this occurs mostly through the action of dedicated transcription factors ( tfs ) , as in the well - known case of the lac operon ( jacob et  al . , 1960 ) . on the other hand , gene expression is continuously adjusted to adapt to varying environmental conditions , leading to quantitative relationships between the molecular content of cells and their growth rates ( scott and  hwa , 2011 ) .  physiological factors ( berthoumieux et  al . , 2013 ) , such as the concentration of rna polymerases ( rnaps ) ( klumpp and hwa , 2008 ) , the regulation of rna degradation ( chen et  al . , 2015 ) , and topological properties of dna ( dorman , 1995 , hatfield and benham , 2002 , travers and muskhelishvili , 2005 ) , are thus known to continuously affect , at a system level , the transcriptional activity of genes . the specificity , if any , of each of these mechanisms with respect to the set of co - regulated genes , nevertheless remains to be understood . in bacteria , the coordination of transcription is strongly related to the linear organization of genomes ( kps et  al . , 2012 ) . at the smallest scale , many co - regulated genes are thus found within operons so that they can be co - transcribed into the same mrnas . despite their apparent simplicity , the operons have nevertheless raised important questions , not only about their determination but also about their definition ( okuda et  al . , 2007 , gell et  al . , 2011 , mazin et  al . , 2014 ) and utility ( de lorenzo and danchin , 2008 , junier , 2014 ) . for instance , although certain operons are easily recognizable by their functional homogeneity ( as , e.g. , for the lac operon ) , many of them are composed of genes whose function appears unrelated ( de lorenzo and danchin , 2008 ) . soon after the seminal work of jacob and monod , studies on operons such as trp also revealed the possibility to  have specific internal regulation of termination ( yanofsky , 2000 ) : mrna - based intrinsic terminators may abort transcription midway , whereas competing mrna secondary structures ( anti - terminators ) may attenuate this effect ( merino and yanofsky , 2005 , santangelo and artsimovitch , 2011 ) . together with the observation that the majority of operons actually contain alternative transcription start sites ( tsss ) ( sharma et  al . , 2010 , cho et  al . , 2014 ) , high - throughput data have thus revealed the presence , inside operons , of differential initiation and termination points ( okuda et  al . , 2007 , gell et  al . , 2011 , nicolas et  al . , 2012 , mazin et  al . , 2014 ) . yet the impact of these internal elements on the genome - wide coordination of transcription has remained unexplored . model systems such as the bacteriophage  further revealed that operons may be part of larger functional genomic units with , in particular , the possibility of having subsequent operons transcribed in one go ( gottesman et  al . trt has actually been shown to be frequent and regulated by dedicated proteins ( stlke , 2002 , nudler and gottesman , 2002 ) , with the so - called  factor playing a major role in many bacteria ( richardson , 2002 ) . transcriptional co - expression has thus been shown to extend beyond operons ( jeong et  al . , 2004 , carpentier et  al . , 2005 , nicolas et  al . , 2012 ) . yet the systematic identification of supra - operonic units and of their regulatory mechanisms remains an open problem . a system - level understanding of transcriptional coordination thus requires abandoning , at least in the first stage , our preconception of the potential units that may come at play . a promising avenue along this line consists in analyzing in detail the genomic properties of proximal genes as a function of their degree of co - expression and to question the structural and regulatory properties that might be associated to the observed patterns ( ma et  al . , 2013 ) . here , we perform such analysis in m.  pneumoniae , a model organism with a reduced genome ( 820 kb ) that offers ideal properties to address questions about the fundamental mechanisms that govern bacterial cell physiology ( gell et  al . , 2009 , in particular , although m.  pneumoniae has two sigma factors ( torres - puig et  al . , 2015 ) , a tiny tf repertoire ( table s1 ) and no  factor ( himmelreich et  al . ,  1996 ) , it shows genome - wide complex specific regulatory patterns in response to different external perturbations ( gell et  al . , 2009 ) . this suggests the existence of fundamental mechanisms ensuring coordination of transcription , different from tfs and from the  factor . to test this hypothesis and to identify associated regulatory mechanisms , we analyzed rna sequencing ( rna - seq ) data obtained from m.  pneumoniae under 115 different conditions ( figure  1a ) . to this end , we built a co - expression measure particularly well