Noncanonical SMC protein in INAUGURAL ARTICLE smegmatis restricts maintenance of

Michael W. Panasa,1,2, Paras Jainb,c,2, Hui Yangc, Shimontini Mitraa, Debasis Biswasd, Alice Rebecca Wattame, Norman L. Letvina,3, and William R. Jacobs, Jr.b,c,4

aDivision of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115; bHoward Hughes Medical Institute and cDepartment of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; dDepartment of Microbiology, Himalayan Institute of Medical Sciences, Swami Ram Nagar, Jolly Grant, Dehradun 248140, India; and eVirginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24061

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2013.

Contributed by William R. Jacobs, Jr., August 1, 2014 (sent for review June 17, 2014; reviewed by Eric J. Rubin) Research on and was revolutionized by the phenotype has not been known. Here we are reporting that the development of a transformation system in the fast-growing mutation mediating the ept phenotype maps to a gene encoding surrogate, . This transformation system a structural maintenance of chromosomes (SMC) homolog, which was made possible by the successful isolation of a M. smegmatis plays an important role in cell division—a fundamental cellular mutant strain mc2155, whose efficient plasmid transformation (ept) process across all domains of life. phenotype supported the replication of Mycobacterium fortuitum Cell division requires DNA replication followed by faithful pAL5000 plasmids. In this report, we identified the EptC gene, the segregation of genetic material to daughter cells. Eukaryotic cells loss of which confers the ept phenotype. EptC shares significant rely on spindle fibers to move homologous chromosomes to the amino acid sequence homology and domain structure with the MukB opposite ends of the cell (5, 6). Segregation in prokaryotes protein of , a structural maintenance of chromosomes resembles that in eukaryotes regarding the basic mechanism and

(SMC) protein. Surprisingly, M. smegmatis has three paralogs of SMC the requirement for SMC proteins. Since the discovery of SMC MICROBIOLOGY proteins: EptC and MSMEG_0370 both share homology with Gram- proteins, extensive studies have led to the understanding of their negative bacterial MukB; and MSMEG_2423 shares homology with roles in chromosome condensation and segregation (7). SMC Gram-positive bacterial SMCs, including the single SMC protein pre- proteins were first functionally characterized in Saccharomyces dicted for Mycobacterium tuberculosis and . cerevisiae when a mutant allele smc1-1 caused a chromosome Purified EptC was shown to bind ssDNA and stabilize negative super- nondisjunction (2:0 segregation) defect at least 10 times more coils in plasmid DNA. Moreover, an EptC–mCherry fusion protein was often than observed in the WT strains (8, 9). SMC proteins are constructed and shown to bind to DNA in live mycobacteria, and to highly conserved in eukaryotes and their homologs are found in prevent segregation of plasmid DNA to daughter cells. To our knowl- almost all Archaea, Gram-positive , and in about 40% edge, this is the first report of impaired plasmid maintenance caused of Gram-negative bacteria. Gram-negative organisms, such as by a SMC homolog, which has been canonically known to assist the segregation of genetic materials. Significance

