Abstract
Stable maintenance and partitioning of the ‘Fertility’ plasmid or the F plasmid in its host Escherichia coli require the function of a ParA superfamily of proteins known as SopA. The mechanism by which SopA mediates plasmid segregation is well studied. SopA is a nucleoid-binding protein and binds DNA in an ATP-dependent but sequence non-specific manner. ATP hydrolysis stimulated by the binding of the SopBC complex mediates the release of SopA from the nucleoid. Cycles of ATP-binding and hydrolysis generate an ATPase gradient that moves the plasmid through a chemophoresis force. Nucleoid binding of SopA thus assumes a central role in its plasmid-partitioning function. However, earlier work also suggests that the F plasmid can be partitioned into anucleate cells, thus implicating nucleoid independent partitioning. Interestingly, SopA is also reported to be associated with the inner membrane of the bacteria. Here, we report the identification of a possible membrane-targeting sequence, a predicted amphipathic helix, at the C-terminus of SopA. Molecular dynamics simulations indicate that the predicted amphipathic helical motif of SopA has weak affinity for membranes. Moreover, we experimentally show that SopA can associate with bacterial membranes, is detectable in the membrane fractions of bacterial lysates, and is sensitive to the membrane potential. Further, unlike the wild-type SopA, a deletion of the C-terminal 29 amino acids results in the loss of F plasmids from bacterial cells.
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References
Abraham MJ, Murtola T, Schulz R et al (2015) Gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2:19–25. https://doi.org/10.1016/j.softx.2015.06.001
Ah-Seng Y, Rech J, Lane D, Bouet JY (2013) Defining the role of ATP hydrolysis in mitotic segregation of bacterial plasmids. PLoS Genet 9:e1003956-14. https://doi.org/10.1371/journal.pgen.1003956
Baxter JC, Funnell BE (2014) Plasmid partition mechanisms. Microbiol Spectr. https://doi.org/10.1128/microbiolspec.plas-0023-2014
Baylon JL, Vermaas JV, Muller MP et al (2016) Atomic-level description of protein-lipid interactions using an accelerated membrane model. Biochim Biophys Acta Biomembr 1858:1573–1583. https://doi.org/10.1016/j.bbamem.2016.02.027
Bouet J-Y (1999) P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities. EMBO J 18:1415–1424. https://doi.org/10.1093/emboj/18.5.1415
Bouet JY, Ah-Seng Y, Benmeradi N, Lane D (2007) Polymerization of SopA partition ATPase: regulation by DNA binding and SopB. Mol Microbiol 63:468–481. https://doi.org/10.1111/j.1365-2958.2006.05537.x
Castaing JP, Bouet JY, Lane D (2008) F plasmid partition depends on interaction of SopA with non-specific DNA. Mol Microbiol 70:1000–1011. https://doi.org/10.1111/j.1365-2958.2008.06465.x
Celler K, Koning RI, Koster AJ, van Wezel GP (2013) Multidimensional view of the bacterial cytoskeleton. J Bacteriol 195:1627–1636
Combet C, Blanchet C, Geourjon C, Deléage G (2000) NPS@: network protein sequence analysis. Trends Biochem Sci 25:147–150
Crooke E (2001) Escherichia coli DnaA protein—phospholipid interactions: in vitro and in vivo. Biochimie 83:19–23. https://doi.org/10.1016/S0300-9084(00)01224-4
Dunham TD, Xu W, Funnell BE, Schumacher MA (2009) Structural basis for ADP-mediated transcriptional regulation by P1 and P7 ParA. EMBO J 28:1792–1802. https://doi.org/10.1038/emboj.2009.120
Ebersbach G, Gerdes K (2005) Plasmid segregation mechanisms. Annu Rev Genet 39:453–479
Eswar N, Webb B, Marti-Renom MA et al (2007) Comparative protein structure modeling using MODELLER. Curr Protoc Protein Sci 50:2.9.1–2.9.31. https://doi.org/10.1002/0471140864.ps0209s50
Ezaki B, Ogura T, Niki H, Hiraga S (1991) Partitioning of a mini-F plasmid into anucleate cells of the mukB null mutant. J Bacteriol 173:6643–6646. https://doi.org/10.1128/jb.173.20.6643-6646.1991
Fink G, Szewczak-Harris A, Löwe J (2016) SnapShot: the bacterial cytoskeleton. Cell 166:522–522
Fung E, Bouet JY, Funnell BE (2001) Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis. EMBO J. https://doi.org/10.1093/emboj/20.17.4901
Funnell BE, Gagnier L (1995) Partition of P1 plasmids in Escherichia coli mukB chromosomal partition mutants. J Bacteriol 177:2381–2386. https://doi.org/10.1128/jb.177.9.2381-2386.1995
