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To investigate how cell elongation impacts extracellular electron transfer (EET) of electroactive microorganisms (EAMs), the division of model EAM Shewanella oneidensis (S. oneidensis) MR‐1 is engineered by reducing the formation of cell divisome. Specially, by blocking the translation of division proteins via anti‐sense RNAs or expressing division inhibitors, the cellular length and output power density are all increased. Electrophysiological and transcriptomic results synergistically reveal that the programmed cell elongation reinforces EET by enhancing NADH oxidation, inner‐membrane quinone pool, and abundance of c‐type cytochromes. Moreover, cell elongation enhances hydrophobicity due to decreased cell‐surface polysaccharide, thus facilitates the initial surface adhesion stage during biofilm formation. The output current and power density all increase in positive correction with cellular length. However, inhibition of cell division reduces cell growth, which is then restored by quorum sensing‐based dynamic regulation of cell growth and elongation phases. The QS‐regulated elongated strain thus enables a cell length of 143.6 ± 40.3 µm (72.6‐fold of that of S. oneidensis MR‐1), which results in an output power density of 248.0 ± 10.6 mW m−2 (3.41‐fold of that of S. oneidensis MR‐1) and exhibits superior potential for pollutant treatment. Engineering cellular length paves an innovate avenue for enhancing the EET of EAMs. [ABSTRACT FROM AUTHOR]

ABSTRACT Escherichia coli has many periplasmic hydrolases to degrade and modify peptidoglycan (PG). However, the redundancy of eight PG endopeptidases makes it challenging to define specific roles to individual enzymes. Therefore, the cellular role of PBP7 (encoded by pbpG) is not clearly defined. In this work, we show that PBP7 localizes in the lateral cell envelope and at midcell. The C‐terminal α‐helix of PBP7 is crucial for midcell localization but not for its activity, which is dispensable for this localization. Additionally, midcell localization of PBP7 relies on the assembly of FtsZ up to FtsN in the divisome, and on the activity of PBP3. PBP7 was found to affect the assembly timing of FtsZ and FtsN in the divisome. The absence of PBP7 slows down the assembly of FtsN at midcell. The ΔpbpG mutant exhibited a weaker incorporation of the fluorescent D‐amino acid HADA, reporting on transpeptidase activity, compared to wild‐type cells. This could indicate reduced PG synthesis at the septum of the ΔpbpG strain, explaining the slower accumulation of FtsN and suggesting that endopeptidase‐mediated PG cleavage may be a rate‐limiting step for septal PG synthesis. [ABSTRACT FROM AUTHOR]

ABSTRACT Bacterial cell division is orchestrated by proteins that assemble in dynamic complexes collectively known as the divisome. Essential monofunctional enzymes with glycosyltransferase or transpeptidase (TPase) activities, FtsW and FtsI respectively, engage in the synthesis of septal peptidoglycan (sPG). Enigmatically, Salmonella has two TPases that can promote cell division independently: FtsI (PBP3) and the pathogen‐specific paralogue PBP3SAL. How Salmonella regulates the assembly of the sPG synthase complex with these two TPases, is unknown. Here, we characterized Salmonella division complexes in wild‐type cells and isogenic mutants lacking PBP3 or PBP3SAL. The complexes were cross‐linked in vivo and pulled down with antibodies recognizing each enzyme. Proteomics of the immunoprecipitates showed that PBP3 and PBP3SAL do not extensively cross‐link in wild type cells, supporting the presence of independent complexes. More than 40 proteins cross‐link in complexes in which these two TPases are present. Those identified with high scores include FtsA, FtsK, FtsQLB, FtsW, PBP1B, SPOR domain‐containing proteins (FtsN, DedD, RlpA, DamX), amidase activators (FtsX, EnvC, NlpD) and Tol‐Pal proteins. Other cross‐linked proteins are the protease Prc, the elongasome TPase PBP2 and, D,D‐endo‐ and D,D‐carboxypeptidases. PBP3 and PBP3SAL localize at midcell and compete for occupying the division complex in response to environmental cues. Thus, a catalytic‐dead PBP3SAL‐S300A variant impairs cell division in a high osmolarity and acidic condition in which it is produced at levels exceeding those of PBP3. Salmonella may therefore exploit an ‘adjustable’ divisome to exchange TPases for ensuring cell division in distinct environments and, in this manner, expand its colonization capacities. [ABSTRACT FROM AUTHOR]

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber‐codon‐suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber‐codon‐suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass‐spectrometry‐based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 μM) include PBPs (PBP1a, KD = 0.07 μM; PBP5 = 0.4 μM); other lytic transglycosylases (SltB2, KD = 0.09 μM; RlpA, KD = 0.4 μM); a type VI secretion system effector (Tse5, KD = 0.3 μM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 μM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm. [ABSTRACT FROM AUTHOR]

The FtsEX membrane complex constitutes an essential component of the ABC transporter superfamily, widely distributed among bacterial species. It governs peptidoglycan degradation for cell division, acting as a signal transmitter rather than a substrate transporter. Through the ATPase activity of FtsE, it facilitates signal transmission from the cytosol across the membrane to the periplasm, activating associated peptidoglycan hydrolases. This review concentrates on the latest structural advancements elucidating the architecture of the FtsEX complex and its interplay with lytic enzymes or regulatory counterparts. The revealed three‐dimensional structures unveil a landscape wherein a precise array of intermolecular interactions, preserved across diverse bacterial species, afford meticulous spatial and temporal control over the cell division process. [ABSTRACT FROM AUTHOR]

