The Origin of Chromosomal Replication Is AsymmetricallyPositioned on the Mycobacterial Nucleoid, and the Timing ofIts Firing Depends on HupB

Joanna Hołówka,a Damian Trojanowski,b Mateusz Janczak,b Dagmara Jakimowicz,a,b Jolanta Zakrzewska-Czerwinskaa,b

aLaboratory of Molecular Biology of Microorganisms, Hirszfeld Institute of Immunology and ExperimentalTherapy, Polish Academy of Sciences, Wrocław, Poland

bDepartment of Molecular Microbiology, Faculty of Biotechnology, University of Wrocław, Wrocław, Poland

ABSTRACT The bacterial chromosome undergoes dynamic changes in response toongoing cellular processes and adaptation to environmental conditions. Among themany proteins involved in maintaining this dynamism, the most abundant is thenucleoid-associated protein (NAP) HU. In mycobacteria, the HU homolog, HupB, pos-sesses an additional C-terminal domain that resembles that of eukaryotic histonesH1/H5. Recently, we demonstrated that the highly abundant HupB protein occupiesthe entirety of the Mycobacterium smegmatis chromosome and that the HupB-binding sites exhibit a bias from the origin (oriC) to the terminus (ter). In this study,we used HupB fused with enhanced green fluorescent protein (EGFP) to perform thefirst analysis of chromosome dynamics and to track the oriC and replication machin-ery directly on the chromosome during the mycobacterial cell cycle. We show thatthe chromosome is located in an off-center position that reflects the unequal divi-sion and growth of mycobacterial cells. Moreover, unlike the situation in E. coli, thesister oriC regions of M. smegmatis move asymmetrically along the mycobacterial nu-cleoid. Interestingly, in this slow-growing organism, the initiation of the next roundof replication precedes the physical separation of sister chromosomes. Finally, weshow that HupB is involved in the precise timing of replication initiation.

IMPORTANCE Although our view of mycobacterial nucleoid organization has evolvedconsiderably over time, we still know little about the dynamics of the mycobacterialnucleoid during the cell cycle. HupB is a highly abundant mycobacterial nucleoid-associated protein (NAP) with an indispensable histone-like tail. It was previously sug-gested as a potential target for antibiotic therapy against tuberculosis. Here, we fusedHupB with enhanced green fluorescent protein (EGFP) to study the dynamics of the my-cobacterial chromosome in real time and to monitor the replication process directly onthe chromosome. Our results reveal that, unlike the situation in Escherichia coli, the nu-cleoid of an apically growing mycobacterium is positioned asymmetrically within the cellthroughout the cell cycle. We show that HupB is involved in controlling the timing ofreplication initiation. Since tuberculosis remains a serious health problem, studies con-cerning mycobacterial cell biology are of great importance.

KEYWORDS bacterial cell cycle, chromosome dynamics, HupB, Mycobacterium,nucleoid-associated proteins (NAPs)

The bacterial chromosome (also called the nucleoid) is a highly organized, dynamicentity (1–6). Chromosomal DNA must be efficiently compacted to fit inside the

small cell compartment, but it must also be available for proteins involved in replica-tion, segregation, and transcription (7, 8). The bacterial chromosome undergoes con-stant topological and architectural changes due to ongoing cellular processes and theneed to adapt to environmental conditions (9). Among the many proteins responsible

Received 24 January 2018 Accepted 21February 2018

Accepted manuscript posted online 12March 2018

Citation Hołówka J, Trojanowski D, Janczak M,Jakimowicz D, Zakrzewska-Czerwinska J. 2018.The origin of chromosomal replication isasymmetrically positioned on themycobacterial nucleoid, and the timing of itsfiring depends on HupB. J Bacteriol 200:e00044-18. https://doi.org/10.1128/JB.00044-18.

Editor Tina M. Henkin, Ohio State University

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to JolantaZakrzewska-Czerwinska,jolanta.zakrzewska@uni.wroc.pl.

