scholarly journals Metabolic Processing of Selenium-based Bioisostere of meso-diaminopimelic Acid in Live Bacteria

2021 ◽  
Author(s):  
Alexis J Apostolos ◽  
Thameez M Koyasseril-Yehiya ◽  
Carolina Santamaria ◽  
José Rogério A Silva ◽  
Jerônimo Lameira ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Turgor Pressure ◽  
Primary Component ◽  
Cellular Growth ◽  
Diaminopimelic Acid ◽  
Common Component ◽  
Growth And Survival ◽  
Native Cell ◽  
Live Bacteria

The bacterial cell wall supports cell shape and prevents lysis due to internal turgor pressure. A primary component of all known bacterial cell walls is the peptidoglycan (PG) layer, which is comprised of repeating units of sugars connected to short and unusual peptides. The various steps within PG biosynthesis are often the target of antibiotics as they are essential for cellular growth and survival. Synthetic mimics of PG have proven to be indispensable tools to study bacterial cell growth and remodeling. Yet, a common component of PG, meso-diaminopimelic acid (m-DAP) at the third position of the stem peptide, remains challenging to build synthetically and is not commercially available. Here, we describe the synthesis and metabolic processing of a selenium-based bioisostere of a m-DAP analogue, selenolanthionine. We show that selenolanthionine is installed within the PG of live bacteria by the native cell wall crosslinking machinery in several mycobacteria species. We envision that this probe will supplement the current methods available for investigating PG crosslinking in m-DAP containing organisms.

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 Immobilization of Phosphatidylserine by Ethanol and Lysozyme on the Cell Surface for Evaluation of Apoptosis-Like Decay in Activated-Sludge Bacteria

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Ben Chen ◽  
Yasi Zhao ◽  
Zemin Li ◽  
Jianxin Pan ◽  
Haizhen Wu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Activated Sludge ◽  
Bacterial Cell ◽  
Cell Membranes ◽  
Bacterial Species ◽  
Accurate Determination ◽  
Bacterial Cells ◽  
Soil Matrix ◽  
Common Component ◽  
Bacterial Decay

ABSTRACT Accurate determination of microbial viability can be crucial in microbe-dominated biosystems. However, the identification of metabolic decay in bacterial cells can be elaborate and difficult. We sought to identify apoptosis-like bacterial processes by using annexin V-fluorescein isothiocyanate (FITC) (AVF), a probe typically used to stain phosphatidylserine (PS) on exposed cell membranes. The bacterial cell wall provides a barrier that is responsible for low efficiency of direct PS staining of decayed bacterial cells. This can be overcome by pretreatment of the bacteria with 70% ethanol, which fixates the bacteria and preserves the PS status, combined with lysozyme treatment to hydrolyze the cell wall. That treatment improved the efficiency of AVF staining considerably, as shown for pure strains of an Ochrobactrum sp. and a Micrococcus sp. Using this method, decayed bacterial cells (induced by starvation) were more strongly stained, indicating externalization of PS to a greater extent than seen for cells harvested at logarithmic growth. A multispecies microbial sludge was artificially decayed by heat treatment or alternating anoxic-oxic treatment, which also induced increased AVF staining, again presumably via decay-related PS externalization. The method developed proved to be efficient for identification of bacterial decay and has potential for the evaluation of multispecies bacterial samples from sources like soil matrix, bioaerosol, and activated sludge. IMPORTANCE Since the externalization of phosphatidylserine (PS) is considered a crucial characteristic of apoptosis, we sought to identify apoptosis-like decay in bacterial cells by PS staining using AVF. We show that this is possible, provided the bacteria are pretreated with ethanol plus lysozyme to remove a physical staining barrier and preserve the original, decay-related externalization of PS. Our work suggests that PS externalization occurs in starved bacteria and this can be quantified with AVF staining, providing a measure of bacterial decay. Since PS is the common component of the lipid bilayer in bacterial cell membranes, this approach also has potential for evaluation of cell decay of other bacterial species.

IN VITRO METABOLISM OF BACTERIAL CELL WALL BY RUMEN PROTOZOA

1984 ◽  
Vol 64 (5) ◽  
pp. 18-19 ◽  
Author(s):  
ANNA M. DENHOLM ◽  
J. R. LING
Keyword(s):  
Cell Wall ◽  
Bacillus Megaterium ◽  
Bacterial Cell ◽  
Bacterial Cell Wall ◽  
Diaminopimelic Acid ◽  
Pimelic Acid ◽  
Rumen Bacteria ◽  
Rumen Protozoa ◽  
Radiolabeled Compounds

Bacillus megaterium GW1, radiolabeled with (DL + meso)-2,6-diamino[G–3H]pimelic acid ([3H]A2pm), was incubated with mixed rumen protozoa, mixed rumen bacteria or alone (control). The protozoa released a large number of radiolabeled compounds into the medium; many of these were peptides. This activity was not found in either rumen bacteria or control incubations. Key words: Protozoa, bacterial cell wall, (DL + meso)-2,6-diaminopimelic acid

scholarly journals The importance of being cross-linked for the bacterial cell wall

