulatus co-expression modules identified are illustrated in figure 3a.
zsummary tends to be more dependent on the module size than medianrank  <cit> ; nevertheless both statistics showed a strong correlation  assessing the preservation of r. capsulatus modules in r. sphaeroides data .
unsurprisingly, there was strong evidence of preservation  of the blue module.
we found low to moderate evidence of preservation  for ten additional modules.
among those, there were modules enriched with proteins implicated in porphyrin and bacteriochlorophyll metabolism , biosynthesis of secondary metabolites , abc transporters , co <dig> fixation , two-component systems , protein secretion , and lysine biosynthesis .figure  <dig>
preservation statistics of
r. capsulatus
modules in
r. sphaeroides
data.
the size of the bubble represents module size in the data subset used to assess module preservation.
the horizontal lines indicate the zsummary.pres thresholds for strong evidence of conservation  and for low to moderate evidence of conservation .
r. capsulatus modules found to be conserved in r. sphaeroides are listed on the right side.
module preservation as a function of module quality;  relationship between the two preservation statistics .
lower medianrank indicates higher preservation.
the orange module, related to rcgta production, is amongst those not preserved in r. sphaeroides.
this is consistent with the fact that no evidence of gta production has been found in r. sphaeroides
<cit> , despite conservation of the gta genes  <cit> .
other non-preserved modules were the pink , darkturquoise , darkred , darkolivegreen , green , turquoise , sienna <dig> , yellowgreen , steelblue , and darkgreen .
there was also no evidence of preservation of the brown module in r. sphaeroides, which was one of the conserved r. capsulatus mrna-protein modules.
assessment of gene-wise conservation of expression
in addition to evaluating module preservation between the two rhodobacter species, we wanted to assess pairwise conservation of expression between orthologs.
therefore, we calculated local network similarity   <cit>  to study the conservation of gene expression between the two species.
this metric was developed for application to expression datasets consisting of unmatched experimental conditions, and it quantifies the similarity of the expression correlations between a pair of orthologs and all other identified orthologs.
we decided to explore the effect of dataset size in lns and obtained the lns within each species by dividing the available data into two different subsets.
we also simulated the null-hypothesis of no conservation by randomizing the ortholog pairs .
lns scores of the null distribution ranged from - <dig>  to  <dig>  with an average very close to zero .
the within-species lns distributions showed a pronounced shift towards positive values .
however, the r. capsulatus distribution was less positive  than that of r. sphaeroides .
this is likely due to the difference in the amounts of transcriptomics data for the two species, as the r. sphaeroides dataset contains eight times as many arrays as the r. capsulatus dataset.figure  <dig>
within-species and between-species expression conservation.
distribution of within-species and between-species lns scores is shifted to right of the null distribution.
within-species show a stronger shift towards positives values;  represents the same data as in  but as the cumulative distribution.
the vertical dashed line indicates the maximum lns score observed in the null distribution.
the lns was then calculated between r. capsulatus and r. sphaeroides orthologs and between the r. capsulatus transcriptomics and proteomics data .
the between-species lns scores of the matched ortholog pairs showed less positive values than the within-species lns, but there were still values to the right of the null distribution such that 30% of ortholog pairs had a positive lns score greater than 100% of the random values .
co-expression of between-species ortholog pairs is expected to be less similar than the within-species co-expression.
furthermore, the orthologs’ functions may have diverged in the different species, in which case the lns should be low to reflect this divergence.
for example, the lns scores of r. capsulatus genes involved in the production of rcgta and their corresponding r. sphaeroides orthologs ranged from - <dig>  to  <dig>  while highly conserved housekeeping genes such as aroa and rada have lns scores of  <dig>  and  <dig> , respectively.
encouragingly, the lns scores between r. capsulatus mrna expression and protein abundance data are also to the right of the null distribution suggesting that the lns metric is sensitive enough to detect conservation of expression in small datasets and between diverse data types.