Garner J, Crooke E (1996) Membrane regulation of the chromosomal replication activity of E.coli DnaA requires a discrete site on the protein. EMBO J 15:2313–2321. https://doi.org/10.1002/j.1460-2075.1996.tb00585.x
Gautier R, Douguet D, Antonny B, Drin G (2008) HELIQUEST: a web server to screen sequences with specific α-helical properties. Bioinformatics 24:2101–2102. https://doi.org/10.1093/bioinformatics/btn392
Gerdes K, Moller-Jensen J, Jensen RB (2000) Plasmid and chromosome partitioning: surprises from phylogeny. Mol Microbiol 37:455–466. https://doi.org/10.1046/j.1365-2958.2000.01975.x
Gitai Z (2006) Plasmid segregation: a new class of cytoskeletal proteins emerges. Curr Biol 16:R133–R136. https://doi.org/10.1016/j.cub.2006.02.007
Hatano T, Yamaichi Y, Niki H (2007) Oscillating focus of SopA associated with filamentous structure guides partitioning of F plasmid. Mol Microbiol 64:1198–1213. https://doi.org/10.1111/j.1365-2958.2007.05728.x
Hu Z, Lutkenhaus J (2003) A conserved sequence at the C-terminus of MinD is required for binding to the membrane and targeting MinC to the septum. Mol Microbiol 47:345–355. https://doi.org/10.1046/j.1365-2958.2003.03321.x
Hu L, Vecchiarelli AG, Mizuuchi K et al (2015) Directed and persistent movement arises from mechanochemistry of the ParA/ParB system. Proc Natl Acad Sci USA 112:E7055–E7064. https://doi.org/10.1073/pnas.1505147112
Hu L, Vecchiarelli AG, Mizuuchi K et al (2017a) Brownian ratchet mechanism for faithful segregation of low-copy-number plasmids. Biophys J 112:1489–1502. https://doi.org/10.1016/j.bpj.2017.02.039
Hu L, Vecchiarelli AG, Mizuuchi K et al (2017b) Brownian ratchet mechanisms of ParA-mediated partitioning. Plasmid 92:12–16. https://doi.org/10.1016/j.plasmid.2017.05.002
Huang J, Rauscher S, Nawrocki G et al (2016) CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat Methods 14:71–73. https://doi.org/10.1038/nmeth.4067
Hwang LC, Vecchiarelli AG, Han YW et al (2013) ParA-mediated plasmid partition driven by protein pattern self-organization. EMBO J 32:1238–1249. https://doi.org/10.1038/emboj.2013.34
Ietswaart R, Szardenings F, Gerdes K, Howard M (2014) Competing ParA structures space bacterial plasmids equally over the nucleoid. PLoS Comput Biol. https://doi.org/10.1371/journal.pcbi.1004009
Inagawa T, Kato J, Niki H et al (2001) Defective plasmid partition in ftsH mutants of Escherichia coli. Mol Genet Genom 265:755–762. https://doi.org/10.1007/s004380100488
Jacob F, Brenner S, Cuzin F (1963) On the regulation of DNA replication in bacteria. Cold Spring Harb Symp Quant Biol 28:329–348. https://doi.org/10.1101/sqb.1963.028.01.048
Karczmarek A, Baselga RMA, Alexeeva S et al (2007) DNA and origin region segregation are not affected by the transition from rod to sphere after inhibition of Escherichia coli MreB by A22. Mol Microbiol 65:51–63. https://doi.org/10.1111/j.1365-2958.2007.05777.x
Katsu T, Tsuchiya T, Fujita Y (1984) Dissipation of membrane potential of Escherichia coli cells induced by macromolecular polylysine. Biochem Biophys Res Commun 122:401–406
Kim S-K, Wang JC (1998) Localization of F plasmid SopB protein to positions near the poles of Escherichia coli cells. Proc Natl Acad Sci USA 95:1523–1527. https://doi.org/10.1073/pnas.95.4.1523
Klauda JB, Venable RM, Freites JA et al (2010) Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J Phys Chem B 114:7830–7843. https://doi.org/10.1021/jp101759q
Larsen RA, Cusumano C, Fujioka A et al (2007) Treadmilling of a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis. Genes Dev 21:1340–1352. https://doi.org/10.1101/gad.1546107
Le Gall A, Cattoni DI, Guilhas B et al (2016) Bacterial partition complexes segregate within the volume of the nucleoid. Nat Commun 7:1–10. https://doi.org/10.1038/ncomms12107
Leibowitz PJ, Schaechter M (1975) The attachment of the bacterial chromosome to the cell membrane. Int Rev Cytol 41:1–28. https://doi.org/10.1016/S0074-7696(08)60964-X
Lenarcic R, Halbedel S, Visser L et al (2009) Localisation of DivIVA by targeting to negatively curved membranes. EMBO J 28:2272–2282. https://doi.org/10.1038/emboj.2009.129