poised to highlight basal co - expression . using a hierarchical clustering framework that is constrained to respect the 1d organization of the genome , we then reveal the existence of three qualitatively distinct levels of co - expression associated to different organizations of adjacent genes and to different properties of intergenic regions . we next show that the degree of co - expression between co - directional genes and operons is tightly related to the capacity of the rnap to transcribe them as if they belonged to the same operon . we then reveal that such trt is both ubiquitous and condition dependent and that it is repressed by dna - bound rnaps , strong intrinsic terminators , and large intergenic distances . we measured the transcriptional activity of 869 m.  pneumoniae genes , of which 701 encode proteins ( lluch - senar et  al . , 2015 ) ,  across 141 conditions ( 282 samples ) . to this end , m.  pneumoniae m129 ( passage 34 , nc_000912 reference genome in the national center for biotechnology information [ ncbi ] ) was grown in modified hayflick medium and transformed by electroporation ( yus et  al . , 2009 ) . rna - seq data were then collected at various stages of the cell growth , after various perturbations and overexpression of different regulators ( table s2 ) . the resulting transcription profiles were generally highly similar , even after shuffling the expression values between the conditions for each gene separately ( light gray distribution in figure  1a ) , showing that genes have mostly stable expression . to focus specifically on basal co - expression we identified a large set of 227 highly similar samples corresponding to 115 conditions ( 112 for which two technical replicates are present ) , the 29 remaining conditions being characterized by a particularly low level of transcription ( table s2 ) . to analyze the basal coordination of transcription between all pairs ( i , j ) of genes , we built a specific measure of transcriptional co - expression , cij , hereafter referred to as  basal correlation . cij quantifies the tendency of the expression of the two genes to systematically vary in parallel ( figure  s1 ) , here among the 227 working samples . specifically , it is equal to the difference between the number of pairs of samples for which the two genes co - vary and the number for which they vary in the opposite direction , normalized by the total number of possible pairs of samples . compared with other correlation measures to which it is related , such as the pearson correlation , cij is well suited to highlight the  basal coordination of genes ( figure  s1 ) . in particular , cij0 indicates that the expression of the two genes varies as many times in the same direction as in the opposite direction . in contrast , cij1 indicates that they always vary in the same direction , whereas cij1 indicates a systematic tendency to vary in the opposite direction . figure  1b shows the resulting heatmap of cij , for which the  genes are sorted according to their genomic position , and each pixel indicates the co - expression level between two genes . one can distinguish the presence of specific contiguous clusters of highly co - expressed genes with , on average , a genomic extension that typically extends up to 10 kbp ( figure  1b , bottom ) . this 10 kbp length scale is larger than the typical length scale of operons , corroborating that the co - expression of proximal genes extend beyond operons . it is actually similar to that found in e.  coli and b.  subtilis when performing a similar analysis of co - expression ( jeong et  al . , 2004 , carpentier et  al . , 2005 , junier and rivoire , 2016 ) . to delineate in a more precise way the relationship between basal coordination of transcription and the established organization of m.  pneumoniae into operons ( see experimental procedures for their definition ) , we analyzed in detail the genomic organization of co - expression . to this end , we developed a hierarchical description of co - expression constrained to respect the linear organization of the genome . briefly , we built a dendrogram in which pairs of adjacent genes were hierarchically fused on the basis of their co - expression ( figure  1c ) . using this dendrogram , we defined domains of the genome , which we call  -domains , as the contiguous domains of genes for which all the adjacent genes have a co - expression larger than  ( figure  1c ) . an analysis of  -domains for all possible values of  reveals that although these domains may coincide with operons at certain values of  , they are generally different . this can be qualitatively appreciated for specific clusters of genes , such as that of the f - atpase machinery ( figure  1c ) . more quantitatively , we evaluated the capacity of  -domains to predict operons using our most recent manual