Abstract To investigate how cell elongation impacts extracellular electron transfer (EET) of electroactive microorganisms (EAMs), the division of model EAM Shewanella oneidensis (S. oneidensis) MR‐1 is engineered by reducing the formation of cell divisome. Specially, by blocking the translation of division proteins via anti‐sense RNAs or expressing division inhibitors, the cellular length and output power density are all increased. Electrophysiological and transcriptomic results synergistically reveal that the programmed cell elongation reinforces EET by enhancing NADH oxidation, inner‐membrane quinone pool, and abundance of c‐type cytochromes. Moreover, cell elongation enhances hydrophobicity due to decreased cell‐surface polysaccharide, thus facilitates the initial surface adhesion stage during biofilm formation. The output current and power density all increase in positive correction with cellular length. However, inhibition of cell division reduces cell growth, which is then restored by quorum sensing‐based dynamic regulation of cell growth and elongation phases. The QS‐regulated elongated strain thus enables a cell length of 143.6 ± 40.3 µm (72.6‐fold of that of S. oneidensis MR‐1), which results in an output power density of 248.0 ± 10.6 mW m−2 (3.41‐fold of that of S. oneidensis MR‐1) and exhibits superior potential for pollutant treatment. Engineering cellular length paves an innovate avenue for enhancing the EET of EAMs.

ABSTRACT Bacterial growth and division rely on intricate regulation of morphogenetic complexes to remodel the cell envelope without compromising envelope integrity. Significant progress has been made in recent years towards understanding the regulation of cell wall metabolic enzymes. However, other cell envelope components play a role in morphogenesis as well. A primary factor required to protect envelope integrity in low osmolarity environments is OpgH, the synthase of osmoregulated periplasmic glucans (OPGs). Here, we demonstrate that OpgH is essential in the α-proteobacterium Caulobacter crescentus. Unexpectedly, depletion of OpgH or attempted complementation with a catalytically dead OpgH variant results in striking asymmetric bulging and cell lysis. These shape defects are accompanied by reduced cell wall synthesis and mislocalization of morphogenetic complexes. Interestingly, overactivation of the CenKR two-component system that has been implicated in cell envelope stress homeostasis in α-proteobacteria phenocopies the morphogenetic defects associated with OpgH depletion. Each of these perturbations leads to an increase in the levels of the elongasome protein, MreB, and decreases in the levels of divisome proteins FtsZ and MipZ as well as OpgH, itself. Constitutive production of OpgH during CenKR overactivation prevents cell bulging, but cells still exhibit morphogenetic defects. We propose that OPG depletion activates CenKR, leading to changes in the expression of cell envelope-related genes, but that OPGs also exert CenKR-independent effects on morphogenesis. Our data establish a surprising function for an OpgH homolog in morphogenesis and reveal an essential role of OpgH in maintaining cell morphology in Caulobacter.IMPORTANCEBacteria must synthesize and fortify the cell envelope in a tightly regulated manner to orchestrate growth and adaptation. Osmoregulated periplasmic glucans (OPGs) are important, but poorly understood, constituents of Gram-negative cell envelopes that contribute to envelope integrity and protect against osmotic stress. Here, we determined that the OPG synthase OpgH plays a surprising, essential role in morphogenesis in Caulobacter crescentus. Loss of OpgH causes asymmetric cell bulging and lysis via misregulation of the localization and activity of morphogenetic complexes. Overactivation of the CenKR two-component system involved in envelope homeostasis phenocopies OpgH depletion, suggesting that depletion of OpgH activates CenKR. Because cell envelope integrity is critical for bacterial survival, understanding how OpgH activity contributes to morphogenesis and maintenance of envelope integrity could aid in the development of antibiotic therapies.

The early phase of bacterial division involves four major core functions: chromosome coordination, Z-ring stabilization, Z-ring anchoring, and regulation of the initiation of late division. Core functions of the early divisome are generally conserved across the Proteobacteria, not necessarily genes. Gene conservation and essentiality cannot be used to define the core early divisome due to the strong epistatic nature of this network and its coevolution with other critical cellular processes. Variation within the early divisome can inform fundamental principles about division and the evolutionary constraints that have shaped division in each lineage. The bacterial divisome is a complex nanomachine that drives cell division and separation. The essentiality of these processes leads to the assumption that proteins with core roles will be strictly conserved across all bacterial genomes. However, recent studies in diverse proteobacteria have revealed considerable variation in the early divisome compared with Escherichia coli. While some proteins are highly conserved, their specific functions and interacting partners vary. Meanwhile, different subphyla use clade-specific proteins with analogous functions. Thus, instead of focusing on gene conservation, we must also explore how key functions are maintained during early division by diverging protein networks. An enhanced awareness of these complex genetic networks will clarify the physical and evolutionary constraints of bacterial division. [ABSTRACT FROM AUTHOR]

Most bacteria divide through a highly conserved process called binary fission, in which there is symmetric growth of daughter cells and the synthesis of peptidoglycan at the mid-cell to enable cytokinesis. During this process, the parental cell replicates its chromosomal DNA and segregates replicated chromosomes into the daughter cells. The mechanisms that regulate binary fission have been extensively studied in several model organisms, including Eschericia coli, Bacillus subtilis, and Caulobacter crescentus. These analyses have revealed that a multi-protein complex called the divisome forms at the mid-cell to enable peptidoglycan synthesis and septation during division. In addition, rod-shaped bacteria form a multi-protein complex called the elongasome that drives sidewall peptidoglycan synthesis necessary for the maintenance of rod shape and the lengthening of the cell prior to division. In adapting to their intracellular niche, the obligate intracellular bacteria discussed here have eliminated one to several of the divisome gene products essential for binary fission in E. coli. In addition, genes that encode components of the elongasome, which were mostly lost as rod-shaped bacteria evolved into coccoid organisms, have been retained during the reductive evolutionary process that some coccoid obligate intracellular bacteria have undergone. Although the precise molecular mechanisms that regulate the division of obligate intracellular bacteria remain undefined, the studies summarized here indicate that obligate intracellular bacteria exhibit remarkable plasticity in their cell division processes. [ABSTRACT FROM AUTHOR]