RESEARCH ARTICLE

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for maintaining chromosome structure, the most abundant are the nucleoid-associatedproteins (NAPs) (10, 11). These small (up to 20-kDa) basic proteins compact chromo-somal DNA into independent topological domains of �10 kb (as established by analysisof the Escherichia coli chromosome) called microdomains (6); various NAPs act to wrapand/or introduce bends (e.g., HU, IHF, and Fis), and to bridge distant chromosomalregions (e.g., H-NS) (11, 12). A growing body of evidence shows that the E. coli NAPs arealso involved in other cellular process (13–16). For example, HU and IHF, which areamong the most highly conserved and abundant NAPs, regulate chromosome replica-tion at the initiation stage (17, 18). Some genera or species also possess uniqueDNA-binding proteins that help coordinate chromosome dynamics with their lifecycles. In the multicellular bacterium Streptomyces coelicolor, two HU-like proteins,HupA and HupS, are responsible for chromosome organization in the vegetativehyphae and during sporulation, respectively (19). Interestingly, the cell cycle regulationof Caulobacter crescentus is dependent on the recently described GapR protein (20), anovel NAP whose DNA-binding activity is regulated by the passage of the replicationfork.

The bacterial cell cycle can be divided into three periods: the time between celldivision and the initiation of chromosome replication in daughter cells (B period),chromosome replication (C period), and the time between the termination of replica-tion and the completion of cell division (D period) (21). In fast-growing E. coli andBacillus subtilis bacteria, those periods overlap, and newborn cells inherit partiallyduplicated chromosomes (21–25). Recent work showed that slow-growing Mycobacte-rium cells can also exhibit an overlap between cell cycle periods. In the majority ofMycobacterium smegmatis cells, after chromosome replication is completed, a newround of replication usually initiates in the mother cell prior to cytokinesis andterminates in the daughter cells (26). Moreover, as seen in fast-growing E. coli cells, asubpopulation of M. smegmatis cells (15%) is capable of starting a new round ofreplication before completing the previous one (i.e., these cells exhibit multiforkreplication) (27). Despite these findings, however, the regulation of chromosomeinitiation in mycobacteria has not yet been explored.

Mycobacteria also exhibit other unusual cell cycle features. In contrast to otherrod-shape bacteria, including E. coli and B. subtilis, mycobacteria elongate apically andoften divide asymmetrically, resulting in unequally sized daughter cells (28–30). Fur-thermore, the daughter cells that inherit the old cell pole (with preassembled elonga-tion machinery) elongate faster than the those with new poles (29). Recent studies ofthe cell cycle in mycobacterium showed that their oriC region is positioned slightlyoff-center with respect to the midcell, and that this is reflected in the asymmetricpositioning of the replication and segregation machineries (replisomes and segro-somes, respectively) (27, 31–34). Replisomes are assembled closer to the old cell pole;they oscillate in the old-pole-proximal cell half during replication, with the processterminating closer to the new cell pole. Soon after the start of replication, the newlyreplicated oriC regions are segregated toward opposite cell poles by ParB and ParA,which are components of the mycobacterial segregation machinery. Interestingly,movement of the ParB complex towards the new pole is faster than movement towardsthe old pole, which contributes to the asymmetry of the mycobacterial cell cycle (34).

Although a recent study examined the localization of particular chromosomalregions (oriC and ter) in mycobacterial cells (35), there is relatively little information onthe overall organization and dynamics of the mycobacterial nucleoid during the cellcycle. It was recently shown that mycobacterial HupB is a homolog of E. coli HU (36, 37)and that its N-terminal domain resembles that of the canonical HU protein, whereas itslong and distinctive C-terminal domain (CTD) contains PAAK/KAAK basic repeats, whichare characteristic of the H1/H5 family of eukaryotic histones (38, 39). The CTD of HupBhas been shown to be essential for its ability to efficiently bind DNA in vivo (40) and invitro (39, 41–46). Interestingly, we observed that the HupB-binding sites display a biasagainst the terminus region, with most of the binding sites localized around oriC.Hence, we proposed that HupB might be involved in organizing the newly replicated

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oriC regions. Moreover, we demonstrated that HupB occupies the whole chromosomeand therefore can be used for real-time nucleoid visualization. In the present study, weperformed real-time analysis of mycobacterial nucleoid dynamics during the cell cycleand examined the involvement of HupB in chromosome replication.