Libante V, Thion L, Lane D (2001) Role of the ATP-binding site of SopA protein in partition of the F plasmid. J Mol Biol 314:387–399. https://doi.org/10.1006/jmbi.2001.5158
Lim GE, Derman AI, Pogliano J (2005) Bacterial DNA segregation by dynamic SopA polymers. Proc Natl Acad Sci USA 102:17658–17663. https://doi.org/10.1073/pnas.0507222102
Lim HC, Surovtsev IV, Beltran BG et al (2014) Evidence for a DNA-relay mechanism in ParABS-mediated chromosome segregation. Elife 2014:1–32. https://doi.org/10.7554/eLife.02758
Lin Z, Mallavia LP (1998) Membrane association of active plasmid partitioning protein A in Escherichia coli. J Biol Chem 273:11302–11312. https://doi.org/10.1074/jbc.273.18.11302
Lutkenhaus J (2012) The ParA/MinD family puts things in their place. Trends Microbiol 20:411–418
Makise M, Mima S, Tsuchiya T, Mizushima T (2001) Molecular mechanism for functional interaction between DnaA protein and acidic phospholipids. Identification of important amino acids. J Biol Chem 276:7450–7456. https://doi.org/10.1074/jbc.M009643200
Milo R, Jorgensen P, Moran U et al (2009) BioNumbers the database of key numbers in molecular and cell biology. Nucleic Acids Res. https://doi.org/10.1093/nar/gkp889
Mól AR, Castro MS, Fontes W (2018) NetWheels: a web application to create high quality peptide helical wheel and net projections. bioRxiv. https://doi.org/10.1101/416347
Ogura T, Hiraga S (1983) Partition mechanism of F plasmid: two plasmid gene-encoded products and a cis-acting region are involved in partition. Cell 32:351–360
Pichoff S, Lutkenhaus J (2005) Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA. Mol Microbiol 55:1722–1734. https://doi.org/10.1111/j.1365-2958.2005.04522.x
Qi Y, Cheng X, Lee J et al (2015) CHARMM-GUI HMMM builder for membrane simulations with the highly mobile membrane-mimetic model. Biophys J 109:2012–2022. https://doi.org/10.1016/j.bpj.2015.10.008
Ravin N, Lane D (1999) Partition of the linear plasmid N15: interactions of N15 partition functions with the sop locus of the F plasmid. J Bacteriol 181:6898–6906. https://doi.org/10.1128/jb.181.22.6898-6906.1999
Ringgaard S, Schirner K, Davis B, Waldor M (2011) A family of ParA-like ATPases promotes cell pole maturation by facilitating polar localization of chemotaxis proteins. Genes Dev 25(14):1544–1555. https://doi.org/10.1101/gad.2061811
Roberts MAJ, Wadhams GH, Hadfield KA et al (2012) ParA-like protein uses nonspecific chromosomal DNA binding to partition protein complexes. Proc Natl Acad Sci USA 109:6698–6703. https://doi.org/10.1073/pnas.1114000109
Salje J, Gayathri P, Löwe J (2010) The ParMRC system: molecular mechanisms of plasmid segregation by actin-like filaments. Nat Rev Microbiol 8:683–692
Salje J, van den Ent F, de Boer P, Löwe J (2011) Direct membrane binding by bacterial actin MreB. Mol Cell 43:478–487. https://doi.org/10.1016/j.molcel.2011.07.008
Sapay N, Guermeur Y, Deléage G (2006) Prediction of amphipathic in-plane membrane anchors in monotopic proteins using a SVM classifier. BMC Bioinform. https://doi.org/10.1186/1471-2105-7-255
Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
Schumacher MA, Ye Q, Barge MT et al (2012) Structural mechanism of ATP-induced polymerization of the partition factor ParF: implications for DNA segregation. J Biol Chem 287:26146–26154. https://doi.org/10.1074/jbc.M112.373696
Srinivasan R, Mishra M, Wu L et al (2008) The bacterial cell division protein FtsZ assembles into cytoplasmic rings in fission yeast. Genes Dev 22:1741–1746. https://doi.org/10.1101/gad.1660908
Strahl H, Hamoen LW (2010) Membrane potential is important for bacterial cell division. Proc Natl Acad Sci USA 107:12281–12286. https://doi.org/10.1073/pnas.1005485107
Surovtsev IV, Campos M, Jacobs-Wagner C (2016) DNA-relay mechanism is sufficient to explain ParA-dependent intracellular transport and patterning of single and multiple cargos. Proc Natl Acad Sci USA 113:E7268–E7276. https://doi.org/10.1073/pnas.1616118113
Szeto TH, Rowland SL, Rothfield LI, King GF (2002) Membrane localization of MinD is mediated by a C-terminal motif that is conserved across eubacteria, archaea, and chloroplasts. Proc Natl Acad Sci USA 99:15693–15698. https://doi.org/10.1073/pnas.232590599
Szeto TH, Rowland SL, Habrukowich CL, King GF (2003) The MinD membrane targeting sequence is a transplantable lipid-binding helix. J Biol Chem 278:40050–40056. https://doi.org/10.1074/jbc.M306876200
