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The recent Covid-19 pandemic has added a huge economic cost to the existing burden of antibiotic resistance and its rapid spread. This has led to increasing concern and efforts among the global health systems to control the spread of antibiotic resistance. Research on the mechanisms underlying antibiotic resistance and the creation of novel antibiotics has received a great deal of attention. Bacteria develop resistance to antibiotics through chromosomal mutations but also often acquire plasmids carrying antibiotic resistance through horizontal gene transfer. However, little is understood about how bacteria retain and maintain the plasmid carrying antibiotic resistance genes through generations. In this article, we describe different strategies by which bacteria maintain and partition plasmids into daughter cells. We present the article in two parts, with the first part covering the historical background of the discovery of plasmids and their role in antibiotic resistance. In the second part, we elaborate on our understanding of its partitioning into daughter cells and maintenance. A detailed understanding of these diverse mechanisms should help us develop novel antibiotics and innovative interventions to tackle the alarming rise of anti-microbial resistance. [ABSTRACT FROM AUTHOR]

Partitioning the replicated genetic material is a crucial process in the cell cycle program of any life form. In bacteria, many plasmids utilize cytoskeletal proteins that include ParM and TubZ, the ancestors of the eukaryotic actin and tubulin, respectively, to segregate the plasmids into the daughter cells. Another distinct class of cytoskeletal proteins, known as the Walker A type Cytoskeletal ATPases (WACA), is unique to Bacteria and Archaea. ParA, a WACA family protein, is involved in DNA partitioning and is more widespread. A centromere-like sequence parS , in the DNA is bound by ParB, an adaptor protein with CTPase activity to form the segregation complex. The ParA ATPase, interacts with the segregation complex and partitions the DNA into the daughter cells. Furthermore, the Walker A motif-containing ParA superfamily of proteins is associated with a diverse set of functions ranging from DNA segregation to cell division, cell polarity, chemotaxis cluster assembly, cellulose biosynthesis and carboxysome maintenance. Unifying principles underlying the varied range of cellular roles in which the ParA superfamily of proteins function are outlined. Here, we provide an overview of the recent findings on the structure and function of the ParB adaptor protein and review the current models and mechanisms by which the ParA family of proteins function in the partitioning of the replicated DNA into the newly born daughter cells. [ABSTRACT FROM AUTHOR]

The ftsH gene of Mycobacterium smegmatis SN2 (MsftsH) was cloned from two independent partial genomic DNA libraries and characterized, along with the identification of ephA and folE as the neighbouring upstream and downstream genes respectively. The genomic organization of the MsftsH locus was found to be identical to that of the Mycobacterium tuberculosis ftsH gene (MtftsH) and similar to that of other bacterial genera, but with divergence in the upstream region. The MsftsH gene is 2.3 kb in size and encodes the AAA (ATPases Associated with diverse cellular Activities) family [Zn.sup.2+]-metalloprotease FtsH (MsFtsH) of 85 kDa molecular mass. This was demonstrated from the expression of the full-length recombinant gene in Escherichia coli JM109 cells and from the identification of native MsFtsH in M. smegmatis SN2 cell lysates by Western blotting with anti-MtFtsH and anti-EcFtsH antibodies respectively. The recombinant and the native MsFtsH proteins were found localized to the membrane of E. coli and M. smegmatis cells respectively. Expression of MsFtsH protein in E. coli was toxic and resulted in growth arrest and filamentation of cells. The MsftsH gene did not complement lethality of a [delta]ftsH3:: kan mutation in E. coli, but when expressed in E. coli cells, it efficiently degraded conventional FtsH substrates, namely [[sigma].sup.32] protein and the protein translocase subunit SecY, of E. coli cells.

Bacterial FtsZ in the fission yeast Schizosaccharomyces pombe is expressed and it is found that FtsZ filaments assemble into cytoplasmic rings. It is concluded that FtsZ rings can assemble in the absence of all other bacterial cytokinetic proteins.

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. [ABSTRACT FROM AUTHOR]

Results demonstrate that the ATPase associated activity family of protein, FtsH acts on the cell-division-initiation protein FtsZ under in vitro conditions. Implication of this finding in the in vivo regulation of FtsZ is discussed.