lns scores are provided in additional file  <dig>
relationship between module preservation statistics
connectivity statistics quantify whether connections between genes in the reference network are similar to those in the test network.
by its definition, lns is a connectivity-based metric.
to relate lns to wgcna module preservation statistics, we obtained the median lns per module .
after comparing the median-lns with wgcna connectivity-based statistics, we found that median-lns correlated best with bicor.kmeall, which is the correlation of the total network module eigengenes connectivity.
a module eigengene  summarizes the expression profile of a module.
the relationship between lns-median and cor.kmeall is shown in figure  <dig>  we observed a pearson correlation between median-lns and cor.kmeall of  <dig>   for the network comparison between the two rhodobacter species, and of  <dig>   for the comparison between r. capsulatus mrna and protein expression.figure  <dig>
relationship between module preservation connectivity statistics.
total network correlation of the module eigengenes connectivity  as a function of the median-lns per module for r. capsulatus modules in r. sphaeroides  and r. capsulatus mrna modules in r. capsulatus proteomics data .
each point represents a module labeled by the colour corresponding to the module name.
the black line is the loess smoothed line.
CONCLUSIONS
using wgcna and functional analysis of r. capsulatus transcriptomics data, we identified distinct groups of co-expressed genes with associations to biological gene sets .
we observed co-expression modules associated with functions known to be co-regulated based on previous studies, such as the production of rcgta, motility and chemotaxis.
these identified co-expression modules will be useful to identify candidate genes for further investigations in r. capsulatus biology, such as the regulation and production of rcgta.
in addition, we distinguished between preserved and non-preserved modules between r. capsulatus and r. sphaeroides.
the module preservation results point to a lack of similarity between the two rhodobacter species for many of the modules, whereas the expression of several metabolic pathways was similar in both species.
we also quantified the conservation of expression of all one-to-one orthologs between these species using the lns metric.
these resources may aid in the identification of functional analog genes  in these bacteria, and comparative transcriptomics studies such as this can be applied to other bacterial species to obtain evidence of gene expression conservation and thereby allow further exploration of gene function.

 species in the genus rhodobacter are purple nonsulfur bacteria found mostly in freshwater environments. a hallmark of purple nonsulfur bacteria is that they display tremendous physiological diversity. genome sequences are available from two rhodobacter species, r. capsulatus and r. sphaeroides, and transcriptional profiling studies have been performed with both species. these two species have been widely studied as model organisms for anoxygenic photosynthesis, carbon and nitrogen fixation, chemotaxis and flagellar motility, and various regulatory systems including quorum sensing, two-component phosphorelays and those responsible for regulation in response to o and light. r. capsulatus is also a model organism for study of a gene transfer agent, rcgta, which is a virus-like particle that packages small segments of the genome of a gta-producing cell that can then be transferred to recipient cells. weighted gene co-expression network analysis has been widely used to analyze transcriptional profiles since its introduction in, and has proved to be a useful approach for the functional annotation of uncharacterized genes. in a recent critical assessment of methods for constructing gene networks wgcna was found to be one of the methods that performed the best for constructing global co-expression networks. after network construction, functional analysis focuses on groups of tightly connected genes instead of single genes. because genes within the same modules tend to maintain a consistent, correlated expression relationship independent of phenotype or experimental condition, such genes are assumed to be functionally associated, and shared regulatory and/or functional pathways may be inferred. in addition, wgcna offers functionality to assess whether gene modules are preserved in other networks. preserved gene modules indicate biological processes that are conserved between species and may be candidates for functional information transfer between species. non-preserved gene modules reflect species-specific modules, which may provide insight into biological processes that have diverged between species. recently, a metric for expression divergence called local network similarity was proposed to assess expression conservation of a pair of orthologs. lns is the correlation between the correlations of the pair of orthologs’ expression and the expression patterns of all other identified orthologs. this metric differs from the module preservation statistics obtained by wgcna in that it is applied