scholarly journals FtsN activates septal cell wall synthesis by forming a processive complex with the septum-specific peptidoglycan synthase in E. coli

2021 ◽  
Author(s):  
Zhixin Lyu ◽  
Atsushi Yahashiri ◽  
Xinxing Yang ◽  
Joshua W McCausland ◽  
Gabriela M Kaus ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Single Molecule ◽  
Spatial Organization ◽  
Genetic Manipulation ◽  
Cell Wall Synthesis ◽  
Single Population ◽  
E Coli ◽  
Ftsz Ring ◽  
Synthesis Activity

The FtsN protein of Escherichia coli and other proteobacteria is an essential and highly conserved bitopic membrane protein that triggers the inward synthesis of septal peptidoglycan (sPG) during cell division. Previous work has shown that the activation of sPG synthesis by FtsN involves a series of interactions of FtsN with other divisome proteins and the cell wall. Precisely how FtsN achieves this role is unclear, but a recent study has shown that FtsN promotes the relocation of the essential sPG synthase FtsWI from an FtsZ-associated track (where FtsWI is inactive) to an sPG-track (where FtsWI engages in sPG synthesis). Whether FtsN works by displacing FtsWI from the Z-track or capturing/retaining FtsWI on the sPG-track is not known. Here we use single-molecule imaging and genetic manipulation to investigate the organization and dynamics of FtsN at the septum and how they are coupled to sPG synthesis activity. We found that FtsN exhibits a spatial organization and dynamics distinct from those of the FtsZ-ring. Single FtsN molecules move processively as a single population with a speed of ~ 9 nm s-1, similar to the speed of active FtsWI molecules on the sPG-track, but significantly different from the ~ 30 nm s-1 speed of inactive FtsWI molecules on the FtsZ-track. Furthermore, the processive movement of FtsN is independent of FtsZ's treadmilling dynamics but driven exclusively by active sPG synthesis. Importantly, only the essential domain of FtsN, a three-helix bundle in the periplasm, is required to maintain the processive complex containing both FtsWI and FtsN on the sPG-track. We conclude that FtsN activates sPG synthesis by forming a processive synthesis complex with FtsWI exclusively on the sPG-track. These findings favor a model in which FtsN captures or retains FtsWI on the sPG-track rather than one in which FtsN actively displaces FtsWI from the Z-track.

scholarly journals Interactions between Penicillin-Binding Proteins (PBPs) and Two Novel Classes of PBP Inhibitors, Arylalkylidene Rhodanines and Arylalkylidene Iminothiazolidin-4-ones

2004 ◽  
Vol 48 (3) ◽  
pp. 961-969 ◽  
Author(s):  
Astrid Zervosen ◽  
Wei-Ping Lu ◽  
Zhouliang Chen ◽  
Ronald E. White ◽  
Thomas P. Demuth ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Wall Synthesis ◽  
Bacterial Strains ◽  
Gram Negative ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Peptidoglycan Biosynthesis ◽  
Vancomycin Resistant ◽  
Inhibitory Activities

ABSTRACT Several non-β-lactam compounds were active against various gram-positive and gram-negative bacterial strains. The MICs of arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones were lower than those of ampicillin and cefotaxime for methicillin-resistant Staphylococcus aureus MI339 and vancomycin-resistant Enterococcus faecium EF12. Several compounds were found to inhibit the cell wall synthesis of S. aureus and the last two steps of peptidoglycan biosynthesis catalyzed by ether-treated cells of Escherichia coli or cell wall membrane preparations of Bacillus megaterium. The effects of the arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-one derivatives on E. coli PBP 3 and PBP 5, Streptococcus pneumoniae PBP 2xS (PBP 2x from a penicillin-sensitive strain) and PBP 2xR (PBP 2x from a penicillin-resistant strain), low-affinity PBP 2a of S. aureus, and the Actinomadura sp. strain R39 and Streptomyces sp. strain R61 dd-peptidases were studied. Some of the compounds exhibited inhibitory activities in the 10 to 100 μM concentration range. The inhibition of PBP 2xS by several of them appeared to be noncompetitive. The dissociation constant for the best inhibitor (Ki = 10 μM) was not influenced by the presence of the substrate.

