Bacterial Cell Division Research Articles

Filamentous Thermosensitive Mutant Z: An Appealing Target for Emerging Pathogens and a Trek on Its Natural Inhibitors.

Simple SummaryAntimicrobial resistance (AMR) is a pressing issue worldwide that must be addressed swiftly. It is driven by spontaneous evolution, bacterial mutation, and the dissemination of resistant genes via horizontal gene transfer. Researchers are working on many novel targets, which can become a pathway to inhibit harmful bacteria. Filamentous Thermosensitive mutant-Z (Fts-Z) is one such bacterial target that has gained popularity amongst scientists due to its conserved nature in bacteria and absence in eukaryotes. The aim of this work was to review the Fts-Z mechanism of action along with current studies on natural inhibitors for Fts-Z.Antibiotic resistance is a major emerging issue in the health care sector, as highlighted by the WHO. Filamentous Thermosensitive mutant Z (Fts-Z) is gaining significant attention in the scientific community as a potential anti-bacterial target for fighting antibiotic resistance among several pathogenic bacteria. The Fts-Z plays a key role in bacterial cell division by allowing Z ring formation. Several in vitro and in silico experiments have demonstrated that inhibition of Fts-Z can lead to filamentous growth of the cells, and finally, cell death occurs. Many natural compounds that have successfully inhibited Fts-Z are also studied. This review article intended to highlight the structural–functional aspect of Fts-Z that leads to Z-ring formation and its contribution to the biochemistry and physiology of cells. The current trend of natural inhibitors of Fts-Z protein is also covered.

Elhik’s Metamathematics and the robot Philia: Internet Communication Protocol Modelling of Observeds according to Observers

Like all robot and IoT oriented mathematical studies this study is also transdisciplinary however most of the study remains in the field of mathematics and philosophy of mathematics due to the aim of mathematical modelling of the observations: a) protein synthesis by the information encoded in DNA and b) communication protocols of hacking software with encryption of IP of computers c) the process of DNA duplication to offspring formation in asexual and sexual reproduction. The ultimate mathematical aim of this study is providing a metamathematical approach for different types of mathematics to be utilized in information sciences by forced or free choice of artificial intelligence driven technological beings and computational systems. This study is biomathematically inspired from protein synthesis, cellular reproduction, DNA duplication, bacterial simple cell division, bacterial sex and zygote formation in human and mammalian fertilization; and all are modelled as moving pieces of quantized information. The ultimate robotic aim is finding an algorithm for data off-springs, thus the formation of the data-offspring in the form of living being off-springs’ DNA is chosen to be mathematically described. For this purpose, a new mathematics with new mathematical operations and a definition to mathematics are postulated, together with twelve postulations about the real-life observations. The introduced Aydan operation is synthesizing fuzzy logic based mathematical existence possibilities into abstract formal logic based abstract mathematical categories to be analyzed with additional color codes for each object, whereas deAydan operation is doing the opposite by analyzing a given synthesis. There is a coexistence of mathematical objects and operators in the biological cells. The co-existence of objects and mathematical operations in a given mathematics M, are axiomatically chosen to be a satisfiability rule for M to be accepted as mathematics in the form of a universal set isolated from other universal sets. Aydan function can be used for hacking other AI by deviating its results by merging them with forced formal logic based analytic input by another hacking software, and deAydan function can be used as anti-hack in the opposite direction. The metamathematical existence degree of a given mathematics is shown to be between zero and infinite dimensions. ELHIK is an abbreviation of the words El Harizmi and Immanuel Kant.

FtsZ filament structures in different nucleotide states reveal the mechanism of assembly dynamics.

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogs and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP γ-phosphate and Mg2+ at the subunit interface, a K+ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signaling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

Inhibition of Filamentous Thermosensitive Mutant-Z Protein in Bacillus subtilis by Cyanobacterial Bioactive Compounds.

