• Users Online: 1040
  • Home
  • Print this page
  • Email this page


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 3  |  Page : 281-286

MtFtsX a predicted membrane domain of ABC transporter complex MtFtsEX of Mycobacterium tuberculosis interacts with the cell division protein MtFtsZ


1 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Asir, Saudi Arabia
2 School of Biological Sciences, National Institute of Science Education and Research, Bhubaneswar, Odisha, India
3 Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru, Karnataka, India

Date of Web Publication12-Sep-2019

Correspondence Address:
Dr Mushtaq Ahmad Mir
King Khalid University, P.O. Box: 960, Postal Code: 61421, Abha, Asir
Saudi Arabia
Dr Parthasarathi Ajitkumar
Department of Microbiology and Cell Biology Indian Institute of Science, Bangalore
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_98_19

Rights and Permissions
  Abstract 


Background: Bacterial cytokinesis is orchestrated by a complex of dozen of proteins called 'divisome' at the mid-cell site. FtsZ, the eukaryotic tubulin homolog, localizes to the mid-cell site where it polymerizes and forms a cytokinetic Z-ring. The Z-ring acts as a docking platform for other proteins to localize. In model organisms, Escherichia coli and Bacillus subtilis, FtsZ is known to interact with several proteins. The role of few of these interactions is known, while of others is yet to be studied. In Mycobacterium tuberculosis, the cell division and its regulation are poorly studied. Although, most of the divisome proteins are conserved in M. tuberculosis, surprisingly the homologues of the protein factors required for membrane association of Z-ring and its stabilization are absent. In E. coli FtsE and FtsX, the constituent ATPase and membrane domains of the ABC transporter complex, localize to the Z-ring immediately after Z-ring stabilizing proteins, ZipA and FtsA. Therefore, investigation of the interaction between MtFtsX and MtFtsZ is demanding. Methods: Bacterial two-hybrid system was used to identify the interaction between MtFtsE and MtFtsZ. This interaction was further confirmed by biochemical methods of Ni2+-NTA agarose pull-down and coimmunoprecipitation. Results and Conclusion: Here, we demonstrated that MtFtsX interacts with MtFtsZ in vivo and ex-vivo. Further, we showed that self-interacting MtFtsX interacts with MtFtsE. However, we did not find any interaction between MtFtsE and MtFtsZ. These results suggest that the membrane domain MtFtsX of the ABC transporter complex 'MtFtsEX' might be the membrane-tethering and stabilizing factor for Z-ring in M. tuberculosis.

Keywords: Cell division, FtsE, FtsX and FtsZ, Mycobacterium tuberculosis


How to cite this article:
Mir MA, Srinivasan R, Ajitkumar P. MtFtsX a predicted membrane domain of ABC transporter complex MtFtsEX of Mycobacterium tuberculosis interacts with the cell division protein MtFtsZ. Int J Mycobacteriol 2019;8:281-6

How to cite this URL:
Mir MA, Srinivasan R, Ajitkumar P. MtFtsX a predicted membrane domain of ABC transporter complex MtFtsEX of Mycobacterium tuberculosis interacts with the cell division protein MtFtsZ. Int J Mycobacteriol [serial online] 2019 [cited 2019 Sep 20];8:281-6. Available from: http://www.ijmyco.org/text.asp?2019/8/3/281/266500




  Introduction Top


Bacterial cell division has been studied to a great detail in  Escherichia More Details coli and Bacillus subtilis, but it remains poorly understood in Mycobacterium tuberculosis. FtsZ, a highly conserved eukaryotic-tubulin homolog in bacteria, is a scaffolding protein that initiates the septum formation at the mid-cell site and remains the main player in the division process, commenced by a complex of several proteins called “divisome.”[1] The function of most of the constituent proteins of divisome is not completely known. The constituent proteins localize to the mid-cell site in a specific order. FtsZ is the first protein, which localizes and polymerizes at the mid-cell site to form the cytokinetic Z-ring.[2] ZipA [3],[4],[5] and FtsA [6],[7] are the immediate proteins in E. coli that localize to mid-cells site to stabilize the Z ring. Thereafter, several other proteins localize to the mid-cell site in a hierarchical order of (FtsE + FtsX) >FtsK > FtsQ >(FtsL + FtsB) >FtsW > FtsI > FtsN > AmiC > EnvC.[8]