The 11 lytic transglycosylases of Pseudomonas aeruginosa have overlapping activities in the turnover of the cell‐wall peptidoglycan. Rare lipoprotein A (RlpA) is distinct among the 11 by its use of only peptidoglycan lacking peptide stems. The spatial localization of RlpA and its interactome within P. aeruginosa are unknown. We employed suppression of introduced amber codons at sites in the rlpA gene for the introduction of the unnatural‐amino‐acids Νζ‐[(2‐azidoethoxy)carbonyl]‐l‐lysine (compound 1) and Nζ‐[[[3‐(3‐methyl‐3H‐diazirin‐3‐yl)propyl]amino]carbonyl]‐l‐lysine (compound 2). In live P. aeruginosa, full‐length RlpA incorporating compound 1 into its sequence was fluorescently tagged using strained‐promoted alkyne‐azide cycloaddition and examined by fluorescence microscopy. RlpA is present at low levels along the sidewall length of the bacterium, and at higher levels at the nascent septa of replicating bacteria. In intact P. aeruginosa, UV photolysis of full‐length RlpA having compound 2 within its sequence generated a transient reactive carbene, which engaged in photoaffinity capture of neighboring proteins. Thirteen proteins were identified. Three of these proteins—PBP1a, PBP5, and MreB—are members of the bacterial divisome. The use of the complementary methodologies of non‐canonical amino‐acid incorporation, photoaffinity proximity analysis, and fluorescent microscopy confirm a dominant septal location for the RlpA enzyme of P. aeruginosa, as a divisome‐associated activity. This accomplishment adds to the emerging recognition of the value of these methodologies for identification of the intracellular localization of bacterial proteins. [ABSTRACT FROM AUTHOR]

The spheroid bacterium Staphylococcus aureus is often used as a model of morphogenesis due to its apparently simple cell cycle. S. aureus has many cell division proteins that are conserved across bacteria alluding to common functions. However, despite intensive study, we still do not know the roles of many of these components. Here, we have examined the functions of the paralogues DivIVA and GpsB in the S. aureus cell cycle. Cells lacking gpsB display a more spherical phenotype than the wild-type cells, which is associated with a decrease in peripheral cell wall peptidoglycan synthesis. This correlates with increased localization of penicillin-binding proteins at the developing septum, notably PBPs 2 and 3. Our results highlight the role of GpsB as an apparent regulator of cell morphogenesis in S. aureus. [ABSTRACT FROM AUTHOR]

Most bacteria divide through a highly conserved process called binary fission, in which there is symmetric growth of daughter cells and the synthesis of peptidoglycan at the mid-cell to enable cytokinesis. During this process, the parental cell replicates its chromosomal DNA and segregates replicated chromosomes into the daughter cells. The mechanisms that regulate binary fission have been extensively studied in several model organisms, including Eschericia coli, Bacillus subtilis, and Caulobacter crescentus. These analyses have revealed that a multi-protein complex called the divisome forms at the mid-cell to enable peptidoglycan synthesis and septation during division. In addition, rod-shaped bacteria form a multi-protein complex called the elongasome that drives sidewall peptidoglycan synthesis necessary for the maintenance of rod shape and the lengthening of the cell prior to division. In adapting to their intracellular niche, the obligate intracellular bacteria discussed here have eliminated one to several of the divisome gene products essential for binary fission in E. coli. In addition, genes that encode components of the elongasome, which were mostly lost as rod-shaped bacteria evolved into coccoid organisms, have been retained during the reductive evolutionary process that some coccoid obligate intracellular bacteria have undergone. Although the precise molecular mechanisms that regulate the division of obligate intracellular bacteria remain undefined, the studies summarized here indicate that obligate intracellular bacteria exhibit remarkable plasticity in their cell division processes.

This article relates personal recollections and starts with the origin of electron microscopy in the sixties of the previous century at the University of Amsterdam. Novel fixation and embedding techniques marked the discovery of the internal bacterial structures not visible by light microscopy. A special status became reserved for the freeze-fracture technique. By freeze-fracturing chemically fixed cells, it proved possible to examine the morphological effects of fixation. From there on, the focus switched from bacterial structure as such to their cell cycle. This invoked bacterial physiology and steady-state growth combined with electron microscopy. Electron-microscopic autoradiography with pulses of [3H] Dap revealed that segregation of replicating DNA cannot proceed according to a model of zonal growth (with envelope-attached DNA). This stimulated us to further investigate the sacculus, the peptidoglycan macromolecule. In particular, we focused on the involvement of penicillin-binding proteins such as PBP2 and PBP3, and their role in division. Adding aztreonam (an inhibitor of PBP3) blocked ongoing divisions but not the initiation of new ones. A PBP3-independent peptidoglycan synthesis (PIPS) appeared to precede a PBP3-dependent step. The possible chemical nature of PIPS is discussed. [ABSTRACT FROM AUTHOR]

The spheroid bacterium Staphylococcus aureus is often used as a model of morphogenesis due to its apparently simple cell cycle. S. aureus has many cell division proteins that are conserved across bacteria alluding to common functions. However, despite intensive study, we still do not know the roles of many of these components. Here, we have examined the functions of the paralogues DivIVA and GpsB in the S. aureus cell cycle. Cells lacking gpsB display a more spherical phenotype than the wild-type cells, which is associated with a decrease in peripheral cell wall peptidoglycan synthesis. This correlates with increased localization of penicillin-binding proteins at the developing septum, notably PBPs 2 and 3. Our results highlight the role of GpsB as an apparent regulator of cell morphogenesis in S. aureus.