RESULTSoriC is localized asymmetrically on the mycobacterial nucleoid. Our previous

studies showed that HupB occupies the entire M. smegmatis chromosome. Here, weused strains producing HupB fused with either green (enhanced green fluorescentprotein [EGFP]) or red (mCherry) fluorescent proteins (FPs) as a tool to visualize themycobacterial chromosome (40). Snapshot analysis of the HupB-EGFP-expressing strainrevealed that the nucleoid is localized asymmetrically within the cell during both theexponential and stationary growth phases (Fig. 1A). To visualize nucleoid localizationduring the cell cycle, we performed time-lapse fluorescence microscopy (TLFM) ofHupB-EGFP-expressing cells stained with the lipophilic styryl dye FM5-95 (to monitorcell division; Fig. 1B). The fluorescence intensity profiles of HupB-EGFP along the longaxis of the cell were measured between two septum formation events in relation to theold cell pole (Fig. 1C; see also Movie S1 in the supplemental material). The resultsshowed that during most stages of the cell cycle (0 to 75% of cell cycle progression, Fig.1C) the chromosome is localized asymmetrically and remains closer to the new cellpole. At the later stages of the cell cycle (76 to 100% of cell cycle progression, Fig. 1C),and prior to cell division, this asymmetry in chromosome localization diminishes (seeFig. 3).

We previously demonstrated that oriC is located slightly off-center (27, 31, 34) but,in contrast to the nucleoid, is situated closer to the old cell pole. Thus, it can beassumed that the positioning of oriC on the mycobacterial nucleoid should be moreasymmetric. To verify this hypothesis, we analyzed the localization of oriC directly onthe nucleoid during the cell cycle. We constructed strains that produced HupB-EGFPand ParB-mCherry fusion proteins (see Fig. S1 in the supplemental material); ParB bindsin the vicinity of oriC (31, 33, 34) and can be used as marker for oriC. TLFM analysis ofHupB-EGFP/ParB-mCherry cells (Fig. 2A and B) confirmed that shortly before theduplication of origin regions (i.e., replication initiation), the ParB focus was positionedcloser to the edge of the chromosome, near the old pole. As replication progressed, onecopy of oriC remained in the old-pole-proximal cell half and moved a short distancetoward the outer edge of the chromosome, while the second copy moved to theopposite edge of the chromosome (Fig. 2B; see also Movie S3 in the supplementalmaterial). Thus, our data confirmed earlier studies, which showed that the partitioningmachinery exhibits asymmetric dynamics in the mycobacterial cell (34).

We recently showed that the replisome is also off-center during chromosomereplication (31). To further localize the replisome within the nucleoid, we constructeda HupB-EGFP/DnaN-mCherry-expressing strain (Fig. S1); DnaN is a subunit of DNApolymerase III that acts as a sliding clamp, so this enabled us to track the replicationmachinery directly on the chromosome (Fig. 2C). TLFM analysis of HupB-EGFP/DnaN-mCherry cells (Fig. 2D; see also Movie S2 in the supplemental material) revealed thatthe replisome(s) was indeed located asymmetrically on the chromosome. At the time ofinitiation, the replisomes were assembled close to the edge of the chromosome in theold-pole-proximal cell half. As replication proceeded, the replisomes split and merged,but the overall positions of the replication forks on chromosome remained significantlyoff-center (Fig. 2D). Shortly before and during the termination of replication, thereplisomes migrated closer to the opposite edge of the chromosome.

Taken together, present work indicates that the subcellular localization of the M.smegmatis chromosome is asymmetric during most of the cell cycle. Moreover, oriCregions (i.e., segrosomes) and the replisomes exhibit significantly off-center positionson the nucleoid at the initial stage of replication.