plasmid segregation | electroporation | DNA topology | partition errors | A mutant strain of Mycobacterium smegmatis mc2155 provided cell division the first plasmid transformation platform for any mycobac- teria. It has since been used extensively as a surrogate host to he genetic bases for signature phenotypes of Mycobacterium study mycobacterial gene functions in Mycobacterium tuber- Ttuberculosis, including acid fast staining, virulence, and sus- culosis and Mycobacterium leprae. In this report, we discovered ceptibility to tuberculosis (TB)-specific drugs were unknown before that the efficient plasmid transformation (ept) phenotype of the generation of a plasmid transformation system in mycobacteria mc2155 is caused by a loss-of-function mutation in a gene, (1). Whereas recombinant DNA technology in Escherichia coli namely eptC, which encodes a protein that shares homology laid the foundation for modern genetic research, analysis of my- with the structural maintenance of chromosomes (SMC) family. cobacterial genes in E. coli was insufficient to elucidate pheno- While SMCs generally promote DNA segregation to daughter types. The profound difference in cell wall composition, lipid cells, we show that EptC selectively inhibits plasmid segrega- metabolism, promoter recognition, and posttranslational modifi- tion and thereby establish a new plasmid restrictive role of this cation between mycobacteria and E. coli greatly limited the use of mycobacterial SMC homolog. E. coli as a surrogate host for mycobacterial gene analysis. Even Author contributions: M.W.P., P.J., H.Y., N.L.L., and W.R.J. designed research; M.W.P., P.J., though plasmid transformation in Streptomyces, phylogenetically H.Y., S.M., and D.B. performed research; M.W.P. and S.M. performed transposon screen- closer than E. coli to Mycobacterium, was established in 1978 (2), ing and complemented mc2155 with eptC; P.J., H.Y., A.R.W., and W.R.J. analyzed data; neither Streptomyces nor E. coli is sensitive to TB-specific drugs and M.W.P., P.J., H.Y., and W.R.J. wrote the paper. such as isoniazid, ethambutol, ethionamide, thioacetazone, or Reviewers included: E.J.R., Harvard School of Public Health. isoxyl. Snapper et al. developed the first plasmid transformation The authors declare no conflict of interest. system for mycobacteria by isolating a Mycobacterium smegmatis See Profile 10.1073/pnas.1407879111. 2 mutant, namely mc 155, that allowed for the replication of My- 1Present address: Department of Microbiology and Immunology, Stanford University, cobacterium fortuitum plasmids (1, 3). This enabled the de- Stanford, CA 94305. velopment of efficient cloning vectors, the recreation of drug- 2M.W.P. and P.J. contributed equally to this work. resistant phenotypes, and important discoveries of the biology of 3Deceased May 28, 2012. 2 mycobacteria. Although mc 155 was first isolated in 1988 and 4To whom correspondence should be addressed. Email: [email protected]. characterized to be a plasmid-specific phenotype in 1990 (4), the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. genetic basis for this efficient plasmid transformation (ept) 1073/pnas.1414207111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1414207111 PNAS Early Edition | 1of8 Downloaded by guest on September 27, 2021 E. coli, do not encode the canonical SMC, but rely on a func- 0.2% acetamide, and (ii) 7H10 plates containing 40 μg/mL kanamycin and 100 tional analog MukB (10). μg/mL hygromycin. The percentage of plasmid-retaining cells after inducing In prokaryotes, chromosome segregation has mainly been EptC expression was determined by dividing the number of colonies obtained studied by genetic and biochemical analyses of the low-copy on plates containing kanamycin, acetamide, and hygromycin by the number of number plasmid DNA (11–13). High-copy number plasmids rely colonies obtained on plates containing only kanamycin and hygromycin. The primarily on passive diffusion for plasmid maintenance (14), which data plotted were obtained from three independent biological replicates. is inapplicable to chromosomal DNA. However, the accurate segregation of low-copy number plasmids requires dedicated par- Single-Cell Imaging Using Microfluidics. Time-lapse microscopy was performed titioning loci consisting of three components: a cis-acting centro- using Cellasic (Millipore) microfluidics system. The images were captured using Elements software (Nikon) and Nikon Ti microscope either at 300× or mere site and two trans-acting proteins ParA and ParB (15). The 1,000× magnification every 30 min. The 7H9 media with 100 μg/mL hygrom- mycobacterial pAL5000 is a low-copy number plasmid (two to five ycin was used as growth media and 7H9 media with 0.2% acetamide was used copies per cell) (16) and, noticeably, does not require a dedicated as induction media without selection, whereas 7H9 media with 100 μg/mL plasmid partitioning system for its segregation in M. smegmatis hygromycin and 0.2% acetamide was used as induction media with selection. mc2155. Thus, maintenance of pAL5000 plasmids by mc2155, but not by its parental strain, motivated us to look for an alteration in Protein Purification. M. smegmatis eptC was PCR amplified and cloned in the host machinery responsible for plasmid segregation. pET14b using NdeI and ClaI sites. EptC was expressed in E. coli BL21 (DE3) In this study, we report that the genetic basis of M. smegmatis pLysS and purified using Ni-Nitrilotriacetatic acid chromatography followed 2 mc 155’s ability to segregate episomal plasmids, and thus assume by heparin Sepharose chromatography. M. smegmatis topoisomerase I was transformability, is a mutation in a gene annotated here as eptC. cloned and purified as described before (21). Electrophoretic mobility shift Biochemical and genetic analyses of EptC demonstrate that the Assays, coupled topoisomerase assays, and plasmid multimerization assays protein belongs to the family of SMC proteins. EptC interferes with were performed using the purified proteins (see SI Materials and Methods faithful segregation of pAL5000 plasmids to daughter M. smegmatis for details). Effect of EptC on plasmid topology in E. coli was determined cells by directly binding to plasmids and modulating plasmid by inducing EptC with 0.1 mM isopropyl-β-D-thiogalactopyranoside (see SI supercoiling status. The appropriate supercoiling status has been Materials and Methods for details). shown crucial for successful plasmid segregation (17). Sequencing and Analysis of Genomic DNA Regions. Genomic DNA was isolated Materials and Methods from M. smegmatis mc26 and mc2155 using the Invitrogen PureLink Genomic DNA Mini Kit and sequenced on a MiSeq desktop sequencer. Data from pre- Bacterial Strains and Plasmids. The bacterial strains and plasmids used in this viously sequenced genomes were obtained from the Pathosystems Resources study are listed in Tables S1 and S2. Integration Center (PATRIC) (22). SEED’s Compare Regions tool (23) offered on PATRIC website was used to examine the genetic neighborhood around the Isolation of M. smegmatis mc26 Transposon Mutants Exhibiting the ept eptABCD region. The presence or absence of specific genes within genomes Phenotype. Two independent mc26 transposon mutant libraries, generated was confirmed by BLAST. Domains found across the proteins of interest were using phAE181 (18, 19), were transformed with 200 ng kanamycin-resistant compared and examined at the Conserved Domain Database (24). The pre- pAL5000 plasmid DNA by electroporation (2.5 kV, 25 mF, and 1,000 ohms). dicted secondary structures of the protein sequences were examined using Mutants exhibiting the ept phenotype were selected on 7H10 plates con- Protein Homology/Analogy Recognition Engine (Phyre V 2.0) (25). taining 20 μg/mL kanamycin and 100 μg/mL hygromycin. Transposon insertion sites were mapped for 28 mutants exhibiting ept phenotype following the Results protocol described in SI Materials and Methods. 2 A Loss-of-Function Mutation in the M. smegmatis EptC Gene Causes the The unmarked deletion mutants of M. smegmatis mc 6ineptC and eptABCD were generated following the protocol described in Jain et al. (20). Efficient Plasmid Transformation Phenotype. M. smegmatis, rather The primers used to generate and confirm the mutations are listed in SI than M. tuberculosis, was chosen as the host to develop plasmid Materials and Methods. transformation in mycobacteria because M. smegmatis is non- pathogenic, an excellent host for , and yields Complementation of eptC. Plasmids expressing eptC, either from the consti- colonies in 3 d rather than 21–28 d. The M. smegmatis ATCC607 2 tutive hsp60 promoter (pYUB1281) or from the acetamide-inducible promoter strain was first obtained and stocked as mc 1, but three distinct

