BACKGROUND
the bacterial transcriptome, as one of the important genome-level information, reflects the global cellular activity .
since the fitness and/or activity of the cells is directly influenced by the environmental perturbation  and genetic disturbance , the evaluation of the environmental and genetic contributions to transcriptome turns to be highly essential.
in recent, the transcriptome analysis  of bacteria  has been performed extensively using a range of experimental and analytical tools  <cit> , to illustrate an overall picture of the biochemical events occurring inside the cells.
in particular, to examine the environmental contributions to bacterial transcriptome, gene expression profiling has been tested across multiple environmental conditions in a high-throughput manner  <cit> .
as a consequence, the intensive studies on global transcriptional activity contributed by environmental perturbations successfully provided conclusions regarding the molecular mechanisms involved in specific pathways and/or regulatory networks  <cit> , as well as the theoretical and/or overall outlook in regard to global dynamic changes in transcriptome  <cit> .
however, the genetic contribution to bacterial transcriptome has been rarely reported.
although the previous studies revealed the potential links between transcriptional regulation and chromosome organization  <cit>  and the relation between changes in dna methylation and chromosomal structure and changes in global gene expression  <cit> , whether and how the genomic scars  influence the global transcriptional activity is unclear.
both genomic and environmental perturbations could initiate a global change in the transcriptome, because both are stressful for living cells.
whether there is any potential linkage between the two contributions is an intriguing question.
to address the question, a comprehensive analysis comparing the effects of genomic and environmental interruptions on the transcriptome is highly required.
such analysis would not only enable a comparison of the internal and external influences on the plasticity of genome-wide transcriptional activity but would also provide a valuable example of a global and conceptual assessment of high-throughput biological data.
therefore, a multi-level survey of genomic expression is highly in demand, to show how genomic disturbance causes the global transcriptional reorganization and whether there is any relationship between the contributions of genomic and environmental interruptions to transcriptome.
as a pilot study, we evaluated these two distinct entities, the genome and the environment, at the level of the transcriptome.
we addressed how genomic and environmental perturbations contribute to the bacterial transcriptome and whether there is any interaction between the two influences.
a multiple deletion e. coli strain, mds <dig>  <cit> , was compared with its wild-type parent strain mg <dig>  <cit>  in this study.
because of its lack of insertion sequences   <cit> , mds <dig> has been widely used in a number of applications  <cit> ; however, its detailed, genome-wide analysis has been rarely reported  <cit> .
the genes used for the comparative transcriptome analysis were selected on the basis of the genome sequences of mds <dig> and mg <dig>  the comparison of mg <dig> to mds <dig> provided insights into genomic disturbance-induced transcriptional reorganization.
the evaluation of the heat shock response  provided insights into environmental perturbation-induced transcriptional changes.
we examined whether the losses of insertion elements in genome interrupt the genome-wide transcriptional activity  and whether these multiple deletions in genome and the heat shock stress exert common or different effects on transcriptional changes.
furthermore, we performed multilevel comparative analyses of chromosomal periodicity, regulatory networks, the localization of differential gene expression, transcriptional sensitivity in response to genomic and environmental perturbations and the epistatic effect, that is, a negatively compensated relationship, on the transcriptome of the genome and the environment.
RESULTS
strains and genes subjected to transcriptome analysis
gene expression was analyzed using a custom, high-density dna microarray  with a precise and wide quantitative range of mrna concentrations  <cit> , as previously described for genome resequencing  <cit>  and transcriptome analysis  <cit> .
first, the wild-type strain mg <dig> and the multiple deletion strain mds <dig>  <cit>  were used to examine whether and how removing the nonessential genomic regions in e. coli contributed to its transcriptome.
repeated experiments were performed with cells grown precisely to mid-exponential phase in minimal medium.
the genes shared between the two strains were determined by comparing their genome sequences .
in total, locus tags  were assigned to  <dig> and  <dig> open reading frames  in mg <dig> and mds <dig>  respectively , which was consistent to the original report  <cit> .
genes with repeated locus tags were removed from the analysis, for a final total of  <dig> genes with locus tags in mg <dig>  excluding the  <dig> deleted genes  <cit>  and  <dig> mutated genes in mg <dig> , a total of  <dig> genes were determined to be common between the two strains.
subsequently, as the heat shock stress is known as one of many external perturbations and showed distinguishable expression profiles  <cit> , heat shock experiments were performed to evaluate how the external disturbance contributed to the transcriptome for comparative analyses.
the average gene expression levels from repeated experiments were plotted in figure 1b and used for further analysis.
we showed that neither genomic reduction nor heat shock altered the shape of the distribution of gene expression .
priority in chromosomal periodicity
the periodicity of genome-wide transcriptional activity was studied to provide a global view of gene expression profiling  <cit> .
the average transcription levels  over the entire genome were plotted for both genomes using a 100-bp sliding window .
the periodic patterns showed a major peak of approximately  <dig> kb  in mg <dig>  consistent with previous reports  <cit> .
however, these periodic patterns were disturbed by heat shock.
the most significant wavelength, as indicated by the red, vertical, broken line in figure  <dig> , shifted from  <dig> to  <dig> kb .
this slight but statistically significant change in the major peak caused a reduction in the number of periods from seven to six .
this finding indicated that the heat shock treatment initiated a reorganization of the transcriptome that gave a high priority to a periodicity of six periods.
interestingly, the reduced genome also showed a fixed periodicity of six periods.
mds <dig> maintained the parental periodogram, with a major peak at  <dig> kb that was accompanied by a decrease in the chromosomal period number .
this reduced period was unchanged in mds <dig> in response to heat shock perturbation .
in addition, when the expression of the  <dig> common genes in mg <dig> was plotted against the mds <dig> genome, an identical chromosomal periodicity of six periods was observed independently of external conditions .
statistical analyses confirmed that the altered periodicity was highly specific for the mds <dig> genome and was not due to random deletions in the genome .
the stable six periods may be have been caused by the presence of six macrodomains in the chromosomal structure, as determined by recombination activity  <cit> .
the results suggested that the absent genetic regions in mds <dig>  may contain a considerable number of genes that are not or rarely transcribed under heat shock conditions, potentially contributing to an additional period.
thus, the multiple deletions not only reduced the genome size but also provided, probably by chance, a steady periodicity to the global transcriptional activity of the e. coli chromosome.
overlaps in perturbed regulatory networks
regulatory network maps comprising the transcription factors  that control more than  <dig> genes  were constructed as shown in figure  <dig>  significant changes in gene expression are represented as gradations according to their fdr values, which were determined using the rank product method  <cit> .