Antibiotic resistance is one of the major growing concerns for public health. Conventional antibiotics act on a few predefined targets and, with time, several bacteria have developed resistance against a large number of antibiotics. The WHO has suggested that antibiotic resistance is at a crisis stage and identification of new antibiotics and targets could be the only approach to bridge the gap. Filamentous Temperature Sensitive-Mutant Z (Fts-Z) is one of the promising and less explored antibiotic targets. It is a highly conserved protein and plays a key role in bacterial cell division by introducing a cytokinetic Z-ring formation. In the present article, the potential of over 165 cyanobacterial compounds with reported antibiotic activity against the catalytic core domain in the Fts-Z protein of the Bacillus subtilis was studied. The identified cyanobacterial compounds were screened using the GLIDE module of Maestro v-2019-2 followed by 100-ns molecular dynamics (MD) simulation. Ranking of the potential compound was performed using dock score and MMGBSA based free energy. The study reported that the docking score of aphanorphine (−6.010 Kcalmol−1) and alpha-dimorphecolic acid (ADMA) (−6.574 Kcalmol−1) showed significant role with respect to the reported potential inhibitor PC190723 (−4.135 Kcalmol−1). A 100 ns MD simulation infers that Fts-Z ADMA complex has a stable conformation throughout the progress of the simulation. Both the compounds, i.e., ADMA and Aphanorphine, were further considered for In-vitro validation by performing anti-bacterial studies against B. subtilis by agar well diffusion method. The results obtained through In-vitro studies confirm that ADMA, a small molecule of cyanobacterial origin, is a potential compound with an antibacterial activity that may act by inhibiting the novel target Fts-Z and could be a great drug candidate for antibiotic development.

Expanded profiling of β-lactam selectivity for penicillin-binding proteins in Streptococcus pneumoniae D39.

Penicillin-binding proteins (PBPs) are integral to bacterial cell division as they mediate the final steps of cell wall maturation. Selective fluorescent probes are useful for understanding the role of individual PBPs, including their localization and activity during growth and division of bacteria. For the development of new selective probes for PBP imaging, several β-lactam antibiotics were screened, as they are known to covalently bind PBP in vivo. The PBP inhibition profiles of 16 commercially available β-lactam antibiotics were evaluated in an unencapsulated derivative of the D39 strain of Streptococcus pneumoniae, IU1945. These β-lactams have not previously been characterized for their PBP inhibition profiles in S.pneumoniae and these data augment those obtained from a library of 20 compounds that we previously reported. We investigated seven penicillins, three carbapenems, and six cephalosporins. Most of these β-lactams were found to be co-selective for PBP2x and PBP3, as was noted in our previous studies. Six out of 16 antibiotics were selective for PBP3 and one molecule was co-selective for PBP1a and PBP3. Overall, this work expands the chemical space available for development of future β-lactam-based probes for specific pneumococcal PBP labeling and these methods can be used for the development of probes for PBP labelling in other bacterial species.

Effect of Thioflavin T on the Elongation Rate of Bacteria.

The growing field of bacterial electrophysiology examines the relationship between bacterial membrane potential and cell division, growth, sporulation, and biofilm formation. These experiments require Nernstian fluorescent dyes to monitor membrane potential. Our research uses single cell imaging to determine if a common fluorescent dye, Thioflavin T (ThT), affects the growth of bacteria. We use a combination of standard growth curve measurements and single cell imaging, both brightfield and fluorescence microscopy, to monitor the growth of Bacillus subtilis and Escherichia coli as a function of ThT concentration. Increased membrane potential (hyperpolarization) leads to increased intracellular accumulation of ThT: High fluorescence intensity is an indicator of hyperpolarization. Blue light is used to hyperpolarize a subpopulation of cells to monitor cellular elongation in response to increased cellular internalization of ThT. Single cell imaging shows that the elongation rates of B. subtilis and E. coli are decreased when these cells are incubated with ThT. At micromolar concentrations of ThT, this effect may be masked in standard growth curves, but is visible with single cell measurements on agarose pads. The increased cellular accumulation of ThT is a standard measure of hyperpolarization in bacterial electrophysiology. Growth curves, a bulk measurement, are typically used to determine suitable concentrations of ThT for use in experiments. Single cell measurements show that cells incubated with ThT have decreased elongation rates. This creates a potential experimental artifact that could lead to misinterpretation of data. Hyperpolarized cells internalize more ThT. This increased intracellular concentration of ThT, rather than the change in membrane potential, could lead to decreased growth. These experiments point toward the importance of single cell measurements to detect subtle changes in cell growth. We hope this research will be useful for other researchers in their choice of dye for the detection of membrane potential.