The new round of replication occurs prior to chromosome separation. Since thenew round of replication in M. smegmatis starts before cytokinesis (26), we examined

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the replication dynamics with respect to the separation of sister chromosomes. Real-time analysis of HupB-EGFP cells stained with FM5-95 showed that septum formationstarted �38 � 15 min (average � standard deviation; n � 78) before the physicalseparation of sister chromosomes (Fig. 3A). To examine whether the next round ofreplication (and consequently the segregation of the oriC regions) starts before sister

FIG 1 Localization of the nucleoid in M. smegmatis cells. (A) Micrographs showing representative HupB-EGFP cellsof the log and stationary growth phases with fluorescence profiles along the long cell axis, as measured from thedistant cell pole. Bars, 2 �m. (B) Micrographs of representative FM5-95-stained HupB-EGFP cells. Bar, 2 �m. (C)Real-time analysis of the relative subcellular localization of the chromosome. Fluorescence profiles were measuredbetween two septum formation events in relation to the old cell pole and approximated at each time point toprovide a model of real-time nucleoid localization inside the cell (op and np indicate the old and new cell poles,respectively; n � 20).

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chromosome separation, we performed TLFM experiments using dual-reporter strainsexpressing HupB-EGFP/DnaN-mCherry and HupB-EGFP/ParB-mCherry. Our results indi-cated that after the completion of chromosome replication, which took 149 � 21 min(average � standard deviation; n � 71) under our experimental conditions, the nextround of replication was initiated at 37 � 18 min (average � standard deviation; n �

142), and the separation of sister chromosomes occurred at 55 � 25 min (average �

standard deviation; n � 65) after replication termination. Thus, the next replicationround began 15 � 15 min (average � standard deviation; median � 20 min; n � 178)before the physical separation of sister chromosomes (Fig. 3B). The segregation of theParB/oriC complexes, which happens just after the initiation of chromosome replication(31), occurred 19 � 16 min (average � standard deviation; median � 20 min; n � 160)before sister chromosome separation (Fig. 3C). Together, our data indicate that theinitiation of a new round of chromosome replication and the subsequent segregationof newly replicated chromosomal regions precede the physical separation of sisterchromosomes, resulting in the inheritance of partially duplicated chromosomes (seeFig. 3).

HupB is involved in regulating replication initiation. In E. coli, HU stimulates theassembly of the prereplication complex at the oriC region (17, 18). Our previous

FIG 2 Relative positioning of the replisome and oriC along the longitudinal axis of the chromosome during the cell cycle. (A, C)Micrographs of representative HupB-EGFP/ParB-mCherry (A) and HupB-EGFP/DnaN-mCherry (C) cells. The white dotted line indicatescell boundaries established using the differential interference contrast (DIC) channel. Bars, 2 �m. (B, D) Graphs present the real-timelocalization of oriC (B) and replisomes (D) on the chromosome (op and np indicate the old and new cell poles, respectively; n � 20).

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ChIP-seq and PALM microscopy results suggested that HupB may also contribute toorganizing the oriC region by binding nearby (40). This led us to question whetherHupB is involved in the timing of replication initiation during the cell cycle. To test this,we constructed strains that harbored (i) a deletion of either the whole hupB gene or thesequence encoding the CTD of HupB and (ii) DnaN-EGFP, which enabled us to monitorthe progression of replication. These strains were designated �hupB/DnaN-EGFP andHupBΔCTD/DnaN-EGFP strains, respectively (Fig. S1). TLFM analysis revealed that strainswith deletion of hupB or producing truncated HupB (HupBΔCTD) exhibited delays in theinitiation of replication compared to control DnaN-EGFP cells (Fig. 4). The average timebetween the termination of the previous round and initiation of the next round ofreplication (B and D phases) (Fig. 4A) was 44 � 22 min for the DnaN-EGFP strain(average � standard deviation; n � 152), whereas those of �hupB/DnaN-EGFP andHupBΔCTD/DnaN-EGFP strains were 58 � 22 min (average � standard deviation; n �

199, P � 4.3 � 10�8) and 59 � 23 min (average � standard deviation; n � 167, P �

7.8 � 10�9), respectively. Density plots presenting the B and D time distributions forthe analyzed strains (Fig. 4B) showed that the percentage of mutant cells in which thenext round of replication starts after 60 min is higher in the mutant cells (29% and 35%for �hupB/DnaN-EGFP and HupBΔCTD/DnaN-EGFP strains, respectively) than in thecontrol cells (13% for DnaN-EGFP). To confirm that this delay was directly connected tothe absence of HupB, we constructed a complementation strain (Fig. S1). Indeed, TLFM

FIG 3 Real-time microscopic analysis of the M. smegmatis cell cycle. (A) TLFM analysis of sister chromosomeseparation in HupB-EGFP cells stained with FM5-95. Green and red triangles indicate sister chromosome separationand septum formation, respectively. Bar, 2 �m. (B, C) TLFM analysis of chromosome replication (B) and segregation(C) relative to sister chromosome separation. Green triangles indicate sister chromosome separation, while redtriangles indicate replication initiation (appearance of DnaN-mCherry fluorescent foci [B]) and duplication of oriC(segregation of ParB-mCherry complexes [C]). The dotted lines indicate cell boundaries established using thedifferential interference contrast (DIC) channel. Bars, 2 �m.

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analysis showed that average B plus D time in the complementation strain was similar(47 � 21 min, n � 169, P � 0.56, with 17% of the population starting the new roundof replication after 60 min) (Fig. 4) to that seen in control DnaN-EGFP cells.

Because HupB seems to be involved in regulating replication initiation, we examinedwhether deletion of hupB affected the frequency of multifork replication. Our analysisshowed that cells lacking HupB showed a lower frequency of reinitiation of chromo-some replication compared to the that of control strain (�5% and 13% of cells,respectively; n � 208 and 282, respectively).

Since HupB organizes the newly replicated oriC proximal regions (40), and deletionof hupB delays replication initiation, we tested whether HupB influences the segrega-tion of newly replicated oriC regions. We constructed an M. smegmatis strain producingParB-mCherry and a dual-reporter strain producing ParB-mNeon/mCherry and DnaN-mCherry/EGFP, and used these strains to track the segregation machinery and simul-taneously observe the replication and segregation machineries in the hupB-deletionbackground (Fig. S1). Our real-time microscopic experiments did not show any signif-icant delays in the segregation or chromosomal localization of newly replicated oriCregions. However, we observed that strains lacking HupB exhibited a longer lagbetween two oriC duplication events (generation time) compared to that of controlParB-mCherry cells (34). The average generation times for �hupB/ParB-mCherry andParB-mCherry strains were 156 � 26 min (n � 143) and 137 � 24 min (n � 146),respectively. Similarly, the strain producing CTD-truncated HupB (HupBΔCTD/DnaN-EGFP/ParB-mCherry) exhibited a prolonged generation time compared to the controlstrain (data not shown).

Taken together, our results indicate that HupB affects the dynamics of replicationinitiation in M. smegmatis, delaying replication initiation and diminishing the frequencyof multifork replication. Moreover, the lack of HupB prolongs the generation time,which is consistent with the slight growth delay seen in the mutant strains (Fig. S1).

DISCUSSION

The architecture and topology of the bacterial chromosome must be constantlyrearranged and adapted during the cell cycle to account for ongoing cellular processes(i.e., replication, segregation, transcription, and translation) (7, 8, 12). Maintaining thetopological homeostasis and architectural flexibility of the nucleoid requires the coop-

FIG 4 Analysis of replication initiation delay in strains lacking the entire hupB gene or expressing a truncated version of HupB lackingits 3= region (which encodes the CTD). (A) Diagrams present the canonical cell cycle and the cell cycle of the mycobacterial controlstrain (wild type) and hupB deletion mutants (B) Density plot presents the distribution of the delay time (i.e., the time measuredbetween replication termination and the initiation of the next round of replication; B and D phases) for the analyzed strains. The areaof the density plot colored red indicates a delay time greater than 60 min.