van den Ent F, Møller-Jensen J, Amos LA et al (2002) F-actin-like filaments formed by plasmid segregation protein ParM. EMBO J 21:6935–6943
Vecchiarelli AG, Han YW, Tan X et al (2010) ATP control of dynamic P1 ParA-DNA interactions: a key role for the nucleoid in plasmid partition. Mol Microbiol 78:78–91. https://doi.org/10.1111/j.1365-2958.2010.07314.x
Vecchiarelli AG, Hwang LC, Mizuuchi K (2013) Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism. Proc Natl Acad Sci USA 110:1390–1397. https://doi.org/10.1073/pnas.1302745110
Vecchiarelli A, Mizuuchi K, Funnell B (2012) Surfing biological surfaces: exploiting the nucleoid for partition and transport in bacteria. Mol Microbiol 86(3):513–523. https://doi.org/10.1111/mmi.12017
Vecchiarelli AG, Neuman KC, Mizuuchi K (2014a) A propagating ATPase gradient drives transport of surface-confined cellular cargo. Proc Natl Acad Sci USA 111:4880–4885. https://doi.org/10.1073/pnas.1401025111
Vecchiarelli AG, Seol Y, Neuman KC, Mizuuchi K (2014b) A moving ParA gradient on the nucleoid directs subcellular cargo transport via a chemophoresis force. Bioarchitecture 4:154–159. https://doi.org/10.4161/19490992.2014.987581
Vermaas JV, Tajkhorshid E (2014) A microscopic view of phospholipid insertion into biological membranes. J Phys Chem B 118:1754–1764. https://doi.org/10.1021/jp409854w
Watanabe E, Inamoto S, Lee MH et al (1989) Purification and characterization of the sopB gene product which is responsible for stable maintenance of mini-F plasmid. MGG Mol Gen Genet 218:431–436. https://doi.org/10.1007/BF00332406
Weiss DS, Chen JC, Ghigo JM et al (1999) Localization of FtsI (PBP3) to the septal ring requires its membrane anchor, the Z ring, FtsA, FtsQ, and FtsL. J Bacteriol 181:508–520. https://doi.org/10.1128/jb.181.2.508-520.1999
Wu LJ, Ishikawa S, Kawai Y et al (2009) Noc protein binds to specific DNA sequences to coordinate cell division with chromosome segregation. EMBO J 28:1940–1952. https://doi.org/10.1038/emboj.2009.144
Zhang H, Schumacher MA (2017) Structures of partition protein para with nonspecific dna and parb effector reveal molecular insights into principles governing walker-box dna segregation. Genes Dev 31:481–492. https://doi.org/10.1101/gad.296319.117
Acknowledgements
The authors thank the members of the members of RS and AS labs for their technical support and helpful suggestions. DM, SP, and NM thank DAE for the financial support and fellowship received. AS thanks NISER-Bhubaneswar for allowing SP to work at IISc-Bangalore. The work was supported by research grants to RS from SERB (EMR/2016/000487), DBT (No. BT/PR15183/BRB/10/1443/2015), and intramural core funding from DAE. A.S. thanks the Indian Institute of Science and the Ministry of Human Resource Development of India for the startup grant and the Department of Science and Technology of India for the early career grant (ECR/2016/001702). This research was also supported by the Department of Biotechnology, Government of India, in the form of IISc-DBT partnership program. Support from FIST program sponsored by the Department of Science and Technology and UGC, Centre for Advanced Studies and Ministry of Human Resource Development, India, is gratefully acknowledged by the authors.. The authors thank Ms. Kirtika Jha for technical help, Drs. Jean-Yves Bouet (LMGM, Toulouse, France, Joe Lutkenhaus (Kansas), Mohan Chandra Joshi (JMI, New Delhi), Ajitkumar (IISc, Bengaluru), Manjula Reddy (CCMB, Hyderabad), Harinarayanan (CDFD, Hyderabad), and Tushar Beuria (ILS, Bhubaneswar) for the generous gifts of strains and plasmid. pDSW210 was originally a gift from Dr. David Weiss. The authors thank Dr. Tirumala K. Chowdary for sharing computational resources and critical reading of the manuscript.
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Mishra, D., Pahujani, S., Mitra, N. et al. Identification of a Potential Membrane-Targeting Sequence in the C-Terminus of the F Plasmid Segregation Protein SopA. J Membrane Biol 254, 243–257 (2021). https://doi.org/10.1007/s00232-020-00157-8
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DOI: https://doi.org/10.1007/s00232-020-00157-8