Impacts of local and global sequence patterns within the disordered tail on assembly and functions of B. subtilis FtsZ

FtsZ is one of the main drivers of bacterial cell division. In the presence of GTP, FtsZ assembles into treadmilling polymers that serve as a platform for assembly of the division machinery. In B. subtilis, the C-terminal tail of FtsZ is encompasses a 50 aa linker (CTL) that is hypervariable across orthologs and a conserved C-terminal peptide (CTP). Deletion of the intrinsically disordered C-terminal tail of FtsZ compromises cell division and leads to a deleterious phenotype in B. subtilis. Biochemical experiments showed that the CTL and the CTP enable autoregulation of FtsZ polymerization and GTPase activity.

The Planctomycetia: an overview of the currently largest class within the phylum Planctomycetes.

The phylum Planctomycetes comprises bacteria with uncommon features among prokaryotes, such as cell division by budding, absence of the bacterial tubulin-homolog cell division protein FtsZ and complex cell plans with invaginations of the cytoplasmic membrane. Although planctomycetes are ubiquitous, the number of described species and isolated strains available as axenic cultures is still low compared to the diversity observed in metagenomes or environmental studies. An increasing interest in planctomycetes is reflected by the recent description of a large number of new species and their increasing accessibility in terms of pure cultures. In this review, data from all taxonomically described species belonging to Planctomycetia, the class with the currently highest number of characterized members within the phylum Planctomycetes, is summarized. Phylogeny, morphology, physiology, ecology and genomic traits of its members are discussed. This comprehensive overview will help to acknowledge several aspects of the biology of these fascinating bacteria.

Cell scientist to watch – Martin Loose

ABSTRACT Martin Loose studied chemistry at the University of Heidelberg, Germany. He then joined Petra Schwille's group at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, where he obtained his PhD degree in 2010 for work on self-organization and pattern formation in the bacterial Min protein system. He then moved to Tim Mitchison's lab at Harvard Medical School, Boston, USA for his postdoc, funded by Human Frontier Science Program (HSFP) and European Molecular Biology Organization (EMBO) long-term fellowships; there, he discovered that the bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns. Martin established his independent research group at the Institute of Science and Technology (IST) Austria in 2015, supported by an European Research Council (ERC) starting grant and HFSP Young Investigator Grant. His lab studies the self-organization of bacterial cell division and small GTPase networks.

Identification of the potential active site of the septal peptidoglycan polymerase FtsW.

SEDS (Shape, Elongation, Division and Sporulation) proteins are widely conserved peptidoglycan (PG) glycosyltransferases that form complexes with class B penicillin-binding proteins (bPBPs, with transpeptidase activity) to synthesize PG during bacterial cell growth and division. Because of their crucial roles in bacterial morphogenesis, SEDS proteins are one of the most promising targets for the development of new antibiotics. However, how SEDS proteins recognize their substrate lipid II, the building block of the PG layer, and polymerize it into glycan strands is still not clear. In this study, we isolated and characterized dominant-negative alleles of FtsW, a SEDS protein critical for septal PG synthesis during bacterial cytokinesis. Interestingly, most of the dominant-negative FtsW mutations reside in extracellular loops that are highly conserved in the SEDS family. Moreover, these mutations are scattered around a central cavity in a modeled FtsW structure, which has been proposed to be the active site of SEDS proteins. Consistent with this, we found that these mutations blocked septal PG synthesis but did not affect FtsW localization to the division site, interaction with its partners nor its substrate lipid II. Taken together, these results suggest that the residues corresponding to the dominant-negative mutations likely constitute the active site of FtsW, which may aid in the design of FtsW inhibitors.