Bacterial cytokinesis starts with the polymerization of the tubulin-like FtsZ, which forms the cell division scaffold. SepF aligns FtsZ polymers and also acts as a membrane anchor for the Z-ring. While in most bacteria cell division takes place at midcell, during sporulation of Streptomyces many septa are laid down almost simultaneously in multinucleoid aerial hyphae. The genomes of streptomycetes encode two additional SepF paralogs, SflA and SflB, which can interact with SepF. Here we show that the sporogenic aerial hyphae of sflA and sflB mutants of Streptomyces coelicolor frequently branch, a phenomenon never seen in the wild-type strain. The branching coincided with ectopic localization of DivIVA along the lateral wall of sporulating aerial hyphae. Constitutive expression of SflA and SflB largely inhibited hyphal growth, further correlating SflAB activity to that of DivIVA. SflAB localized in foci prior to and after the time of sporulation-specific cell division, while SepF co-localized with active septum synthesis. Foci of FtsZ and DivIVA frequently persisted between adjacent spores in spore chains of sflA and sflB mutants, at sites occupied by SflAB in wild-type cells. Taken together, our data show that SflA and SflB play an important role in the control of growth and cell division during Streptomyces development. • Discovery of the SepF-like proteins SflA and SflB as novel cell division control proteins in Streptomyces. • Over-expression of SflA or SflB reduces growth and prevents attachment of surface-grown colonies. • Deletion of the genes for SflA or SflB results in unique premature germination of spores in the spore chains of Streptomyces. • Premature germination is caused by the ectopic localization of DivIVA in sflA and sflB mutants. • SflA and SflB ensure the disassembly of DivIVA and FtsZ after completion of cell division. [ABSTRACT FROM AUTHOR]

Cytokinesis is an essential process in bacterial cell division, and it involves more than 25 essential/non-essential cell division proteins that form a protein complex known as a divisome. Central to the divisome are the proteins FtsB and FtsL binding to FtsQ to form a complex FtsQBL, which helps link the early proteins with late proteins. The FtsQBL complex is highly conserved as a component across bacteria. Pathogens like Vibrio cholerae, Mycobacterium ulcerans, Mycobacterium leprae, and Chlamydia trachomatis are the causative agents of the bacterial Neglected Tropical Diseases Cholera, Buruli ulcer, Leprosy, and Trachoma, respectively, some of which seemingly lack known homologs for some of the FtsQBL complex proteins. In the absence of experimental characterization, either due to insufficient resources or the massive increase in novel sequences generated from genomics, functional annotation is traditionally inferred by sequence similarity to a known homolog. With the advent of accurate protein structure prediction methods, features both at the fold level and at the protein interaction level can be used to identify orthologs that cannot be unambiguously identified using sequence similarity methods. Using the FtsQBL complex proteins as a case study, we report potential remote homologs using Profile Hidden Markov models and structures predicted using AlphaFold. Predicted ortholog structures show conformational similarity with corresponding E. coli proteins irrespective of their level of sequence similarity. Alphafold multimer was used to characterize remote homologs as FtsB or FtsL, when they were not sufficiently distinguishable at both the sequence or structure level, as their interactions with FtsQ and FtsW play a crucial role in their function. The structures were then analyzed to identify functionally critical regions of the proteins consistent with their homologs and delineate regions potentially useful for inhibitor discovery. [ABSTRACT FROM AUTHOR]

The spatiotemporal regulation of cell division is a fundamental issue in cell biology. Bacteria have evolved a variety of different systems to achieve proper division site placement. In many cases, the underlying molecular mechanisms are still incompletely understood. In this study, we investigate the function of the cell division regulator MipZ from Caulobacter crescentus, a P-loop ATPase that inhibits the polymerization of the treadmilling tubulin homolog FtsZ near the cell poles, thereby limiting the assembly of the cytokinetic Z ring to the midcell region. We show that MipZ interacts with FtsZ in both its monomeric and polymeric forms and induces the disassembly of FtsZ polymers in a manner that is not dependent but enhanced by the FtsZ GTPase activity. Using a combination of biochemical and genetic approaches, we then map the MipZ--FtsZ interaction interface. Our results reveal that MipZ employs a patch of surface-exposed hydrophobic residues to interact with the C-terminal region of the FtsZ core domain. In doing so, it sequesters FtsZ monomers and caps the (+)-end of FtsZ polymers, thereby promoting their rapid disassembly. We further show that MipZ influences the conformational dynamics of interacting FtsZ molecules, which could potentially contribute to modulating their assembly kinetics. Together, our findings show that MipZ uses a combination of mechanisms to control FtsZ polymerization, which may be required to robustly regulate the spatiotemporal dynamics of Z ring assembly within the cell. [ABSTRACT FROM AUTHOR]

Salt stress significantly impedes high cell-density culture by suppressing the reproductive capacity of lactic acid bacteria. This study examined the effects of salt stress conditions (2% w/v NaCl) on Lactobacillus bulgaricus sp1.1. Although DNA replication and division ring localization remained unaffected, the down-regulation of the ftsZ and dnaA genes inhibited division ring assembly. Consequently, bacteria exhibited filamentation, increased cell length, and accumulated chromosomes and division rings, which obstructed cell division. Under salt stress, sp1.1 experienced several transcriptional changes. The serine/threonine kinase gene was identified as a significant regulator of bacterial division under salt stress. Exogenous addition of serine and threonine could stimulate the expression of Ser/Thr kinase gene, promote the assembly of division ring, and enhance the dividing ability of the bacterium, which led to a density of 7.93 log CFU/mL under the salt stress, which was 6.92 times higher than that of the untreated group. This study provides a new perspective for high-density cultivation of lactic acid bacteria. • The division ring of strain sp1.1 is incomplete under salt stress. • Gene Ser/Thr kinase is a key gene affecting bacterial division under salt stress. • Ser and Thr promote cell division by increasing the expression of Ser/Thr kinase gene. [ABSTRACT FROM AUTHOR]

