Galactose repressor mediated intersegmental
By microscopic analysis of fluorescent-labeled GalR , a regulonspecific transcription factor in Escherichia coli , we observed that GalR is present in the cell as aggregates ( one to three fluorescent foci per cell ) in nongrowing cells . 
To investigate whether these foci represent GalR-mediated association of some of the GalR specific DNA binding sites ( gal operators ) , we used the chromosome conformation capture ( 3C ) method in vivo . 
Our 3C data demonstrate that , in stationary phase cells , many of the operators distributed around the chromosome are interacted . 
By the use of atomic force microscopy , we showed that the observed remote chromosomal interconnections occur by direct interactions between DNA-bound GalR not involving any other factors . 
Mini plasmid DNA circles with three or five operators positioned at defined loci showed GalR-dependent loops of expected sizes of the intervening DNA segments . 
Our findings provide unique evidence that a transcription factor participates in organizing the chromosome in a threedimensional structure . 
We believe that these chromosomal connections increase local concentration of GalR for coordinating the regulation of widely separated target genes , and organize the chromosome structure in space , thereby likely contributing to chromosome compaction . 
The genes involved in D-galactose metabolism and regulation with their cognate promoters constitute a regulon , which is regulated by Gal repressor ( GalR ) in Escherichia coli . 
Purified GalR is a homodimer of a 37-kDa subunit ( 1 ) . 
GalR is also known to form oligomers and higher-order structures , which give rise to paracrystals , at less than 0.2 M salt concentrations both in the absence and presence of DNA ( 1 ) . 
GalR represses gene transcription by binding to speci c operator DNA sequences that fi are associated with the gene promoters ( 2 ) . 
There are at least ve known promoters each associated with one or two GalR fi binding operators , located at 17.0 ( galE operon ) , 48.2 ( mgl operon ) , 48.2 ( galS ) , 64.1 ( galR ) , and 66.5 ( galP ) min on the chromosome . 
So far only three of the cognate promoters of -- galE , galS , and galP contain two operators ( 2 ) . 
We have pre - -- viously shown that GalR bound to the two operators , which encompass two promoters , P1 and P2 , in the galE operon , associates to form a DNA loop ( 3 ) . 
Whereas simple DNA binding represses the P1 promoter , only DNA looping represses the P2 promoter . 
We proposed that the GalR ( dimers ) bound to the regulon operators located around the chromosome associate in some order giving rise to a specific 3D network of D-galactose metabolism-related genes to better coordinate the regulation of the functionally related promoters and to maintain higher local concentrations of GalR around the operators , . 
The interactions may also help compaction of the chromosome . 
The most likely way to bring distant regulatory loci together is through interactions between DNA-bound proteins . 
Here we demonstrate , by both in vivo and in vitro methods , that operator-bound GalR located around the chromosome interact with each other . 
We observed that there are greater interactions in nongrowing cells than in growing cells . 
We used fluorescent Venus labeled GalR to trace location of GalR in cell ( 4 ) , Chromosome Conformation 
Capture ( 3C ) analysis to determine the aggregation of distally located DNA-bound GalR in vivo ( 5 ) , and atomic force microscopy ( AFM ) to visualize DNA-bound GalR -- GalR interactions in vitro ( 6 , 7 ) . 
The implication of the results is discussed . 
Results
Fluorescence Microscopy Analysis of GalR-Venus . 
Elf et al. demonstrated location of the LacI repressor protein bound to its DNA target in the lac operon ( 4 ) . 
This was accomplished by using a fusion of the LacI protein to a modified rapidly maturing YFP fluorescent protein ( Venus ) , and observing the cells under a fluorescent microscope . 
We used an anologous approach to find out the location of DNA-bound GalR around the chromosome . 
We genetically fused the Venus gene sequence to a single copy chromosomal GalR gene at the carboxy-end to generate galR-venus ( Fig. 1A ) . 
Any potential effect of the fusion of Venus to GalR on the GalR expression level was examined by Western blot . 
As shown in Fig . 
S1 , the expression level of the fusion protein under various growth conditions was almost identical . 