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eration of many proteins, including topoisomerases and NAPs (11, 47). The HU-likeproteins are the most highly conserved and abundant NAPs in bacteria (36, 37). Themycobacterial homolog of HU, HupB, possesses an additional CTD that is uniqueamong Actinobacteria. The CTD domain contains positively charged amino acid resi-dues, and resembles that of the eukaryotic histone H1/H5 (38, 39, 41). We previouslydemonstrated that HupB occupies the entire M. smegmatis chromosome and that itsbinding activity requires the CTD (40). Since little is known about mycobacterialchromosome dynamics during the cell cycle, we decided to use HupB-FP, along withoriC and markers of chromosomal replication, to monitor the localizations of theseplayers in relation to chromosome organization during the cell cycle. This work wasparticularly interesting given the elongation mode and asymmetrical cell division ofmycobacteria, which lead to distinct subpopulations of cells that differ in size (28–30).Additionally, due to the possible role of HupB in organizing the oriC region, weexamined whether HupB affects the dynamics of replication initiation.

In contrast to what is seen in E. coli (1, 48–51), we found that the nucleoid of anapically growing mycobacterium is positioned asymmetrically within the cell, closer tothe new cell pole (Fig. 1C; see Movie S1 in the supplemental material). The newbornmycobacterial cell inherits an old pole and creates a new pole, at which the elongationmachinery must be assembled (28, 29). Thus, the nucleoid occupies region closer to thenew cell pole, which is probably related to asymmetric segregation of the oriC regions(34). Additionally, due to the asymmetric division and the difference in the elongationrates of the old and new cell poles (29), the daughter cells are unequal in size andexhibit heterogeneity in chromosome condensation, with shorter cells having morecondensed chromosomes than longer cells (Fig. 1B and 3A).

Our studies revealed that, in mycobacterial cells, oriC is positioned within theold-pole-proximal half of the nucleoid, often close to its edge (Fig. 2A and B). Shortlyafter replication initiation, one of the oriC regions moves only slightly to remain in theold-pole-proximal cell half, while the second oriC travels along the entire chromosometo the opposite edge of the chromosome (Fig. 2A and B). Thus, the oriC regions aresegregated on the nucleoid in an asymmetrical manner. This is consistent with theprevious reports that segregation is asymmetrical in mycobacterial cells (33, 34), and itpresumably reflects the specific orientation of the chromosome (i.e., ori-ter subcellularpositioning) (32, 35). However, further studies will be required to validate this possi-bility. We also found that replisomes are assembled close to the chromosome edge,closer to the old cell pole (Fig. 2C and D). As replication proceeds, the replisomesfrequently split and merge, but remain in the old-pole cell half (27, 31, 32). Prior to thetermination, they migrate to the opposite chromosome edge, closer to the new cellpole.

In bacteria, chromosome replication (phase C) takes place throughout most of thecell cycle. Fast-growing E. coli and B. subtilis cells shorten their doubling times byinitiating the new round of the replication before the previous round has finished (i.e.,they undergo multifork replication) (22–24, 52, 53). Recent work showed that M.smegmatis compensates for its elongated replication time (more than 2 h) by initiatingchromosome replication before cytokinesis (26) and having a relatively large fraction ofcells (up to 15%) undergoing multifork chromosome replication (28). In this, M. smeg-matis resembles other actinobacterial species, such as Corynebacterium glutamicum(54). Our experiments revealed that, in mycobacteria, the initiation of the next repli-cation round and the start of septum formation precede sister chromosome separationby �20 min and 40 min, respectively (Fig. 3A and 5). Thus, mycobacterial daughter cellsinherit partially replicated chromosomes (Fig. 5), presumably as a strategy to compen-sate for the elongated replication time (phase C).