overall, both genome reduction and heat shock exerted broad effects on transcriptional reorganization: the expression levels of a number of genes were changed .
a small number of regulators showed remarkable, independent transcriptional changes; for example, gadx was induced only by genome reduction  and rpod only by heat shock .
it indicated that genome structural changes might activate the primary activator of the gad system but fairly influenced the global transcriptional factor .
in addition, a comparable number of regulators were disturbed by both perturbations in a correlated manner: those induced by genome reduction were also induced by heat shock.
the changes in these overlapping perturbed regulators occurred in either the same or the reverse direction; e.g., the expression level of phob was upregulated by both genome reduction and heat shock , whereas, the expression level of gade was upregulated by genome reduction but repressed by heat shock .
to identify these regulators  upstream of genes with marked changes in transcription induced by either genome reduction or heat shock, a gene set enrichment analysis  was applied  <cit> .
among the  <dig> regulatory networks controlled by  <dig> sigma factors and  <dig> tfs,  <dig> and  <dig> networks showed clear transcriptional changes  associated with genome reduction and heat shock, respectively .
heat shock interrupted approximately equal numbers of the induced and repressed regulatory networks , whereas genome reduction triggered many more upregulated than downregulated networks .
moreover, most of the regulatory networks that responded to heat shock were highly sensitive to genome reduction as well .
these overlapping regulatory networks  showed a marked tendency to undergo transcriptional changes in the same direction  in response to genomic and heat shock perturbations.
the exception  was found in the network controlled by gadx, where the downstream genes were induced by genome reduction but suppressed by heat shock .
the information pertaining to transcription factors , sigma factors and the downstream genes under their control is from regulondb.
the transcriptional changes in the downstream genes were evaluated with gene set enrichment analysis   <cit> , and the disturbed regulatory networks were determined accordingly .
perturbation and direction refer to the type of disturbance to the transcriptome and the directionality of the changes in gene expression, respectively.
locus tag, name of tf, size and fdr q-val represent the gene id, the corresponding regulator gene, the number of regulated downstream genes and the statistical significance, respectively.
taken together, the transcriptional reorganization observed in the regulatory networks was not only secondary to the expression changes in the regulators  but also occurred in networks controlled by the regulators of steady expression .
the latter case suggested either weakened transcriptional control by the regulators or a high level of sensitivity in the downstream genes .
the discovery of regulatory networks  that were universally involved in both perturbations indicated that transcriptional reorganization responds similarly to both endogenous and exogenous disruptions.
genome location dependency and gene category enrichment of highly responsive genes
genes with significant transcriptional changes  among the  <dig> common genes were also determined with the rank product method.
in all,  <dig> and  <dig> genes were identified that showed either up- or downregulated expression in response to genome reduction  or heat shock , respectively .
the gene names, gene categories, locus tags, and directions of the transcriptional changes are summarized in additional file 3: table s <dig>  and the genome locations are marked in additional file 1: figure s <dig>  an analysis of the distances between the degs and the genome scars caused by genome reduction showed that the degs_gr, but not the degs_hs, were located much closer to the nearest genome scar than were the genes with conserved expression   .
this tendency indicated that the genomic disturbance caused by genome reduction brought about considerable transcriptional changes in neighboring genes.
in particular, the transcriptional changes were highly significant in the direction of upregulation , which suggests that the removal of the dispensable genetic segment might increase the expression efficiency of the neighboring genes.
we assumed that the deleted genes most likely perturbed the local superhelical density and thereby affected the transcriptional activity of the neighboring genes, although the functions of the deleted genes near the deletion junctions or mutations remain to be further examined.
the gene categories that were enriched in the degs were analyzed to address the question of whether the genomic and heat shock perturbations had affected any specific gene categories .
the heat map  illustrates that the heat shock affected only a limited number of gene categories  with high significance .
however, the genome reduction influenced a relatively large number of gene categories with diverse directions in transcriptional changes; for example, the transporter genes  and the rna-coding genes  were highly enriched in the degs_gr in the directions of down-  and upregulation , respectively.
only the “unknown function”  category was significantly enriched in both the degs_gr and the degs_hs .
this result suggested that the genes with unknown functions were highly sensitive to both genomic and environmental perturbations, as might result from a loose control of gene expression.
the genes in the factor category , of which most are heat shock proteins , were not only enriched in the upregulated degs_hs , as extensively reported, but were also detected in the upregulated degs_gr .
as a result, factor  was the most highly enriched category among the  <dig> overlapping genes .
intriguingly, some well-known molecular chaperones, such as clpb and ibpa, were highly responsive to both perturbations , indicating a universal response mechanism to internal and external stressors in these genes.
positive correlations in changes of gene expression
the directions of the transcriptional changes demonstrated by the degs were further analyzed, in particular, on the gene expression patterns by means of correlations.
in a global view, the degs_gr  also showed remarkable transcriptional changes in response to heat shock , as evidenced by the significantly lower correlation of the degs_gr  compared with that  of the  <dig> common genes .
similarly, the degs_hs showed large transcriptional changes in response to genomic perturbation ; their correlation  was significantly lower than that  of the  <dig> common genes .
furthermore, the upregulated degs_hs  were also induced by genome reduction , and the upregulated degs_gr  were also induced by heat shock .
these correlated patterns of changes in gene expression deduced from the overall evaluation of the degs  implied that the overlapping genes  might play a crucial role in producing such transcriptional fluctuation because not all the degs changed significantly when moving to the other condition.
among the  <dig> overlapping genes ,  <dig> and  <dig> genes with up- or downregulated expression in response to genome reduction were up- or downregulated by heat shock in the same direction, respectively.
only a single gene  reversed the direction of its transcription; i.e., it was induced by heat shock but repressed by genome reduction.
the overall directional similarities implied that the genes that were sensitive to genomic disturbance tended to be highly responsive to external perturbation and vice versa.
furthermore, the transcriptional changes of the  <dig> genes that were induced by genome reduction were plotted against those that were induced by heat shock .
a clear, positive correlation between the transcriptional changes produced by the genetic and environmental contributions was observed .
in other words, larger degrees of transcriptional fluctuation triggered by genetic interruption were correlated with greater changes in gene expression induced by environmental perturbation.
the positive correlation between these two contributions to the transcriptome implies somehow a linkage in transcriptional reorganization in response to both genetic and environmental stresses.
antagonistic epistasis in the transcriptome
theoretically, the positive correlation in the transcriptional changes triggered by the genetic and environmental interruptions  potentially had a tendency to amplify the magnitude of the changes in gene expression that occurred in response to both genetic and environmental perturbations.