(Pace) (pYUB1492) were used to complement EptC. The mCherry gene was colonial morphotypes were discovered when the stock was grown cloned in frame with a 13-aa linker sequence (KLPARASDPPVAT) downstream on solid media. The predominant orange rough morphotype was 2 of eptC to express the EptC–mCherry fusion protein (pYUB1513). The Pace-eptC- stocked as mc 6 and used for protoplast transfection of myco- mCherry cassette was also expressed from the pAL5000 plasmid (pYUB1512). DNA and the generation of the first shuttle An in-frame N-terminal deletion was generated in eptC (eptCΔNTD) after phasmid (26). Shuttle phasmids made from TM4 and L5 myco- digesting pYUB1512 with PmlI and NdeI followed by end-filling and ligation proved that (i) E. coli propagated DNA was not (pYUB1515). restricted in mycobacteria, (ii) the kanamycin-resistant gene is a suitable marker for the selection of transfectants in mycobacteria, Measurement of Transformation Efficiency. M. smegmatis strains (see Fig. 2, and (iii) integrating shuttle phasmids expressing kanamycin re- where the strains are presented) were transformed with 200 ng pYUB1062 sistance could be successfully transformed and maintained by using electroporation (2.5 kV, 25 mF, and 1,000 ohms). The notation (U) and mc26. After numerous failed transformation attempts to in- (I) indicate that the competent cells were made after growing the cells in the troduce the episomal pAL5000-derived plasmid in M. smegmatis absence, (U), or presence of 0.2% acetamide, (I). Transformations on the mc26, one transformation yielded three colonies, one of which mc26, mc23412, and mc23422 background were plated on two types of 2 μ was stocked as mc 154. This successful transformation inspired us plates: (i) 100 g/mL hygromycin and 0.2% acetamide plates; and (ii) 100 to hypothesize that mc2154 acquired a mutation that permitted μg/mL hygromycin plates without acetamide. Transformations on mc23414, 2 2 plasmid maintenance. To test this hypothesis, a plasmid-cured mc 3416, and mc 3425 background were plated on two types of plates: (i)40 2 μ μ derivative of mc 154 was shown to routinely exhibit transfor- g/mL kanamycin, 100 g/mL hygromycin, and 0.2% acetamide plates; and (ii) 5 μ μ mation efficiency greater than 10 cfu per μg DNA. The plas- 40 g/mL kanamycin and 100 g/mL hygromycin without acetamide. Trans- 2 2 formation efficiency was defined as the number of transformant colonies midless version of mc 154 was stocked as mc 155. Subsequent obtained per microgram of plasmid DNA. studies led us to conclude that the ept phenotype was a plasmid- specific phenomenon (4). Measurement of Plasmid Retention. M. smegmatis strains mc23414 and mc23416 To test whether the ept phenotype was due to loss of gene were transformed with pYUB1062. Transformations were plated on plates function(s), two independent transposon libraries of M. smegmatis 2 containing 40 μg/mL kanamycin and 100 μg/mL hygromycin. The colonies mc 6 were created. Transposon mutants bearing hygromycin- obtained were grown in 7H9 media containing 40 μg/mL kanamycin and 100 marked insertion were subsequently transformed with a kanamy-

μg/mL hygromycin to an OD600 of 0.6. Tenfold serial dilutions were plated on cin-marked episomal plasmid and selected for kanamycin and (i)7H10platescontaining40μg/mL kanamycin, 100 μg/mL hygromycin, and hygromycin coresistance (18). Screening of ∼20,000 independent

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1414207111 Panas et al. Downloaded by guest on September 27, 2021 2 transposon mutants in M. smegmatis mc 6 resulted in 28 candi- 2 eptA eptB eptC eptD 2 A mc 6 mc 6 [Wild Type]

dates exhibiting the ept phenotype. Twenty-four of these 28 INAUGURAL ARTICLE mc23412 mc26ΔeptC::γδ γδ p mutants had a transposon-disrupted MSMEG_1281 which map- ace eptC mc23414 mc26ΔeptC::γδ attP :: P :eptC ped to the four independent transposon insertion sites shown in γδ L5 ace pace null 2 mc23416 mc 6ΔeptC::γδ attP :: P :null Fig. 1A. Three of the four remaining transposon mutants had γδ L5 ace acquired a spontaneous point mutation within the MSMEG_1281 mc23422 mc26ΔeptABCD::γδ γδ pace (Table S1). The one remaining mutant had neither a transposon 2 eptC 2 mc 3425 mc 6ΔeptABCD::γδ attPL5:: Pace:eptC disruption nor a spontaneous mutation in MSMEG_1281, and was γδ

not further analyzed. eptA eptB1 eptC1 eptD 2 2 mc2155 mc2155 Whole-genome sequencing of mc 6 and mc 155 frozen stocks p hsp60 null mc23418 mc2155 attP :: P :null from the laboratory of W.R.J. revealed two SNPs, with one p L5 ace hsp60 eptC 2 2 relevant SNP at position 1368003, which is in close proximity to mc 3419 mc 155 attPL5:: Pace:eptC the transposon insertion that resulted in the ept phenotype (Fig. 1B and SI Text). At this position, the parental M. smegmatis mc26 B 6 strain has nucleotide A, whereas the ept mutant strain mc2155 10 Express functional EptC 2 No functional EptC has nucleotide T. Reannotating the region based on mc 6 se- 10 5 quence showed a four-gene operon structure, with the former 4 10 gene’s stop codon overlapping with the latter gene’s start codon. 3 This operon is referred to as eptABCD. The A→T substitution in 10

2 2 [cfu/ μ g] mc 155 at the eptB/eptC junction of the parental mc 6 converts 10 2 1 the stop codon TAA in eptB into a leucine codon TTA. This 10

substitution within the operon resulted in an extended EptB Efficiency Transformation 1 2 6 2 protein with an additional 48 aa and a shortened EptC protein (I) 2 mc 2 341 2 3422 2 155 2 3418 2 3419 (Fig. 1B). These findings suggest that mc 155 does not make the mc 2 3414 (I) 2 3416 mc 2 3425 (I) 2 3414 (U) 2 3416 (U) 2 3425 (U) mc c c mc mc parental EptB or EptC. The transposon insertion in eptC and the m mc mc mc m mc whole-genome sequencing results led us to hypothesize that loss Fig. 2. Deletion of eptC in M. smegmatis mc26 is sufficient to confer the ept of EptC function was sufficient to confer the ept phenotype. 2 To test the sufficiency, we precisely deleted eptC and eptABCD, phenotype. (A) Schematics of the M. smegmatis mc 6 and M. smegmatis mc2155-derived strains. (B) Deletion of eptC in M. smegmatis mc26(mc23412) generating the unmarked deletion strains M. smegmatis mc23412