Localization, Assembly, and Activation of the Escherichia coli Cell Division Machinery.

Decades of research, much of it in Escherichia coli, have yielded a wealth of insight into bacterial cell division. Here, we provide an overview of the E. coli division machinery with an emphasis on recent findings. We begin with a short historical perspective into the discovery of FtsZ, the tubulin homolog that is essential for division in bacteria and archaea. We then discuss assembly of the divisome, an FtsZ-dependent multiprotein platform, at the midcell septal site. Not simply a scaffold, the dynamic properties of polymeric FtsZ ensure the efficient and uniform synthesis of septal peptidoglycan. Next, we describe the remodeling of the cell wall, invagination of the cell envelope, and disassembly of the division apparatus culminating in scission of the mother cell into two daughter cells. We conclude this review by highlighting some of the open questions in the cell division field, emphasizing that much remains to be discovered, even in an organism as extensively studied as E. coli.

The coiled-coil domain of Escherichia coli FtsLB is a structurally detuned element critical for modulating its activation in bacterial cell division

The FtsLB complex is a key regulator of bacterial cell division, existing in either an off state or an on state, which supports the activation of septal peptidoglycan synthesis. In Escherichia coli, residues known to be critical for this activation are located in a region near the C-terminal end of the periplasmic coiled-coil domain of FtsLB, raising questions about the precise role of this conserved domain in the activation mechanism. Here, we investigate an unusual cluster of polar amino acids found within the core of the FtsLB coiled coil. We hypothesized that these amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is a “detuned” structural element. In addition, we identified suppressor mutations within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. We propose a revised structural model of the tetrameric FtsLB (named the “Y-model”) in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the hydrophilic terminal moieties of the polar amino acids remain more favorably exposed to water than in the original four-helix bundle model (“I-model”). We propose that a shift in this architecture, dependent on its marginal stability, is involved in activating the FtsLB complex and triggering septal cell wall reconstruction.

Residue 49 of AtMinD1 Plays a Key Role in the Guidance of Chloroplast Division by Regulating the ARC6-AtMinD1 Interaction

Chloroplasts evolved from a free-living cyanobacterium through endosymbiosis. Similar to bacterial cell division, chloroplasts replicate by binary fission, which is controlled by the Minicell (Min) system through confining FtsZ ring formation at the mid-chloroplast division site. MinD, one of the most important members of the Min system, regulates the placement of the division site in plants and works cooperatively with MinE, ARC3, and MCD1. The loss of MinD function results in the asymmetric division of chloroplasts. In this study, we isolated one large dumbbell-shaped and asymmetric division chloroplast Arabidopsis mutant Chloroplast Division Mutant 75 (cdm75) that contains a missense mutation, changing the arginine at residue 49 to a histidine (R49H), and this mutant point is located in the N-terminal Conserved Terrestrial Sequence (NCTS) motif of AtMinD1, which is only typically found in terrestrial plants. This study provides sufficient evidence to prove that residues 1–49 of AtMinD1 are transferred into the chloroplast, and that the R49H mutation does not affect the function of the AtMinD1 chloroplast transit peptide. Subsequently, we showed that the point mutation of R49H could remove the punctate structure caused by residues 1–62 of the AtMinD1 sequence in the chloroplast, suggesting that the arginine in residue 49 (Arg49) is essential for localizing the punctate structure of AtMinD11–62 on the chloroplast envelope. Unexpectedly, we found that AtMinD1 could interact directly with ARC6, and that the R49H mutation could prevent not only the previously observed interaction between AtMinD1 and MCD1 but also the interaction between AtMinD1 and ARC6. Thus, we believe that these results show that the AtMinD1 NCTS motif is required for their protein interaction. Collectively, our results show that AtMinD1 can guide the placement of the division site to the mid chloroplast through its direct interaction with ARC6 and reveal the important role of AtMinD1 in regulating the AtMinD1-ARC6 interaction.