for instance, the gene expression levels of dnaj were approximately  <dig>  and  <dig> folds up-regulated due to genome reduction and heat shock, respectively.
owing to the positive correlation, the change in the expression of dnaj was assumed to be multiplied, up to  <dig> folds, by the two simultaneously occurred perturbations.
actually, it was a 21-fold increase, a highly suppressed value, in the transcriptional change.
to understand such repressed change in gene expression, the genetic concepts of epistasis and additivity were employed, as represented schematically in figure 6a.
these concepts are widely used to determine whether the final fitness or output is simply the sum of the effects of two genes  or whether one gene partially inhibits  or enhances  the effects of the other gene.
the extended definition of epistasis could be applied to the interactions among the chemicals and molecules in highly complex biochemical reactions  <cit> .
here, the two analyzed effects were the transcriptional changes contributed by genome reduction  and heat shock  individually, and the final output was defined as the transcriptional changes that were produced when the genomic and heat shock perturbations occurred simultaneously , i.e., the reduced genome undergoing heat shock .
the slopes represent the transcriptional changes  perturbed by the genome and environment in an additive or epistatic manner .
the difference in the slopes, defined as α, represented the relative magnitude of the difference  between the additive and simultaneous contributions.
accordingly, the relationship between the contributions of genome reduction and heat shock to transcriptome was subsequently analyzed in a genomic view.
considering the difference in the number of genes in mg <dig> and mds <dig>  the common genes  shared with both strains were applied in the following analyses.
the transcriptional changes due to these two independent contributors  were added, and the sum was considered to be the additive contribution .
the changes in gene expression between mds42_heatshock and mg <dig> represented simultaneous genetic and environmental interruptions .
the additive contribution was then plotted against the simultaneous contribution, as shown in figure 6b.
the simultaneous contribution showed a smaller magnitude of transcriptional changes than did the additive contribution  , approximately 30% suppressive effect .
it strongly indicated a negatively compensated relationship between the genomic and environmental contributions to the changes in the transcriptome, as so-called “antagonistic epistasis”.
moreover, the magnitude of the compensatory effect became larger  in the gene groups of the degs_gr and the degs_hs , suggesting that the negative epistasis became more significant, approximately 40%.
both the genetic and environmental perturbations alone triggered fluctuations in the transcriptome, whereas either perturbation repressed or limited the fluctuation caused by the other when both occurred simultaneously.
these results indicated that negative epistasis may be universally involved in biological processes, although the underlying mechanisms and theoretical principles remain to be determined.
discussion
this study demonstrated a considerable interaction between the genetic and environmental contributions to the changes in the transcriptome, as evidenced by the identical preferred chromosomal periodicity, the directional similarities in the transcriptional changes, the overlaps between the perturbed regulatory networks, the positive correlation in transcriptional changes induced by the genetic and environmental contributions and the negative epistasis.
thus, our multilevel comparative analysis provided a global comparison of the effects of genomic and heat shock perturbations to the e. coli transcriptome.
a more comprehensive evaluation based on the additional experiments under cross multiple conditions and a cross validation using the fruitful reported data sets are essential to figure out the common features of the genomic and environmental contributions to the reorganization in transcriptome.
the transcriptome analysis caught the genome reduction effect.
the deleted regions were assumed to have crucial functions, on the basis of our novel observations of the reduced genome, along with the previous reports of its advantages in various applications  <cit>  but its limited evolvability  <cit> .
as these regions were largely occupied by genes that were derived from the is/phage, that encoded structural components, or that had unknown or predicted functions, it highlighted the potential importance of these genes.
we observed that the deletions altered the transcriptional efficiency, the stability of transcriptional patterns and the specificity of transcriptional changes.
the high average levels of gene expression  and the strong significance of the degs_gr upregulation  indicated that the elimination of the redundant genome sequence may save materials and energy, leading to increases in the transcriptional efficiency of the remaining genes.
the observation that the fixed chromosomal periodicity  was identical to the number of chromosomal macrodomains  <cit>  indicated that the global transcriptional pattern may be stabilized by the complete removal of the flexible is/phage regions.
the slight but significant increase in the cell growth rate most likely resulted from this steady expression pattern, which potentially accelerated cell division  <cit> .
the significant enrichment of genes of unknown function in the degs_gr and degs_hs  suggested that these genes are easily disturbed.
additional analyses revealed an apparent bias in the genomic positions of the unknown function genes; these genes were preferentially located in areas near the deleted regions , which explained the finding of genomic scar dependency in the degs_gr .
the genomic distribution of the functionally undefined genes reflected shared gene organization strategies  <cit>  that were most likely shaped by evolution.
taken together, the data suggest the possibility of a relationship between the identities of the deleted genes , the chromosomal distribution of the deleted genes, the global expression patterns , and the cell growth .
the genomic and environmental contributions to transcriptome reorganization indicated a negative epistatic relationship , that is, a negatively compensative relation of the transcriptional changes caused by the two contributors.
such relationship would inhibit the transcriptional fluctuation that was amplified by the positive correlation between the changes in gene expression triggered by the genetic and environmental interruptions .
epistasis has largely been studied within the framework of genetics, i.e., how a mutation in one gene masks the phenotypic effect of a mutation in another gene  <cit> .
the present study was the first to compare the contributions of the genome and the environment to the transcriptome and to include an analysis of the antagonistic  epistasis involved in the reorganization of transcriptome.
because mds <dig> was synthetically constructed and is therefore not a product of evolution in the wild nature, the antagonistic epistatic relationship between the genomic and environmental contributions suggested a universal innate principle of cell biology.
this principle could be described as the need to restrict the consequences of two positively correlated effects to physiologically tolerable limits.
the magnitude of the compensatory effect detected in the degs was larger than that in the  <dig> common genes  strongly supported this theory.
the adage that the sum is less than its parts  appears to be applicable to the ability of living organisms to survive in severe environments for millions of years and to an earlier report of the robustness and/or plasticity of living systems  <cit> , although more data are required to support this hypothesis.