Here we report the discovery of a bacterial DNA-segregating actinlike protein (BtParM) from Bacillus thuringiensis, which forms novel antiparallel, two-stranded, supercoiled, nonpolar helical filaments, as determined by electronmicroscopy. The BtParMfilament features of supercoiling and forming antiparallel double-strands are unique within the actin fold superfamily, and entirely different to the straight, double-stranded, polar helical filaments of all other known ParMs and of eukaryotic F-actin. The BtParM polymers show dynamic assembly and subsequent disassembly in the presence of ATP. BtParR, the DNA-BtParM linking protein, stimulated ATP hydrolysis/phosphate release by BtParM and paired two supercoiled BtParM filaments to form a cylinder, comprised of four strands with inner and outer diameters of 57 Å and 145 Å, respectively. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable features to the eukaryotic chromosomesegregating microtubule. [ABSTRACT FROM AUTHOR]

Introduction: Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. Methods: Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ32P-GTP synthesised by NDK from GDP and γ32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. Results: NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK’s NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK. Conclusion: Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented. [ABSTRACT FROM AUTHOR]

Successful cytokinesis requires proper assembly of the contractile actomyosin ring, its stable positioning on the cell surface and proper constriction. Over the years, many of the key molecular components and regulators of the assembly and positioning of the actomyosin ring have been elucidated. Here we show that cell geometry and mechanics play a crucial role in the stable positioning and uniform constriction of the contractile ring. Contractile rings that assemble in locally spherical regions of cells are unstable and slip towards the poles. By contrast, actomyosin rings that assemble on locally cylindrical portions of the cell under the same conditions do not slip, but uniformly constrict the cell surface. The stability of the rings and the dynamics of ring slippage can be described by a simple mechanical model. Using fluorescence imaging, we verify some of the quantitative predictions of the model. Our study reveals an intimate interplay between geometry and actomyosin dynamics, which are likely to apply in a variety of cellular contexts. [ABSTRACT FROM AUTHOR]

Summary: The identity of protease(s), which would degrade bacterial cell division protein FtsZ in vivo, remains unknown. However, we had earlier demonstrated that Escherichia coli metalloprotease FtsH degrades E. coli cell division protein FtsZ in an ATP- and Zn2+-dependent manner in vitro. In this study, we examined FtsH protease-mediated degradation of FtsZ in vitro in detail using seven different deletion mutants of FtsZ as the substrates, which lack different extents of specific regions at the N- or C-terminus. FtsH protease assay in vitro on these mutants revealed that FtsH could degrade all the seven deletion mutants irrespective of the deletions or the extent of deletions at the N- or C-terminus. These observations indicated that neither the N-terminus nor the C-terminus was required for the degradation of FtsZ, like already known in the case of the FtsH substrate σ 32 protein. The recombinant clones expressing full-length FtsZ protein and FtsZ deletion mutant proteins would be useful in investigating the possibility of FtsZ as a potential in vivo substrate for FtsH in ftsH-null cells carrying ftsH suppressor function and ectopically expressed FtsH protease. [Copyright &y& Elsevier]

Summary: Proteins structurally related to eukaryotic actins have recently been identified in several prokaryotic organisms . These actin-like proteins (MreB and ParM) and the deviant Walker A ATPase (SopA) play a key role in DNA segregation and assemble into polymers in vitro and in vivo . MreB also plays a role in cellular morphogenesis . Whereas the dynamic properties of eukaryotic actins have been extensively characterized, those of bacterial actins are only beginning to emerge . We have established the fission yeast Schizosaccharomyces pombe as a cellular model for the functional analysis of the Escherichia coli actin-related protein MreB. We show that MreB organizes into linear bundles that grow in a symmetrically bidirectional manner at 0.46 ± 0.03 μm/min, with new monomers and/or oligomers being added along the entire length of the bundle. Organization of linear arrays was dependent on the ATPase activity of MreB, and their alignment along the cellular long axis was achieved by sliding along the cortex of the cylindrical part of the cell. The cell ends appeared to provide a physical barrier for bundle elongation. These experiments provide new insights into the mechanism of assembly and organization of the bacterial actin cytoskeleton. [Copyright &y& Elsevier]

Summary: In eukaryotic cells microtubules and actin filaments help to generate the spatial organization of the cytoplasm that is required for polarity and shape. Recent work in fission yeast demonstrates that changing cell shape in turn reorganizes the cytoskeleton and cell polarization machinery. [Copyright &y& Elsevier]

Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines. [ABSTRACT FROM AUTHOR]

Reports on a study which reveals that a C-terminal deletion mutant of Mycobacterium tuberculosis FtsZ shows fast polymerization in vitro. Formation of a ring structure by the FtsZ proteins; Differences in the polymerization and GTPase activities in MtFtsZ; Earliest time at which long polymers of full-length MtFtsZ.