scholarly journals Regeneration of Escherichia coli Giant Protoplasts to Their Original Form

Life ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 24 ◽  
Author(s):  
Kazuhito V. Tabata ◽  
Takao Sogo ◽  
Yoshiki Moriizumi ◽  
Hiroyuki Noji
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Colony Formation ◽  
Cell Walls ◽  
Microscopic Investigation ◽  
Original Form ◽  
Cell Wall Synthesis ◽  
E Coli ◽  
The Relationship

The spheroplasts and protoplasts of cell wall-deficient (CWD) bacteria are able to revert to their original cellular morphologies through the regeneration of their cell walls. However, whether this is true for giant protoplasts (GPs), which can be as large as 10 μm in diameter, is unknown. GPs can be prepared from various bacteria, including Escherichia coli and Bacillus subtilis, and also from fungi, through culture in the presence of inhibitors for cell wall synthesis or mitosis. In this report, we prepared GPs from E. coli and showed that they can return to rod-shaped bacterium, and that they are capable of colony formation. Microscopic investigation revealed that the regeneration process took place through a variety of morphological pathways. We also report the relationship between GP division and GP volume. Finally, we show that FtsZ is crucial for GP division. These results indicate that E. coli is a highly robust organism that can regenerate its original form from an irregular state, such as GP.

scholarly journals Deficiency in l-Serine Deaminase Interferes with One-Carbon Metabolism and Cell Wall Synthesis in Escherichia coli K-12

2010 ◽  
Vol 192 (20) ◽  
pp. 5515-5525 ◽  
Author(s):  
Xiao Zhang ◽  
Ziad W. El-Hajj ◽  
Elaine Newman
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Division ◽  
Amino Acid ◽  
Cell Wall Synthesis ◽  
E Coli ◽  
Amino Acid Mixtures ◽  
Glycine Cleavage ◽  
High Level ◽  
K 12

ABSTRACT Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes l-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S l-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of l-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C1 units and interferes with cell wall synthesis. We suggest that at high concentrations, l-serine decreases synthesis of UDP-N-acetylmuramate-l-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high l-serine is overcome in several ways: by a large concentration of l-alanine, by overproducing MurC together with a low concentration of l-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.

Sites of Adsorption of the Bacteriophages T3 and T5 on the Wall of Escherichia Coli B.

1967 ◽  
Vol 25 ◽  
pp. 96-97
Author(s):  
Manfred E. Bayer
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Electron Microscope ◽  
Uranyl Acetate ◽  
E Coli ◽  
Receptor Sites ◽  
Rigid Cell Wall ◽  
Host Cell Surface ◽  
Bacterial Viruses ◽  
Escherichia Coli B

Bacterial viruses adsorb specifically to receptors on the host cell surface. Although the chemical composition of some of the cell wall receptors for bacteriophages of the T-series has been described and the number of receptor sites has been estimated to be 150 to 300 per E. coli cell, the localization of the sites on the bacterial wall has been unknown.When logarithmically growing cells of E. coli are transferred into a medium containing 20% sucrose, the cells plasmolize: the protoplast shrinks and becomes separated from the somewhat rigid cell wall. When these cells are fixed in 8% Formaldehyde, post-fixed in OsO4/uranyl acetate, embedded in Vestopal W, then cut in an ultramicrotome and observed with the electron microscope, the separation of protoplast and wall becomes clearly visible, (Fig. 1, 2). At a number of locations however, the protoplasmic membrane adheres to the wall even under the considerable pull of the shrinking protoplast. Thus numerous connecting bridges are maintained between protoplast and cell wall. Estimations of the total number of such wall/membrane associations yield a number of about 300 per cell.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