CONCLUSIONS
this study investigated the relationship between the genetic and environmental contributions to the bacterial transcriptome.
intriguingly, in addition to similarities and overlaps in periodicity, regulatory networks, degs and directionality, both positive correlations and negative epistasis between the genomic and environmental contributions to the global transcriptional activity were observed.
these results linked the genome and the environment at the level of the transcriptome and revealed the epistatic nature of the genome-wide transcriptional reorganization that occurs universally in living cells in response to both endogenous and exogenous disturbances.
materials and methods
strains and culture conditions
the wild-type e. coli strain mg <dig> was provided by the national bioresource project, national institute of genetics, shizuoka, japan.
the reduced-genome e. coli strain mds <dig> was purchased from scarab genomics.
the genome sequences of mg <dig> and mds <dig> were retrieved from the information deposited in the genbank and ddbj databanks and have accession ids of u <dig> and ap <dig>  respectively.
when comparing the two deposited genome sequences, we found that  <dig> genes presented in mg <dig> were absent in mds <dig> , consistent with the original report  <cit> .
in addition,  <dig> genes showed mismatched sequences between two genomes .
thus, a total of  <dig> genes  of perfect matched sequences were determined to be common between the two strains.
the bacterial cells were cultured in  <dig> ml of m <dig> minimal medium 2so <dig>   <dig> μm feso4·7h2o,  <dig> μm thiamine hydrochloride,  <dig> μm mgso4·7h2o and  <dig> mm glucose) at 37°c with shaking at  <dig> rpm in a br-21fp air incubator .
cell culture conditions for the precise measurement of mrna levels and growth rates
the cell cultures were established from a glycerol stock with two preculture steps, and serial transfers were performed during the exponential phase.
the dilution rate needed to keep the final cell concentration at approximately 1-2 ×  <dig> cells/ml was calculated according to the growth rate observed during the previous day.
the initial cell concentration  was determined on the basis of the concentration of the preculture used for transfer and the dilution rate.
the final cell concentration  was measured using a flow cytometric analysis  of the remainder of the cell cultures that had been sampled for microarray analysis.
the growth rate was calculated according to the following formula: ln/t, where ct, c <dig>  and t represent the final and initial cell concentrations  and the culture time , respectively.
this formula has been previously applied in the quantitative evaluation on bacterial growth in exponential phase  <cit> .
heat shock experiment
as previous studies showed that heat shock response in e. coli was evidently induced within a few minutes  after the temperature upshift from 30–37°c to 42–50°c  <cit> , the condition for heat shock experiment was determined as a 5-min incubation following the temperature upshift from 37°c to 45°c  <cit> .
exponentially growing cells at approximately  <dig> cells/ml were rapidly transferred to an adjacent water bath incubator  set at 45°c.
following a 5-min incubation at 45°c, the cell culture was immediately poured into a cold phenol-ethanol solution to prepare the samples for microarray analysis.
each heat shock experiment was performed separately to enable the precise control of the timing of the heat shock to ensure that the mrna levels accurately reflected the stress response.
microarrays and data extraction
a high-density dna microarray covering the entire genome of the e. coli w <dig> strain was utilized  <cit>  as described elsewhere  <cit> .
the sample preparation, the microarray analysis with the affymetrix genechip system, and the data extraction based on the finite hybridization  model  <cit>  were performed as previously described  <cit> .
to avoid any significant errors resulting from the diverse signal intensities of the genechips , only array results with highly similar distributions of probe fluorescence intensities were included in the subsequent expression analysis.
the results of  <dig> arrays for the two strains under the two conditions were used .
the raw data from these  <dig> microarrays were deposited in the ncbi gene expression omnibus database under the geo series accession number gse <dig> .
the gene names are based on the genome information for w <dig> and represent the genes in common between strains w <dig> and mg <dig>  <cit> .
the entire dataset of gene names and categories is from genobase, japan .
computational analyses and graphics
the transcription levels were determined as the log-scale mrna concentrations .
the transcriptional changes  were calculated as the difference between the two transcription levels.
although several methods for the determination of chromosomal periodicity have been reported  <cit> , a simple approach was used here.
each expression value that had been determined with the method described in the previous section was projected onto the genome site that corresponded to the gene position, using 100-base bins.
next, the series of expression levels along the genome was smoothed with a moving average of  <dig> bins .
the periodicity was calculated using a standard fourier transform, and the significance of the periodicity was assessed with the chi-squared test.
the approximate line of the periodicity was calculated using the highest peak  of the periodogram and was fitted by minimizing the square error between the approximate line and the series of expression values.
gene set enrichment analysis  was performed according to the original report  <cit>  using the available online tools .
the tf and sigma factor gene regulation datasets were from regulondb.
to compare the responses of mg <dig> and mds <dig>  we limited the gene sets to those genes that were included among the  <dig> common genes.
the pre-ranked gene lists used as the input for gsea comprised genes filtered by the absolute value of their expression difference between mg1655_hs and mg <dig> .
the regulatory networks were visualized using gephi software .
subsequently, the bioconductor software package rankprod  <cit> , which is based on the rank product method, was employed to identify the differential gene expression caused by genome reduction and the heat shock response.
the rank product method is a nonparametric statistical method derived from biological reasoning that detects items that are consistently ranked high or low in a number of lists.
an advantage of this method is its ability to identify biologically relevant expression changes among a relatively small number of samples  <cit> .
finally, the statistical analyses, with the exception of gsea, were performed using r software  <cit>  , and the array plot  of the gene categories was constructed using mathematica  <dig> .
abbreviations
e. coli: escherichia coli; tf: transcription factor; bp: base pair; kb: kilobase; gr: genome reduction; hs: heat shock; degs: differentially expressed genes; fdr: false discovery rate; gsea: gene set enrichment analysis.
competing interests
the authors declare that there is no competing interest.
authors’ contributions
bwy and ty conceived the research; bwy designed and performed the experiments; bwy, ss and ty analyzed the data; fk, hm and ty provided the analytic tools; and bwy, ss and ty wrote the manuscript.
all the authors read and approved the final manuscript for publication.
supplementary material
additional file 1
figure s <dig>  chromosomal locations of the degs.
figure s <dig>  statistical analysis.
figure s <dig>  genes of unknown function.
figure s <dig>  a close-up view of the transition in major peaks.
click here for file
additional file 2
table s <dig>  detailed results of the gene set enrichment analysis.
click here for file
additional file 3
table s <dig>  list of the degs.
click here for file
additional file 4
table s <dig>  analysis of the enriched gene categories.
click here for file
acknowledgements
we thank junko asada, natsuko yamawaki, shingo suzuki, naoaki ono and chikara furusawa for technical assistance and analytical tools.
we thank the anonymous reviewers for helping us to improve the clarity of the manuscript.
bwy was supported by the project of novel and innovative r&d making use of brain structures, from the ministry of internal affairs and communications, japan.