If this hierarchical order remains the same in M. tuberculosis, then the immediate player recruited to mid-cell site after FtsZ would be the FtsE-FtsX complex. This possibility is because the structural homologs of ZipA and FtsA are absent in M. tuberculosis, although the presence of their functional homologs cannot be ruled out. While E. coli FtsX interacts with FtsA and FtsQ,[9] FtsE is known to interact with FtsZ in FtsX-independent manner.[10] The E. coli FtsEX deletion strain displays a lethal division defect in low osmolar growth media, which is partially or fully suppressed by restoring the osmolarity of the medium,[11] or by overexpression of FtsZ, FtsN, or FtsP (SufI)[11],[12] proteins. Although the actual role of FtsEX in cell division is not clear, recent studies have unveiled an unprecedented role of FtsEX in the latter stages of cell division. FtsE-FtsX complex regulates the cell wall hydrolases at the division site in E. coli[13] and M. tuberculosis.[14] In this study, we show that MtFtsX interacts with MtFtsZ and thus probably playing the role of the membrane anchor for the cytokinetic Z-ring at mid-cell site.


  Methods Top


Cloning of MtftsE, MtftsX and MtftsZ genes

The genes were polymerase chain reaction (PCR) amplified from either genomic DNA of M. tuberculosis H37 Ra or MTCY270 cosmid (kind gift from Stewart Cole) and were individually cloned in expression as well as in two-hybrid vectors. The recombinant plasmid pBAD33-MtftsE was generated by mobilizing XbaI fragment of MtftsE along with ribosome binding site (RBS) from pET20b-MtftsE[15] into pBAD33[16] at XbaI restriction site. The MtftsX gene amplified by PCR using primers MtftsX1 and MtftsX2 was cloned in phosphate-buffered saline (PBS) (KS) at BamHI and XbaI restriction sites. After sequencing to rule out any mutation in the open reading frame (ORF), the gene MtftsX was subcloned under T5 promoter in pQE30 at BamHI and SacI restriction sites to obtain pQE30-MtftsX. The MtftsZ gene was cloned in pET20b (+) at EcoRV and NotI restriction sites to obtain recombinant plasmid pET20b-MtftsZ. Subsequently, the plasmid was digested with XbaI restriction enzyme to release 1.28 kb fragment. The fragment containing MtftsZ gene and an RBS upstream of it was later cloned at the XbaI site in pBAD33,[16] to obtain pBAD33-MtftsZ.

For the in vivo interaction studies using bacterial two-hybrid system, the genes were cloned in pT18 and pT25 vectors.[17] MtftsX gene was PCR amplified from M. tuberculosis H37 Ra genomic DNA, using MtXpT18/25f and MtXpT18/25r primers, and cloned in pT18 or pT25 vector at KpnI/HindIII or BamHI/KpnI restriction sites, respectively, to generate pT18-MtftsX and pT25-MtftsX. The recombinant plasmid pT25-MtftsE was obtained from pT25-MtftsEX by digesting the latter with KpnI and SmaI restriction enzymes to remove MtftsX gene. The vector backbone harboring MtftsE gene was end-filled and ligated. However, the recombinant plasmid pT18-MtftsE was obtained by cloning MtftsE gene amplified from M. tuberculosis H37 Ra genomic DNA using MtEpT18/25f and MtEpT18/25r primers into pT18 at KpnI and HindIII restriction sites. The MtftsEX ORF was PCR amplified from M. tuberculosis H37 Ra genomic DNA using primers MtEpT18/25f and MtXpT18/25r and cloned into pT25 at BamHI and KpnI restriction sites. Similarly, the MtftsZ gene amplified from MTCY270 cosmid DNA using the primer pairs MtZpT25f and MtZpT25r and MtZpT18f and MtZpT18r was cloned in pT25 and pT18 vectors,[17] respectively, at the corresponding BamHI/KpnI and KpnI/XhoI sites, respectively, to generate pT25-MtftsZ and pT18-MtftsZ recombinant plasmids. pT18-Zip and pT25-Zip and pT18 and pT25[17] were used as the positive and negative controls, respectively. The primer sequences are given in [Table 1] and the plasmids generated in this study are listed in [Table 2]. All the clones generated in this study were sequenced on both the strands to rule out any point mutation in the ORFs. For all the PCRs, Pfu DNA polymerase (Promega) was used. T4 DNA polymerase and (NEB) T4 DNA ligase (NEB) were used in end-filling and ligation reactions, respectively. The recombinant plasmid pET-MtftsZ-PFH was generated by replacing Hnef in pET-Hnef-PFH [18] (a kind gift from Zhao, LJ) by MtftsZ.
Table 1: Oligonucleotides used in the study