> MICROBIOLOGY 2 Δ γδ 2 2 Δ γδ resulted in 1,000-fold increase in the transformation efficiency of pAL5000 (mc 6 eptC:: )andmc3422 (mc 6 eptABCD:: ) (Fig. 2A). Δ 2 2 2 2 plasmids. eptC was conditionally expressed to complement eptC (mc 3414) Like mc 155, both mc 3412 and mc 3422 were efficiently trans- 2 and ΔeptABCD (mc 3425) under the acetamidase promoter (Pace). The absence formed with the pAL5000-derived plasmids (Fig. 2B). Comple- and presence of the inducing agent are denoted by “(U)” for uninduced and mentation of both eptC and eptABCD deletions by a chromosomal “(I)” for induced, respectively. Complementation of mc23412 with functional integrated EptC gene alone under the control of an acetamidase eptC [mc23414 (I)] reduced the transformation efficiency of M. smegmatis 2 2 R 2 2 promoter (mc 3414: mc 3412::attPL5::PaceeptC,Kan ;andmc3425: by >1,000-fold from the level observed in mc 155 to that comparable with 2 R 2 2 mc 3422::attPL5::PaceeptC,Kan ) resulted in the reduction of trans- mc 6. The M. smegmatis ΔeptABCD strain complemented with eptC [mc 3425 formation efficiency by greater than 1,000-fold (Fig. 2B). The (I)] also showed extremely low transformation frequency, as observed in the 2 2 Δ γδ ability of eptC to complement not only the single gene deletion parental mc 6. M. smegmatis mc 3416 ( eptC:: attPL5::Pace:null) was used as but also the operon deletion showed that the expression of eptC acontrol.M. smegmatis mc2155 encodes eptB1 and eptC1 alleles of mc26 eptB alone was sufficient to inhibit pAL5000 plasmid transformation in and eptC, respectively. Complementation of M. smegmatis mc2155 with WT M. smegmatis. No difference in the transformation efficiency was eptC expressed from the Hsp60 promoter (mc23419) displayed only 0.3% transformation efficiency of the control strain (mc23418). Error bars depict standard deviation of the mean to a bar graph in three biological replicates.

ept1 ept6 A ept7 ept8 ept5 2 2 eptA eptB eptC 270 1611 3027 3321 eptD observed for the control strain mc 3416 (mc 3412::attPL5::Pace, M. smegmatis mc2 6 KanR) under similar conditions (Fig. 2B). Furthermore, comple- MukF MukE NTPase MukB SbcCD mentation of mc2155 by the EptC gene from M. smegmatis mc26 smtA mukF mukB E. coli mukE also resulted in loss of the ept phenotype, confirming that the absence of a functional EptC conferred the ept phenotype in mc2155 (Fig. 2B). These precise deletion and complementation B eptA eptB eptC eptD studies, together with our whole-genome sequencing results, led us to conclude that loss-of-function mutation in mc26 EptC gene con- ferred the ept phenotype. ...A G D E - EptB M. smegmatis mc2 6 ……GGCGATGAGTAAAACGATCTCTCGGACAGCCGAACTGAG…… M S K T I S R T A E L... EptC Bioinformatic Analysis of EptC Reveals Homology to SMC Proteins. Sequence analysis by Phyre 2 software of the region encom- ...A G D E L N D L S D.... D R - EptB1 passing eptC revealed that EptA, EptB, and EptC proteins share M. smegmatis mc2 155 ……GGCGATGAGTTAAACGATCTCTCGGAC //GACCGGTGA…… M S - EptC1 secondary structure homology to MukF, MukE, and MukB, re- spectively [refs. 10, 25, and 27; Fig. 1A and Table S3]. MukBEF Fig. 1. M. smegmatis mc26 ept mutants have a nonfunctional EptC gene. belongs to the SMC family and plays an important role in chro- (A) The operon structure of eptABCD is shown. The orientation of the trans- mosome condensation and segregation in E. coli (7, 28). E. coli poson insertions in eptC of the M. smegmatis mc26 mutants exhibiting the ept and its close relatives (gamma proteobacteria) use the protein phenotype is denoted by blue arrows. The numbers indicate the nucleotide MukB in place of a canonical SMC. Although MukB has limited position of transposon insertions in eptC, with position 1 marking the begin- sequence homology to SMC proteins, it does share a five-domain ning of eptC coding sequences. The domains identified by bioinformatic structure common to all SMC family members (7, 28–30). The N analysis are matched to their respective homolog in E. coli.(B) Comparison of – the eptABCD locus between mc26andmc2155. A single A→T point mutation, terminus of EptC (residues 22 80) contains a P-loop NTPase core domain with a conserved walkerA motif. Such an NTPase which resulted in eptC1 [K3Stop(Ochre)], eptB1 [Stop(Ochre)241L] mutation at the protein level, was responsible for the ept phenotype in M. smegmatis mc2155. core domain is characteristic of all SMC proteins. The C terminus The point mutation in mc2155 resulted in a translation read-through of EptB of EptC shows similarity to the SbcCD_C domain, which harbors by 48 aa and introduced a premature stop codon to EptC. the ATPase dimerization region (30, 31). The N and C termini

Panas et al. PNAS Early Edition | 3of8 Downloaded by guest on September 27, 2021 of EptC are separated by a very long coiled coil region with MSMEG_2423 MSMEG_0370 EptC [cd03278] [smart00968][cd03278] [pfam13555] [pfam13558] [pfam13555] [pfam13558] a noncoil disruption of ∼200 aa in the middle (Fig. S1). A coiled coil region separated by a hinge is a hallmark of SMC proteins [TIGR02168] [COG4913] [COG4913] Weak Strong (32). EptD shows similarity to a conserved hypothetical protein homology homology