to a pair of genes instead of to a gene module. lns and wgcna may also be applied to diverse datasets such as mrna expression and protein abundance data. observations of low to moderate correlations between mrna expression and protein abundance data are recurrent in the literature, indicating that network-based metrics of similarity may be more suitable to compare these two types of data. in this study, we constructed an r. capsulatus gene co-expression network, and took advantage of the module preservation functionality in wgcna to identify r. capsulatus gene modules preserved in a collection of published r. sphaeroides mrna expression data, and in a r. capsulatus proteomics dataset. in addition, we calculated lns for all pairs of orthologs between the two rhodobacter species, and we also applied this metric to assess whether r. capsulatus genes and proteins have similar co-expression relationships in the protein abundance and mrna expression data. we also related lns to wgcna module preservation statistics and investigated the effect of the size of the datasets in lns. in sum, we produced comparative transcriptomics resources to guide further functional studies of r. capsulatus, and, to the best of our knowledge, performed the first application of network-based expression preservation metrics between transcriptomics and proteomics data.  r. capsulatusco-expression network we used gene expression experiments encompassing different conditions and/or mutant strains for the genes on the r. capsulatus microarrays to construct a gene co-expression network using wgcna. a total of gene co-expression modules were identified. to assess the stability of modules, we performed a resampling analysis of cluster robustness as described in. the results of cluster stability analysis indicated that module assignments were reasonably stable with many of the modules being identified in most resampled data sets. the modules varied in size from to genes with an average size of genes. a total of genes out of the genes represented on the microarrays were assigned to modules. thirty-seven modules had enrichment of at least one type of biological gene set, and modules were related to at least one biological pathway, which indicated that the modules were biologically meaningful. some modules of interest are discussed below to illustrate the validity of this analysis. one gene co-expression module containing genes was associated with the production of rcgta. this module was enriched with the rcgta gene cluster. it also contained the endolysin and holin genes required for rcgta release, and genes predicted to be involved in dna uptake and recombination, with two genes annotated as related to competence and three genes associated with dna repair and protection and incorporation of dna received from rcgta particles. there were also two genes encoding predicted signal transduction proteins, rcc encoding a sensor domain protein and rcc encoding a diguanylate cyclase/phosphodiesterase, which had previously been identified as affected by the loss of the response regulator ctra similar to the rcgta gene cluster. the trends for genes in this module were increased expression in the stationary phase relative to logarithmic phase, reduced expression in the ctra and gtai mutants but not in the ccka mutant, and greatly increased expression in the rcgta overproducer strain, de.figure expression profiles of genes in selected co-expression modules across all conditions and/or mutant strains. heatmap colours indicate robust z-scores. colours on top of the columns refer to clusters of conditions/mutant strains highlighted in figure  numbers below the columns correspond to the indices of the conditions mutant strains described in table  orange co-expression module; pink co-expression module; darkturquoise co-expression module. signal transduction and transcriptional regulation proteins affected by the loss of the response regulator protein ctra are significantly over-represented among the genes forming the pink module, with out of the previously identified proteins in this module. the pink module showed a significant enrichment of genes involved in chemotaxis, two-component systems, and flagellar assembly. this module also contains all r. capsulatus proteins containing a methyl-accepting chemotaxis protein signalling domain. genes in the pink module showed significantly lower expression in both the ccka and ctra strains. this corresponds to previous work that demonstrated that ctra and ccka are required for expression of flagellar and chemotaxis genes. genes within this module have also been shown to be involved in control of motility and expression of the rcgta genes. the darkturquoise module also contained an over-representation of flagellar genes, and visually showed a very similar expression profile as the pink module. the one exception was that expression of genes in the darkturquoise module was elevated in the de strain in the transition and stationary growth phases while expression of genes in the pink module was not. the median expression profiles of the pink and darkturquoise modules reciprocally correlate the most with each other. correlations