Click here to view
Table 2: Plasmid constructs used in the study

Click here to view


Bacterial two-hybrid assay

Bacterial two-hybrid analysis was carried out by streaking the adenylate cyclase deficient (cya-) E. coli strain, DHP1, and harboring recombinant two-hybrid vectors expressing T18 and T25 fusions of fts genes of various combinations or Zip fusions (positive control) or empty vectors (negative control) on MacConkey agar plates containing 1% maltose. The plates were incubated for 24–30 h at 30°C. The pink color of the bacterial streaks indicates that the two proteins interact, whereas the streak that does not produce pink color indicates that the two proteins of interest do not interact. Plates were scanned, and Adobe Photoshop was used to adjust the brightness and contrast of the images.

Ni 2+-nanoparticle tracking analysis-agarose pull down, co-immunoprecipitation, and western blotting

For Ni 2+-nanoparticle tracking analysis (NTA), pull-down and co-immunoprecipitation (Co-IP) experiments, the whole cell lysates were used. In brief, E. coli cells harboring pQE30-MtFtsE and pBAD33-MtFtsZ grown to an O.D600 of 0.4–0.6 at 37°C were induced with 1 mM IPTG and 0.1% arabinose, respectively, for 1 h. Cells were harvested by centrifugation and lysed by sonication in ice-cold lysis buffer (50 mM Hepes-KOH, pH 7.7, 50 mM KCl, and 1 mM PMSF) and the suspension thus generated was subjected to centrifugation at 10,000 rpm. The cell debris (pellet) was discarded and the supernatant fraction (whole cell lysate) was saved at -80oC for further use. The samples were maintained at 4°C throughout lysis and centrifugation.

For Ni 2+-NTA pull-down experiments, the Ni 2+-NTA agarose beads incubated with whole cell lysates for 30 min at 4°C were washed thrice with the 500 μl ice-cold lysis buffer. Subsequently, the beads were boiled in 1 × sample loading buffer for 5 min in a boiling water bath and loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. For Co-IP, the whole cell lysates were incubated overnight with appropriate primary antibodies (1:100 dilution) at 4°C. Subsequently, protein A-sepharose beads were added and incubated further for 3 h under similar conditions. The protein A-sepharose beads were washed thrice with 500 μl each of ice-cold lysis buffer. After boiling the beads in 1 × sample buffer, the samples were fractionated on 10% SDS-PAGE. After fractionation, the proteins were transferred to polyvinylidene fluoride membrane for western blotting. The membrane was blocked for 2 h with 1x PBS buffer containing 0.2% Tween 20 and 5% skimmed milk. After blocking, the membrane was further incubated for 2 h with primary antibodies specific to the protein of interest. The anti-MtFtsZ and anti-MtFtsE polyclonal antibodies were used at 1:10,000 dilution, whereas anti-polyhistidine monoclonal antibodies were used at 1:3000 dilution. The membrane was washed thrice with 1x PBS containing 0.2% Tween 20 and incubated with appropriate secondary antibodies of either horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) or HRP-conjugated anti-mouse IgG antibodies for 2 h. Both the antibodies were used at 1:10,000 dilution. After three washes with 1x PBS containing 0.2% Tween 20, the blot was developed using enhanced chemiluminescent detection system (Sigma).