 the bacterial transcriptome, as one of the important genome-level information, reflects the global cellular activity. since the fitness and/or activity of the cells is directly influenced by the environmental perturbation and genetic disturbance, the evaluation of the environmental and genetic contributions to transcriptome turns to be highly essential. in recent, the transcriptome analysis of bacteria has been performed extensively using a range of experimental and analytical tools, to illustrate an overall picture of the biochemical events occurring inside the cells. in particular, to examine the environmental contributions to bacterial transcriptome, gene expression profiling has been tested across multiple environmental conditions in a high-throughput manner. as a consequence, the intensive studies on global transcriptional activity contributed by environmental perturbations successfully provided conclusions regarding the molecular mechanisms involved in specific pathways and/or regulatory networks, as well as the theoretical and/or overall outlook in regard to global dynamic changes in transcriptome. however, the genetic contribution to bacterial transcriptome has been rarely reported. although the previous studies revealed the potential links between transcriptional regulation and chromosome organization and the relation between changes in dna methylation and chromosomal structure and changes in global gene expression, whether and how the genomic scars influence the global transcriptional activity is unclear. both genomic and environmental perturbations could initiate a global change in the transcriptome, because both are stressful for living cells. whether there is any potential linkage between the two contributions is an intriguing question. to address the question, a comprehensive analysis comparing the effects of genomic and environmental interruptions on the transcriptome is highly required. such analysis would not only enable a comparison of the internal and external influences on the plasticity of genome-wide transcriptional activity but would also provide a valuable example of a global and conceptual assessment of high-throughput biological data. therefore, a multi-level survey of genomic expression is highly in demand, to show how genomic disturbance causes the global transcriptional reorganization and whether there is any relationship between the contributions of genomic and environmental interruptions to transcriptome. as a pilot study, we evaluated these two distinct entities, the genome and the environment, at the level of the transcriptome. we addressed how genomic and environmental perturbations contribute to the bacterial transcriptome and whether there is any interaction between the two influences. a multiple deletion e. coli strain, mds, was compared with its wild-type parent strain mg in this study. because of its lack of insertion sequences, mds has been widely used in a number of applications; however, its detailed, genome-wide analysis has been rarely reported. the genes used for the comparative transcriptome analysis were selected on the basis of the genome sequences of mds and mg the comparison of mg to mds provided insights into genomic disturbance-induced transcriptional reorganization. the evaluation of the heat shock response provided insights into environmental perturbation-induced transcriptional changes. we examined whether the losses of insertion elements in genome interrupt the genome-wide transcriptional activity and whether these multiple deletions in genome and the heat shock stress exert common or different effects on transcriptional changes. furthermore, we performed multilevel comparative analyses of chromosomal periodicity, regulatory networks, the localization of differential gene expression, transcriptional sensitivity in response to genomic and environmental perturbations and the epistatic effect, that is, a negatively compensated relationship, on the transcriptome of the genome and the environment.  strains and genes subjected to transcriptome analysis gene expression was analyzed using a custom, high-density dna microarray with a precise and wide quantitative range of mrna concentrations, as previously described for genome resequencing and transcriptome analysis. first, the wild-type strain mg and the multiple deletion strain mds were used to examine whether and how removing the nonessential genomic regions in e. coli contributed to its transcriptome. repeated experiments were performed with cells grown precisely to mid-exponential phase in minimal medium. the genes shared between the two strains were determined by comparing their genome sequences. in total, locus tags were assigned to and open reading frames in mg and mds respectively, which was consistent to the original report. genes with repeated locus tags were removed from the analysis, for a final total of genes with locus tags in mg excluding the deleted genes and mutated genes in mg, a total of genes were determined to be common between the two strains. subsequently, as the heat shock stress is known as one of many external perturbations and showed distinguishable expression profiles, heat shock experiments were performed to evaluate how the external disturbance contributed to the transcriptome for comparative analyses. the average gene expression levels from repeated experiments were plotted in figure 1b and used for further analysis. we showed that neither genomic reduction nor heat shock altered the shape of the distribution of gene expression. priority in chromosomal periodicity the periodicity of genome-wide transcriptional activity was studied to provide a global view of gene expression profiling. the average transcription levels over the entire genome were plotted for both genomes using a 100-bp sliding window. the periodic patterns showed a major peak of approximately kb in mg consistent with previous reports. however, these periodic patterns were disturbed by heat shock. the most significant wavelength, as indicated by the red, vertical, broken line in figure, shifted from to kb. this slight but statistically significant change in the major peak caused a reduction in the number of periods from seven to six. this finding indicated that the heat shock treatment initiated a reorganization of the transcriptome that gave a high priority to a periodicity of six periods. interestingly, the reduced genome also showed a fixed periodicity of six periods. mds maintained the parental periodogram, with a major peak at kb that was accompanied by a decrease in the chromosomal period number. this reduced period was unchanged in mds in response to heat shock perturbation. in addition, when the expression of the common genes in mg was plotted against the mds genome, an identical chromosomal periodicity of six periods was observed independently of external conditions. statistical analyses confirmed that the altered periodicity was highly specific for the mds genome and was not due to random deletions in the genome. the stable six periods may be have been caused by the presence of six macrodomains in the chromosomal structure, as determined by recombination activity. the results suggested that the absent genetic regions in mds may contain a considerable number of genes that are not or rarely transcribed under heat shock conditions, potentially contributing to an additional period. thus, the multiple deletions not only reduced the genome size but also provided, probably by chance, a steady periodicity to the global transcriptional activity of the e. coli chromosome. overlaps in perturbed regulatory networks regulatory network maps comprising the transcription factors that control more than genes were constructed as shown in figure significant changes in gene expression are represented as gradations according to their fdr values, which were determined using the rank product method. overall, both genome reduction and heat shock exerted broad effects on transcriptional reorganization: the expression levels of a number of genes were changed. a small number of regulators showed remarkable, independent transcriptional changes; for example, gadx was induced only by genome reduction and rpod only by heat shock. it indicated that genome structural changes might activate the primary activator of the gad system but fairly influenced the global transcriptional factor. in addition, a comparable number of regulators were disturbed by both perturbations in a correlated manner: those induced by genome reduction were also induced by heat shock. the changes in these overlapping perturbed regulators occurred in either the same or the reverse direction; e.g., the expression level of phob was upregulated by both genome reduction and heat shock, whereas, the expression level of gade was upregulated by genome reduction but repressed by heat shock. to identify these regulators upstream of genes with marked changes in transcription