between module median expression profiles are shown in additional file in addition to flagellar genes, the darkturquoise module contained an over-representation of gas vesicle genes. in total, the orange, pink and darkturquoise modules represent 84% of what was previously identified as the ctra “regulon”. several modules showed patterns of expression that were most affected by the culture growth medium. this included the darkred and orangered modules that showed a relative decrease in expression in rcv medium and the cyan, greenyellow and paleturquoise modules that showed increased expression in rcv medium. not surprisingly, these modules contained many genes involved in transport and various aspects of metabolism such as sugar and vitamin biochemistry. two modules, midnightblue and salmon which showed high relative expression across all strains and conditions contained many of the genes required for phototrophic growth. this included genes encoding the photosynthetic reaction centre, the light-harvesting complexes i and ii, and bacetriochlorophyll and carotenoid pigment biosynthesis proteins. high expression of these genes is expected because all rna samples used in the microarray experiments came from cultures grown photoheterotrophically, and these genes are well characterized for their global regulation by several key regulators. the darkgreen module contained genes responsible for synthesis of the capsule, rcc0181- and rcc01958- required as an rcgta receptor. this module showed increased expression in strain de and decreased gene expression in the gtai mutant in the logarithmic phase when grown in rcv medium. this module also contains one of the r. capsulatus crispr-associated gene clusters. the skyblue module could also be implicated as affected by quorum sensing because of decreased expression levels in the gtai mutant relative to wild type. this module included another one of the genes required for capsule synthesis, rcc01932, and rcc01955- which are located adjacent to the genes in the darkgreen module mentioned above that are involved in quorum sensing-dependent capsule production. three modules, skyblue, turquoise and violet, showed lower relative expression across all strains and conditions. these modules obviously represent genes with low or no expression under the conditions of these experiments, and the turquoise module was the largest of all modules, with genes. of note in the turquoise module is a large number of prophage genes, representing distinct uncharacterized prophage regions as well as the majority of the genes of rcapmu. this module also includes genes for nitrogen fixation and several alternative sigma factors. as a result of this gene co-expression network analysis, 99% of the r. capsulatus genes described as “hypothetical protein” were assigned to modules. these uncharacterized genes might now be putatively implicated in specific biological processes to guide functional characterization. gene module assignments are provided in additional file we also tested whether genes in certain modules were preferentially packaged in the rcgta particles using the available rcgta packaging microarray data. no modules were found to be over-represented in the rcgta-packed dna but we observed a strong inverse correlation between the content of plasmid genes in a module and the intensity measurements detected in the rcgta dna. this is expected as the rcgta dna was isolated from de which lacks the ~100-kb plasmid present in the genome-sequenced strain, sb. although the plasmid genes were distributed amongst different modules, the darkmagenta module showed the largest proportional plasmid gene content of all co-expression modules, at 60% plasmid-borne genes. the darkmagenta and three other modules that contained > plasmid genes, purple, royalblue and turquoise, combined to contain 64% of all plasmid genes on the arrays, indicating widespread co-regulation of the plasmid-borne genes. to confirm that the modules with a large number of plasmid genes were still identifiable in the absence of the samples from the plasmid-lacking de strain, we assessed whether such modules were reproducible in the data subset without the de data. indeed, there was moderate to strong evidence that all modules were present in the subset of data without the de samples. thus, modules are robust to the lack of signal for the plasmid genes in the de data. to explore similarity of expression profiles based on conditions and/or mutant strains, we performed a hierarchical cluster analysis with multiscale bootstrap resampling using the pvclust r package. the dendrogram obtained indicated that gene expression profiles form groups based on rcv growth medium, the de strain, and culture growth phase.figure dendrogram showing hierarchical clustering of conditions and/or mutant strains based on gene expression profiles. values at branches are approximately unbiased percentage values and bootstrap percentage values. bp values indicate the percentage of bootstrap replicates in which a given cluster appears. leaf numbers correspond to the indices of the conditions/mutant strains described in table  coloured boxes indicate branches