Controlled lysis of bacterial cells utilizing mutants with defective synthesis of D-alanine

1988 ◽  
Vol 34 (3) ◽  
pp. 256-261 ◽  
Author(s):  
Michael P. Heaton ◽  
Robert B. Johnston ◽  
Thomas L. Thompson
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Walls ◽  
Alanine Racemase ◽  
Growth Media ◽  
Bacterial Cells ◽  
Cell Wall Formation ◽  
Fermentation Processes ◽  
Intracellular Enzymes ◽  
Dense Cell

An alanine racemase (EC 5.1.1.1) mutant (Dal−) of Bacillus subtilis required small amounts of D-alanine to synthesize an osmotically stable cell wall in certain growth media. Investigation of the conditions which caused lysis in hypotonic media revealed that in addition to complex media, such as nutrient broth and acid-hydrolyzed casein, glycine inhibited stable cell wall formation. D-Alanine prevented the glycine inhibition. Up to 99% lysis occurred in both dilute and dense cell suspensions (optical densities up to 110) within 2.5 h after adding 1% glycine to late log phase cultures. Intracellular enzymes recovered from the lysate were as active as those from lysozyme-disrupted cells. No amino acid tested other than glycine induced lysis. Dal− mutants can be used for controlled lysis of bacterial cells to facilitate the isolation of normal intracellular constituents and bioengineered products from fermentation processes. Cell walls of most bacteria contain D-alanine; thus, this strategy should be applicable to a wide variety of microorganisms.

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.

Sequential Changes Associated with Cell Wall Formation and Fusion in the Vascular Cambium

IAWA Journal ◽  
1981 ◽  
Vol 2 (4) ◽  
pp. 151-162 ◽  
Author(s):  
A.M. Catesson ◽  
J.C. Roland
Keyword(s):  
Cell Wall ◽  
Electron Microscope ◽  
Cell Walls ◽  
Microscope Level ◽  
Cell Wall Formation ◽  
Characteristic Sequence ◽  
Electron Microscope Level ◽  
Starting Point ◽  
Cell Wall Development ◽  
Cambial Zone

Cytochemical techniques and mild extractions were used at the electron microscope level for the study of the cambial zone of several hardwoods and one softwood. The maturation processes of the primary radial and tangential cell walls involve a progressive disappearance of their initial heterogeneity. The buttress-like zone joining these walls appears to be the starting point for a characteristic sequence of changes and intra-wall rearrangement. Topochemical results have suggested an alternative to the 'emboxing concept' of cell wall development.

scholarly journals Exocytosis of the silicified cell wall of diatoms involves extensive membrane disintegration

2021 ◽  
Author(s):  
Diede de Haan ◽  
Hadas Peled-Zehavi ◽  
Yoseph Addadi ◽  
Oz Ben Joseph ◽  
Lior Aram ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Membrane Dynamics ◽  
Cell Wall Formation ◽  
Mineral Phase ◽  
Wall Formation ◽  
Unicellular Algae ◽  
Silica Cell ◽  
Membrane Bound ◽  
Organelle Membrane

Diatoms are unicellular algae that are characterized by their silica cell walls. The silica elements form intracellularly in a membrane-bound organelle, and are exocytosed after completion. How diatoms maintain membrane homeostasis during the exocytosis of these large and rigid silica elements is a long-standing enigma. We studied membrane dynamics during cell wall formation and exocytosis in the diatom Stephanopyxis turris, using live-cell confocal microscopy and advanced electron microscopy. Our results provide detailed information on the ultrastructure and dynamics of the silicification process, showing that during cell wall formation, the organelle membranes tightly enclose the mineral phase, creating a precise mold of the delicate geometrical patterns. Surprisingly, during exocytosis of the mature silica elements, the proximal organelle membrane becomes the new plasma membrane, and the distal membranes gradually disintegrate into the extracellular space without any noticeable endocytic retrieval or extracellular repurposing. These observations suggest that diatoms evolved an extraordinary exocytosis mechanism in order to secrete their cell wall elements.

scholarly journals Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

2021 ◽  
Vol 17 (3) ◽  
pp. e1009468
Author(s):  
Joshua A. F. Sutton ◽  
Oliver T. Carnell ◽  
Lucia Lafage ◽  
Joe Gray ◽  
Jacob Biboy ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Walls ◽  
Ex Vivo ◽  
Cell Wall Structure ◽  
Wall Structure ◽  
Bacterial Cells ◽  
Human Macrophages ◽  
Structure And Dynamics

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which correlated with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.