Cytokinesis is an essential process in bacterial cell division, and it involves more than 25 essential/non-essential cell division proteins that form a protein complex known as a divisome. Central to the divisome are the proteins FtsB and FtsL binding to FtsQ to form a complex FtsQBL, which helps link the early proteins with late proteins. The FtsQBL complex is highly conserved as a component across bacteria. Pathogens like Vibrio cholerae, Mycobacterium ulcerans, Mycobacterium leprae, and Chlamydia trachomatis are the causative agents of the bacterial Neglected Tropical Diseases Cholera, Buruli ulcer, Leprosy, and Trachoma, respectively, some of which seemingly lack known homologs for some of the FtsQBL complex proteins. In the absence of experimental characterization, either due to insufficient resources or the massive increase in novel sequences generated from genomics, functional annotation is traditionally inferred by sequence similarity to a known homolog. With the advent of accurate protein structure prediction methods, features both at the fold level and at the protein interaction level can be used to identify orthologs that cannot be unambiguously identified using sequence similarity methods. Using the FtsQBL complex proteins as a case study, we report potential remote homologs using Profile Hidden Markov models and structures predicted using AlphaFold. Predicted ortholog structures show conformational similarity with corresponding E. coli proteins irrespective of their level of sequence similarity. Alphafold multimer was used to characterize remote homologs as FtsB or FtsL, when they were not sufficiently distinguishable at both the sequence or structure level, as their interactions with FtsQ and FtsW play a crucial role in their function. The structures were then analyzed to identify functionally critical regions of the proteins consistent with their homologs and delineate regions potentially useful for inhibitor discovery.

Cell wall synthesis in bacteria is determined by two protein complexes: the elongasome and divisome. The elongasome is coordinated by the actin homolog MreB while the divisome is organized by the tubulin homolog FtsZ. While these two systems must coordinate with each other to ensure that elongation and division are coregulated, this cross talk has been understudied. Using the MreB depolymerizing agent, A22, we found that multiple gene deletions result in cells exhibiting increased sensitivity to MreB depolymerization. One of those genes encodes for EnvC, a part of the divisome that is responsible for splitting daughter cells after the completion of cytokinesis through the activation of specific amidases. Here we show this increased sensitivity to A22 works through two known amidase targets of EnvC: AmiA and AmiB. In addition, suppressor analysis revealed that mutations in enzyme 1 of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can suppress the effects of A22 in both wild‐type and envC deletion cells. Together this work helps to link elongation, division, and metabolism. [ABSTRACT FROM AUTHOR]

Bacterial growth and division rely on intricate regulation of morphogenetic complexes to remodel the cell envelope without compromising envelope integrity. Significant progress has been made in recent years towards understanding the regulation of cell wall metabolic enzymes. However, other cell envelope components play a role in morphogenesis as well. A primary factor required to protect envelope integrity in low osmolarity environments is OpgH, the synthase of osmoregulated periplasmic glucans (OPGs). Here, we demonstrate that OpgH is essential in the α-proteobacterium Caulobacter crescentus . Unexpectedly, depletion of OpgH or attempted complementation with a catalytically dead OpgH variant results in striking asymmetric bulging and cell lysis. These shape defects are accompanied by reduced cell wall synthesis and mislocalization of morphogenetic complexes. Interestingly, overactivation of the CenKR two-component system that has been implicated in cell envelope stress homeostasis in α-proteobacteria phenocopies the morphogenetic defects associated with OpgH depletion. Each of these perturbations leads to an increase in the levels of the elongasome protein, MreB, and decreases in the levels of divisome proteins FtsZ and MipZ as well as OpgH, itself. Constitutive production of OpgH during CenKR overactivation prevents cell bulging, but cells still exhibit morphogenetic defects. We propose that OPG depletion activates CenKR, leading to changes in the expression of cell envelope-related genes, but that OPGs also exert CenKR-independent effects on morphogenesis. Our data establish a surprising function for an OpgH homolog in morphogenesis and reveal an essential role of OpgH in maintaining cell morphology in Caulobacter .IMPORTANCEBacteria must synthesize and fortify the cell envelope in a tightly regulated manner to orchestrate growth and adaptation. Osmoregulated periplasmic glucans (OPGs) are important, but poorly understood, constituents of Gram-negative cell envelopes that contribute to envelope integrity and protect against osmotic stress. Here, we determined that the OPG synthase OpgH plays a surprising, essential role in morphogenesis in Caulobacter crescentus . Loss of OpgH causes asymmetric cell bulging and lysis via misregulation of the localization and activity of morphogenetic complexes. Overactivation of the CenKR two-component system involved in envelope homeostasis phenocopies OpgH depletion, suggesting that depletion of OpgH activates CenKR. Because cell envelope integrity is critical for bacterial survival, understanding how OpgH activity contributes to morphogenesis and maintenance of envelope integrity could aid in the development of antibiotic therapies., Competing Interests: The authors declare no conflict of interest.