Modification of 5-methylphenanthridium from benzothiazoles to indoles as potent FtsZ inhibitors: Broadening the antibacterial spectrum toward vancomycin-resistant enterococci.

The death caused by pathogenic bacteria has always been a severe threat to mankind. The prevalence of drug resistance among bacteria underscores an urgent goal for new antibacterial agents with novel mode of action. Here we first designed and synthesized a class of benzothiazolyl-5-methylphenanthridium derivatives and evaluated their antibacterial activity. On this basis, we further designed and synthesized another class of novel indolyl-5-methylphenanthridium derivatives by optimizing the benzothiazolyl-5-methylphenanthridium core and evaluated their antibacterial activity targeting the bacterial cell division protein FtsZ. The results showed that the indolyl-5-methylphenanthridium derivatives had greatly improved activity against various drug-resistant bacterial strains including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus (VRE). Among them, compound C5 displayed excellent antibacterial activity against susceptible (MIC=1μg/mL), methicillin-resistant and clinical isolated S.aureus (MIC=2μg/mL). With low hemolytic activity towards mice red blood cells, C5 exhibited good antibacterial effect invivo in preliminary pharmacodynamic assay. More importantly, C5 was difficult to induce bacterial resistance. Further mechanism studies proved that C5 could inhibit bacterial cell division by promoting FtsZ polymerization, leading to disorderly polymerization and disordered knots. Therefore, our findings suggest that this class of novel indolyl-5-methylphenanthridium derivatives are promising for future antibacterial agents.

The discovery of natural 4'-demethylepipodophyllotoxin from renewable Dysosma versipellis species as a novel bacterial cell division inhibitor for controlling intractable diseases in rice

Natural products (NPs) are inexhaustible wellsprings for new agrochemicals discovery. To excavate novel natural products-based antibacterial leads or candidates potentially targeting FtsZ (filamentous temperature-sensitive protein Z) which is widely regarded as a promising antibacterial target for novel drug discovery, herein, a rapid and efficient screening platform was initially established to disclose the potential FtsZ inhibitors from a variety of commercial natural products. Screening results revealed that 4'-demethylepipodophyllotoxin (DMEP) from renewable Dysosma versipellis (D. versipellis) species could distinctly decrease the XooFtsZ’ GTPase activity and afforded a ponderable bacteriostatic effect (EC50 =38.7 mg L-1) against Xanthomonas oryzae pv. oryzae (Xoo). Further explorations found that DMEP could disorganize the assembly dynamics of XooFtsZ, and thereby yielding long and filamentous Xoo cells from the transmission electron microscopic and fluorescence microscopic imaging. Molecular docking study showed that DMEP displayed strong interactions with Asp38 and Arg205 residues of XooFtsZ, indicating that a natural potential FtsZ inhibitor was discovered gradually. The following bioassay result against rice bacterial blight in vivo presented good curative activity (50.0% at 200 mg L−1) and protective activity (46.8% at 200 mg L−1). Given that natural DMEP has many sites for structural modification, therefore, it can concurrently serve as a novel lead compound for exploring highly bioactive FtsZ inhibitors based on the molecular skeleton of DMEP.

Bacterial minicells to the rescue: cyto-Immunotherapy for the treatment of late stage cancers with minimal to no toxicity.

Bacterial minicells to the rescue: cyto-Immunotherapy for the treatment of late stage cancers with minimal to no toxicity.

Molecular basis of dimerization of lytic transglycosylase revealed by the crystal structure of MltA from Acinetobacter baumannii.