We also tested whether the fusion would affect the binding of GalR to its target DNA . 
The results showed that the gene-reg-ulatory DNA-binding activity of the GalR-Venus fusion on the regulation of two promoters of the gal operon was the same as that of WT GalR ( 8 ) ( Fig . 
S2 ) . 
We concluded that the fusion of Venus did not fundamentally alter the normal GalR property . 
Moreover , a single dimer of GalR-Venus fusion bound to DNA in the cell can not be observed under the conditions used , as opposed to findings by Xie et al , who observed single molecules of LacI-Venus fusion protein ( 5 ) using an EMCCD camera with their microscope set-up that is reportedly capable of detecting single photons , at the expense of resolution ( 5 ) . 
They greatly increased the exposure time and calculated the midpoint of the fluorescence signal to assign `` enhanced localization '' for each focus . 
In our setup , exposure times were ∼ 1 s. Without a single-photon sensitive microscope , we hoped to detect clusters comprising multiple molecules of fluorescent GalR in our microscope if GalR molecules aggregate . 
As shown in Fig. 1B , when the stationary phase GalR-Venus cells were observed under a fluorescence microscope , 277 of 284 counted cells grown in minimal medium displayed 1 -- 3 distinguishable fluorescent foci . 
The distribution of the number of foci per cell is shown in Fig. 1C . 
We rarely observed cells with four or more foci . 
At the resolution limits of our setup , we conclude that the foci represent diffraction-limited localization events . 
Of course , independent localizations that are closer than 200 nm apart will appear as a single focus in our diffraction-limited setup . 
As mentioned above , all of our fluorescence experiments were done 
Author contributions : Z.Q. , R.E. , and S.A. designed research ; Z.Q. , E.K.D. , and P.E. performed research ; S.A. analyzed data ; and Z.Q. and S.A. wrote the paper . 
The authors declare no conflict of interest.
1Present address : Epidemiology Division , Tel Aviv Sourasky Medical Center , Tel Aviv 64239 , Israel . 
2To whom correspondence should be addressed . 
E-mail : sadhya@helix.nih.gov . 
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10 . 
1073/pnas .1208595109 / - / DCSupplemental . 
with GalR-Venus fusion located at the chromosome in normal position that generates ∼ 60 -- 70 dimers in the absence of D-ga-lactose under the conditions used ( 37 °C ) as determined by Western blot . 
Under the conditions used we can not resolve whether each spot has many subnucleoid entities . 
There are ∼ 100 GalR dimers present in E. coli cells grown in minimal medium ( 9 ) . 
It is likely that DNA-bound GalR associate to generate few GalR aggregates . 
We have previously characterized a GalR mutant ( GalRT322R ) , which normally binds to the operators but inefficient in forming tetra - and higher-order oligomers ( 3 ) . 
We generated a GalRT322RVenus mutant and first tested its gene-regulatory activity the same way as the WT ( Fig . 
S2 ) . 
The results showed that although GalRT322R-Venus repressed the P1 promoter of the gal operon by binding to a single cognate operator locus OE , it is defective in tetramerization-mediated repression of the P2 promoter of the gal operon by DNA looping . 
Therefore , if GalR-Venus foci are caused by GalR multimerization , then we do not expect to observe fluorescent foci in cells carrying the GalRT322R-Venus fusion protein and grown under the same conditions . 
Consistently , we observed fluorescent foci in only four of 185 cells that contained the mutant GalR fusion . 
These results support the hypothesis that the foci observed with GalR-Venus were generated by association of two or more GalR dimers presumably bound to DNA . 
Furthermore , when log phase cells carrying the WT GalR-Venus fusion protein were inspected , the fluorescent foci were significantly reduced , caused by fast DNA replication fork of the chromosome presumably interfering with the intrachromosomal contacts ( Fig. 1B ) . 
Moreover , we also investigated the effect of D-galactose ( the inducer of gal operon ) on the observed foci . 
As shown in Fig . 
S3 , both in stationary phase and log phase , no significant difference could be found between the cells treated with and without D-galactose . 