This article relates personal recollections and starts with the origin of electron microscopy in the sixties of the previous century at the University of Amsterdam. Novel fixation and embedding techniques marked the discovery of the internal bacterial structures not visible by light microscopy. A special status became reserved for the freeze-fracture technique. By freeze-fracturing chemically fixed cells, it proved possible to examine the morphological effects of fixation. From there on, the focus switched from bacterial structure as such to their cell cycle. This invoked bacterial physiology and steady-state growth combined with electron microscopy. Electron-microscopic autoradiography with pulses of [3H] Dap revealed that segregation of replicating DNA cannot proceed according to a model of zonal growth (with envelope-attached DNA). This stimulated us to further investigate the sacculus, the peptidoglycan macromolecule. In particular, we focused on the involvement of penicillin-binding proteins such as PBP2 and PBP3, and their role in division. Adding aztreonam (an inhibitor of PBP3) blocked ongoing divisions but not the initiation of new ones. A PBP3-independent peptidoglycan synthesis (PIPS) appeared to precede a PBP3-dependent step. The possible chemical nature of PIPS is discussed.

Compared with the extensive knowledge on cell division in model eukaryotes and bacteria, little is known about how archaea divide. Interestingly, both endosomal sorting complex required for transport (ESCRT)-based and FtsZ-based cell division systems are found in members of the Archaea. In the past couple of years, several studies have started to shed light on FtsZ-based cell division processes in members of the Euryarchaeota. In this review we highlight recent findings in this emerging field of research. We present current knowledge of the cell division machinery of halophiles which relies on two FtsZ proteins, and we compare it with that of methanobacteria, which relies on only one FtsZ. Finally, we discuss how these differences relate to the distinct cell envelopes of these two archaeal model systems. Archaea employ either endosomal sorting complex required for transport (ESCRT)-III homologs or FtsZ homologs as main players in cell division. Two types of FtsZ-based cell division exist in archaea: one-FtsZ-based, occurs in a few lineages, and the other, more common, type, which is two-FtsZ-based. FtsZ homologs have different functions in the two-FtsZ-based cell division: FtsZ1 is involved mainly in ring assembly while FtsZ2 is involved in constriction. The one-FtsZ-based system co-occurs in the presence of a cell wall made of pseudopeptidoglycan and MreB homologs in some methanogens. SepF is the anchor for FtsZ in archaea. The two FtsZs are the result of an ancient duplication followed by loss of one or the other FtsZ during archaeal diversification. [ABSTRACT FROM AUTHOR]

Simple Summary: Antimicrobial 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. [ABSTRACT FROM AUTHOR]

The synthesis of a peptidoglycan septum is a fundamental part of bacterial fission and is driven by a multiprotein dynamic complex called the divisome. FtsW and FtsI are essential proteins that synthesize the peptidoglycan septum and are controlled by the regulatory FtsBLQ subcomplex and the activator FtsN. However, their mode of regulation has not yet been uncovered in detail. Understanding this process in detail may enable the development of new compounds to combat the rise in antibiotic resistance. In this review, recent data on the regulation of septal peptidoglycan synthesis is summarized and discussed. Based on structural models and the collected data, multiple putative interactions within FtsWI and with regulators are uncovered. This elaborates on and supports an earlier proposed model that describes active and inactive conformations of the septal peptidoglycan synthesis complex that are stabilized by these interactions. Furthermore, a new model on the spatial organization of the newly synthesized peptidoglycan and the synthesis complex is presented. Overall, the updated model proposes a balance between several allosteric interactions that determine the state of septal peptidoglycan synthesis. [ABSTRACT FROM AUTHOR]

The penicillin-binding proteins are the enzyme catalysts of the critical transpeptidation crosslinking polymerization reaction of bacterial peptidoglycan synthesis and the molecular targets of the penicillin antibiotics. Here, we report a combined crystallographic, small-angle X-ray scattering (SAXS) in-solution structure, computational and biophysical analysis of PBP1 of Staphylococcus aureus (saPBP1), providing mechanistic clues about its function and regulation during cell division. The structure reveals the pedestal domain, the transpeptidase domain, and most of the linker connecting to the “penicillin-binding protein and serine/threonine kinase associated” (PASTA) domains, but not its two PASTA domains, despite their presence in the construct. To address this absence, the structure of the PASTA domains was determined at 1.5 Å resolution. Extensive molecular-dynamics simulations interpret the PASTA domains of saPBP1 as conformationally mobile and separated from the transpeptidase domain. This conclusion was confirmed by SAXS experiments on the full-length protein in solution. A series of crystallographic complexes with β-lactam antibiotics (as inhibitors) and penta-Gly (as a substrate mimetic) allowed the molecular characterization of both inhibition by antibiotics and binding for the donor and acceptor peptidoglycan strands. Mass-spectrometry experiments with synthetic peptidoglycan fragments revealed binding by PASTA domains in coordination with the remaining domains. The observed mobility of the PASTA domain in saPBP1 could play a crucial role for in vivo interaction with its glycosyltransferase partner in the membrane or with other components of the divisome machinery, as well as for coordination of transpeptidation and polymerization processes in the bacterial divisome.

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (K D  < 0.5 μM) include PBPs (PBP1a, K D  = 0.07 μM; PBP5 = 0.4 μM); other lytic transglycosylases (SltB2, K D  = 0.09 μM; RlpA, K D  = 0.4 μM); a type VI secretion system effector (Tse5, K D  = 0.3 μM); and a regulatory protease for alginate biosynthesis (AlgO, K D  < 0.4 μM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm., (© 2024 The Protein Society.)

