scholarly journals On the Architecture of the Gram-Negative Bacterial Murein Sacculus

2000 ◽  
Vol 182 (20) ◽  
pp. 5925-5930 ◽  
Author(s):  
David Pink ◽  
Jeremy Moeller ◽  
Bonnie Quinn ◽  
Manfred Jericho ◽  
Terry Beveridge
Keyword(s):  
Gram Negative Bacteria ◽  
Defect Structures ◽  
Waste Products ◽  
Gram Negative ◽  
Large Molecules ◽  
Glycan Chain ◽  
Pass Through ◽  
Murein Sacculus ◽  
Chain Lengths ◽  
Peptidoglycan Layer

ABSTRACT The peptidoglycan network of the murein sacculus must be porous so that nutrients, waste products, and secreted proteins can pass through. Using Escherichia coli and Pseudomonas aeruginosa as a baseline for gram-negative sacculi, the hole size distribution in the peptidoglycan network has been modeled by computer simulation to deduce the network's properties. By requiring that the distribution of glycan chain lengths predicted by the model be in accord with the distribution observed, we conclude that the holes are slits running essentially perpendicular to the local axis of the glycan chains (i.e., the slits run along the long axis of the cell). This result is in accord with previous permeability measurements of Beveridge and Jack and Demchik and Koch. We outline possible advantages that might accrue to the bacterium via this architecture and suggest ways in which such defect structures might be detected. Certainly, large molecules do penetrate the peptidoglycan layer of gram-negative bacteria, and the small slits that we suggest might be made larger by the bacterium.

scholarly journals The Rcs Phosphorelay Is a Cell Envelope Stress Response Activated by Peptidoglycan Stress and Contributes to Intrinsic Antibiotic Resistance

2008 ◽  
Vol 190 (6) ◽  
pp. 2065-2074 ◽  
Author(s):  
Mary E. Laubacher ◽  
Sarah E. Ades
Keyword(s):  
Outer Membrane ◽  
Single Molecule ◽  
Stress Responses ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Envelope Stress ◽  
Peptidoglycan Layer

ABSTRACTGram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identifyEscherichia colistress responses activated following inhibition of specific PBPs by the β-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance ofE. colito β-lactam antibiotics.

scholarly journals AmpN-AmpG Operon Is Essential for Expression of L1 and L2 β-Lactamases in Stenotrophomonas maltophilia

2010 ◽  
Vol 54 (6) ◽  
pp. 2583-2589 ◽  
Author(s):  
Yi-Wei Huang ◽  
Cheng-Wen Lin ◽  
Rouh-Mei Hu ◽  
Yu-Tzu Lin ◽  
Tung-Ching Chung ◽  
...  
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Gene Dosage ◽  
Gram Negative Bacteria ◽  
Beta Lactamase ◽  
Gene Dosage Effect ◽  
Gram Negative ◽  
E Coli ◽  
Murein Sacculus ◽  
Lactamase Gene ◽  
L1 And L2

ABSTRACT AmpG is an inner membrane permease which transports products of murein sacculus degradation from the periplasm into the cytosol in Gram-negative bacteria. This process is linked to induction of the chromosomal ampC beta-lactamase gene in some members of the Enterobacteriaceae and in Pseudomonas aeruginosa. In this study, the ampG homologue of Stenotrophomonas maltophilia KJ was analyzed. The ampG homologue and its upstream ampN gene form an operon and are cotranscribed under the control of the promoter P ampN. Expression from P ampN was found to be independent of β-lactam exposure and ampN and ampG products. A ΔampN allele exerted a polar effect on the expression of ampG and resulted in a phenotype of null β-lactamase inducibility. Complementation assays elucidated that an intact ampN-ampG operon is essential for β-lactamase induction. Consistent with ampG of Escherichia coli, the ampN-ampG operon of S. maltophilia did not exhibit a gene dosage effect on β-lactamase expression. The AmpG permease of E. coli could complement the β-lactamase inducibility of ampN or ampG mutants of S. maltophilia, indicating that both species have the same precursor of activator ligand(s) for β-lactamase induction.

scholarly journals An OregonGreen488-labelled d-amino acid for visualizing peptidoglycan by super-resolution STED nanoscopy

Microbiology ◽  
2020 ◽  
Vol 166 (12) ◽  
pp. 1129-1135 ◽  
Author(s):  
Bill Söderström ◽  
Alessandro Ruda ◽  
Göran Widmalm ◽  
Daniel O. Daley
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Chemical Synthesis ◽  
Super Resolution ◽  
Molecular Probes ◽  
Stimulated Emission ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Peptidoglycan Synthesis ◽  
Peptidoglycan Layer