induced by either genome reduction or heat shock, a gene set enrichment analysis was applied. among the regulatory networks controlled by sigma factors and tfs, and networks showed clear transcriptional changes associated with genome reduction and heat shock, respectively. heat shock interrupted approximately equal numbers of the induced and repressed regulatory networks, whereas genome reduction triggered many more upregulated than downregulated networks. moreover, most of the regulatory networks that responded to heat shock were highly sensitive to genome reduction as well. these overlapping regulatory networks showed a marked tendency to undergo transcriptional changes in the same direction in response to genomic and heat shock perturbations. the exception was found in the network controlled by gadx, where the downstream genes were induced by genome reduction but suppressed by heat shock. the information pertaining to transcription factors, sigma factors and the downstream genes under their control is from regulondb. the transcriptional changes in the downstream genes were evaluated with gene set enrichment analysis, and the disturbed regulatory networks were determined accordingly. perturbation and direction refer to the type of disturbance to the transcriptome and the directionality of the changes in gene expression, respectively. locus tag, name of tf, size and fdr q-val represent the gene id, the corresponding regulator gene, the number of regulated downstream genes and the statistical significance, respectively. taken together, the transcriptional reorganization observed in the regulatory networks was not only secondary to the expression changes in the regulators but also occurred in networks controlled by the regulators of steady expression. the latter case suggested either weakened transcriptional control by the regulators or a high level of sensitivity in the downstream genes. the discovery of regulatory networks that were universally involved in both perturbations indicated that transcriptional reorganization responds similarly to both endogenous and exogenous disruptions. genome location dependency and gene category enrichment of highly responsive genes genes with significant transcriptional changes among the common genes were also determined with the rank product method. in all, and genes were identified that showed either up- or downregulated expression in response to genome reduction or heat shock, respectively. the gene names, gene categories, locus tags, and directions of the transcriptional changes are summarized in additional file 3: table s and the genome locations are marked in additional file 1: figure s an analysis of the distances between the degs and the genome scars caused by genome reduction showed that the degs_gr, but not the degs_hs, were located much closer to the nearest genome scar than were the genes with conserved expression. this tendency indicated that the genomic disturbance caused by genome reduction brought about considerable transcriptional changes in neighboring genes. in particular, the transcriptional changes were highly significant in the direction of upregulation, which suggests that the removal of the dispensable genetic segment might increase the expression efficiency of the neighboring genes. we assumed that the deleted genes most likely perturbed the local superhelical density and thereby affected the transcriptional activity of the neighboring genes, although the functions of the deleted genes near the deletion junctions or mutations remain to be further examined. the gene categories that were enriched in the degs were analyzed to address the question of whether the genomic and heat shock perturbations had affected any specific gene categories. the heat map illustrates that the heat shock affected only a limited number of gene categories with high significance. however, the genome reduction influenced a relatively large number of gene categories with diverse directions in transcriptional changes; for example, the transporter genes and the rna-coding genes were highly enriched in the degs_gr in the directions of down- and upregulation, respectively. only the “unknown function” category was significantly enriched in both the degs_gr and the degs_hs. this result suggested that the genes with unknown functions were highly sensitive to both genomic and environmental perturbations, as might result from a loose control of gene expression. the genes in the factor category, of which most are heat shock proteins, were not only enriched in the upregulated degs_hs, as extensively reported, but were also detected in the upregulated degs_gr. as a result, factor was the most highly enriched category among the overlapping genes. intriguingly, some well-known molecular chaperones, such as clpb and ibpa, were highly responsive to both perturbations, indicating a universal response mechanism to internal and external stressors in these genes. positive correlations in changes of gene expression the directions of the transcriptional changes demonstrated by the degs were further analyzed, in particular, on the gene expression patterns by means of correlations. in a global view, the degs_gr also showed remarkable transcriptional changes in response to heat shock, as evidenced by the significantly lower correlation of the degs_gr compared with that of the common genes. similarly, the degs_hs showed large transcriptional changes in response to genomic perturbation; their correlation was significantly lower than that of the common genes. furthermore, the upregulated degs_hs were also induced by genome reduction, and the upregulated degs_gr were also induced by heat shock. these correlated patterns of changes in gene expression deduced from the overall evaluation of the degs implied that the overlapping genes might play a crucial role in producing such transcriptional fluctuation because not all the degs changed significantly when moving to the other condition. among the overlapping genes, and genes with up- or downregulated expression in response to genome reduction were up- or downregulated by heat shock in the same direction, respectively. only a single gene reversed the direction of its transcription; i.e., it was induced by heat shock but repressed by genome reduction. the overall directional similarities implied that the genes that were sensitive to genomic disturbance tended to be highly responsive to external perturbation and vice versa. furthermore, the transcriptional changes of the genes that were induced by genome reduction were plotted against those that were induced by heat shock. a clear, positive correlation between the transcriptional changes produced by the genetic and environmental contributions was observed. in other words, larger degrees of transcriptional fluctuation triggered by genetic interruption were correlated with greater changes in gene expression induced by environmental perturbation. the positive correlation between these two contributions to the transcriptome implies somehow a linkage in transcriptional reorganization in response to both genetic and environmental stresses. antagonistic epistasis in the transcriptome theoretically, the positive correlation in the transcriptional changes triggered by the genetic and environmental interruptions potentially had a tendency to amplify the magnitude of the changes in gene expression that occurred in response to both genetic and environmental perturbations. for instance, the gene expression levels of dnaj were approximately and folds up-regulated due to genome reduction and heat shock, respectively. owing to the positive correlation, the change in the expression of dnaj was assumed to be multiplied, up to folds, by the two simultaneously occurred perturbations. actually, it was a 21-fold increase, a highly suppressed value, in the transcriptional change. to understand such repressed change in gene expression, the genetic concepts of epistasis and additivity were employed, as represented schematically in figure 6a. these concepts are widely used to determine whether the final fitness or output is simply the sum of the effects of two genes or whether one gene partially inhibits or enhances the effects of the other gene. the extended definition of epistasis could be applied to the interactions among the chemicals and molecules in highly complex biochemical reactions. here, the two analyzed effects were the transcriptional changes contributed by genome reduction and heat shock individually, and the final output was defined as the transcriptional changes that were produced when the genomic and heat shock perturbations occurred simultaneously, i.e., the reduced genome undergoing heat shock. the slopes represent the transcriptional changes perturbed by the genome and environment in an additive or epistatic manner. the difference in the slopes, defined as α, represented the relative magnitude of the difference