The distribution of the number of foci per cell cultured to stationary phase with D-galactose is shown in Fig. 1C . 
Because D-galactose does not effectively induce the gal operon located at 17.0 min of the chromosome in stationary phase cells ( 10 ) , D-galactose may not break GalR-mediated bridges detected here under similar conditions . 
Alternatively , the GalRmediated intrachromosomal links in stationary phase cells may have more complex structure/compositions to be sensitive to D-galactose . 
The fluorescence data strongly suggest that the GalR binding sites along the chromosome are brought together through GalR polymerization . 
In addition , Xie et al reported that the signal intensities for single foci that they measured occurred in discrete quanta , suggesting that these investigators were measuring single molecules of fluorophores ( 5 ) . 
As the intensities of our foci do not appear to occur in such discrete quanta , we are not concluding that we are detecting single molecules at each focus . 
Intersegmental Chromosomal Associations . 
The intersegmental chromosomal connections by DNA-bound GalR as suggested by the above fluorescent labeling of GalR experiment can be tested in vivo by the 3C method or its refinements ( 5 ) . 
Such techniques have been used in chromosomal 3D structure analysis in yeast and human ( 11 -- 13 ) . 
Fig . 
S4 shows the principle of the 3C method as adapted from Dekker et al. ( 5 ) . 
First , we performed the 3C analysis to test whether the operators of GalR are physically connected to each other in stationary phase cells . 
We designed appropriate primers listed in Table S1 for studying any connections between four of the operator loci located at 17.0 , 48.2 , 64.1 , and 66.5 min around the E. coli chromosome ( Fig. 2A ) . 
We also designed 22 other primers ( also listed in Table S1 ) each proximate to binding sites of FruR , MalT , PurR , TyrR , and H-NS transcription factors as likely negative controls for interactions with GalR sites ( shown in Fig. 2A ) . 
We used EcoRI for 3C analysis , as its digestion sites are appropriately located in the vicinity of all chosen DNA targets . 
The efficiency of digestion was estimated by the amount of PCR products obtained with primers around individual EcoRI sites . 
After digestion , we detected very little DNA amplification of these restriction sites reflecting successful digestions , although the PCR amplifications were abundant without EcoRI treatments . 
Typical results with the PDF/PDR primer pair are shown in Fig. 3A ( lanes 2 , 3 , 5 , and 6 ) . 
There were very few amplification products when treated with DNA ligase after digestion , showing that the digested two ends could not be ligated and restore the original DNA sequence in noticeable amounts ( Fig. 3A , lanes 1 and 4 ) . 
In addition , we found that the digestion efficiencies in cross-linked and non -- cross-linked samples were somewhat different ( Fig. 3B ) . 
In the non -- cross-linked samples , DNA was more or less completely digested in 1 h , whereas in the cross-linked samples , the maximum digestion efficiencies reached to ∼ 80 % in 4 h. Thus , we set the digestion time as 4 h . 
The PCF/PCR primer pair was used as the internal control of template for a DNA segment containing no EcoRI site . 
We performed 3C analysis between GalR and GalR or GalR and non-GalR DNA targets for potential contacts between them in stationary phase cells . 
Interaction efficiency between two sites was defined by normalizing the ratios of the amount of PCR products between the two in the cross-linked and non -- cross-linked samples compared with the internal controls ( 13 ) . 
We arbitrarily set a threefold change or higher , to assign a positive interaction between a given pair of targets . 
Among 94 combinations tested , we found contacts among 30 of them . 
Sample PCR results are shown in Fig. 4A and summarized in Fig. 4B . 
There were no visible signals among the remaining 64 combinations . 
Surprisingly , three of the four GalR targets showed positive signals when matched not only against each other but also with six of the non-GalR targets that were not expected to contact GalR targets . 
For example , primers P14 , P24 , and P26 close to GalR targets gave interaction signals when paired against P9 and P12 primers designed to test contacts with PurR and TyrR targets , respectively . 
GalR-Mediated Intrachromosomal Contacts . 