annotation of operons as the ground truth ( table s1 ) . to this end , we made  vary from 1 to 1 and we assessed both the specificity and the sensitivity of predictions . the resulting area under the receiver operating curve ( auc ) , which summarizes the balance between specificity and sensitivity by a single value , was equal to 0.76 ( figure  1d ) . -domains are thus not perfect predictors ( in which case the auc would have been equal to 1 ) , corroborating the necessity to analyze co - expression properties independently of our knowledge of operons , at least in the first stage . to better understand the hierarchical properties of co - expression , we analyzed the relative orientations of adjacent genes as a function of their co - expression ( 846 pairs analyzed for which expression values were available for the two genes ) ( figure  2a ) . we also analyzed the tendency of co - directional genes to overlap ( figure  2b ) , as well as the intergenic distances between the non - overlapping ones ( figure  2c ) . notably , the three properties ( relative orientation , overlapping , and distance ) suggest a similar three - level organization of co - expression . specifically , for co - expression > 0.6 ( strong co - expression ) , 227 of 234 pairs of genes are co - directional , with a high proportion ( 52% ) of overlapping cases ( p4.1011 , hypergeometric test ) . for co - expression between 0.3 and 0.6 ( moderate co - expression , 375 pairs ) , genes significantly tend to be co - directional ( p7.107 ) and to overlap ( p7.105 ) , all the more that co - expression is large . at this level , although intergenic distances between non - overlapping genes are larger than those with strong co - expression , they remain relatively small . plotting the co - expression level of all pairs of non - overlapping co - directional genes as a function of their intergenic distance actually reveals the existence of a 100  bp length scale below which typical co - expression is larger than 0.3 and above which co - expression is low and statistically insensitive to distances ( figure  2d ) . finally , below 0.3 ( low co - expression , 237 pairs ) , there is no enrichment for a specific relative orientation of genes ( p 1 , binomial test of the hypothesis that the probability of co - directionality is equal to 0.5 ) . moreover , intergenic distances between co - directional genes are large , exceeding 100  bp and reaching typically 400  bp at very low co - expression ( figure  2c ) . performing the same analyses using pearson correlation led qualitatively to the same findings ( figure  s2 ) . the sharp delineation of the three different regimes as well as their correspondence between the different properties ( relative orientation , overlapping , and distance ) is less clear , though , than those obtained using the basal correlation . because a significant number of operons have moderate co - expression levels and because trt pervades the transcriptomes of bacteria ( wade and grainger , 2014 ) , we wondered whether trt could explain the co - expression levels of co - directional genes belonging to distinct operons . we thus investigated the tendency of all adjacent co - directional genes belonging to two different operons to be transcribed as a single transcript ( 268 pairs analyzed ) . we examined the variation of expression in their intergenic region as a function of the variation of expression of the downstream gene ( figure  3a ) . our rationale was that trt , if present , should leave a trace on the expression of the intergenic region that precedes the downstream gene . we thus compared the co - expression between the downstream gene and the sense ( 5 3 ) intergenic region ( co - expression cs in figure  3a ) with that between the two genes ( co - expression c ) ; as a control , we considered the anti - sense ( 3 5 ) region ( figure  3a , right ) ( co - expression ca ) . to prevent any bias arising from the transcription of the utrs inside operons , for this analysis , we defined intergenic regions as the sequences that separate the transcription termination site ( tts ) of the upstream gene and the tss of the downstream gene . for co - expression levels larger than 0.3 , we observed that the degree of co - expression between co - directional adjacent operons was higher when there was co - expression with the sense intergenic region ( figure  3a ) ; in contrast , it did not show any dependency with the anti - sense expression ( figure  3a , right ) . the same analyses , but considering genes that are separated by more than 100  bp ( figure  3b ) , or using the pearson correlation ( figure  s2e ) , led to the same conclusions . next , to explicitly demonstrate the role of trt in basal coordination of transcription , we first studied the efficiency ,  , of trt extending between two co - directional