  Results Top


MtFtsX interacts with MtFtsZ ex vivo

To determine whether the M. tuberculosis cell division proteins MtFtsX and MFtsZ interact with each other, Ni-NTA-agarose pull down and co-immunoprecipitation approaches were employed. Both the proteins were coexpressed in E. coli M15 cells. The MtFtsX-6x His protein was expressed from pQE30-MtftsX [[Figure 1]a, lane 2] and MtFtsZ was expressed from pBAD33-MtftsZ [[Figure 1]b, lane 2]. As expected, no expression of MtFtsX-6x His and MtFtsZ was detected in E. coli M15 cells harboring plasmid vectors pQE30 and pBAD33 [[Figure l]a and [Figure l]b, lane 1]. The MtFtsX-6x His protein was pulled down with Ni 2+-NTA agarose beads from the whole cell lysate prepared from the cells coexpressing both MtFtsX-His and MtFtsZ. As shown in [Figure 1]c, lane 2, the MtFtsZ protein came down along with MtFtsX-6x His. To rule out the possibility of MtFtsZ coming down by interacting with Ni 2+-NTA agarose beads rather than interacting with MtFtsX-6x His, the whole cell lysate prepared from the E. coli M15 cells expressing MtFtsZ alone [[Figure 1]b, lane 3] was incubated with Ni 2+-NTA agarose beads. As shown in [Figure 1]c, lane 1, anti-MtFtsZ western blotting of the Ni 2+-NTA agarose eluate detected a mild intensity band of MtFtsZ, which is several fold lower than MtFtsZ band in lane 2, suggesting that the MtFtsX interacts with MtFtsZ. The amount of whole cell lysate used was normalized to the amount of MtFtsZ of coexpressed sample.
Figure 1: (a and b) Western blot of whole cell lysates of Escherichia coli M15 cells either expressing MtFtsZ or MtFtsX-His or both. (a) Anti-polyhistidine Western blot of whole cell lysates of Escherichia coli cells either coexpressing MtFtsZ and MtFtsX-His (lane 2) or expressing MtFtsX alone (lane 3). (b) Anti-MtFtsZ Western blot of whole cell lysates of Escherichia coli cells either coexpressing MtFtsZ and MtFtsX-His (lane 2) or expressing MtFtsZ alone (lane 3). Lane 1 in a and b contain whole cell lysates of Escherichia coli cells harboring empty vectors. (c and d) Interaction between MtFtsX and MtFtsZ. (c) Anti-MtFtsZ Western blot of Ni2+-NTA agarose pulled down proteins from whole cell lysates of Escherichia coli cells either expressing MtFtsZ alone (lane 1) or coexpressing both the MtFtsX-His and MtFtsZ (lane 2). (d) Anti-polyhistidine Western blot of immunoprecipitate from the whole cell lysates of Escherichia coli cell expressing either both the MtFtsZ and MtFtsX-His (lane 2) or MtFtsX-His alone (lane 3). For the control, whole cell lysates of Escherichia coli cells coexpressing MtFtsZ and MtFtsX-His was incubated with protein-A-sepharose beads only (lane 1). For immunoprecipitation, affinity purified anti-MtFtsZ antibodies raised in rabbit were used. IP: Immunoprecipitation: PD: Pull-down; WB: Western blot

Click here to view


Reciprocally, when MtFtsZ was immunoprecipitated with affinity purified anti-MtFtsZ polyclonal antibodies from the MtFtsX-6x His and MtFtsZ coexpressed whole cell lysate, the MtFtsX-6x His co-immunoprecipitated along with MtFtsZ [[Figure 1]d, lane 2]. To rule out the possibility of the cross-reactivity of anti-MtFtsZ antibodies with MtFtsx-6x His, which could be the reason for Co-IP of MtFtsX-6x His with MtFtsZ, the whole cell lysate of the cells expressing MtFtsX-6x His alone [[Figure 1]a, lane 3] was incubated with anti-MtFtsZ antibodies. As shown in [[Figure 1]d, lane 3 a very-low-intensity MtFtsX band was detected when this immunoprecipitate was western blotted using anti-polyhistidine monoclonal antibodies. [[Figure 1]d, lane 3], suggesting that the MtFtsX-6x His coimmunoprecipitated with MtFtsZ rather than pulled down directly by anti-MtFtsZ polyclonal antibodies. Similar to the agarose beads [[Figure 1]c, lane 1], sepharose beads also showed a very minimal nonspecific binding to MtFtsX-6x His protein [[Figure 1]d, lanes 1 and 3]. The whole cell lysate containing MtFtsX-6x His was normalized to the MtFtsX-6x His of coexpressed whole cell lysate. Thus, the Co-IP confirmed the interaction of MtFtsX with MtFtsZ ex vivo.