Peptidoglycan digestion by murein-degrading enzymes is a critical process in bacterial cell growth and/or cell division. The membrane-bound lytic murein transglycosylase A (MltA) is a murein-degrading enzyme; it catalyzes the cleavage of the β-1,4-glycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycans. Although substrate recognition and cleavage by MltA have been examined by previous structural and mutagenesis studies, the overall mechanism of MltA in conjunction with other functionally related molecules on the outer membrane of bacterial cells for peptidoglycan degradation has remained elusive. In this study, the crystal structure of MltA from the virulent human pathogen Acinetobacter baumannii is characterized and presented. The study indicated that MltA from A.baumannii forms homodimers via an extra domain which is specific to this species. Furthermore, the working mechanism of MltA with various functionally related proteins on the bacterial outer membrane was modeled based on the structural and biochemical analysis.

Correlative Super-Resolution Optical and Atomic Force Microscopy Reveals Relationships Between Bacterial Cell Wall Architecture and Synthesis in Bacillus subtilis.

Understanding how bacteria grow and divide requires insight into both the molecular-level dynamics of ultrastructure and the chemistry of the constituent components. Atomic force microscopy (AFM) can provide near molecular resolution images of biological systems but typically provides limited chemical information. Conversely, while super-resolution optical microscopy allows localization of particular molecules and chemistries, information on the molecular context is difficult to obtain. Here, we combine these approaches into STORMForce (stochastic optical reconstruction with atomic force microscopy) and the complementary SIMForce (structured illumination with atomic force microscopy), to map the synthesis of the bacterial cell wall structural macromolecule, peptidoglycan, during growth and division in the rod-shaped bacterium Bacillus subtilis. Using “clickable” d-amino acid incorporation, we fluorescently label and spatially localize a short and controlled period of peptidoglycan synthesis and correlate this information with high-resolution AFM of the resulting architecture. During division, septal synthesis occurs across its developing surface, suggesting a two-stage process with incorporation at the leading edge and with considerable in-filling behind. During growth, the elongation of the rod occurs through bands of synthesis, spaced by ∼300 nm, and corresponds to denser regions of the internal cell wall as revealed by AFM. Combining super-resolution optics and AFM can provide insights into the synthesis processes that produce the complex architectures of bacterial structural biopolymers.

A high-frequency single nucleotide polymorphism in the MtrB sensor kinase in clinical strains of Mycobacterium tuberculosis alters its biochemical and physiological properties.

The DNA polymorphisms found in clinical strains of Mycobacterium tuberculosis drive altered physiology, virulence, and pathogenesis in them. Although the lineages of these clinical strains can be traced back to common ancestor/s, there exists a plethora of difference between them, compared to those that have evolved in the laboratory. We identify a mutation present in ~80% of clinical strains, which maps in the HATPase domain of the sensor kinase MtrB and alters kinase and phosphatase activities, and affects its physiological role. The changes conferred by the mutation were probed by in-vitro biochemical assays which revealed changes in signaling properties of the sensor kinase. These changes also affect bacterial cell division rates, size and membrane properties. The study highlights the impact of DNA polymorphisms on the pathophysiology of clinical strains and provides insights into underlying mechanisms that drive signal transduction in pathogenic bacteria.

Elongation at Midcell in Preparation of Cell Division Requires FtsZ, but Not MreB nor PBP2 in Caulobacter crescentus.

Controlled growth of the cell wall is a key prerequisite for bacterial cell division. The existing view of the canonical rod-shaped bacterial cell dictates that newborn cells first elongate throughout their side walls using the elongasome protein complex, and subsequently use the divisome to coordinate constriction of the dividing daughter cells. Interestingly, another growth phase has been observed in between elongasome-mediated elongation and constriction, during which the cell elongates from the midcell outward. This growth phase, that has been observed in Escherichia coli and Caulobacter crescentus, remains severely understudied and its mechanisms remain elusive. One pressing open question is which role the elongasome key-component MreB plays in this respect. This study quantitatively investigates this growth phase in C. crescentus and focuses on the role of both divisome and elongasome components. This growth phase is found to initiate well after MreB localizes at midcell, although it does not require its presence at this subcellular location nor the action of key elongasome components. Instead, the divisome component FtsZ seems to be required for elongation at midcell. This study thus shines more light on this growth phase in an important model organism and paves the road to more in-depth studies.