in the dendrogram of conditions/mutant strains having in common rcv growth medium, the de strain, and culture growth phase. network based comparison of transcript levels and protein abundance we explored the preservation of co-expression between r. capsulatus mrnas and proteins by applying the modulepreservation function from wgcna. this function, using a permutation test, assessed whether the module nodes identified in r. capsulatus gene co-expression network remained connected in the protein co-expression network and whether the connectivity pattern between nodes in both networks was similar. a composite preservation statistic, zsummary, can be used to evaluate whether modules are preserved. a zsummary >  indicates that there is weak to moderate evidence of preservation and zsummary >  indicates that there is strong evidence that the module is preserved. note that module preservation can be assessed using the protein co-expression network without cluster detection. we realized that the small sample size of the proteomics dataset might reduce the statistical power to pinpoint preserved modules; however, it seemed possible that strongly conserved biological signals could be identified. indeed, we found evidence of preservation of two gene modules in the protein abundance data: the blue module, which was enriched with a number of housekeeping functions, and especially with ribosomal proteins, and the brown module, which was enriched in genes related to iron transport. additional file shows preservation statistics of r. capsulatus mrna modules in the protein co-expression network. this suggests that network-based analysis may be suitable for identifying preservation of global expression between transcriptomics and proteomics data. comparison of these two data types has frequently yielded mixed results with reports of low to moderate correlations. a network-based analysis with a larger sample size of protein abundance data is needed to corroborate and further extend our finding. comparative transcriptomics in rhodobacterspecies we investigated the preservation of global co-expression between r. capsulatus and r. sphaeroides using the network-based statistics calculated by the modulepreservation function from wgcna. there are two main network-based statistics found to accurately distinguish preserved from unpreserved modules: zsummary and medianrank. these statistics are calculated twice: once to assess whether modules are reproducible in the reference data subset consisting only of genes in common with the test dataset, and the second time to evaluate the conservation of the modules in the test data subset. the quality statistics are a complementary approach to the cluster stability analysis to assess the robustness of the identified modules. one-to-one orthologs between the species have been identified by reciprocal best match, and we calculated the module preservation statistics in the data subsets containing these orthologous genes between the r. capsulatus and r. sphaeroides co-expression networks. the quality and preservation of the r. capsulatus co-expression modules identified are illustrated in figure 3a. zsummary tends to be more dependent on the module size than medianrank; nevertheless both statistics showed a strong correlation assessing the preservation of r. capsulatus modules in r. sphaeroides data. unsurprisingly, there was strong evidence of preservation of the blue module. we found low to moderate evidence of preservation for ten additional modules. among those, there were modules enriched with proteins implicated in porphyrin and bacteriochlorophyll metabolism, biosynthesis of secondary metabolites, abc transporters, co fixation, two-component systems, protein secretion, and lysine biosynthesis.figure preservation statistics of r. capsulatus modules in r. sphaeroides data. the size of the bubble represents module size in the data subset used to assess module preservation. the horizontal lines indicate the zsummary.pres thresholds for strong evidence of conservation and for low to moderate evidence of conservation. r. capsulatus modules found to be conserved in r. sphaeroides are listed on the right side. module preservation as a function of module quality; relationship between the two preservation statistics. lower medianrank indicates higher preservation. the orange module, related to rcgta production, is amongst those not preserved in r. sphaeroides. this is consistent with the fact that no evidence of gta production has been found in r. sphaeroides, despite conservation of the gta genes. other non-preserved modules were the pink, darkturquoise, darkred, darkolivegreen, green, turquoise, sienna, yellowgreen, steelblue, and darkgreen. there was also no evidence of preservation of the brown module in r. sphaeroides, which was one of the conserved r. capsulatus mrna-protein modules. assessment of gene-wise conservation of expression in addition to evaluating module preservation between the two rhodobacter species, we wanted to assess pairwise conservation of expression between orthologs. therefore, we calculated local network similarity to study the conservation of gene expression between the