Bacterial cell shape is determined by the peptidoglycan (PG) layer. The cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a filamentous strain with ovoid-shaped cells connected together with incomplete cell constriction. When deprived of combined nitrogen in the growth medium, about 5–10% of the cells differentiate into heterocysts, cells devoted to nitrogen fixation. It has been shown that PG synthesis is modulated during heterocyst development and some penicillin-binding proteins (PBPs) participating in PG synthesis are required for heterocyst morphogenesis or functioning. Anabaena has multiple PBPs with functional redundancy. In this study, in order to examine the function of PG synthesis and its relationship with heterocyst development, we created a conditional mutant of mraY , a gene necessary for the synthesis of the PG precursor, lipid I. We show that mraY is required for cell and filament integrity. Furthermore, when mraY expression was being limited, persistent septal PG synthetic activity was observed, resulting in increase in cell width. Under non-permissive conditions, filaments and cells were rapidly lysed, and no sign of heterocyst development within the time window allowed was detected after nitrogen starvation. When mraY expression was being limited, a high percentage of heterocyst doublets were found. These doublets are formed likely as a consequence of delayed cell division and persistent septal PG synthesis. MraY interacts with components of both the elongasome and the divisome, in particular those directly involved in PG synthesis, including HetF, which is required for both cell division and heterocyst formation. [ABSTRACT FROM AUTHOR]

Bacterial cell shape is determined by the peptidoglycan (PG) layer. The cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a filamentous strain with ovoid-shaped cells connected together with incomplete cell constriction. When deprived of combined nitrogen in the growth medium, about 5–10% of the cells differentiate into heterocysts, cells devoted to nitrogen fixation. It has been shown that PG synthesis is modulated during heterocyst development and some penicillin-binding proteins (PBPs) participating in PG synthesis are required for heterocyst morphogenesis or functioning. Anabaena has multiple PBPs with functional redundancy. In this study, in order to examine the function of PG synthesis and its relationship with heterocyst development, we created a conditional mutant of mraY, a gene necessary for the synthesis of the PG precursor, lipid I. We show that mraY is required for cell and filament integrity. Furthermore, when mraY expression was being limited, persistent septal PG synthetic activity was observed, resulting in increase in cell width. Under non-permissive conditions, filaments and cells were rapidly lysed, and no sign of heterocyst development within the time window allowed was detected after nitrogen starvation. When mraY expression was being limited, a high percentage of heterocyst doublets were found. These doublets are formed likely as a consequence of delayed cell division and persistent septal PG synthesis. MraY interacts with components of both the elongasome and the divisome, in particular those directly involved in PG synthesis, including HetF, which is required for both cell division and heterocyst formation.

Bacterial cells in their natural environments encounter rapid and large changes in external osmolality. For instance, enteric bacteria such as Escherichia coli experience a rapid decrease when they exit from host intestines. Changes in osmolality alter the mechanical load on the cell envelope, and previous studies have shown that large osmotic shocks can slow down bacterial growth and impact cytoplasmic diffusion. However, it remains unclear how cells maintain envelope integrity and regulate envelope synthesis in response to osmotic shocks. In this study, we developed an agarose pad-based protocol to assay envelope stiffness by measuring population-averaged cell length before and after a hyperosmotic shock. Pad-based measurements exhibited an apparently larger length change compared with single-cell dynamics in a microfluidic device, which we found was quantitatively explained by a transient increase in division rate after the shock. Inhibiting cell division led to consistent measurements between agarose pad-based and microfluidic measurements. Directly after hyperosmotic shock, FtsZ concentration and Z-ring intensity increased, and the rate of septum constriction increased. These findings establish an agarose pad-based protocol for quantifying cell envelope stiffness, and demonstrate that mechanical perturbations can have profound effects on bacterial physiology.

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity.

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 E scherichia 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. [ABSTRACT FROM AUTHOR]

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity. [ABSTRACT FROM AUTHOR]

Bacterial cells in their natural environments encounter rapid and large changes in external osmolality. For instance, enteric bacteria such as Escherichia coli experience a rapid decrease when they exit from host intestines. Changes in osmolality alter the mechanical load on the cell envelope, and previous studies have shown that large osmotic shocks can slow down bacterial growth and impact cytoplasmic diffusion. However, it remains unclear how cells maintain envelope integrity and regulate envelope synthesis in response to osmotic shocks. In this study, we developed an agarose pad-based protocol to assay envelope stiffness by measuring population-averaged cell length before and after a hyperosmotic shock. Pad-based measurements exhibited an apparently larger length change compared with single-cell dynamics in a microfluidic device, which we found was quantitatively explained by a transient increase in division rate after the shock. Inhibiting cell division led to consistent measurements between agarose pad-based and microfluidic measurements. Directly after hyperosmotic shock, FtsZ concentration and Z-ring intensity increased, and the rate of septum constriction increased. These findings establish an agarose pad-based protocol for quantifying cell envelope stiffness, and demonstrate that mechanical perturbations can have profound effects on bacterial physiology. [ABSTRACT FROM AUTHOR]

Bacterial growth and cell division are coordinated with hydrolysis of the peptidoglycan (PG) layer of the cell wall, but the mechanisms of regulation of extracellular PG hydrolases are not well understood. Here we report the biochemical, structural, and genetic analysis of the Mycobacterium tuberculosis homolog of the transmembrane PG-hydrolase regulator, FtsX. The purified FtsX extracellular domain binds the PG peptidase Rv2190c/RipC N-terminal segment, causing a conformational change that activates the enzyme. Deletion of ftsEX and ripC caused similar phenotypes in Mycobacterium smegmatis, as expected for genes in a single pathway. The crystal structure of the FtsX extracellular domain reveals an unprecedented fold containing two lobes connected by a flexible hinge. Mutations in the hydrophobic cleft between the lobes reduce RipC binding in vitro and inhibit FtsX function in M. smegmatis. These studies suggest how FtsX recognizes RipC and support a model in which a conformational change in FtsX links the cell division apparatus with PG hydrolysis.