with unknown function (33). M. smegmatis Whereas we discovered EptC, a SMC protein highly similar to M. vanbaalenii those in Gram-negative bacteria, others have found and stu- M. canetti M. kansasii died the canonical Gram-positive SMC in M. smegmatis (34). M. marinum To comprehensively understand the SMC composition in the M. abscessus M. fortuitum Mycobacterium , we examined the subtilis SMC, M. avium E. coli MukB, and M. smegmatis EptC protein sequence against M. ulcerans whole-genome sequences of all Mycobacterium species, known M. leprae M. tuberculosis to date, by Basic Local Alignment Search Tool (BLAST), using Bacillus subtillus blastP algorithm. In contrast to the fact that most prokaryotes Esherichia coli encode a single SMC (29), the M. smegmatis genome encodes Streptococcus mutans three SMC paralogs: EptC, MSMEG_370, and MSMEG_2423. Fig. 3. Organization of SMCs across the Mycobacterium genus and in The canonical SMC in M. smegmatis is MSMEG_2423 (MsSMC) other model bacterial systems. Two additional paralogs of EptC, namely and is 42% similar to BsSMC. Noticeably, this is the SMC found MSMEG_0370 and MSMEG_2423, were identified in M. smegmatis. EptC and in all mycobacteria known to date and has the signature hinge MSMEG_370 share 38% similarity to each other and 28% and 25% similarity region, which is missing in the other two SMC paralogs (29, 34). to MsSMC (MSMEG_2423), respectively. The organization of SMCs across MsSMC shares 82% similarity with the M. tuberculosis protein species is listed with M. smegmatis at the top. The protein equivalent (elon- encoded by Rv2922c, the only SMC present in M. tuberculosis gated equal signs) and domain equivalent (outlined squares) in other species (MtSMC). Most other mycobacterial species possess at least two are shown below the M. smegmatis SMC arrangement. The color of the SMC paralogs: one similar to the canonical BsSMC/MsSMC and bottom line in an elongated equal sign indicates the level of homology of that the other similar to MSMEG_0370. Only four of the mycobac- particular protein to its equivalent in M. smegmatis (darker indicates a higher terial genomes have a third paralog of SMC equivalent to M. level of homology). The same applies to the color of the squares discussed smegmatis EptC (Fig. 3). Notably, M. canetti, with two SMCs below. The absolute E values are provided in Dataset S1. Three conserved reported in a recently published sequence (35), is an exception to domains were identified in EptC: (i) a P loop containing region of AAA domain tubercle bacilli’s single SMC organization in their genomes. (pfam13555; squares with blue outline); (ii) uncharacterized protein con- served in bacteria (COG4913; region in between two outlined squares); and Taken together, our bioinformatic analysis allowed us to con- (iii) putative exonuclease SbcCD, C subunit (pfam13558; squares with yellow clude that eptC encoded a noncanonical SMC protein. outline). All three domains of EptC were also identified in MSMEG_0370. However, the third EptC paralog, MSMEG_2324, has a completely different EptC Is a Functional SMC Protein that Prevents Plasmid Segregation. domain structure from EptC. MSMEG_2324 has (i) the ATP-binding cassette SMC proteins are known to bind ssDNA and to modulate DNA domain of barmotin (cd03278; squares with red outline), and this domain was superhelicity status. To test that eptC encodes a functional SMC, we present at both ends of the protein; (ii) a chromosome segregation protein overexpressed and purified the EptC protein from E. coli.EMSAs SMC domain (TIGR02168; regions bounded by the two neighboring outlined carried out using single-stranded oligonucleotides revealed that squares); and (iii) Flexible Hinge Domain in SMC proteins (smart00968; EptC directly bound to DNA in a dose-dependent fashion. At squares with purple outline). The first ATP-binding cassette domain of lower concentrations, slow-migrating and faster-migrating EptC– MSMEG_2423 (cd03278) contains a region similar to pfam13555 found in EptC DNA complexes were observed. An increase in EptC concentra- and MSMEG_0370. tion resulted in the disappearance of the fast-migrating complex and increased intensity of slower-migrating complex. At even higher EptC concentrations, the mobility of the EptC–DNA com- electrophoresis. The effect of EptC binding on plasmid DNA was plex was further retarded (Fig. 4A).Adecreaseinmobilityathigher not influenced by the presence of ATP in the assay (Fig. S3). The concentrations suggested that EptC oligomerized on DNA. EMSA increased negative supercoils on 1D gel and the resolution performed with other 37–45-mer single-stranded oligonucleotides revealed by the 2D electrophoresis proved that EptC was not only having independent sequences under similar conditions showed bioinformatically but also functionally homologous to MukB. similar mobility patterns (Fig. S2). This consistent mobility pattern We next examined if EptC bound to double-stranded linear irrespective of oligonucleotide sequences showed that like other DNA and thus could bring two DNA molecules into close prox- SMCs, EptC bound to ssDNA in a dose-dependent but sequence- imity, which is a third function of SMCs. Two linearized plasmids, independent fashion. one derived from pAL5000 and the other plasmid lacking Efficient segregation of low-copy plasmids in E. coli is affected pAL5000 origin (control plasmid) were used in an EptC-assisted by plasmid topology (36), and SMC proteins are well known to ligation assay (41). As the concentration of EptC increased, a clear alter DNA topology after binding to DNA (7, 28, 37). A coupled pattern of plasmid multimerization was observed in pAL5000 topoisomerase assay was used to examine the effect of EptC on origin-containing plasmid. Plasmid multimerization peaked at DNA topology (38). Negative DNA supercoils are the substrate 0.6 μM EptC, and then decreased as EptC concentration in the of topoisomerase I used in assay (21). If EptC’s net effect is the reaction mixtures exceeded 0.6 μM (Fig. 4C). A similar pattern of stabilization of negative supercoiling like the MukB proteins plasmid multimerization was observed when the reaction was (39), then EptC binding to DNA would restrict topoisomerase performed using a plasmid lacking the pAL5000 origin (Fig. S4A). I from accessing and relaxing negative supercoils and would be Because many SMC proteins are capable of altering DNA to- manifested as a net inhibition of topoisomerase I activity in the pology outside of their original biological system, we made use of presence of EptC. If EptC functions like BsSMC and eukaryotic the T7 promoter so that EptC was expressed in E. coli BL21(DE3) condensing/SMC by stabilizing positive supercoils (12, 38), com- strain but not in DH5α strain. The very same EptC-encoding pensatory negative supercoils would be introduced in the plasmid plasmid was then isolated from the expressing and the non- after EptC binding, which would be relaxed by topoisomerase I. In expressing E. coli strains. Comparing the topology of plasmids this scenario, addition of EptC will display a net effect of stimu- from these two sources, we found that expression of M. smegmatis lation in topoisomerase I activity. A 1D coupled topoisomerase EptC led to significantly more plasmid multimers in E. coli (Fig. assay showed increased negative supercoiling of plasmid DNA 4D). The increase in plasmid multimerization in both M. smegmatis with increasing concentrations of EptC (12, 40) (Fig. 4B). The and E. coli showed EptC’s third hallmark SMC function of increase in negative supercoiling was confirmed when the top- bringing two DNA molecules into close proximity within and oisomers obtained from the 1D assay were resolved by 2D gel outside its species of origin. It is worth mentioning that the