between the additive and simultaneous contributions. accordingly, the relationship between the contributions of genome reduction and heat shock to transcriptome was subsequently analyzed in a genomic view. considering the difference in the number of genes in mg and mds the common genes shared with both strains were applied in the following analyses. the transcriptional changes due to these two independent contributors were added, and the sum was considered to be the additive contribution. the changes in gene expression between mds42_heatshock and mg represented simultaneous genetic and environmental interruptions. the additive contribution was then plotted against the simultaneous contribution, as shown in figure 6b. the simultaneous contribution showed a smaller magnitude of transcriptional changes than did the additive contribution, approximately 30% suppressive effect. it strongly indicated a negatively compensated relationship between the genomic and environmental contributions to the changes in the transcriptome, as so-called “antagonistic epistasis”. moreover, the magnitude of the compensatory effect became larger in the gene groups of the degs_gr and the degs_hs, suggesting that the negative epistasis became more significant, approximately 40%. both the genetic and environmental perturbations alone triggered fluctuations in the transcriptome, whereas either perturbation repressed or limited the fluctuation caused by the other when both occurred simultaneously. these results indicated that negative epistasis may be universally involved in biological processes, although the underlying mechanisms and theoretical principles remain to be determined. discussion this study demonstrated a considerable interaction between the genetic and environmental contributions to the changes in the transcriptome, as evidenced by the identical preferred chromosomal periodicity, the directional similarities in the transcriptional changes, the overlaps between the perturbed regulatory networks, the positive correlation in transcriptional changes induced by the genetic and environmental contributions and the negative epistasis. thus, our multilevel comparative analysis provided a global comparison of the effects of genomic and heat shock perturbations to the e. coli transcriptome. a more comprehensive evaluation based on the additional experiments under cross multiple conditions and a cross validation using the fruitful reported data sets are essential to figure out the common features of the genomic and environmental contributions to the reorganization in transcriptome. the transcriptome analysis caught the genome reduction effect. the deleted regions were assumed to have crucial functions, on the basis of our novel observations of the reduced genome, along with the previous reports of its advantages in various applications but its limited evolvability. as these regions were largely occupied by genes that were derived from the is/phage, that encoded structural components, or that had unknown or predicted functions, it highlighted the potential importance of these genes. we observed that the deletions altered the transcriptional efficiency, the stability of transcriptional patterns and the specificity of transcriptional changes. the high average levels of gene expression and the strong significance of the degs_gr upregulation indicated that the elimination of the redundant genome sequence may save materials and energy, leading to increases in the transcriptional efficiency of the remaining genes. the observation that the fixed chromosomal periodicity was identical to the number of chromosomal macrodomains indicated that the global transcriptional pattern may be stabilized by the complete removal of the flexible is/phage regions. the slight but significant increase in the cell growth rate most likely resulted from this steady expression pattern, which potentially accelerated cell division. the significant enrichment of genes of unknown function in the degs_gr and degs_hs suggested that these genes are easily disturbed. additional analyses revealed an apparent bias in the genomic positions of the unknown function genes; these genes were preferentially located in areas near the deleted regions, which explained the finding of genomic scar dependency in the degs_gr. the genomic distribution of the functionally undefined genes reflected shared gene organization strategies that were most likely shaped by evolution. taken together, the data suggest the possibility of a relationship between the identities of the deleted genes, the chromosomal distribution of the deleted genes, the global expression patterns, and the cell growth. the genomic and environmental contributions to transcriptome reorganization indicated a negative epistatic relationship, that is, a negatively compensative relation of the transcriptional changes caused by the two contributors. such relationship would inhibit the transcriptional fluctuation that was amplified by the positive correlation between the changes in gene expression triggered by the genetic and environmental interruptions. epistasis has largely been studied within the framework of genetics, i.e., how a mutation in one gene masks the phenotypic effect of a mutation in another gene. the present study was the first to compare the contributions of the genome and the environment to the transcriptome and to include an analysis of the antagonistic epistasis involved in the reorganization of transcriptome. because mds was synthetically constructed and is therefore not a product of evolution in the wild nature, the antagonistic epistatic relationship between the genomic and environmental contributions suggested a universal innate principle of cell biology. this principle could be described as the need to restrict the consequences of two positively correlated effects to physiologically tolerable limits. the magnitude of the compensatory effect detected in the degs was larger than that in the common genes strongly supported this theory. the adage that the sum is less than its parts appears to be applicable to the ability of living organisms to survive in severe environments for millions of years and to an earlier report of the robustness and/or plasticity of living systems, although more data are required to support this hypothesis.  this study investigated the relationship between the genetic and environmental contributions to the bacterial transcriptome. intriguingly, in addition to similarities and overlaps in periodicity, regulatory networks, degs and directionality, both positive correlations and negative epistasis between the genomic and environmental contributions to the global transcriptional activity were observed. these results linked the genome and the environment at the level of the transcriptome and revealed the epistatic nature of the genome-wide transcriptional reorganization that occurs universally in living cells in response to both endogenous and exogenous disturbances. materials and methods strains and culture conditions the wild-type e. coli strain mg was provided by the national bioresource project, national institute of genetics, shizuoka, japan. the reduced-genome e. coli strain mds was purchased from scarab genomics. the genome sequences of mg and mds were retrieved from the information deposited in the genbank and ddbj databanks and have accession ids of u and ap respectively. when comparing the two deposited genome sequences, we found that genes presented in mg were absent in mds, consistent with the original report. in addition, genes showed mismatched sequences between two genomes. thus, a total of genes of perfect matched sequences were determined to be common between the two strains. the bacterial cells were cultured in ml of m minimal medium 2so μm feso4·7h2o, μm thiamine hydrochloride, μm mgso4·7h2o and mm glucose) at 37°c with shaking at rpm in a br-21fp air incubator. cell culture conditions for the precise measurement of mrna levels and growth rates the cell cultures were established from a glycerol stock with two preculture steps, and serial transfers were performed during the exponential phase. the dilution rate needed to keep the final cell concentration at approximately 1-2 ×  cells/ml was calculated according to the growth rate observed during the previous day. the initial cell concentration was determined on the basis of the concentration of the preculture used for transfer and the dilution rate. the final cell concentration was measured using a flow cytometric analysis of the remainder of the cell cultures that had been sampled for microarray analysis. the growth rate was calculated according to the following formula: ln/t, where ct, c and t represent the final and initial cell concentrations and the culture time, respectively. this formula has been previously applied in the quantitative evaluation on bacterial growth in exponential phase. heat shock experiment as