RodZ is required for determination of cell shape in rod-shaped bacterium, such as Escherichia coli. RodZ is a transmembrane protein and forms a supramolecular complex called the Rod complex with other proteins, such as MreB-actin and peptidoglycan synthesis enzymes (for e.g., PBP2). Deletion of the rodZ gene changes the cell shape from rod to round or ovoid. Another supramolecular complex called divisome that controls cell division mainly consists of FtsZ-tubulin. MreB directly interacts with FtsZ and this interaction is critical to trigger a transition from cell elongation to cell division. Recently, we found that RodZ also directly interacts with FtsZ, and RodZ recruits MreB to the divisome. Formation of the division ring, called Z ring, is delayed if RodZ does not interact with FtsZ, indicating that RodZ might facilitate the formation of the Z ring during the cell division process. In this mini-review, we have summarized the roles of RodZ in cell elongation and cell division, especially based on our recent study.

Proper control of cell division in the intracellular pathogen Mycobacterium tuberculosis is central to its growth, survival, pathogenesis, and resistance to antibiotics. Nevertheless, the divisome components and mechanisms by which mycobacteria regulate their cell cycle are not entirely understood. Here we demonstrate that the previously uncharacterized Rv0954 protein localizes to the mid-cell during cell division and interacts with the division-related proteins LamA, PbpA, and PknH. Deletion of rv0954 did not result in alterations in cell morphology or sensitivity to cell wall-targeting antibiotics but transposon mutagenesis demonstrated genetic interactions with genes related to cell division. This work suggests that Rv0954 participates in cell division and reveals potential components of the mycobacterial divisome for future investigation.

ABSTRACT Spatiotemporal regulation of septal peptidoglycan (PG) synthesis is achieved by coupling assembly and activation of the synthetic enzymes (FtsWI) to the Z ring, a cytoskeletal element that is required for division in most bacteria. In Escherichia coli, the recruitment of the FtsWI complex is dependent upon the cytoplasmic domain of FtsL, a component of the conserved FtsQLB complex. Once assembled, FtsWI is activated by the arrival of FtsN, which acts through FtsQLB and FtsA, which are also essential for their recruitment. However, the mechanism of activation of FtsWI by FtsN is not clear. Here, we identify a region of FtsL that plays a key role in the activation of FtsWI which we designate AWI (activation of FtsWI) and present evidence that FtsL acts through FtsI. Our results suggest that FtsN switches FtsQLB from a recruitment complex to an activator with FtsL interacting with FtsI to activate FtsW. Since FtsQLB and FtsWI are widely conserved in bacteria, this mechanism is likely to be also widely conserved. IMPORTANCE A critical step in bacterial cytokinesis is the activation of septal peptidoglycan synthesis at the Z ring. Although FtsN is the trigger and acts through FtsQLB and FtsA to activate FtsWI the mechanism is unclear. Here, we find an essential role for FtsL in activating septal peptidoglycan (PG) synthesis and find that it acts on FtsI. Our results suggest a model where FtsWI is recruited in an inactive form by FtsQLB, and upon the arrival of FtsN, FtsQLB undergoes a conformational change so that a region of FtsL, which we designate the AWI domain, becomes available to interact with FtsI and activate the FtsWI complex. This mechanism for activation of the divisome has similarities to the activation of the elongasome and is likely to be widely conserved in bacteria.

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.

The synthesis of a peptidoglycan septum is a fundamental part of bacterial fission and is driven by a multiprotein dynamic complex called the divisome. FtsW and FtsI are essential proteins that synthesize the peptidoglycan septum and are controlled by the regulatory FtsBLQ subcomplex and the activator FtsN. However, their mode of regulation has not yet been uncovered in detail. Understanding this process in detail may enable the development of new compounds to combat the rise in antibiotic resistance. In this review, recent data on the regulation of septal peptidoglycan synthesis is summarized and discussed. Based on structural models and the collected data, multiple putative interactions within FtsWI and with regulators are uncovered. This elaborates on and supports an earlier proposed model that describes active and inactive conformations of the septal peptidoglycan synthesis complex that are stabilized by these interactions. Furthermore, a new model on the spatial organization of the newly synthesized peptidoglycan and the synthesis complex is presented. Overall, the updated model proposes a balance between several allosteric interactions that determine the state of septal peptidoglycan synthesis.

Cell division in bacteria is mediated by a multiprotein assembly called the divisome. A major function of this machinery is the synthesis of the peptidoglycan (PG) cell wall that caps the daughter poles and prevents osmotic lysis of the newborn cells. Recent studies have implicated a complex of FtsW and FtsI (FtsWI) as the essential PG synthase within the divisome; however, how PG polymerization by this synthase is regulated and coordinated with other activities within the machinery is not well understood. Previous results have implicated a conserved subcomplex of division proteins composed of FtsQ, FtsL, and FtsB (FtsQLB) in the regulation of FtsWI, but whether these proteins act directly as positive or negative regulators of the synthase has been unclear. To address this question, we purified a five-member Pseudomonas aeruginosa division complex consisting of FtsQLB-FtsWI. The PG polymerase activity of this complex was found to be greatly stimulated relative to FtsWI alone. Purification of complexes lacking individual components indicated that FtsL and FtsB are sufficient for FtsW activation. Furthermore, support for this activity being important for the cellular function of FtsQLB was provided by the identification of two division-defective variants of FtsL that still form normal FtsQLB-FtsWI complexes but fail to activate PG synthesis. Thus, our results indicate that the conserved FtsQLB complex is a direct activator of PG polymerization by the FtsWI synthase and thereby define an essential regulatory step in the process of bacterial cell division. [ABSTRACT FROM AUTHOR]