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.1414207111 Panas et al. Downloaded by guest on September 27, 2021 EptC EptC A EptC/DNA 0 5 10 20 40 B INAUGURAL ARTICLE EptC/DNA 0 5 10 20 40 80 80 Protein DNA Complex EptC expression A + inducer No Selection Cell division

1 2 3 4 5 6 7 8 } Cell division Fluorescent cell 1 2 3 4 5 +TopoI CDEptC Non-Fluorescent − 0 0.1 0.2 0.4 0.6 0.8 1.0 2.0 EptC (μM) population − + + + + + + + + DNA ligase EptC − + }Multimers B -Trimer } Concatemers i ii iii iv -Dimer - Nicked

-Monomer - Supercoiled }Topoisomers

Bright Field Fluorescence Bright Field Fluorescence Fig. 4. EptC is a functional SMC homolog of MukB. (A) EMS demonstrating

the ability of EptC to bind DNA directly in a dose-dependent manner. Three Pace C eptCΔNTD−mCherry picomoles of ssDNA oligonucleotide were incubated with increasing amounts of purified EptC. Lanes: 1, ssDNA; 2–5, ssDNA with increasing concentrations 0.0h 6.0h 18 h 30 h of EptC. (B) Topoisomerase I-coupled DNA-binding assays showing that the EptC had a net effect of stabilizing negative supercoils. Lanes: 1, negatively supercoiled DNA alone; 2, DNA with topoisomerase I; 3–7, DNA with a con- stant amount of topoisomerase I but increasing amount of EptC; 8, DNA with EptC. (C) EptC-assisted plasmid ligation assay demonstrating that EptC brought DNA molecules in close proximity, and the multimer product peaked at 0.6 μM. (D) Expression of EptC increasing the amount of plasmid multimers in the cell. MICROBIOLOGY

Pace expression of EptC in presence of selection does not result in D eptC−mCherry

growth arrest. At the same time, the ratio of another compatible 0.0h 6.0h 18 h 30 h plasmid present in the EptC expressing strain does not change in the absence of selection marker (Fig. S4B). These experiments led us to conclude that the expression of M. smegmatis EptC in E. coli does not result in plasmid loss. Having established the DNA-binding and topology modula- tion properties of EptC, we proceeded to characterize the cellular function of EptC in the live mycobacterial system. To examine the effect of EptC expression on plasmid segregation, red fluo- rescent mCherry fusion proteins were made with full-length EptC (EptC–mCherry) and N-terminal-deleted EptC (EptCΔNTD- E 100 Express functional EptC mCherry) under the control of the acetamidase promoter. These No functional EptC two fusion protein expression vectors were transformed into the 10 EptC deletion strain of mc26, and hygromycin-resistant trans- 1 0.1 formants were selected. We reasoned that induction of EptC % Plasmid Retention would lead to loss of the following: (i) the same expression plasmid, 0.01 (ii) red fluorescence, and (iii) hygromycin resistance. In the pres- 3416 (I) 2 3414 (I) 2 3416 (U) 2 2 3414 (U) c c c ence of the inducer but absence of hygromycin, hygromycin-sen- m mc m m sitive, nonfluorescent cells would proliferate (Fig. 5A). Both expression vectors yielded hygromycin-resistant colonies. Fig. 5. Expression of EptC stimulates the loss of episomal plasmids in the Addition of acetamide led to transient expression of EptC and absence of selection. (A) Schematic representation of the assay. (B) EptC- mCherry forms localized foci in the cell which was not the case when mCherry red fluorescence in transformants harboring both constructs. was expressed alone (compare i and ii with iii and iv) when observed at 1,000× Notably, we observed localization of mCherry signals within cells magnification. (C) Time lapse microscopy during regulatable expression of harboring both constructs, implying that the DNA binding and N-terminal-truncated EptC (EptCΔNTD). Nonfluorescent cells were not ob- the inhibition of plasmid segregation roles of EptC could be served in the cells expressing EptCΔNTD in the absence of hygromycin, the decoupled. EptC/EptCΔNTP-mCherry proteins formed two dis- selection marker on the plasmid. In D,(i) EptC-mCherry was expressed from an

crete foci approximately at a quarter length from the two poles. In episomal plasmid under Pace;(ii) without induction, continuous cell division the cells relatively longer to others, EptC/EptCΔNTD-mCherry with basal level mCherry expression in daughter cells indicated faithful plasmid was observed at three or four loci distributed asymmetrically segregation. EptC expression was then induced by adding acetamide at the within the cells (Fig. 5 B, i and ii). In contrast, expression of end of 6 h, and selection for the plasmid (hygromycin) was removed from the mCherry alone did not show localized signals within a cell (Fig. media; (iii) at the end of 11 h (5 h after induction of EptC), nonfluorescent cells 5 B, iii and iv). The distribution of EptC loci is consistent with arose from the fluorescent cell. At 18 h, nonfluorescent cells were clearly visi- ble; and (iv) at the end of 30 h, the majority of cells were nonfluorescent. (Scale the distribution of SMC/MukB protein in other prokaryotes (37). μ Considering the universal importance of the NTPase domain bars, 10 m.) (E) Expression of eptC triggers the loss of the preexisting pAL5000 plasmid in mc23414. Of the mc23414 cells, which had been pretransformed with in SMC proteins (7), we expected N-terminal-truncated EptC to a pAL5000 plasmid, only one-thousandth of the population were able to be defective in causing plasmid loss. Thus, we expected to ob- maintain the pAL5000 plasmid upon induction of eptC expression from an serve fluorescent cells proliferating in the presence of both the integrated location. We used hygromycin resistance, the antibiotic resistance inducer acetamide and the selection marker hygromycin. After marker on the pAL5000 plasmid, as an indicator for the presence of pAL5000 the induction of EptC expression, cells expressing EptCΔNTD- episomal plasmid. Error bars depict standard deviation of the mean to a bar mCherry continued to grow and all of the progenies were red graph in three biological replicates.