Ultrastructure of a palmelloid-forming strain of Chlamydomonas eugametos

1978 ◽  
Vol 56 (19) ◽  
pp. 2348-2356 ◽  
Author(s):  
Kazuo Nakamura ◽  
Douglas F. Bray ◽  
Emile B. Wagenaar
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Transitional Region ◽  
Chloroplatinic Acid ◽  
Cell Wall Formation ◽  
Body Structure ◽  
Wild Type ◽  
Adhesive Properties ◽  
Chlamydomonas Eugametos ◽  
Unicellular Green Alga

A palmelloid-forming mutant of the unicellular green alga Chlamydomonas eugametos has been studied ultrastructurally. The repetition, within the palmelloid envelope, of four-celled groups surrounded by wall layers suggests that normal asexual cytokinesis occurs but successive cell generations are unable to separate. Individual palmelloid cells are smaller than wild-type cells and possess flagella which are short (1–2 μm) and occasionally bulbous at the tip but appear normal with regard to internal microtubular, transitional region, and basal body structure. The association of granules with the outer surface of the palmelloid envelope and the tendency of palmelloids to form large aggregates in culture indicate a change in the adhesive properties of the cell walls of this mutant. A comparison of the ultrastructure of mutant palmelloids with previously described chloroplatinic-acid-induced palmelloids shows that the two types differ in both flagellar development and in the extent of cell wall formation.

scholarly journals SaccuFlow: High-throughput Analysis Platform to Investigate Bacterial Cell Wall Interactions

2021 ◽  
Author(s):  
Alexis J Apostolos ◽  
Noel J Ferraro ◽  
Brianna E Dalesandro ◽  
Marcos Pires
Keyword(s):  
Cell Wall ◽  
High Throughput ◽  
Bacterial Cell ◽  
Cell Walls ◽  
Turgor Pressure ◽  
Principal Component ◽  
Bacterial Cells ◽  
High Throughput Analysis ◽  
Peptidoglycan Biosynthesis ◽  
Analysis Platform

Bacterial cell walls are formidable barriers that protect bacterial cells against external insults and oppose internal turgor pressure. While cell wall composition is variable across species, peptidoglycan is the principal component of all cell walls. Peptidoglycan is a mesh-like scaffold composed of crosslinked strands that can be heavily decorated with anchored proteins. The biosynthesis and remodeling of peptidoglycan must be tightly regulated by cells because disruption to this biomacromolecule is lethal. This essentiality is exploited by the human innate immune system in resisting colonization and by a number of clinically relevant antibiotics that target peptidoglycan biosynthesis. Evaluation of molecules or proteins that interact with peptidoglycan can be a complicated and, typically, qualitative effort. We have developed a novel assay platform (SaccuFlow) that preserves the native structure of bacterial peptidoglycan and is compatible with high-throughput flow cytometry analysis. We show that the assay is facile and versatile as demonstrated by its compatibility with sacculi from Gram-positive bacteria, Gram-negative bacteria, and mycobacteria. Finally, we highlight the utility of this assay to assess the activity of sortase from Staphylococcus aureus against potential anti-virulence agents.

scholarly journals Remodeling of Crossbridges Controls Peptidoglycan Cross-linking Levels in Bacterial Cell Walls

2019 ◽  
Author(s):  
Sean E. Pidgeon ◽  
Alexis J. Apostolos ◽  
Marcos M. Pires
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Bacterial Species ◽  
Physiological Role ◽  
Primary Component ◽  
Acyl Donor ◽  
Bacterial Cells ◽  
Structural Variations ◽  
Acyl Acceptor ◽  
Donor And Acceptor