adjacent operons , say , x  and y with x preceding y ( figure  s3a ) . was defined as the ratio between the rna - seq expression levels measured at the tss of y and at the stop codon of the last cistron of x ( independently whether this was associated to a well - defined terminator ) . we thus assumed that the rna - seq level just preceding y would result from the trt of x and would be representative of the basal level of y. according to this model , for which we provide below an experimental validation , the overall expression of y is thus equal to the sum of its basal level coming from trt , plus some contribution from its own tss ( figure  s3a ) . we analyzed seven pairs of genes with various degrees of correlations and distances : two pairs with strong correlation , including one overlapping case ( mpn155a - mpn155 ) ; four pairs with moderate correlation and distances larger than 100  bp , including two pairs with an intermediate gene located on the opposite strand of their intergenic region ; and one pair with low co - expression . as shown in figure  s3b ,  was close to 1  and varied little for pairs with strong correlation , but also for  the pair mpn160-mpn161 ( highest moderate correlation , c=0.5 ) except during heat shock . for the other pairs , in particular , for the pair mpn161-mpn162 ( low correlation ) , we observed a 2-fold variation during cold shock that poorly correlated to the expression variation of the downstream gene . we then tested the validity of our trt - based model of transcriptional coordination by confronting predictions of the model ( using rna - seq data ) to direct measurements of transcripts using real - time quantitative pcr ( qpcr ) . specifically , for the aforementioned seven pairs , we measured the level of transcripts extending between the genes during cold shock , heat shock , and exponential growth ( control ) . as shown in figure  s3c , the variations of extended trt measured by real - time qpcr were qualitatively ( quantitatively in most cases ) similar to those predicted by the model . notably , this was true for the cases with an intermediate gene on the opposite strand . we thus conclude that trt is ubiquitous and can explain , in principle , many of the significant co - expression levels of co - directional adjacent operons . these results also suggest that large pieces of genomes that extend beyond operons may be transcribed en bloc . an instructive example concerns the ribosome - encoding genes : these genes are surrounded by transcription - related genes and other biological pathways , apparently forming altogether a large domain containing more than 50 genes ( corresponding to 15 manually annotated operons ) with a high level of background expression ( figure  3c ) . although some of this background is expected to result from the strong tendency of these promoters to initiate transcription , as demonstrated by our real - time qpcr analysis ( figure  s3 ) it also results from trt extending between operons . in this context , it is important to recognize that large domains of coordinated expression , in which several genes and operons may be transcribed in a row , remain compatible with the very presence of operons . this can be seen by the decrease of expression at the end of certain operons or by the presence of steep fold changes at their promoter ( vertical gray lines in figure  3c ) . the pair mpn155a - mpn155 ( strong basal co - expression ) provides a good example of this effect as it shows extended trt between the two corresponding operons ( figures s3b and s3c ) but also a sharp tss at the downstream gene at late exponential phase ( in red in figure  3c ) and at stationary phase ( figure  s3b ) . rna - seq data and real - time qpcr show that trt may vary not only along the genome but also among conditions ( figure  s3 ) . the ten - gene ( four - operon ) domain containing the heat shock gene ( grpe ) provides an insightful example of such variations ( figure  5c ) , with the operon containing grpe differentiating into two sub - operons during heat shock and distinct operons becoming transcribed as a single operon during cold shock ( figures s3b and s3c ) . notably , both our rna - seq analysis and our real - time qpcr measurements further suggest that trt is globally enhanced during cold shock ( table s3 ; figure  s3 ) , in accord with reports in e.  coli and b.  subtilis of the anti - terminator role of cspa cold - shock proteins ( bae et  al . , 2000 , stlke , 2002 ) . to systematically quantify trt variations among conditions , we analyzed the behavior of the ttss internal to pairs of co - directional genes belonging to different operons ( 233 ttss analyzed ) , independently of whether a well - defined terminator was associated to the tts . for each tts , we computed the variation of its downstream expression ( down ) as