Panas et al. PNAS Early Edition | 5of8 Downloaded by guest on September 27, 2021 fluorescent, no matter whether hygromycin was present (Movie been shown in E. coli that the correct supercoiling status is a pre- S1) or absent (Fig. 5C and Movie S2). Retention of the episomal requisite for plasmid segregation (17). Taking the established plamid expressing N-terminal-truncated EptC is consistent with evidence in the literature and our own experimental results to- the hypothesis that full-length EptC is required to interfere with gether, we favor the second mechanism proposed above. How- pAL5000 plasmid maintenance in M. smegmatis. ever, we do not preclude the possibilities that EptC expression In contrast, cells expressing the full-length EptC–mCherry lost level (51, 52) or an out-of-phase organization of the L and H sites plasmids for survival and division in the absence of hygromycin. of the pAL500 plasmid in the presence of EptC may secondarily Five hours after adding the inducer but not the selection agent, contribute to plasmid loss. daughter cells from the parental fluorescent cell became non- Elimination of foreign DNA by bacterial cells is well docu- fluorescent and continued proliferating. (Fig. 5D and Movie S3). mented in the literature. Many bacterial species have acquired Our time-lapse microscopy showed that plasmidless bacteria restriction–modification (RM) systems to destroy foreign DNA. resulting from induced EptC expression were killed by hygro- Phages have evolved to escape such DNA degradation either by mycin (Fig. S5B and Movie S4), which were quantified by plating reducing the number of certain restriction sites (53) or by meth- in the presence and absence of hygromycin (Fig. 5E and Fig. S5C). ylating or alkylating the DNA bases (54). As a counterphage No difference in the number of colonies was observed when the strategy, certain bacteria encode nucleases to target the phage 10-fold serial dilutions plated on 40 μg/mL kanamycin and 100 modified DNA. Plasmids have also developed effective ways such μg/mL hygromycin plates (EptC expression was not induced) were as encoding methylase to disable the host RM system thus ensure compared with the same dilutions plated on 40 μg/mL kanamycin their own maintenance in the host (55). The F plasmid, a low-copy and 0.2% acetamide (EptC expression was induced but loss of the number plasmid, prevents its loss from the cell by harboring the episomal plasmid was permitted) plates (Fig. S5C). No difference ccdAB plasmid addiction module to eliminate the postsegregated in the number of cells retaining plasmid was observed for the cell population devoid of F plasmid (56). Various plasmid ad- control strain mc23416. diction modules are found in the bacterial chromosomes as well. The genetic, biochemical, and imaging analysis clearly showed However, it is still under debate whether these modules are ac- that EptC was a functional SMC homolog, and it prevented plas- quired to gain fitness under stress conditions (57) or to protect the mid segregation. cell from toxic effects after the curing of plasmids harboring ad- diction modules (58). As a corollary to the mechanism used by Discussion F plasmid to ensure its presence in all of the progenies of the host, Our work has revealed inhibition of plasmid segregation caused the chromosomal toxin–antitoxin (TA) modules guard against the by the SMC homolog EptC, contrary to the conventional un- deletion of important genomic loci (59). Noticeably, eptABCD derstanding of SMC proteins’ role in assisting the partitioning operon is flanked by two of the three TA modules present in and maintenance of genetic materials. It is noteworthy that in- M. smegmatis to prevent loss of the operon (60). Our findings on tegration proficient vectors (42) transforms equally well in mc26 eptC point toward another unprecedented mechanism by which and mc2155. No homolog to eptC was found in genomes of slow bacterial progenies rid extrachromosomal DNA. growing mycobacteria, which explains slow growers’ permissive- SMC proteins often work in conjunction with various topo- ness to transformation (4, 43–45). As one of the three SMC isomerases. In B. subtilis, SMC interacts with topoisomerase IV paralogs encoded by M. smegmatis, eptC is the least represented to influence chromosome supercoiling and segregation (61). In SMC homolog among Mycobacterium species, actinomyces and E. coli, the dimerization domain of MukB physically interacts with environmental bacteria. MSMEG_2423 encodes for the most the C-terminal domain of the ParC subunit of topoisomerase IV to conserved SMC protein across the Mycobacterium genus. Con- modulate topoisomerase IV activity (62). M. tuberculosis encodes sidered the canonical SMC in mycobacteria, MSMEG_2423 and a single SMC, a type I topoisomerase topA (TopoIA), and a type II its equivalent are closer to BsSMC than to E. coli MukB. Sur- topoisomerase gyrBA (DNA gyrase). The two topoisomerases in prisingly, deletion of this universal SMC (MSMEG_2423) in M. tuberculosis are present across the entire Mycobacterium genus M. smegmatis does not result in the phenotypes characteristic of and thus are considered the minimum combination necessary to SMC deletion in other species (34, 46, 47). Besides inhibiting function. M. smegmatis and Mycobacterium vanbaalenii, both hav- plasmid segregation, noncanonical SMC proteins such as EptC ing three paralogs of SMC in their genomes, have topoisomerase may be capable of functionally compensating for the loss of IV equivalent (63) and a type IB topoisomerase (TopoIB), in ad- MSMEG_2423 in M. smegmatis. dition to the minimum combination. Mycobacterium kansasii has The biochemical property of EptC and the requirement for two SMC paralogs, the minimum topoisomerase combination and pAL5000 plasmid replication led us to propose possible mech- an extra TopoIB. also has two SMC paralogs anisms by which EptC inhibits plasmid segregation. The minimal but preserves the minimum combination of topoisomerases. Tak- region required for pAL5000 plasmid replication and mainte- ing together the facts above, mycobacterial species with the maxi- nance consists of two proteins, RepA (307-aa residues) and RepB mum number of SMCs also have the maximum number of (119 residues), and a 435bp cis element sequence containing the topoisomerases. Among the four Mycobacterium species that en- pAL5000 origin (48). The RepA and RepB proteins support code EptC or its homologs in their genomes (Fig. 3), three have plasmid replication in trans. The 435bp cis element contains low- extra topoisomerases in addition to the minimum combination of (L) and high- (H) affinity binding sites for RepB, and harbors topA and gyrBA. Reports on interactions between SMCs and topo- several palindromic sequences (48, 49). RepB binding to these isomerases in mycobacteria are lacking. However, the coexistence sites causes local bending as well as a long-range polar curvature. of extra SMC(s) and topoisomerase(s) may inspire work to bridge The curvature is essential for the in-phase alignment of the L and this gap in knowledge. H sites to form a looped structure (50). Based on knowledge from Our results have elucidated that the molecular genetic basis literature and our recent findings, several models for the mecha- for the ept phenotype of M. smegmatis mc2155 is a loss-of-function nism of plasmid loss caused by EptC can be proposed: (i) Non- mutation mapped to a gene encoding EptC, a noncanonical SMC specific DNA binding activity of EptC interferes with RepB protein. Induced eptC expression in M. smegmatis lacking eptABCD binding; (ii) the alteration in supercoiling by EptC interferes with operon was sufficient to cause plasmid loss (Fig. 2). This result the in-phase alignment of the L and H sites or the ability of RepB allowed us to envision the use of eptC as a genetic tool to cure to introduce long-range polar curvature; (iii) the expression level pAL5000 plasmid in other mycobacterial species including of partition proteins has been proved crucial for the faithful seg- M. tuberculosis and bacillus Calmette–Guérin, which naturally lack regation in more than one scenario (51, 52), and EptC disturbs the the eptABCD operon. Currently, the main means to cure plasmid in optimal expression level of partitioning proteins; and (iv) EptC mycobacteria has been to repeatedly passage the plasmid-bearing competes with other SMCs to interact with topoisomerases, re- strain in media with novobiocin (64). Expression of eptC from a stricting the type of interaction necessary for segregation. It has regulatable promoter can not only eliminate the time consuming