To confirm the participation of GalR in intrachromosomal contacts mentioned above , we performed the 3C assays in a ΔgalR mutant strain ( 14 ) . 
Compared with the interaction frequency in the WT , the de-letion of the GalR encoding gene removed most of the observed interactions , both between any GalR-GalR targets and most but not all of GalR and non-GalR targets ( Fig. 3C , lanes 1 -- 4 ) . 
The results show that GalR indeed mediates most of the interactions observed here ( Fig. 2B ) . 
A GalR homolog , GalS , binds specifically to all GalR targets tested but with different affinities ( 15 , 16 ) . 
We investigated the effect of GalS on the GalR-mediated intrachromosomal contacts in a ΔgalS strain ( 17 ) . 
The results showed that , in the absence of GalS , the interaction frequency of the connections did not diminish but rather frequently enhanced ( Fig. 3C , lanes 5 and 6 ) , which is reminiscent of two previous observations : DNA-bound GalS , unlike DNA-bound GalR , does not associate ( 15 ) , and more GalR are made in ΔgalS cells ( 16 ) . 
We believe that the enhancement of GalR-mediated interactions observed in many cases in ΔgalS strain occurs because GalS competes with GalR for binding to DNA sites in WT , thus reducing the potency of GalR-mediated bridges in DNA ; in the absence of GalS and presence of an extra amount of GalR , the GalR-mediated connections are higher . 
Numerous GalR Binding Sites in the Chromosome . 
As shown above , three loci , galP ( 66.52 min ) , galR ( 64.11 min ) , and mgl-galS ( 48.22 min ) interacted not only with each other but presumably also with or near targets of FruR , MalT , PurR , TyrR , and HNS . 
Two models may explain the latter results . 
( i ) The latter binding proteins collaborate with GalR to form bridging complexes . 
( ii ) There are GalR binding sites not identified previously near the non-GalR targets . 
These ideas were tested by 3C analysis in strains with deleted binding proteins , ΔfruR , ΔmalT , ΔpurR , ΔtyrR , or Δhns . 
We found no significant change in the observed PCR products in the deletion strains compared with WT ( Fig . 
S5 ) . 
On the other hand , in experiments in which one non-GalR site was tested against another non-GalR site , many linkages observed in the WT strain were not found in the ΔgalR strain ( Fig. 3D ) . 
The latter observations suggested that the second hypothesis is a more likely reason for interactions involving the so-called nonGalR sites . 
DNA sequence search indeed revealed the existence of 91 potential GalR binding sites around the chromosome ( with zero or one mismatch with the consensus bases in the gal operator sequence : TGNAANCGNTTNCA ( 2 ) . 
In fact , there is at least one potential GalR-binding site between each EcoRI restriction site at a non-GalR locus tested and the cognate primer sequence used in the 3C assays . 
The potential GalR-binding sites identified by 3C approach are shown in Table 1 . 
We do not know whether these newly identified potential GalR binding sites actually bind GalR and are involved in regulation of specific genes . 
Nonetheless , our results show that a transcription factor for the gal regulon connects distal segments in the bacterial chromosome . 
Curiously enough , we did not observe any contacts between the primer ( P1 ) designed for the GalR-regulated gal operon ( 17 min ) and other primers tested by the 3C assays . 
A priori , this was an unexpected observation . 
As mentioned earlier , the gal operon contains two operators , and GalR binds to both and associates , generating locally a DNA loop of 113 bp ( 18 , 19 ) . 
Given that GalR can form polymers , why GalR bound to these sites does not participate in the GalR-mediated chromosomal interconnections identified above remains to be investigated . 
Having established that the transcription factor GalR forms a chromosomal network in stationary phase cells , we tested whether these chromosomal interconnections exist in growing cells . 
It appears that more than 80 % of the observed contacts disappear when cells are growing exponentially ( Fig . 
S6A ) . 
These results are consistent with the hypothesis that moving DNA replication forks may disrupt the connections and that reassociation may be a slow process . 
Of the five signals in the WT that survived in growing cells , perhaps because these associations may have faster kinetics , only one was sensitive to the presence of D-galactose during logarithmic growth ( Fig . 