a function of the variation of its upstream expression ( up ) in response to perturbations ( 96 perturbations tested with respect to 19 controls ; figure  4a ) . using this approach , we could identify at least six types of ttss ( figure  4b ) . the three first types concern ttss for which a statistically significant positive correlation exists between the values of down and up that are computed over the different perturbations ( see legend of figure  4 for details of the statistical analyses ) . they are respectively defined by downup ( stable trt , in red in figures 4b and 4c ) , downup ( stable trt plus some activation , in orange ) , and downup ( stable trt plus some repression , in yellow ) . for these tsss , trt thus tends to be maintained at a similar level , irrespective of the conditions . notably , these correspond to 85% of the total amount of ttss  ( 50% if only considering downup ) and appear to account for all strong co - expression levels ( figure  4c ) . the two  next types correspond to activation only , with a majority of down0 ( in blue ) , and to repression only , with a majority of down0 ( in cyan ) , irrespective of the value of up . together with the ttss having independent or no apparent statistically significant variations of down ( in green ) , these three last types contribute mainly to low co - expression levels . altogether , these results thus corroborate both the ubiquity of trt and its major role in basal co - expression . the observation of pairs having both low co - expression and stable trts also suggest that trt does not systematically extend to the next operon . finally , to apprehend whether trt is  stochastic  or specifically regulated , we analyzed the behavior of the ttss upon each perturbation . first , in accord with our real - time qpcr experiments ( figure  s3 ) , we observed that the variations of trt ( activation or  repression ) for a given perturbation tend to be the same for all ttss , suggesting that trt is specifically regulated at a genome - wide level . these include cold shock , osmotic shock , and novobiocin treatments , whereas low ph and heat stresses tend to repress trt . finally , by identifying the ttss and the corresponding perturbations for which the variation of trt was extreme ( supplemental experimental procedures ) , we found that conditions for which a large number ( 12 ) of ttss had an extreme behavior were strongly enriched in novobiocin ( gyrase inhibitor ) perturbations ( p8107 , hypergeometric test ) and strongly depleted in single - gene perturbations ( p2104 ) ( table s3 ) . because novobiocin targets topoisomerases and , hence , modify dna supercoiling , these results suggest that the mechanical properties of dna and its interaction with rnaps might play a crucial role in trt variations ( see the following discussion for further details ) . our observations of a trt that depends strongly on conditions , with operons that can be transcribed uniformly , en bloc ( super - operons ) , or differentially ( sub - operons ) , raise at least two fundamental questions : what mechanisms are responsible ( 1 ) for promoting an operon - like transcription of adjacent genes and ( 2 ) for preventing it ? in answer to the first issue , using co - expression levels as a proxy of transcription en bloc , the results in figures 2c and 2d  suggest that compactness , with a distance between open reading frames smaller than 100  bp , may be required for efficient operon - like co - expression . notably this length scale corresponds to the typical distance that is usually considered for operon prediction ( mcclure et  al . , 2013 ) . we note , nevertheless , that pairs of genes with intergenic regions larger than 100  bp can have a high level of trt ( figures 3 and s3 ) . our analysis also shows that compactness alone is not sufficient , because a substantial number ( 52 ) of pairs of co - directional genes with low co - expression levels are separated by less than 100  bp ( among which 17 pairs concern overlapping genes ) . in answer to the second question , let us first mention that although compactness properties call for an important role of distances on the capacity of the rnap to transcribe multiple genes in a row , co - expression does not depend primarily on distances when these exceed 100  bp ( figure  2d ) . to better understand the differences between intermediate co - expression levels and low co - expression levels , we thus investigated the possible impact of  -independent intrinsic terminators found within mrna sequences (  -dependent termination is absent in m.  pneumonia ) . canonical intrinsic terminators consist of an rna hairpin followed by a u tract , a combination that is believed to favor the disruption of the mrna - dna template hybridization necessary for the rnap to process transcription ( peters et  al . , 2011 ) . we thus evaluated the presence , in the intergenic regions of all pairs of co - directional genes , of rna hairpins that were immediately followed by u tracts of various lengths ( nu2 ) ; as a control , we considered intergenic regions that were translated by an arbitrary amount of base pairs , which allowed us  handling distance effects of intergenic regions , a longer sequence being more likely to contain an rna hairpin ( figures s4a and s4b ) , independently whether the latter plays a functional role . we found that more than 15% of the gene pairs with low co - expression contained an rna hairpin with nu4 ( figure  5a ) , a proportion that was highly significant with respect to the control ( p4.103 , two - sided t test with unequal variances ) . note here that the absence of an enrichment of terminators with shorter u tracts ( nu<4 ) corroborates previous observations that long u tracts are needed to have efficient termination ( chen et  al . , 2013 ) . similar trends were observed for intermediate co - expression levels , although involving a lower fraction ( typically half ) of gene pairs , in accord with the fact that at this  level , trt is expected to occur in a larger subset of conditions . according to the above analysis , more than 80% of the co - directional gene pairs with low co - expression do not contain any strong intrinsic terminator in their intergenic region . non - perfect u tracts or more complex termination signals that are yet to be identified might explain part of this low co - expression . the action of nucleoid - associated proteins ( naps ) such as h - ns in e.  coli ( singh et  al . , 2014 ) could also be invoked . data from our lab nevertheless show that m.  pneumoniae contains only one nap , ihf ( gene mpn529 ) , with a low copy number ( < 100 ) . to better apprehend the mechanisms related to the repression of trt , we thus performed chromatin immunoprecipitation sequencing ( chip - seq ) analysis of the -subunit of the rnap . consistent with results obtained in e.  coli ( mooney et  al . , 2009 ) , chip - seq profiles from cells in the stationary phase revealed the presence of well - defined peaks corresponding to preferentially rnap occupancy domains ( rpod ) ( figures 5b and 5c ; table s4 ) . notably , we found that the majority of gene pairs with low co - expression contain rpods in their intergenic regions ( figure  5b ) . for intermediate co - expression , rpods are less present but remain over - compared to strong co - expression . note that in contrast to hairpins , the presence of rpods does not depend on the intergenic distances ( figure  s4c ) , meaning that larger intergenic distances can not simply explain these results . because the average level of transcription strongly depends on rna degradation , we eventually compared the half - lives of transcripts between adjacent genes . we found a remarkable correlation between the degree of transcriptional coordination and the similarity of half - lives ( figure  6 ) . using a correlation measure well poised to quantify basal co - expression and applying it to rna - seq data obtained in more than 100 different conditions , we have revealed the existence in m.  pneumoniae of three distinct levels of basal coordination of transcription ( strong , moderate , and low ) , corresponding to three qualitatively different properties for the relative orientations and intergenic regions of adjacent genes . in accord with the major role of operons in the coordination of gene expression , we have found that strong basal co - expression requires adjacent genes to be co - directional . we have also found the existence of a 100  bp length scale , below which an operon - like behavior appears to be quasi - systematic and above which co - expression depends strongly on the sequence and structural properties of the intergenic region . in particular , although pairs of adjacent genes with low basal co - expression do not show any preferential relative orientations , 70% of the intergenic region of the co - directional pairs either contain a domain preferentially occupied by rnaps ( rpods ) or strong terminators ( 55% and 15% of the cases , respectively ) . by focusing specifically on co - directional adjacent genes , we have further revealed that the coordination of transcription is tightly related to the tendency of proximal genes to be transcribed en bloc , even though these genes may have not been categorized to belong to the same operon . three extreme scenarios can then be considered ( figure  s5a ) , which is in accord with our observation of the three qualitatively distinct co - expression levels . the transcription en bloc may be systematic ( i.e. , it occurs with probability close to 1 in all conditions ) , in which case the genes behave as canonical operons ( green light in figure  s5a ) . or