two species. this metric was developed for application to expression datasets consisting of unmatched experimental conditions, and it quantifies the similarity of the expression correlations between a pair of orthologs and all other identified orthologs. we decided to explore the effect of dataset size in lns and obtained the lns within each species by dividing the available data into two different subsets. we also simulated the null-hypothesis of no conservation by randomizing the ortholog pairs. lns scores of the null distribution ranged from - to with an average very close to zero. the within-species lns distributions showed a pronounced shift towards positive values. however, the r. capsulatus distribution was less positive than that of r. sphaeroides. this is likely due to the difference in the amounts of transcriptomics data for the two species, as the r. sphaeroides dataset contains eight times as many arrays as the r. capsulatus dataset.figure within-species and between-species expression conservation. distribution of within-species and between-species lns scores is shifted to right of the null distribution. within-species show a stronger shift towards positives values; represents the same data as in but as the cumulative distribution. the vertical dashed line indicates the maximum lns score observed in the null distribution. the lns was then calculated between r. capsulatus and r. sphaeroides orthologs and between the r. capsulatus transcriptomics and proteomics data. the between-species lns scores of the matched ortholog pairs showed less positive values than the within-species lns, but there were still values to the right of the null distribution such that 30% of ortholog pairs had a positive lns score greater than 100% of the random values. co-expression of between-species ortholog pairs is expected to be less similar than the within-species co-expression. furthermore, the orthologs’ functions may have diverged in the different species, in which case the lns should be low to reflect this divergence. for example, the lns scores of r. capsulatus genes involved in the production of rcgta and their corresponding r. sphaeroides orthologs ranged from - to while highly conserved housekeeping genes such as aroa and rada have lns scores of and, respectively. encouragingly, the lns scores between r. capsulatus mrna expression and protein abundance data are also to the right of the null distribution suggesting that the lns metric is sensitive enough to detect conservation of expression in small datasets and between diverse data types. lns scores are provided in additional file relationship between module preservation statistics connectivity statistics quantify whether connections between genes in the reference network are similar to those in the test network. by its definition, lns is a connectivity-based metric. to relate lns to wgcna module preservation statistics, we obtained the median lns per module. after comparing the median-lns with wgcna connectivity-based statistics, we found that median-lns correlated best with bicor.kmeall, which is the correlation of the total network module eigengenes connectivity. a module eigengene summarizes the expression profile of a module. the relationship between lns-median and cor.kmeall is shown in figure  we observed a pearson correlation between median-lns and cor.kmeall of for the network comparison between the two rhodobacter species, and of for the comparison between r. capsulatus mrna and protein expression.figure relationship between module preservation connectivity statistics. total network correlation of the module eigengenes connectivity as a function of the median-lns per module for r. capsulatus modules in r. sphaeroides and r. capsulatus mrna modules in r. capsulatus proteomics data. each point represents a module labeled by the colour corresponding to the module name. the black line is the loess smoothed line.  using wgcna and functional analysis of r. capsulatus transcriptomics data, we identified distinct groups of co-expressed genes with associations to biological gene sets. we observed co-expression modules associated with functions known to be co-regulated based on previous studies, such as the production of rcgta, motility and chemotaxis. these identified co-expression modules will be useful to identify candidate genes for further investigations in r. capsulatus biology, such as the regulation and production of rcgta. in addition, we distinguished between preserved and non-preserved modules between r. capsulatus and r. sphaeroides. the module preservation results point to a lack of similarity between the two rhodobacter species for many of the modules, whereas the expression of several metabolic pathways was similar in both species. we also quantified the conservation of expression of all one-to-one orthologs between these species using the lns metric. these resources may aid in the identification of functional analog genes in these bacteria, and comparative transcriptomics studies such as this can be applied to other bacterial species to obtain evidence of gene expression conservation and thereby allow further exploration of gene function. 