The peptidoglycan (PG) layer, a crucial component of the tripartite E.coli envelope, is required to maintain cellular integrity, protecting the cells from mechanical stress resulting from intracellular turgor pressure. Thus, coordinating synthesis and hydrolysis of PG during cell division (septal PG) is crucial for bacteria. The FtsEX complex directs septal PG hydrolysis through the activation of amidases; however, the mechanism and regulation of septal PG synthesis are unclear. In addition, how septal PG synthesis and hydrolysis are coordinated has remained unclear. Here, we have shown that overexpression of FtsE leads to a mid-cell bulging phenotype in E.coli , which is different from the filamentous phenotype observed during overexpression of other cell division proteins. Silencing of the common PG synthesis genes murA and murB reduced bulging, confirming that this phenotype is due to excess PG synthesis. We further demonstrated that septal PG synthesis is independent of FtsE ATPase activity and FtsX. These observations and previous results suggest that FtsEX plays a role during septal PG hydrolysis, whereas FtsE alone coordinates septal PG synthesis. Overall, our study findings support a model in which FtsE plays a role in coordinating septal PG synthesis with bacterial cell division. IMPORTANCE The peptidoglycan (PG) layer is an essential component of the E.coli envelope that is required to maintain cellular shape and integrity. Thus, coordinating PG synthesis and hydrolysis at the mid-cell (septal PG) is crucial during bacterial division. The FtsEX complex directs septal PG hydrolysis through the activation of amidases; however, its role in regulation of septal PG synthesis is unclear. Here, we demonstrate that overexpression of FtsE in E.coli leads to a mid-cell bulging phenotype due to excess PG synthesis. This phenotype was reduced upon silencing of common PG synthesis genes murA and murB . We further demonstrated that septal PG synthesis is independent of FtsE ATPase activity and FtsX. These observations suggest that the FtsEX complex plays a role during septal PG hydrolysis, whereas FtsE alone coordinates septal PG synthesis. Our study indicates that FtsE plays a role in coordinating septal PG synthesis with bacterial cell division., Competing Interests: The authors declare no conflict of interest.

MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod-shaped bacteria. Despite belonging to the actin family, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we provide insights into the effect of nucleotide state on membrane binding of Spiroplasma citri MreB5 (ScMreB5). Filaments of ScMreB5WT and an ATPase-deficient mutant, ScMreB5E134A, assemble independently of the nucleotide state. However, capture of the filament dynamics revealed that efficient filament formation and organization through lateral interactions are affected in ScMreB5E134A. Hence, the catalytic glutamate functions as a switch, (a) by sensing the ATP-bound state for filament assembly and (b) by assisting hydrolysis, thereby potentially triggering disassembly, as observed in other actins. Glu134 mutation and the bound nucleotide exhibit an allosteric effect on membrane binding, as observed from the differential liposome binding. We suggest that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis among actins has been repurposed in MreBs for modulating filament organization on the membrane., (© 2022 Pande et al.)

Elastic forces within DNA drive the segregation of chromosomes in bacteria., (Copyright © 2014, Srinivasan and Balasubramanian.)

We examined whether C-terminal residues of soluble recombinant FtsZ of Mycobacterium tuberculosis (MtFtsZ) have any role in MtFtsZ polymerization in vitro. MtFtsZ-deltaC1, which lacks C-terminal extreme Arg residue (underlined in the C-terminal extreme stretch of 13 residues, DDDDVDVPPFMRR), but retaining the penultimate Arg residue (DDDDVDVPPFMR), polymerizes like full-length MtFtsZ in vitro. However, MtFtsZ-deltaC2 that lacks both the Arg residues at the C-terminus (DDDDVDVPPFM), neither polymerizes at pH 6.5 nor forms even single- or double-stranded filaments at pH 7.7 in the presence of 10 mM CaCl(2). Neither replacement of the penultimate Arg residue, in the C-terminal Arg deletion mutant DDDDVDVPPFMR, with Lys or His or Ala or Asp (DDDDVDVPPFMK/H/A/D) enabled polymerization. Although MtFtsZ-deltaC2 showed secondary and tertiary structural changes, which might have affected polymerization, GTPase activity of MtFtsZ-deltaC2 was comparable to that of MtFtsZ. These data suggest that MtFtsZ requires an Arg residue as the extreme C-terminal residue for polymerization in vitro. The polypeptide segment containing C-terminal 67 residues, whose coordinates were absent from MtFtsZ crystal structure, was modeled on tubulin and MtFtsZ dimers. Possibilities for the influence of the C-terminal Arg residues on the stability of the dimer and thereby on MtFtsZ polymerization have been discussed.