MtFtsX interacts with MtFtsZ in vivo

To verify the interaction of MtFtsX with MtFtsZ in vivo, we took advantage of bacterial two-hybrid system, which is based on the reconstitution of signal transduction pathway in adenylate cyclase deficient (cya) E. coli strain (DHP1).[17] MtFtsZ and MtFtsX were expressed as fusion proteins of T18 and T25 fragments of adenylate cyclase. Interaction of T18 and T25 fragments causes functional complementation of adenylate cyclase. As a result, the DHP1 colonies will appear pink on MacConky agar plate. The DHP1 cells expressing MtFtsX-T18 and T25-MtFtsZ fusion proteins appeared as pink colonies on MacConkey agar plate [Figure 2]a, suggesting that the MtFtsZ and MtFtsX proteins interact with each other. Surprisingly, the reciprocal fusion proteins, namely, T25-MtFtsX and MtFtsZ-T18 did not interact [Figure 2]a. It indicated that the blocking of C-terminus of MtFsZ abolished its interaction with MtFtsX, which is in agreement with the fact that C-terminus of MtFtsZ is involved in interaction with other cell division proteins, like ZipA in E. coli.[19],[20] Dysfunction of the T25-MtFtsX cannot be the reason for noninteraction of T25-MtFtsX with MtFtsZ-T18 as the same fusion protein is interacting with MtFtsX-T18 [Figure 2]b. Moreover, the nonstability of the MtFtsZ-T18 was also ruled out, as the fusion was found intact when coexpressed with T25-MtFtsX in the DHP1 cells [Figure 3]. No interaction was observed between T18 and T25 fragments of adenylate cyclase [Figure 2]a and [Figure 2]b as expected, whereas the positive control Zip fusions of T18 and T25 interacted as expected.
Figure 2: Interaction of MtFtsX with MtFtsZ and MtFtsE in vivo. (a) Interaction of MtFtsX with MtFtsZ. Escherichia coli DHPI cells expressing T18 and T25 fusions of MtFtsE, MtFtsX, and MtFtsZ in various combinations were streaked on MacConkey agar plate containing 1% maltose. (b) Interaction of MtFtsX with self and with MtFtsE. Escherichia coli DHPI cells expressing T18 and T25 fusions of MtFtsX and MtFtsE in various combinations were streaked on MacConkey agar plate containing 1% maltose. For the positive control cells expressing zinc finger protein fusions of T18 and T25 and for negative control cells harboring empty two-hybrid vectors, T18 and T25 were streaked on the same plates. The plates were scanned after 24 h of incubation at 37°C

Click here to view
Figure 3: Expression profile of MtFtsE and MtFtsZ protein expressed from two-hybrid vectors. (a and b) Western blot of T25 and T18 fusions of MtFtsE. Anti-MtFtsE western blot of the whole cell lysates of DHP1 cells expressing individually T25-MtFtsE (a, lane 3), MtFtsE-T18 (b, lane 2) and of M15 cells expressing MtFtsE from recombinant plasmid pQE30-MtftsE (a, lane 2 and b, lane 3). (c and d) Western blot of T25 and T18 fusions of MtFtsZ. Anti-MtFtsZ western blot of whole cell lysates of DHP1 cells expressing individually MtFtsZ-T18 (c, lane 2), T25-MtFtsZ (d, lane 3) and of Escherichia coli JM109 cells expressing MtFtsZ-PFH (c, lane 3 and d, lane 2). Lane 1 contains whole cell lysates of the DHP1 cells harboring two-hybrid vectors T18 and T25. MtFtsZ-PFH is expressed from pET-MtftsZ- PFH