In E. coli, HU stimulates the assembly of the prereplication complex (17, 18). SinceHupB preferentially occupies oriC-proximal regions (40), we examined the influence ofHupB on the timing of replication initiation. Our microscopic analysis revealed thatstrains lacking HupB exhibited delays in replication initiation. The time between thetermination of the ongoing replication round and the initiation of the next round was

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prolonged in �hupB/DnaN-EGFP cells compared to those of control DnaN-EGFP cellsand the complementation strain (Fig. 4). Interestingly, HupBΔCTD/DnaN-EGFP cells alsoshowed a delay in replication initiation (�59 min). This is consistent with the previousobservation (40) that HupBΔCTD does not effectively bind DNA. We further observedthat overlapping replication rounds were less frequent in the strain lacking HupBcompared to in the control strain (�5% and 13%, respectively). This suggests that HupBis involved in controlling the initiation of chromosome replication. Our previousChIP-seq analysis (40) showed that HupB binds in the vicinity of oriC, suggesting thatit may affect the organization/topology of the nearby region (55) to facilitate theformation of the prereplication complex. Thus, we cannot exclude the possibility that,similarly to the situation in Helicobacter pylori (56), the modulation of local DNAtopology is an important factor regulating the formation of prereplication complex.Future work will be needed to elucidate the relevant mechanism(s) of action.

Interestingly, cells lacking HupB did not show any delay in the initial stage of thesegregation of newly replicated oriC regions after replisome assembly (data not shown).The time between replisome assembly and oriC duplication (�4 min) (31) is short, andthere is little distance between the oriC regions at this stage, so we were unable toobserve these subtle differences using epifluorescence microscopy. However, thegeneration time (that between two consecutive duplications of the oriC region) wasprolonged in cells devoid of either HupB or its CTD in comparison to in cells of thecontrol strain (�156 min and 169 min, respectively, versus 137 min). That may be aconsequence of the delayed initiation of the next replication round and/or a result ofdisturbed segregation/organization of newly replicated oriC proximal regions. Sincemycobacterial HupB presumably interacts with topoisomerase I (TopA) (45), the lack ofHupB may disturb the topological homeostasis of the chromosome. In Streptomyces,TopA is recruited to the ParB complexes during sporulation (57, 58), which suggeststhat HupB may play a role in the segregation/organization of oriC regions. Thus, as wepreviously suggested, HupB may be involved in organizing newly replicated regionsand (potentially) in coordinating chromosome replication with segregation.

Taken together, our present work shows that the HupB-EGFP fusion protein allowedus to successfully analyze mycobacterial chromosome dynamics during the cell cycleand to track replication and oriC positioning directly on the nucleoid. In contrast towhat is seen in E. coli, the oriC region(s) is asymmetrically positioned on the mycobac-terial nucleoid. Our results demonstrate that the initiation of the next round ofreplication, followed by duplication of the oriC regions, precedes the physical separa-tion of sister chromosomes. Moreover, we show that HupB is responsible for the precisetiming of replication initiation. Strains lacking HupB exhibited prolonged generationtimes, which may reflect the delay in replication initiation and/or some disruptionduring the course of segregation.

MATERIALS AND METHODSDNA manipulations, bacterial strains, and culture conditions. DNA manipulations were carried

out using standard protocols (59). Reagents and enzymes were obtained from Thermo Scientific, Roth,

FIG 5 Scheme of the M. smegmatis cell cycle. Yellow and red triangles represent the movements during phase C of replisomes and oriC,respectively; green triangles indicate the time at which sister chromosomes start to separate prior to cell division during the D plus B phases.