previous studies showed that heat shock response in e. coli was evidently induced within a few minutes after the temperature upshift from 30–37°c to 42–50°c, the condition for heat shock experiment was determined as a 5-min incubation following the temperature upshift from 37°c to 45°c. exponentially growing cells at approximately cells/ml were rapidly transferred to an adjacent water bath incubator set at 45°c. following a 5-min incubation at 45°c, the cell culture was immediately poured into a cold phenol-ethanol solution to prepare the samples for microarray analysis. each heat shock experiment was performed separately to enable the precise control of the timing of the heat shock to ensure that the mrna levels accurately reflected the stress response. microarrays and data extraction a high-density dna microarray covering the entire genome of the e. coli w strain was utilized as described elsewhere. the sample preparation, the microarray analysis with the affymetrix genechip system, and the data extraction based on the finite hybridization model were performed as previously described. to avoid any significant errors resulting from the diverse signal intensities of the genechips, only array results with highly similar distributions of probe fluorescence intensities were included in the subsequent expression analysis. the results of arrays for the two strains under the two conditions were used. the raw data from these microarrays were deposited in the ncbi gene expression omnibus database under the geo series accession number gse. the gene names are based on the genome information for w and represent the genes in common between strains w and mg. the entire dataset of gene names and categories is from genobase, japan. computational analyses and graphics the transcription levels were determined as the log-scale mrna concentrations. the transcriptional changes were calculated as the difference between the two transcription levels. although several methods for the determination of chromosomal periodicity have been reported, a simple approach was used here. each expression value that had been determined with the method described in the previous section was projected onto the genome site that corresponded to the gene position, using 100-base bins. next, the series of expression levels along the genome was smoothed with a moving average of bins. the periodicity was calculated using a standard fourier transform, and the significance of the periodicity was assessed with the chi-squared test. the approximate line of the periodicity was calculated using the highest peak of the periodogram and was fitted by minimizing the square error between the approximate line and the series of expression values. gene set enrichment analysis was performed according to the original report using the available online tools. the tf and sigma factor gene regulation datasets were from regulondb. to compare the responses of mg and mds we limited the gene sets to those genes that were included among the common genes. the pre-ranked gene lists used as the input for gsea comprised genes filtered by the absolute value of their expression difference between mg1655_hs and mg. the regulatory networks were visualized using gephi software. subsequently, the bioconductor software package rankprod, which is based on the rank product method, was employed to identify the differential gene expression caused by genome reduction and the heat shock response. the rank product method is a nonparametric statistical method derived from biological reasoning that detects items that are consistently ranked high or low in a number of lists. an advantage of this method is its ability to identify biologically relevant expression changes among a relatively small number of samples. finally, the statistical analyses, with the exception of gsea, were performed using r software, and the array plot of the gene categories was constructed using mathematica. abbreviations e. coli: escherichia coli; tf: transcription factor; bp: base pair; kb: kilobase; gr: genome reduction; hs: heat shock; degs: differentially expressed genes; fdr: false discovery rate; gsea: gene set enrichment analysis. competing interests the authors declare that there is no competing interest. authors’ contributions bwy and ty conceived the research; bwy designed and performed the experiments; bwy, ss and ty analyzed the data; fk, hm and ty provided the analytic tools; and bwy, ss and ty wrote the manuscript. all the authors read and approved the final manuscript for publication. supplementary material additional file 1 figure s chromosomal locations of the degs. figure s statistical analysis. figure s genes of unknown function. figure s a close-up view of the transition in major peaks. click here for file additional file 2 table s detailed results of the gene set enrichment analysis. click here for file additional file 3 table s list of the degs. click here for file additional file 4 table s analysis of the enriched gene categories. click here for file acknowledgements we thank junko asada, natsuko yamawaki, shingo suzuki, naoaki ono and chikara furusawa for technical assistance and analytical tools. we thank the anonymous reviewers for helping us to improve the clarity of the manuscript. bwy was supported by the project of novel and innovative r&d making use of brain structures, from the ministry of internal affairs and communications, japan.
the transcriptome analysis showed the following: there was a reorganization of the transcriptome in accordance with preferred chromosomal periodicity upon genomic or heat shock perturbation; there was a considerable overlap between the perturbed regulatory networks and the categories enriched for differentially expressed genes following genome reduction and heat shock; the genes sensitive to genome reduction tended to be located close to genomic scars, and some were also highly responsive to heat shock; and the genomic and environmental contributions to the transcriptome displayed not only a positive correlation but also a negatively compensated relationship. both large deletions in genome and heat shock stress would lead to alterations in the gene expression profile; however, whether there is any potential linkage between these disturbances to the transcriptome have not been discovered. the contributions of genome reduction and heat shock to the escherichia coli transcriptome were evaluated at multiple levels. transcriptomenegative epistasisgenome reductionchromosomal periodicityregulatory networktranscriptional changegenomic interruptionenvironmental perturbationheat shockdirectionality here, the relationship between the genomic and environmental contributions to the transcriptome was analyzed by comparing the transcriptomes of the bacterium escherichia coli grown in regular and transient heat shock conditions. the observations of overlapping perturbed networks, directional similarity in transcriptional changes, positive correlation and epistatic nature linked the two contributions and suggest somehow a crosstalk guiding transcriptional reorganization in response to both genetic and environmental disturbances in bacterium e. coli.
 both large deletions in genome and heat shock stress would lead to alterations in the gene expression profile; however, whether there is any potential linkage between these disturbances to the transcriptome have not been discovered. here, the relationship between the genomic and environmental contributions to the transcriptome was analyzed by comparing the transcriptomes of the bacterium escherichia coli grown in regular and transient heat shock conditions.  the transcriptome analysis showed the following: there was a reorganization of the transcriptome in accordance with preferred chromosomal periodicity upon genomic or heat shock perturbation; there was a considerable overlap between the perturbed regulatory networks and the categories enriched for differentially expressed genes following genome reduction and heat shock; the genes sensitive to genome reduction tended to be located close to genomic scars, and some were also highly responsive to heat shock; and the genomic and environmental contributions to the transcriptome displayed not only a positive correlation but also a negatively compensated relationship.  the contributions of genome reduction and heat shock to the escherichia coli transcriptome were evaluated at multiple levels. the observations of overlapping perturbed networks, directional similarity in transcriptional changes, positive correlation and epistatic nature linked the two contributions and suggest somehow a crosstalk guiding transcriptional reorganization in response to both genetic and environmental disturbances in bacterium e. coli. transcriptomenegative epistasisgenome reductionchromosomal periodicityregulatory networktranscriptional changegenomic interruptionenvironmental perturbationheat shockdirectionality 