it occurs from time to time , meaning that it can take place in specific conditions and be absent in others . variations may also occur in a given condition , because transcription may terminate with a certain probability due , for example , to the presence of an intrinsic terminator . in this case , gene expression may present staircase - like patterns ( gell et  al . , 2009 ) ( orange light ) . finally , transcription en bloc can  never  occur , in which case genes must be considered to belong to different transcriptional units ( red light ) . in accord with the rich zoology of operon - related structures that have been described over the past decade ( okuda et  al . , 2007 , gell et  al . , 2009 , cho et  al . , 2009 , nicolas et  al . , 2012 , mazin et  al . , 2014 ) and with the ubiquitous presence of pervasive transcription ( wade and grainger , 2014 ) , our findings thus indicate that operon - like behaviors are often stochastic and condition dependent , with frequencies of occurrence that depend on  intergenic sequences . in particular , transcriptional initiation may often occur on top of a background level of continuous expression . in this context , we surmise that one of the most fundamental mechanism for the coordination of transcription relies on a high probability to have specific large domains of genes that are transcribed in a row , independently of the fact that these domains may contain several internal entry points and exit points for the rnaps ( see figure  s5b for a schematic representation of this model ) . these internal landmarks might then be used by the bacterium to adapt to a wide range of conditions ( see , e.g. , figure  5c ) . they might also contribute to the activation of a given domain ( see the following discussion ) . our scenario implies , on one hand , the existence of two mechanisms internal to the domains , which are a priori necessary to maintain a proper balance between transcripts . first , there should exist a mechanism that enhances the transcription of upstream genes whenever transcription is initiated within the domain , in order to avoid a gradient of transcripts along the domain ( with downstream genes in larger quantity than upstream genes ) . although at this stage we have no direct evidence of such phenomenon , this prediction suits the proposal , in bacteria , of a control of gene expression by dna supercoiling ( dorman , 1995 , hatfield and benham , 2002 , travers and muskhelishvili , 2005 ) . it is also in accord with our observation of a strong impact of novobiocin ( a gyrase inhibitor ) treatment on trt properties ( table s3 ) . the negative supercoiling that is generated upstream of the transcribing rnaps might indeed enhance the initiation of the upstream genes ( meyer and beslon , 2014 ) . considering that these effects can propagate all the way up to the borders of the domain because of the long - range nature of the transmission of supercoiling constraints ( krasilnikov et  al . , 1999 ) , an internal initiation event should in principle be able to activate the expression of the whole domain ( figure  s5b ) , in particular without the additional action of tfs . second , produced transcripts should have similar degradation rates , which we confirmed by analyzing rna half - lives ( figure  6 ) . well - defined domains of basal co - expression require , on the other hand , the ability , upstream , to prevent the activation of genes and , downstream , to terminate the transcription process . supposing that the upstream activation is mainly the result of supercoiling transmission , stalled rnaps , as suggested by the presence of rpods ( reppas et  al . , 2006 , mooney et  al . , 2009 ) , could act as topological barriers ( higgins , 2014 ) ( see figure  5c for a suggestive example ) . downstream , in addition to the possibility of rpod roadblocks , strong intrinsic terminators are expected to play an important role in terminating transcription ( figure  5 ) . other mechanisms can be contemplated , such as anti - sense transcription ( lybecker et  al . , 2014 ) or the action of small rnas , although recent work from our lab shows that the latter have little impact on gene expression ( llorns - rico et  al . , 2016 ) . here , and more particularly in the absence of the  factor and of naps , which have been shown to prevent pervasive transcription ( singh et  al . , 2014 ) , the mechanisms at the core of the basal coordination of transcription in m.  pneumoniae thus appear to rely solely on the physical entities ( rnap and mrna ) and mechanical properties of the transcription process itself . although a strong terminator can efficiently prevent trt , it may  prevent co - expression only partially . this can be seen for  instance by the adjacent genes 5s rrna ( mpnr03 ) and mpn095 , which is the unique pair showing both strong co - expression ( c=0.68 ) and the presence of a strong intergenic terminator . although some specific processing of rrna might occur , overriding of the terminal signal , as suggested by the high level of co - expression with the sense intergenic region ( cs=0.55 ) , might explain the strong co - expression . local concentration effects of rnaps might also contribute , more particularly because of the high expression level of the 5s rrna . in such situations , intergenic distances might play a crucial role in the isolation of adjacent genes . specifically , compared to the 2030  nm size of the rnap , the 130  bp that separate the tts of the 5s rrna from the tss of mpn095 correspond to a maximal spatial distance of 45  nm ; 400  bp , the typical distance for pairs of genes with co - expression close to 0 ( figure  2c ) , correspond to 135  nm . our scenario reckons with the intrinsic stochastic nature of transcriptional initiation , with the capacity of the rnap to transcribe multiple operons in one go ( santangelo and artsimovitch , 2011 ) , and with the possible role of supercoiling to transmit regulatory properties , especially in a bacterium that is depleted in tfs ( zhang and baseman , 2011 , dorman , 2011 ) . it also opens new roads to understand the existence of preferential regions and promoters for the binding of rnaps ( reppas et  al . , 2006 , mooney et  al . , 2009 ) and suggests that a large part of the specific basal coordination of  transcription might rely exclusively on the interplay among rnap , dna , and mrna . importantly , our findings appear to hold in a wide range of  bacterial species . a similar three - level organization of co - expression , with the same properties of relative orientations and of intergenic distances ( including the existence of a 100  bp length scale ) , is indeed observed both in e.  coli and in b.  subtilis , ( figure  s6 ) . domains of proximal genes that are conserved in phylogenetically distant bacteria have also been shown to correspond , both in e.  coli and in b.  subtilis , to domains of highly co - expressed genes and operons where trt is particularly enhanced ( junier and rivoire , 2016 ) . finally , we note that  -independent terminators , as well as attenuators of these terminators through , for example , the action of riboswitches , are often conserved among distant bacteria ( vitreschak et  al . , 2004 , merino and yanofsky , 2005 ) . together with the dynamical interplay between dna and rnaps , they may thus correspond to ancestral mechanisms upon which the basal functioning of bacteria has been tinkered . in particular , tfs and other types of gene control such as the invertible dna switches of bacteroides ( kuwahara et  al . , 2004 ) may represent evolutionary solutions dedicated to specific needs related to the lifestyle of each bacterium . rna isolation was performed using mirneasy kits from qiagen , and an in - column dnase treatment was included . rna was measured using a nanodrop ( thermo ) , and integrity was confirmed in a 6000 nano chip bioanalyzer ( agilent ) . we then used the truseq stranded mrna sample prep kit v2 ( illumina ) to obtain a paired - end strand - specific rna - seq library . see table s2 for further details of conditions . chip - seq of rnap ( tap - tagged ; see khner et  al . , 2009 ) was performed as previously described ( yus et  al . , 2012 ) . we identified all mrna tsss from their associated tssrnas ( yus et  al . , 2012 ) . we distinguished productive promoters from short tssrnas as explained previously ( llorns - rico et  al . , regarding 3 sites , we used strand - specific deep sequencing and tiling array data to define approximately their positions ( gell et  al . , 2009 ) . we then used these data to refine our previously published operon map ( gell et  al . , 2009 ) ( updated map in table s1 ) . cells were collected in the indicated conditions , and rna was purified as described above . retrotranscription and real - time qpcr of 800 base long regions were done in one step with the gotaq 1-step rt - qpcr system ( promega ) . oligos ( table s5 ) were used at 0.15  m , and 25  ng total rna was used as a template . potential intrinsic terminators were defined as a rna hairpin immediately followed by a u tract . rna hairpins were identified as described previously ( mathews et  al . , 1999 ) . rpods were identified by the presence of significant peaks ( see supplemental experimental procedures ) in the chip - seq data of the rnap -subunit ( gene mpn191 ) at 6 and 96  hr . rna half - lives were determined using a dna gyrase inhibitor ( novobiocin ) , which alters the chromosomal supercoiling releasing the rnap , thus stopping transcription ( dorman , 2011 ) . after novobiocin treatment , rna was extracted at different time points , and rna - seq was performed to determine transcript levels .