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and Sigma-Aldrich unless otherwise indicated. All plasmids used to construct the M. smegmatis mc2 155mutant strains were propagated in the E. coli DH5� strain. E. coli cells were grown in LB broth or on LBagar plates (Difco) supplemented with the proper antibiotic(s) (ampicillin 100 �g/ml and/or kanamycin50 �g/ml) and/or other compounds (0.004% X-Gal and 0.5 mM isopropyl-�-D-1-thiogalactopyranoside[IPTG]) according to standard procedures. M. smegmatis mc2 155 strains were grown in 7H9 brothsupplemented with 10% oleic acid-albumin-dextrose-catalase (OADC; BD) and 0.05% Tween 80 or on7H10 agar plates (Difco) supplemented with 10% OADC, 0.5% glycerol, and 0.004% X-Gal and/orkanamycin (50 �g/ml) or 2% sucrose. The utilized strains, plasmids and oligonucleotides are listed inTable S1 in the supplemental material.

Construction of M. smegmatis mc2 155 mutant strains. The allelic replacement of the ParB-encoding gene with parB-mcherry/parB-mneon-green and that of the DnaN-encoding gene with dnaN-egfp/dnaN-mcherry were performed as described by Parish and Stoker (60). M. smegmatis cells weretransformed with 200 ng of NaOH-EDTA-treated plasmid DNA (61), and unmarked mutants were selectedaccording to the previously described procedure (60). The correct allelic replacement was confirmed byPCR, and fluorescent fusion proteins were verified by Western blotting. To construct the complemen-tation strain, we used the mycobacteriophage L5-based integration-proficient vector pMV306 (Kanr) (62).M. smegmatis electrocompetent cells were transformed with pMV306 derivatives, and recombinants wereselected using kanamycin. Transformants were analyzed by PCR and Western blotting. The growth ratesof the mutant strains were monitored under different conditions for 48 h using a Bioscreen incubator.

Microscopy. Snapshot imaging was performed using log phase (optical density at 600 nm[OD600] � 0.6 to 0.7) or late stationary phase (OD600, �2) cells. M. smegmatis cultures were grownovernight in liquid medium, centrifuged (6,000 rpm for 5 min), resuspended in PBS, and smeared ontomicroscope slides. Dried samples were mounted with 5 �l of phosphate-buffered saline (PBS)-glycerol(1:1) solution and examined with a Zeiss Axio Imager Z1 fluorescence microscope equipped with a 100�objective. Pictures were analyzed using Fiji software and R software (R Foundation for StatisticalComputing, Vienna, Austria; http://www.r-project.org), including the ggplot2 package (63). For allmeasurements, a two-sided parametric Student t test was applied. To avoid generation of false assump-tions in the case of nonnormal distributions, the statistical significance of differences in measured valueswas confirmed with the nonparametric two-sided Wilcoxon test with minimum 0.995 confidenceintervals.

Time-lapse microscopy. For real-time analysis, early log phase (OD600 � 0.2 to 0.4) M. smegmatiscultures grown in liquid medium were used. For membrane staining, cells were first incubated for 15 minwith FM5-95 (0.5 �g/ml) and then plated on solid medium containing FM5-95 (0.5 �g/ml). Experimentswere performed by culturing cells either on an IBIDI �-dish (35 mm, low) with solid medium or in liquidmedium using an ONIX microfluidic system. Images were recorded at 10-min intervals using a DeltaVision Elite inverted microscope equipped with a 100� oil immersion objective. Data were analyzedusing Fiji software and the Peaks and ggplot2 packages of the R software (R package version 0.2 [M.Morhac, 2012]) (63).

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/JB.00044-18.

SUPPLEMENTAL FILE 1, PDF file, 0.2 MB.SUPPLEMENTAL FILE 2, AVI file, 0.1 MB.SUPPLEMENTAL FILE 3, AVI file, 0.1 MB.SUPPLEMENTAL FILE 4, AVI file, 0.1 MB.

ACKNOWLEDGMENTSWe are grateful to Agnieszka Strzałka and Rafał Kociatkiewicz for assisting with data

analysis using the R statistical programming language.This study was supported by the National Science Center, Poland (MAESTRO grant

2012/04/A/NZ1/00057). The cost of publication was supported by the Wroclaw Centreof Biotechnology, under the Leading National Research Center (KNOW) program, 2014to 2018.

We declare that we have no competing financial interests.

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