S6B ) . 
GalR-Mediated Looping in Vitro by Atomic Force Microscopy . 
We have previously used AFM to observe GalR - and LacI-mediated DNA loops ( 7 ) . 
In this research , we engineered five different operators for GalR binding in two mini DNA circles ( pMini-1 and pMini-2 ) with discrete distances between binding sites . 
The map of pMini-1 and pMini-2 are shown in Fig. 5 A and C. Purified mini DNA circles were mixed with or without GalR protein as described in SI Materials and Methods . 
Samples were then scanned by an atomic force microscope . 
The AFM images of DNA without the protein show plectonomic structures with tight superhelical stretches of DNA and occasional loops because of crossing over of two double helical chains ( Fig. 5 B and D , Upper ) . 
In the presence of GalR , we observed 71 molecules of GalR-mediated looped-out DNA of 246 total pMini-1 molecules and 33 of 154 total pMini-2 molecules inspected . 
We also counted the numbers of observed loops per GalR-DNA complexes . 
For pMini-1 , 30 % of DNA molecules formed loops ( 71 of 246 ) , in which 12.6 % contain two loops , 9.7 % three loops , 5.7 % four loops , and 0.8 % five loops . 
In pMini-2 , almost 20 % formed loops ( 33 of 154 ) , in which 9.1 % contain two loops , 3.2 % three loops , 7.8 % four loops , and 1.3 % five loops ( Fig. 5E ) . 
GalR mediation in the loop formation was inferred by measuring the height and width of the overlapping DNA chains ( 6 ) . 
DNA loops frequently emanate from one or more such taller and broader globular particles at the DNA crossover points as shown by black arrows in Fig. 5 B and D , Lower Left . 
We assume that these particles are oligomers of DNA-bound GalR . 
To establish that oligomerization of DNA-bound GalR generates looping of the DNA intervals , the contour lengths of large numbers of loops in both DNA alone and DNA/GalR samples were traced and the DNA lengths measured ( samples are shown in Fig. 5 B and D. ) . 
Without protein , the loops sizes were , as expected , random because of DNA crossovers . 
We note that the observed DNA crossover points in the DNA-only samples are of different volumes and are not large enough to account for at least a GalR dimer . 
Volume estimates of the `` cores '' in the GalR plus samples suggest that oligomers ( up to octamers ) connect two or more gal operators sites . 
We also found that each DNA mol-ecule may contain more than one GalR core particle ( Fig. 5D , Lower Left ) . 
This may be the result of group of DNA binding sites -- in the current case , two and three -- get together with GalR independent of each other . 
To confirm the GalR mediated formation of core particle , we used the GalRT322R , which is incapable of tetramerization , to see segmental interactions by AFM analysis . 
We observed the dimeric GalR bound to DNA circles when incubated with GalRT322R , as indicated by red arrows in the lower right panel of Fig. 5 B and D. However , we also observed that only two loops per molecule at most ( 49 of 526 for pMini-1 and 12 of 205 for pMini-2 ) , whereas for the WT GalR , even five loops per molecule could be found ( shown in lower panel of Fig. 5 B , D , and E ) . 
The formation of two loops per molecule , which means that GalR is tetramerized , may be because of the residual tetra-merization activity in the mutant GalR . 
Moreover , the GalRT322R protein did not show any DNA-bound GalR that contains higher than tetrameric structure , unlike the DNA-bound WT GalR . 
To further examine the bridging of GalR binding sites , we constructed a smaller 648 bp DNA circle ( pMini-3 ) , which contained only three operators one from previously known sites -- and two from the newly proposed operators from 3C studies described above . 
It was important to include the latter two because direct GalR binding to these presumed operators has not been directly demonstrated . 
The operators were separated by 100 , 200 , and 300 bp in pMini-3 ( Fig. 6A ) . 
AFM analysis of pMini-3 alone showed , as expected , mostly plectonomic DNA ( Fig. 6B , Upper ) . 
The plasmid in the presence of GalR clearly showed 1 -- 3 loops of DNA per molecule . 