Fluorescent d-amino acids (FDAAs) are molecular probes that are widely used for labelling the peptidoglycan layer of bacteria. When added to growing cells they are incorporated into the stem peptide by a transpeptidase reaction, allowing the timing and localization of peptidoglycan synthesis to be determined by fluorescence microscopy. Herein we describe the chemical synthesis of an OregonGreen488-labelled FDAA (OGDA). We also demonstrate that OGDA can be efficiently incorporated into the PG of Gram-positive and some Gram-negative bacteria, and imaged by super-resolution stimulated emission depletion (STED) nanoscopy at a resolution well below 100 nm.

scholarly journals Mechanistic aspects of maltotriose-conjugate translocation to the Gram-negative bacteria cytoplasm

2018 ◽  
Vol 2 (1) ◽  
pp. e201800242 ◽  
Author(s):  
Estelle Dumont ◽  
Julia Vergalli ◽  
Jelena Pajovic ◽  
Satya P Bhamidimarri ◽  
Koldo Morante ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Small Molecule ◽  
Single Channel ◽  
Efflux Pumps ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Gram Negative ◽  
Entry Pathway ◽  
Pass Through ◽  
Novel Antibiotics

Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic molecules. An approach to overcome this challenge is to hijack uptake pathways so that bacterial transporters shuttle the antibiotic to the cytoplasm. Here, we have characterized maltodextrin–fluorophore conjugates that can pass through both the outer and inner membranes mediated by components of theEscherichia colimaltose regulon. Single-channel electrophysiology recording demonstrated that the compounds permeate across the LamB channel leading to accumulation in the periplasm. We have also demonstrated that a maltotriose conjugate distributes into both the periplasm and cytoplasm. In the cytoplasm, the molecule activates the maltose regulon and triggers the expression of maltose binding protein in the periplasmic space indicating that the complete maltose entry pathway is induced. This maltotriose conjugate can (i) reach the periplasmic and cytoplasmic compartments to significant internal concentrations and (ii) auto-induce its own entry pathwayviathe activation of the maltose regulon, representing an interesting prototype to deliver molecules to the cytoplasm of Gram-negative bacteria.

scholarly journals Using a bacteriocin structure to engineer a phage lysin that targets Yersinia pestis

2012 ◽  
Vol 40 (6) ◽  
pp. 1503-1506 ◽  
Author(s):  
Petra Lukacik ◽  
Travis J. Barnard ◽  
Susan K. Buchanan
Keyword(s):  
Streptococcus Pneumoniae ◽  
Cell Surface ◽  
Yersinia Pestis ◽  
Outer Membrane ◽  
Bacterial Culture ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Phage Lysin ◽  
Phage Lysins ◽  
Peptidoglycan Layer

Purified phage lysins present an alternative to traditional antibiotics and work by hydrolysing peptidoglycan. Phage lysins have been developed against Gram-positive pathogens such as Bacillus anthracis and Streptococcus pneumoniae, where the peptidoglycan layer is exposed on the cell surface. Addition of the lysin to a bacterial culture results in rapid death of the organism. Gram-negative bacteria are resistant to phage lysins because they contain an outer membrane that protects the peptidoglycan from degradation. We solved crystal structures of a Yersinia pestis outer-membrane protein and the bacteriocin that targets it, which informed engineering of a bacterial–phage hybrid lysin that can be transported across the outer membrane to kill specific Gram-negative bacteria. This work provides a template for engineering phage lysins against a wide variety of bacterial pathogens.

scholarly journals Microbiota of gills and antibiotic susceptibility patterns of bacteria isolates from Clarias gariepinus in different holding facilities

2020 ◽  
Vol 18 (3) ◽  
pp. 119-128
Author(s):  
D.A. Adah ◽  
L Saidu ◽  
S.J. Oniye ◽  
S.A. Adah ◽  
O.B. Daodu ◽  
...  
Keyword(s):  
Clarias Gariepinus ◽  
Bacillus Species ◽  
Antibiotics Resistance ◽  
Aeromonas Species ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Waste Products ◽  
Gram Negative ◽  
Opportunistic Bacteria ◽  
Susceptibility Patterns