in addition, using local network similarity, a recently proposed metric for expression divergence, we assessed the expression conservation of between-species pairs of orthologs, and within-species gene-protein expression profiles. we identified r. capsulatus modules enriched with genes for ribosomal proteins, porphyrin and bacteriochlorophyll anabolism, and biosynthesis of secondary metabolites to be preserved in r. sphaeroides whereas modules related to rcgta production and signalling showed lack of preservation in r. sphaeroides. in addition, we demonstrated that network statistics may also be applied within-species to identify congruence between mrna expression and protein abundance data for which simple correlation measurements have previously had mixed results. functional analysis of gene modules can then associate co-expressed genes with biological pathways, and network statistics can determine the degree of module preservation in related networks. comparative transcriptomicsmodule preservationgene-protein expression conservationrhodobacter capsulatusrhodobacter sphaeroidesissue-copyright-statement© the author 2014 in this paper, we constructed an r. capsulatus gene co-expression network, performed functional analysis of identified gene modules, and investigated preservation of these modules in r. capsulatus proteomics data and in r. sphaeroides transcriptomics data. we identified two r. capsulatus gene modules preserved in the protein abundance data. we also identified several gene modules preserved between both rhodobacter species, which indicate that these cellular processes are conserved between the species and are candidates for functional information transfer between species. many gene modules were non-preserved, providing insight into processes that differentiate the two species. our analyses provide new sources of information for functional annotation in r. capsulatus because uncharacterized genes in modules are now connected with groups of genes that constitute a joint functional annotation. representative strains of two rhodobacter species, r. capsulatus and r. sphaeroides, have had their genomes fully sequenced and both have been the subject of transcriptional profiling studies. investigation of the module gene contents and expression profiles revealed patterns that were validated based on previous studies supporting the biological relevance of these modules. the analysis identified gene co-expression modules in r. capsulatus. gene co-expression networks can be used to identify modules of genes with similar expression profiles. the genus rhodobacter contains purple nonsulfur bacteria found mostly in freshwater environments.
 the genus rhodobacter contains purple nonsulfur bacteria found mostly in freshwater environments. representative strains of two rhodobacter species, r. capsulatus and r. sphaeroides, have had their genomes fully sequenced and both have been the subject of transcriptional profiling studies. gene co-expression networks can be used to identify modules of genes with similar expression profiles. functional analysis of gene modules can then associate co-expressed genes with biological pathways, and network statistics can determine the degree of module preservation in related networks. in this paper, we constructed an r. capsulatus gene co-expression network, performed functional analysis of identified gene modules, and investigated preservation of these modules in r. capsulatus proteomics data and in r. sphaeroides transcriptomics data.  the analysis identified gene co-expression modules in r. capsulatus. investigation of the module gene contents and expression profiles revealed patterns that were validated based on previous studies supporting the biological relevance of these modules. we identified two r. capsulatus gene modules preserved in the protein abundance data. we also identified several gene modules preserved between both rhodobacter species, which indicate that these cellular processes are conserved between the species and are candidates for functional information transfer between species. many gene modules were non-preserved, providing insight into processes that differentiate the two species. in addition, using local network similarity, a recently proposed metric for expression divergence, we assessed the expression conservation of between-species pairs of orthologs, and within-species gene-protein expression profiles.  our analyses provide new sources of information for functional annotation in r. capsulatus because uncharacterized genes in modules are now connected with groups of genes that constitute a joint functional annotation. we identified r. capsulatus modules enriched with genes for ribosomal proteins, porphyrin and bacteriochlorophyll anabolism, and biosynthesis of secondary metabolites to be preserved in r. sphaeroides whereas modules related to rcgta production and signalling showed lack of preservation in r. sphaeroides. in addition, we demonstrated that network statistics may also be applied within-species to identify congruence between mrna expression and protein abundance data for which simple correlation measurements have previously had mixed results. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords comparative transcriptomicsmodule preservationgene-protein expression conservationrhodobacter capsulatusrhodobacter sphaeroidesissue-copyright-statement© the author 2014 