We found that each loop-containing DNA molecule , unlike the previous two plasmids , contained only one GalR core per molecule . 
This is because three operators per DNA would not allow formation of more than one GalR core . 
We measured contour lengths of GalR-bound and unbound molecules of pMini-3 . 
Based on the measured contour lengths , the inferred binding patterns of GalR to pMini-3 are shown in Fig. 6C . 
The contour lengths of the plasmids carrying protein are highly consistent with the expected values from the binding site loci . 
Discussion We used two independent in vivo methods , microscopic visuali-zation of fluorescent-labeled GalR , and conformation capture of chromosome by cross-linking , to locate DNA bound GalR protein in stationary phase cell . 
Results from these two experiments presented here suggest that in stationary phase cells , the E. coli chromosome is partially condensed by the DNA sequence-spe-cific GalR transcription factor that connects remote segments of DNA in 3D space . 
Incidentally , we identified many more previously unknown GalR binding sites that participate in such interconnections . 
We confirmed the connections between remote DNA sites by GalR in the absence of other factors in vitro by direct visualization of the interactions by AFM . 
Although the mechanism , the interface , and the energetics of a small ( ∼ 100-bp ) DNA loop formation by association of two DNA-bound GalR dimers are known in detail ( 20 , 21 ) , the frequency or the stability of the remote intersegmental chromosomal multiconnections presumably by multimeric GalR dimers remains unknown at this stage , although the connections are neither rare nor transient . 
Given the current findings with GalR , we believe that other proteins in the cell may make similar intrachromosomal connections and may also contribute to 3D folding . 
The implication of our finding of remote intersegmental chromosomal connections is of importance . 
GalR-mediated intrachromosomal connections may serve at least two functions : The `` togetherness '' of gal regulon members should increase the local concentration of GalR around the distant gal regulon promoters and thus coordinate regulation of the functionally regulated gene products , as argued by Dröge and Müller-Hill ( 22 ) . 
One way to increase the local concentration of DNA binding proteins is to have their genes located next to their DNA targets . 
Another putative role of GalR-mediated chromosomal connections may be architectural ; it may incidentally help chromosomal compaction . 
We note that specific biological roles of many putative GalR binding sites discovered here remain unknown ; either they are part of yet-to-be discovered GalR regulated genes and members of the gal regulon , or they serve purely an architectural role . 
We note that a ΔgalR strain does not have any effect on cell growth except a change in intermediary metabolites ( 23 ) . 
We are currently investigating the significance of associations of DNA-GalR complexes in stationary phase cells . 
Our modified 3C method developed based upon the principle of Dekker et al. ( 6 ) has been used by Wang et al. ( 24 ) to show some intersegmental chromosomal contacts based on the nucleoid protein HNS . 
The E. coli chromosome has been shown to contain several kinds of topographical arrangements . 
( i ) It contains six `` macro-domains '' with defined boundaries , four of which are structured and two of which are nonstructured ( 25 -- 28 ) . 
Attempts to have site-specific recombination between att sites engineered into the macrodomains showed that chromosomal inversions within and between domains frequently have physiological consequences , and sometimes they are not permissible , putting some limits to chromosomal rearrangements ( plasticity ) ( 29 ) . 
( ii ) The chromosome may be organized into supercoiled topological domains ( 30 -- 34 ) . 
( iii ) Between replication cycles , the chromosome is 
1 . 
Majumdar A , Rudikoff S , Adhya S ( 1987 ) Purification and properties of Gal repressor : pL-galR fusion in pKC31 plasmid vector . 
J Biol Chem 262:2326 -- 2331 . 
2 . 
Weickert MJ , Adhya S ( 1993 ) The galactose regulon of Escherichia coli . 
Mol Microbiol 10:245 -- 251 . 
3 . 
Geanacopoulos M , Adhya S ( 2002 ) Genetic analysis of GalR tetramerization in DNA looping during repressosome assembly . 
J Biol Chem 277:33148 -- 33152 . 
4 . 