Click here to view


MtFtsX interacts with MtFtsE in vivo

FtsX is predicted to be the membrane component of the ABC transporter type protein complex FtsE-FtsX. Earlier, by pull-down assays, we showed that MtFtsX interacts with MtFtsE.[15] In the present study, we wanted to know whether these proteins interact in vivo. The bacterial two-hybrid system confirmed that MtFtsX-T18 and T25-MtFtsE interacted very well in vivo [Figure 2]b. Surprisingly, the reciprocal protein fusions (MtFtsE-T18 and T25-MtFtsX) did not interact [Figure 2]b. One possibility could be the instability of the fusion protein, MtFtsE-T18. Western blotting of the whole cell lysate of DHPI coexpressing MtFtsE-T18 and T25-MtTtsX ruled out this possibility as the MtFtsE-T18 fusion was intact [Figure 3]. However, the intactness of the T25-MtFtsX fusion-proteins could not be ascertained due to the lack of antibodies against MtFtsX protein. We believe that the T25-MtFtsX fusion would be intact and functional, as it interacted very well with MtFtsX-T18 [Figure 2]b. However, blockage of interaction due to the presence of fusion partner T18 at the C-terminus of MtFtsE or T25 on the N-terminus of MtFsX cannot be ruled out.

Earlier, we showed that the MtFtsE and MtFtsX exist as a complex on the membrane of mycobacterial cells.[15] Therefore, we were curious to know whether MtFtsE, independent of MtFtsX, would interact with MtFtsZ. Pull-down assays did not detect any interaction between MtFtsE and MtFtZ (data not shown). Bacterial two-hybrid system analysis also did not detect any interaction between MtFtsE and MtFtsZ, even when reciprocal T18 and T25 fusions of MtFtsE and MtFtsZ were expressed in DHP1 cells [Figure 2]a. We did not attempt to examine the interaction of MtFtsE with MtFtsZ independent of MtFtsX, as it is not in the scope of this study.


  Discussion Top


FtsZ, the eukaryotic tubulin homolog and highly conserved protein across diverse bacterial genera, localizes to the mid-cell site during bacterial cell division to initiates septum formation by forming cytokinetic Z-ring. FtsZ, being the cytosolic protein, needs additional factors to tether and stabilize Z-ring. In E. coli, FtsA (the actin homolog) and FtsA stabilize the Z-ring. Several other FtsZ-interacting proteins, namely, ZapA, SepF, EzrA, etc., have been identified in E. coli and B. subtilis.[21],[22],[23] The absence of these proteins in M. tuberculosis raises the question as to how Z-ring in M. tuberculosis is tethered to membrane and stabilized. In E. coli, the FtsEX complex, which is predicted to be the ABC transporter, is recruited relatively early to the divisome.[8] FtsE is the nucleotide-binding domain, while FtsX encompasses the transmembrane domain of the complex. Recruitment of FtsX to the mid-cell site is accomplished by interaction with FtsA and FtsQ,[8] while FtsE interacts directly with the FtsZ.[10]

In M. tuberculosis, we have earlier shown that MtFtsE and MtFtsX exist as a complex in the membrane, although their precise subcellular localization is not yet known. Since the proteins, which stabilize the Z-ring at mid-cell site, are lacking in M. tuberculosis, we believe that MtFtsEX complex might interact with MtFtsZ and thus could probably tether and stabilize septal Z-ring. In support of this hypothesis, we showed that MtFtsX interacts with MtFtsZ. Further, bacterial two-hybrid interaction studies revealed that C-terminus of MtFtsZ is involved in interaction with MtFtsX, which is in agreement with the fact that several regulatory proteins interact FtsZ at its C-terminus in other bacterial systems. FtsW believed to bridge cell division and septal peptidoglycan synthesis in M. tuberculosis interacts with FtsZ at its C-terminus.[24] FtsA of Caulobacter crescentus,[25] ZipA and ZapD of E. coli,[26] and SepF [22] and EzrA of B. subtillis[21] interact with their cognate FtsZ at its C-terminus.

FtsE and FtsX exist as a complex in the membrane of E. coli cells.[27] Although FtsX does not interact directly with FtsZ,[9] it remains associated with the later through interaction with FtsE.[10] Contrary to this, MtFtsE in M. tuberculosis remains associated with MtFtsZ through interaction of the latter with MtFtsX (this study). These results imply that the FtsEX complex interacts with FtsZ either through FtsE or FtsX. Homologs of FtsA and ZipA being absent in M. tuberculosis[1],[28],[29] suggest that the direct interaction of MtFtsX with MtFtsZ might serve the purpose of stabilization of septal Z-ring in M. tuberculosis, which needs further investigation.