1999 ◽  
Vol 181 (13) ◽  
pp. 3981-3993 ◽  
Author(s):  
Sylvia A. Denome ◽  
Pamela K. Elf ◽  
Thomas A. Henderson ◽  
David E. Nelson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Binding Proteins ◽  
Kanamycin Resistance ◽  
Open Reading Frames ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Peptidoglycan Biosynthesis ◽  
Kanamycin Resistance Cassette ◽  
Genes Encoding

ABSTRACT The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

scholarly journals Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment

2020 ◽  
Vol 203 (2) ◽  
pp. e00463-20
Author(s):  
Amit Bhambhani ◽  
Isabella Iadicicco ◽  
Jules Lee ◽  
Syed Ahmed ◽  
Max Belfatto ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Gene Product ◽  
Fluorescent Protein ◽  
Lytic Bacteriophage ◽  
Cell Wall Synthesis ◽  
Content Type ◽  
Ftsz Ring ◽  
Green Fluorescent

ABSTRACTPrevious work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

Analysis of the Crushing Effect about Ultrasound on E. coli and B. subtilis

2012 ◽  
Vol 260-261 ◽  
pp. 1017-1021
Author(s):  
Xin Ying Wang ◽  
Yong Tao Liu ◽  
Min Hui ◽  
Ji Fei Xu
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Bacterial Cell ◽  
Logarithmic Phase ◽  
Growth Stages ◽  
Bacterial Cells ◽  
E Coli ◽  
Different Growth Stages ◽  
Main Factor

Escherichia coli and Bacillus subtilis as objects of the study, ultrasonic fragmentation acted on the bacterial cells in different growth stages, results showed that, it’s similar to the crushing effect of ultrasound on E. coli and B. subtilis cells of different growth stages, the highest crushing rate in the logarithmic phase, reached to 95.8% and 94.3% respectively, the crushing rate of adjustment phase is lowest, maintained at around 60%, the crushing rate stability cell was centered, which can be achieved 90%. The structure of the bacterial cell wall didn’t the main factor to decide the ultrasonic fragmentation effect, but different growth periods of bacterial cells did the determinant.

scholarly journals The Transcriptional Factors MurR and Catabolite Activator Protein Regulate N-Acetylmuramic Acid Catabolism in Escherichia coli

2008 ◽  
Vol 190 (20) ◽  
pp. 6598-6608 ◽  
Author(s):  
Tina Jaeger ◽  
Christoph Mayer
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Sole Source ◽  
Lag Phase ◽  
Face To Face ◽  
E Coli ◽  
Catabolite Activator Protein ◽  
Activator Protein ◽  
High Level ◽  
First Time

ABSTRACT The MurNAc etherase MurQ of Escherichia coli is essential for the catabolism of the bacterial cell wall sugar N-acetylmuramic acid (MurNAc) obtained either from the environment or from the endogenous cell wall (i.e., recycling). High-level expression of murQ is required for growth on MurNAc as the sole source of carbon and energy, whereas constitutive low-level expression of murQ is sufficient for the recycling of peptidoglycan fragments continuously released from the cell wall during growth of the bacteria. Here we characterize for the first time the expression of murQ and its regulation by MurR, a member of the poorly characterized RpiR/AlsR family of transcriptional regulators. Deleting murR abolished the extensive lag phase observed for E. coli grown on MurNAc and enhanced murQ transcription some 20-fold. MurR forms a stable multimer (most likely a tetramer) and binds to two adjacent inverted repeats within an operator region. In this way MurR represses transcription from the murQ promoter and also interferes with its own transcription. MurNAc-6-phosphate, the substrate of MurQ, was identified as a specific inducer that weakens binding of MurR to the operator. Moreover, murQ transcription depends on the activation by cyclic AMP (cAMP)-catabolite activator protein (CAP) bound to a class I site upstream of the murQ promoter. murR and murQ are divergently orientated and expressed from nonoverlapping face-to-face (convergent) promoters, yielding transcripts that are complementary at their 5′ ends. As a consequence of this unusual promoter arrangement, cAMP-CAP also affects murR transcription, presumably by acting as a roadblock for RNA polymerase.

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