Elf J , Li GW , Xie XS ( 2007 ) Probing transcription factor dynamics at the single-mole-cule level in a living cell . 
Science 316:1191 1194 . 
-- 5 . 
Dekker J , Rippe K , Dekker M , Kleckner N ( 2002 ) Capturing chromosome conformation . 
Science 295:1306 -- 1311 . 
6 . 
Lyubchenko YL , Shlyakhtenko LS , Aki T , Adhya S ( 1997 ) Atomic force microscopic demonstration of DNA looping by GalR and HU . 
Nucleic Acids Res 25:873 -- 876 . 
7 . 
Virnik K , et al. ( 2003 ) `` Antiparallel '' DNA loop in gal repressosome visualized by atomic force microscopy . 
J Mol Biol 334:53 -- 63 . 
8 . 
Lewis DE , Geanacopoulos M , Adhya S ( 1999 ) Role of HU and DNA supercoiling in transcription repression : Specialized nucleoprotein repression complex at gal promoters in Escherichia coli . 
Mol Microbiol 31:451 -- 461 . 
9 . 
Tokeson JP ( 1989 ) Ultrainduction of the Escherichia coli galactose operon . 
PhD Dissertation ( Howard Univ , Washington , DC ) . 
10 . 
Adhya S ( 1967 ) Control of synthesis of the galactose metabolizing enzymes of Escherichia coli : I. Polarity in the galactose operon : II . 
The glucose effect and the ga-lactose enzymes . 
PhD Dissertation ( Univ of Wisconsin , Madison , WI ) . 
11 . 
Dekker J ( 2008 ) Mapping in vivo chromatin interactions in yeast suggests an extended chromatin ber with regional variation in compaction . 
J Biol Chem 283:34532 -- 34540 . 
fi 12 . 
Lieberman-Aiden E , et al. ( 2009 ) Comprehensive mapping of long-range interactions reveals folding principles of the human genome . 
Science 326:289 293 . 
-- 13 . 
Singh BN , Ansari A , Hampsey M ( 2009 ) Detection of gene loops by 3C in yeast . 
Methods 48:361 -- 367 . 
14 . 
Tokeson JP , Garges S , Adhya S ( 1991 ) Further inducibility of a constitutive system : Ultrainduction of the gal operon . 
J Bacteriol 173:2319 -- 2327 . 
15 . 
Geanacopoulos M , Adhya S ( 1997 ) Functional characterization of roles of GalR and GalS as regulators of the gal regulon . 
J Bacteriol 179:228 -- 234 . 
16 . 
Semsey S , et al. ( 2009 ) Dominant negative autoregulation limits steady-state repression levels in gene networks . 
J Bacteriol 191:4487 -- 4491 . 
17 . 
Golding A , Weickert MJ , Tokeson JP , Garges S , Adhya S ( 1991 ) A mutation defining ultrainduction of the Escherichia coli gal operon . 
J Bacteriol 173:6294 -- 6296 . 
18 . 
Aki T , Adhya S ( 1997 ) Repressor induced site-specific binding of HU for transcriptional regulation . 
EMBO J 16:3666 -- 3674 . 
condensed into a filament extending from one cellular pole to the other ( 35 ) . 
The two ends of the filaments are connected by a stretched-out DNA segment that includes the `` terminus . '' 
How the topography of the `` macro-domains , '' `` topological loops , '' the `` filament , '' and the currently demonstrated remote `` segmental connections '' in stationary phase cells reconcile with each other in the bacterial nucleoid remains a challenging question , and we do not know whether the different chromosome conformations discussed above are present in both log and stationary phase cells . 
Materials and Methods
Protocols for fluorescent microscopy , AFM , and 3C analysis used in this study are described in SI Materials and Methods . 
Cells used for FM and 3C analysis were cultured in M63 minimal medium . 
Constructions of GalR-Venus and GalRT322R-Venus strains and mini circles used for AFM assay are described in detail also in the SI Materials and Methods . 
All the primers used for constructions are listed in Table S2 . 
ACKNOWLEDGMENTS . 