The role of FtsX in cell division has begun to unveil. Its interaction with MtFtsZ in M. tuberculosis and the regulation of cell wall hydrolases and their regulators in diverse bacterial genera, namely, M. tuberculosis, E. coli, and S. pneumonia,[13],[14],[30] suggest that the ubiquitous transmembrane component FtsX of the divisome links the cytokinetic ring with the PG hydrolysis during bacterial cell division.

Financial support and sponsorship

MAM was supported by the grant GRP-221-38 from Deanship of Scientific Research, King Khalid University, Abha, Saudi Arabia. PA was supported by the ICMR research Grant No. 63/72/2000-BMS and the work was carried out using infrastructural facilities provided by the DBT-supported Genomics Initiative on Microbial Pathogens–Structural Genomics Initiative in the Division of Biological Sciences, Indian Institute of Science, the DST-FIST and UGC-CAS at the Department of Microbiology and Cell Biology, Indian Institute of Science.

Conflicts of interest

There are no conflicts of interest.

Authors Contribution

MAM and PA designed the experiments. MAM wrote the manuscript and performed the experiments. PA reviewed the manuscript. RS generated a recombinant plasmid pBAD33-MtftsZ.



 
  References Top

1.
Hett EC, Rubin EJ. Bacterial growth and cell division: A mycobacterial perspective. Microbiol Mol Biol Rev 2008;72:126-56.  Back to cited text no. 1
    
2.
Bi EF, Lutkenhaus J. FtsZ ring structure associated with division in Escherichia coli. Nature 1991;354:161-4.  Back to cited text no. 2
    
3.
Hale CA, de Boer PA. Direct binding of FtsZ to ZipA, an essential component of the septal ring structure that mediates cell division in E. coli. Cell 1997;88:175-85.  Back to cited text no. 3
    
4.
Liu Z, Mukherjee A, Lutkenhaus J. Recruitment of ZipA to the division site by interaction with FtsZ. Mol Microbiol 1999;31:1853-61.  Back to cited text no. 4
    
5.
RayChaudhuri D. ZipA is a MAP-tau homolog and is essential for structural integrity of the cytokinetic FtsZ ring during bacterial cell division. EMBO J 1999;18:2372-83.  Back to cited text no. 5
    
6.
Ma X, Sun Q, Wang R, Singh G, Jonietz EL, Margolin W. Interactions between heterologous FtsA and FtsZ proteins at the FtsZ ring. J Bacteriol 1997;179:6788-97.  Back to cited text no. 6
    
7.
Pichoff S, Lutkenhaus J. Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA. Mol Microbiol 2005;55:1722-34.  Back to cited text no. 7
    
8.
Schmidt KL, Peterson ND, Kustusch RJ, Wissel MC, Graham B, Phillips GJ, et al. A predicted ABC transporter, FtsEX, is needed for cell division in Escherichia coli. J Bacteriol 2004;186:785-93.  Back to cited text no. 8
    
9.
Karimova G, Dautin N, Ladant D. Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysis. J Bacteriol 2005;187:2233-43.  Back to cited text no. 9
    
10.
Corbin BD, Wang Y, Beuria TK, Margolin W. Interaction between cell division proteins FtsE and FtsZ. J Bacteriol 2007;189:3026-35.  Back to cited text no. 10
    
11.
Reddy M. Role of FtsEX in cell division of Escherichia coli: Viability of FtsEX mutants is dependent on functional sufI or high osmotic strength. J Bacteriol 2007;189:98-108.  Back to cited text no. 11
    
12.
Samaluru H, SaiSree L, Reddy M. Role of sufI (FtsP) in cell division of Escherichia coli: Evidence for its involvement in stabilizing the assembly of the divisome. J Bacteriol 2007;189:8044-52.  Back to cited text no. 12
    
13.
Yang DC, Peters NT, Parzych KR, Uehara T, Markovski M, Bernhardt TG. An ATP-binding cassette transporter-like complex governs cell-wall hydrolysis at the bacterial cytokinetic ring. Proc Natl Acad Sci U S A 2011;108:E1052-60.  Back to cited text no. 13
    