2019 ◽  
Author(s):  
Garima Rani ◽  
Issan Patri
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Turgor Pressure ◽  
Length Distribution ◽  
Peptide Bond ◽  
Vital Role ◽  
Bacterial Cell Wall ◽  
Cross Linking ◽  
Strand Length

AbstractThe bacterial cell wall is primarily composed of a mesh of stiff glycan strands cross-linked by peptide bridges and is essential for safeguarding the cell. The structure of the cell wall has to be stiff enough to bear the high turgor pressure and sufficiently tough to ensure protection against failure. Here we explore the role of various design features of the cell in enhancing the toughness of the cell wall. We explain how the glycan strand length distribution and the degree of cross-linking can play a vital role in ensuring that the cell wall offers sufficient resistance to propagation of cracks. We suggest a possible mechanism by which peptide bond hydrolysis can also help mitigate this risk of failure. We also study the reinforcing effect of MreB on the cell wall and conclude that the cross-linked structure of the cell wall plays the more important role in safeguarding against mechanical failure due to cracking.

scholarly journals Facile Synthesis and Metabolic Incorporation of m-DAP Bioisosteres Into Cell Walls of Live Bacteria

2020 ◽  
Author(s):  
Alexis J. Apostolos ◽  
Julia M. Nelson ◽  
Marcos M. Pires
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Cell Walls ◽  
Drug Targets ◽  
Solid Phase ◽  
Bacterial Species ◽  
Building Blocks ◽  
Structural Features ◽  
Diaminopimelic Acid ◽  
Bacterial Cells

AbstractBacterial cell walls contain peptidoglycan (PG), a scaffold that provides proper rigidity to resist lysis from internal osmotic pressure and a barrier to protect cells against external stressors. It consists of repeating sugar units with a linkage to a stem peptide that becomes highly crosslinked by cell wall transpeptidases (TP). Because it is an essential component of the bacterial cell, the PG biosynthetic machinery is often the target of antibiotics. For this reason, cellular probes that advance our understanding of PG biosynthesis and its maintenance can be powerful tools to reveal novel drug targets. While synthetic PG fragments containing L-Lysine in the 3rd position on the stem peptide are easier to access, those with meso-diaminopimelic acid (m-DAP) pose a severe synthetic challenge. Herein, we describe a solid phase synthetic scheme based on the widely available Fmoc-protected L-Cysteine building block to assemble meso-cystine (m-CYT), which mimics key structural features of m-DAP. To demonstrate proper mimicry of m-DAP, cell wall probes were synthesized with m-CYT in place of m-DAP and evaluated for their metabolic processing in live bacterial cells. We found that m-CYT-based cell wall probes were properly processed by TPs in various bacterial species that endogenously contain m-DAP in their PG. We anticipate that this strategy, which is based on the use of inexpensive and commercially available building blocks, can be widely adopted to provide greater accessibility of PG mimics for m-DAP containing organisms.

scholarly journals Viral lysis of bacteria: an important source of dissolved amino acids and cell wall compounds

2006 ◽  
Vol 86 (3) ◽  
pp. 605-612 ◽  
Author(s):  
Mathias Middelboe ◽  
Niels O.G. Jørgensen
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Organic Matter ◽  
Organic Carbon ◽  
Bacterial Cell ◽  
Bacterial Cell Wall ◽  
Diaminopimelic Acid ◽  
Wall Material ◽  
Cell Wall Material ◽  
Viral Lysis

Viral infection of bacteria causes release of dissolved organic matter (DOM), which is available for bacterial uptake. In aquatic environments, this virus-mediated transformation of living cells into dissolved and colloidal organic matter may be a quantitatively important process in the pelagic recycling of carbon and nutrients, but little is known about the amount, composition, or bioavailability of viral lysates. By using a model system of a marine bacterium (Cellulophaga sp.) and a virus specific to this bacterium, the present study provides a first quantification of the input of dissolved free and combined amino acids (DFAA and DCAA) and bacterial cell wall compounds following viral lysis. The DCAA constituted 51–86% of the total virus-mediated organic carbon release of 1087–1825 μg C l−1 (estimated biomass of the lysed bacteria), whereas DFAA and glucosamine each accounted for 2–3% of total lysate-C. The viral particles themselves constituted 4–6% of the released organic carbon, and altogether, the applied analyses thus identified 53–92% of the released lysates. Approximately 12% of the identified compounds were derived from bacterial cell wall peptidoglycan, including various D-isomers of DFAA and DCAA, glucosamine and diaminopimelic acid (DAPA). Although a portion of this cell wall material may have entered the pool of refractory material, a significant fraction of some peptidoglycan-derived components, e.g. 83% of the released D-DFAA, were removed from the dissolved phase during the last part of the incubations, suggesting that part of the cell wall material were utilized by the developing virus-resistant Cellulophaga population. Therefore, we suggest that virus-mediated DOM is a source of a variety of organic compounds, which contribute significantly to the pool of rapidly recycling material in the ocean.

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