We thank Mark Umbarger ( Harvard Medical School ) for kindly providing the basic 3C protocol ; Ximiao He ( National Cancer Institute ) for help with DNA sequence search ; and Robert Weisberg , Richard Losick , Gene-Wei Li , and Donald Court for help and discussions . 
This research was supported by the Intramural Research Program of the National Institutes of Health , National Cancer Institute , Center for Cancer Research . 
19 . 
Lewis DE , Adhya S ( 2002 ) In vitro repression of the gal promoters by GalR and HU depends on the proper helical phasing of the two operators . 
J Biol Chem 277 : 2498 -- 2504 . 
20 . 
Geanacopoulos M , et al. ( 1999 ) GalR mutants defective in repressosome formation . 
Genes Dev 13:1251 -- 1262 . 
21 . 
Lia G , et al. ( 2003 ) Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein ( HU ) - mediated DNA looping . 
Proc Natl Acad Sci USA 100 : 11373 -- 11377 . 
22 . 
Dröge P , Müller-Hill B ( 2001 ) High local protein concentrations at promoters : Strat-egies in prokaryotic and eukaryotic cells . 
Bioessays 23:179 -- 183 . 
23 . 
Lee SJ , et al. ( 2009 ) Cellular stress created by intermediary metabolite imbalances . 
Proc Natl Acad Sci USA 106:19515 -- 19520 . 
24 . 
Wang W , Li GW , Chen C , Xie XS , Zhuang X ( 2011 ) Chromosome organization by a nucleoid-associated protein in live bacteria . 
Science 333:1445 -- 1449 . 
25 . 
Boccard F , Esnault E , Valens M ( 2005 ) Spatial arrangement and macrodomain organization of bacterial chromosomes . 
Mol Microbiol 57:9 -- 16 . 
26 . 
Espeli O , Mercier R , Boccard F ( 2008 ) DNA dynamics vary according to macrodomain topography in the E. coli chromosome . 
Mol Microbiol 68:1418 -- 1427 . 
27 . 
Lesterlin C , Mercier R , Boccard F , Barre FX , Cornet F ( 2005 ) Roles for replichores and macrodomains in segregation of the Escherichia coli chromosome . 
EMBO Rep 6 : 557 -- 562 . 
28 . 
Valens M , Penaud S , Rossignol M , Cornet F , Boccard F ( 2004 ) Macrodomain organization of the Escherichia coli chromosome . 
EMBO J 23:4330 -- 4341 . 
29 . 
Esnault E , Valens M , Espéli O , Boccard F ( 2007 ) Chromosome structuring limits ge-nome plasticity in Escherichia coli . 
PLoS Genet 3 : e226 . 
30 . 
Deng S , Stein RA , Higgins NP ( 2005 ) Organization of supercoil domains and their reorganization by transcription . 
Mol Microbiol 57:1511 -- 1521 . 
31 . 
Hardy CD , Cozzarelli NR ( 2005 ) A genetic selection for supercoiling mutants of Escherichia coli reveals proteins implicated in chromosome structure . 
Mol Microbiol 57 : 1636 -- 1652 . 
32 . 
Noom MC , Navarre WW , Oshima T , Wuite GJ , Dame RT ( 2007 ) H-NS promotes looped domain formation in the bacterial chromosome . 
Curr Biol 17 : R913 -- R914 . 
33 . 
Postow L , Hardy CD , Arsuaga J , Cozzarelli NR ( 2004 ) Topological domain structure of the Escherichia coli chromosome . 
Genes Dev 18:1766 -- 1779 . 
34 . 
Sinden RR , Ussery DW ( 1992 ) Analysis of DNA structure in vivo using psoralen photobinding : Measurement of supercoiling , topological domains , and DNA-protein interactions . 
Methods Enzymol 212:319 -- 335 . 
35 . 
Wiggins PA , Cheveralls KC , Martin JS , Lintner R , Kondev J ( 2010 ) Strong intranucleoid interactions organize the Escherichia coli chromosome into a nucleoid filament . 
Proc Natl Acad Sci USA 107:4991 -- 4995 .