ABSTRACTCell walls are barriers found in almost all known bacterial cells. These structures establish a controlled interface between the external environment and vital cellular components. A primary component of cell wall is a highly crosslinked matrix called peptidoglycan (PG). PG crosslinking, carried out by transglycosylases and transpeptidases, is necessary for proper cell wall assembly. Transpeptidases, targets of β-lactam antibiotics, stitch together two neighboring PG stem peptides (acyl-donor and acyl-acceptor strands). We recently described a novel class of cellular PG probes that were processed exclusively as acyl-donor strands. Herein, we have accessed the other half of the transpeptidase reaction by developing probes that are processed exclusively as acyl-acceptor strands. The critical nature of the crossbridge on the PG peptide was demonstrated in live bacterial cells and surprising promiscuity in crossbridge primary sequence was found in various bacterial species. Additionally, acyl-acceptor probes provided insight into how chemical remodeling of the PG crossbridge (e.g., amidation) can modulate crosslinking levels, thus establishing a physiological role of PG structural variations. Together, the acyl-donor and -acceptor probes will provide a versatile platform to interrogate PG crosslinking in physiologically relevant settings.SYNOPSIS TOC

scholarly journals Exo-β-N-acetylmuramidase NamZ of Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases

2021 ◽  
Author(s):  
Maraike Müller ◽  
Matthew Calvert ◽  
Isabel Hottmann ◽  
Robert Maria Kluj ◽  
Tim Teufel ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Structural Model ◽  
Specific Cell ◽  
Bacterial Cells ◽  
Growth Defects ◽  
Founding Member ◽  
Putative Active Site ◽  
Starvation Conditions

ABSTRACTEndo-β-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that potentially lyse bacterial cells. They catalyze an endo-lytic cleavage of the peptidoglycan, the structural component of the bacterial cell wall; i.e. they hydrolyze glycosidic N-acetylmuramic acid (MurNAc)-β-1,4-N-acetylglucosamine (GlcNAc)-bonds within the heteroglycan backbone of peptidoglycan. In contrast, little is known about exo-β-N-acetylmuramidases, catalyzing an exo-lytic cleavage of β-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We identified ybbC of B. subtilis, renamed namZ, as encoding the reported exo-β-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl β-MurNAc and the peptidoglycan-derived disaccharide MurNAc-β-1,4-GlcNAc. Together with the exo-β-N-acetylglucosaminidase NagZ and the exo-muramoyl-L-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. NamZ is a member of the DUF1343 protein family of unknown function and shows no significant sequence identity with known glycosidases. A structural model of NamZ revealed a putative active site located in a cleft within the interface of two subdomains, one of which constituting a Rossmann-fold-like domain, unusual for glycosidases. On this basis, we propose that NamZ represents the founding member of a novel family of peptidoglycan hexosaminidases, which is mainly present in the phylum Bacteroidetes and, less frequently, within Firmicutes (Bacilli, Clostridia), Actinobacteria and Gammaproteobacteria.

scholarly journals Cell-wall thickening in Bacillus subtilis. Comparison of thickened and normal walls

1970 ◽  
Vol 120 (1) ◽  
pp. 159-170 ◽  
Author(s):  
R. C. Hughes ◽  
P. J. Tanner ◽  
Elaine Stokes
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Walls ◽  
Teichoic Acid ◽  
Cellular Growth ◽  
Wall Thickening ◽  
Complete Medium ◽  
Bacillus Subtilis 168 ◽  
Normal Wall

1. Incubation of Bacillus subtilis 168 trp in a glucose–amino acids–salts medium lacking tryptophan leads to an inhibition of cellular growth without affecting cell-wall synthesis. The cell walls increased approximately two- to three-fold in thickness and at the same time the amount of mucopeptide in the cells measured chemically increased to about the same extent. 2. Synthesis of mucopeptide and teichoic acid as measured by the extent of incorporation of radioactivity continued linearly for approximately 1h and then stopped. No reason was found for the strictly limited synthesis of the wall polymers. 3. The initial rates of incorporation of [32P]Pi or [3H]alanine into teichoic acid and of 3H-labelled amino acids into mucopeptide were not appreciably inhibited by the addition of chloramphenicol to the glucose–amino acids–salts medium. 4. There was no selective turnover of the mucopeptide synthesized by the cells in a medium lacking tryptophan on resumption of growth in a complete medium. 5. Wall synthesis taking place during the thickening process was similar to normal wall synthesis proceeding in growing cells. Walls of different thicknesses prepared from cells incubated for various times in incomplete medium did not differ qualitatively in composition. The products of autolysis of thickened walls were isolated and the analyses indicated a close similarity in the details of their mucopeptide structure compared with the mucopeptide of cells growing in the exponential phase.

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