14.
Mavrici D, Marakalala MJ, Holton JM, Prigozhin DM, Gee CL, Zhang YJ, et al. Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, ripC. Proc Natl Acad Sci U S A 2014;111:8037-42.  Back to cited text no. 14
    
15.
Mir MA, Rajeswari HS, Veeraraghavan U, Ajitkumar P. Molecular characterisation of ABC transporter type FtsE and FtsX proteins of Mycobacterium tuberculosis. Arch Microbiol 2006;185:147-58.  Back to cited text no. 15
    
16.
Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 1995;177:4121-30.  Back to cited text no. 16
    
17.
Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A 1998;95:5752-6.  Back to cited text no. 17
    
18.
Zhao LJ, Narayan O. A gene expression vector useful for protein purification and studies of protein-protein interaction. Gene 1993;137:345-6.  Back to cited text no. 18
    
19.
Haney SA, Glasfeld E, Hale C, Keeney D, He Z, de Boer P. Genetic analysis of the Escherichia coli FtsZ. ZipA interaction in the yeast two-hybrid system. Characterization of FtsZ residues essential for the interactions with ZipA and with FtsA. J Biol Chem 2001;276:11980-7.  Back to cited text no. 19
    
20.
Hale CA, Rhee AC, de Boer PA. ZipA-induced bundling of FtsZ polymers mediated by an interaction between C-terminal domains. J Bacteriol 2000;182:5153-66.  Back to cited text no. 20
    
21.
Singh JK, Makde RD, Kumar V, Panda D. A membrane protein, ezrA, regulates assembly dynamics of FtsZ by interacting with the C-terminal tail of FtsZ. Biochemistry 2007;46:11013-22.  Back to cited text no. 21
    
22.
Król E, van Kessel SP, van Bezouwen LS, Kumar N, Boekema EJ, Scheffers DJ. Bacillus subtilis sepF binds to the C-terminus of FtsZ. PLoS One 2012;7:e43293.  Back to cited text no. 22
    
23.
Galli E, Gerdes K. FtsZ-zapA-zapB interactome of Escherichia coli. J Bacteriol 2012;194:292-302.  Back to cited text no. 23
    
24.
Datta P, Dasgupta A, Bhakta S, Basu J. Interaction between FtsZ and FtsW of Mycobacterium tuberculosis. J Biol Chem 2002;277:24983-7.  Back to cited text no. 24
    
25.
Din N, Quardokus EM, Sackett MJ, Brun YV. Dominant C-terminal deletions of FtsZ that affect its ability to localize in caulobacter and its interaction with FtsA. Mol Microbiol 1998;27:1051-63.  Back to cited text no. 25
    
26.
Schumacher MA, Huang KH, Zeng W, Janakiraman A. Structure of the Z ring-associated protein, zapD, bound to the C-terminal domain of the tubulin-like protein, FtsZ, suggests mechanism of Z ring stabilization through FtsZ cross-linking. J Biol Chem 2017;292:3740-50.  Back to cited text no. 26
    
27.
de Leeuw E, Graham B, Phillips GJ, ten Hagen-Jongman CM, Oudega B, Luirink J. Molecular characterization of Escherichia coli FtsE and FtsX. Mol Microbiol 1999;31:983-93.  Back to cited text no. 27
    
28.
Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998;393:537-44.  Back to cited text no. 28
    
29.
Slayden RA, Knudson DL, Belisle JT. Identification of cell cycle regulators in Mycobacterium tuberculosis by inhibition of septum formation and global transcriptional analysis. Microbiology 2006;152:1789-97.  Back to cited text no. 29
    
30.
Sham LT, Barendt SM, Kopecky KE, Winkler ME. Essential pcsB putative peptidoglycan hydrolase interacts with the essential FtsXSpn cell division protein in Streptococcus pneumoniae D39. Proc Natl Acad Sci U S A 2011;108:E1061-9.  Back to cited text no. 30
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Methods
Results
Discussion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed25    
    Printed0    
    Emailed0    
    PDF Downloaded6    
    Comments [Add]    

Recommend this journal