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1414207111 Panas et al. Downloaded by guest on September 27, 2021 and laborious passages in slow-growing mycobacteria but also biofilm formation in M. smegmatis (87). The useful transposon 2

decrease the probability of selecting for secondary mutations in (IS1096) was identified from the genome of mc 155 (88) and used INAUGURAL ARTICLE DNA gyrase or in the ATPase domain of MutL-like proteins, whose to generate valuable auxotrophic mutants of bacillus Calmette– activity is also inhibited by novobiocin (65). Guérin (89) and the first diverse genetic libraries in M. tuberculosis Despite the delay in knowing the molecular basis for the (90). M. smegmatis mc2155 was also used to characterize a unique transformability of M. smegmatis mc2155, this strain provided conjugation system (91). the first plasmid transformation platform for any Mycobacterium Besides advances in TB chemotherapy, M. smegmatis mc2155 (1, 4). Having a plasmid transformation system allowed for the has been used to study fundamental cellular processes, such as development of facile cloning vectors to make bacillus Calmette– cell division (92) and dynamics of mycobacterial persistence (93). Guérin a recombinant vaccine (16), to complement genetic dele- There is no doubt that part of the reason for the usefulness of tions, and to study mycobacterial promoters (66). M. smegmatis mc2155 in these discoveries is the strain’s ability to stably maintain mc2155 itself has the potential to be an effective vaccine strain. foreign plasmids and express antibiotic resistance and fluorescent 2 When the esx-3 gene in mc 155 is replaced with the esx-3 ortholog genes, allowing for selection and imaging. The growing interest in from M. tuberculosis, the cross-complemented M. smegmatis strain M. smegmatis has extended beyond its role as a model organism to was proven safe and capable of offering remarkable immune understanding the plasticity and novelty in its metabolic network protection (67). Perhaps more importantly, M. smegmatis has (94). It is evident that the importance of this organism is gaining proven to be a more suitable surrogate host than conventional host recognition in the Mycobacterium research community. organisms such as E. coli, B. subtilis,orStreptomyces for investigating 2 Given the variability of SMC organizations across species the molecular biology of mycobacteria. Isolation of mc 155 allowed within the Mycobacterium genus, it is hopeful that further studies for the identification of genes involved in isoniazid (68–71), ethi- on mycobacterial SMCs may improve our understanding about onamide (72, 73), isoxyl (74, 75) or thioacetazole (75), ethambutol striking and fundamentally important differences, such as growth (76–78), capreomycin (79), streptomycin (80), fuoroquinolone rate, virulence, and the persistence phenotype, between organ- (81), and kanamycin (79) resistance. The ability to transform and isms in this genus. express genes in M. smegmatis allowed the discovery of the first auxiliary secA2 secretion system (82), a novel way to study iron ACKNOWLEDGMENTS. We thank Brian Weinrick for his performing the metabolism (83), and the analyses of unique carbohydrate antigens whole-genome sequencing. We also thank Travis Hartman, Catherine (84). Bedaquiline, the only new TB drug approved by the Food Vilcheze, Christopher Kerantzas, Howard Takiff, Michael Keogh, and Birgit and Drug Administration in the past 40 y, was discovered through Korioth-Schmitz for providing valuable suggestions in manuscript editing. ascreeningonM. smegmatis (85). M. smegmatis was used to show Funding for this work was provided by Bill and Melinda Gates Foundation Grant OPP38614 and National Institutes of Health/National Institute of Allergy MICROBIOLOGY that lysogenization by bxb1 disrupted a GroEL-1 gene involved in and Infectious Diseases (NIH/NIAID) Grants AI26170 and AI106543-01. A.R.W. biofilm formation (86). A promising new drug targeting persisters was supported by NIH/NIAID Contract HSN272200900040C. P.J. is the recipient has been identified from screening for compounds inhibiting of a Potts Memorial Foundation research fellowship.

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