Gill is a key respiratory and excretory organ in fish as it provides oxygen need for survival and excretes waste products. However, gills can be infected with pathogenic and opportunistic bacteria leading to increasing fish morbidity and mortality. This study was carried out to isolate, estimate and identify bacteria on the gills of Clarias gariepinus reared in different holding facilities. The susceptibility patterns of the bacteria were also studied using 10 antibiotics commonly used in pisciculture in Nigeria. A total of 84 bacteria belonging to 12 genera were isolated from the gills of 75 Clarias gariepinus. Gram-negative bacteria isolated included Salmonella species (3.6%), Pseudomonas species (7.1%), Aeromonas species (2.4%), Escherichia coli (13.1%), Proteus species (11.9%) Klebsiella species (3.6%), Citrobacter species (4.8%), and Shigella species (3.6%). Gram-positive Corynebacterium species (3.6 %), Staphylococcus species (20.3%), Bacillus species (19.0%) and Streptococcus species (7.1%) were also isolated. The result showed varying bacteria species when considering the different holding facilities. Greater than 50 % of Gram-positive and Gram-negative bacteria isolated were resistant to 5 and 6 different antibiotics respectively while greater than 80 % of all the bacteria were resistant to ≥ 3 antibiotics. The presence of these bacteria in fish predict subsequent impediment in pisciculture and may lead to socioeconomic losses, environmental contaminations and high public health risk. This study calls for concern and an urgent intervention on antibiotic stewardship among fish farmers. Keywords: Antibiotics resistance, Clarias gariepinus, Fish farms, Gills microbiota, Kaduna state

scholarly journals Peptidoglycan Remodeling EnablesEscherichiacoliTo Survive Severe Outer Membrane Assembly Defect

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Niccolò Morè ◽  
Alessandra M. Martorana ◽  
Jacob Biboy ◽  
Christian Otten ◽  
Matthias Winkle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Cross Links ◽  
Peptidoglycan Layer

ABSTRACTGram-negative bacteria have a tripartite cell envelope with the cytoplasmic membrane (CM), a stress-bearing peptidoglycan (PG) layer, and the asymmetric outer membrane (OM) containing lipopolysaccharide (LPS) in the outer leaflet. Cells must tightly coordinate the growth of their complex envelope to maintain cellular integrity and OM permeability barrier function. The biogenesis of PG and LPS relies on specialized macromolecular complexes that span the entire envelope. In this work, we show thatEscherichia colicells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG. This PG remodeling program relies mainly on the activities of the stress response LDT, LdtD, together with the major PG synthase PBP1B, its cognate activator LpoB, and the carboxypeptidase PBP6a. Our data support a model according to which these proteins cooperate to strengthen the PG in response to defective OM synthesis.IMPORTANCEIn Gram-negative bacteria, the outer membrane protects the cell against many toxic molecules, and the peptidoglycan layer provides protection against osmotic challenges, allowing bacterial cells to survive in changing environments. Maintaining cell envelope integrity is therefore a question of life or death for a bacterial cell. Here we show thatEscherichia colicells activate the LD-transpeptidase LdtD to introduce 3-3 cross-links in the peptidoglycan layer when the integrity of the outer membrane is compromised, and this response is required to avoid cell lysis. This peptidoglycan remodeling program is a strategy to increase the overall robustness of the bacterial cell envelope in response to defects in the outer membrane.

scholarly journals Unique Regions of the Polysaccharide Copolymerase Wzz2fromPseudomonas aeruginosaAre Essential for O-Specific Antigen Chain Length Control

2019 ◽  
Vol 201 (15) ◽  
Author(s):  
Steven M. Huszczynski ◽  
Chelsea Coumoundouros ◽  
Phi Pham ◽  
Joseph S. Lam ◽  
Cezar M. Khursigara
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Chain Length ◽  
Virulence Factor ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
O Antigen ◽  
Chain Lengths ◽  
Insight Into

ABSTRACTThe outer leaflet of the outer membrane of nearly all Gram-negative bacteria contains lipopolysaccharide (LPS). The distal end of LPS may be capped with O antigen, a long polysaccharide that can range from a few to hundreds of sugars in length. The chain length of the polysaccharide has many implications for bacterial survival and consequently is tightly controlled. In the Wzx/Wzy-dependent route of O antigen synthesis, one or more Wzz proteins determine the chain length via an unknown mechanism. To gain insight into this mechanism, we identified and characterized important regions of two Wzz proteins inPseudomonas aeruginosaserotype O13, which confer the production of “long” (Wzz1) and “very long” (Wzz2) chain lengths, respectively. We found that compared to Wzz1, Wzz2has distinct amino acid insertions in the central α-helices (insα6and insα7) and in membrane-distal (insL4) and -proximal (insIL) loops. When these regions were deleted in Wzz2, the mutant proteins conferred drastically shortened chain lengths. Within these regions we identified several conserved amino acid residues that were then targeted for site-directed mutagenesis. Our results implicate an RTE motif in loop 4 and a “hot spot” of charged and polar residues in insα7in the function of Wzz2. We present evidence that the functionally important residues of insα7are likely involved in stabilizing Wzz through coiled-coil interactions.IMPORTANCEO antigen is an important virulence factor presented on the cell surface of Gram-negative bacteria that is critical for bacterial physiology and pathogenesis. However, some aspects of O antigen biosynthesis, such as the mechanisms for determining polysaccharide chain length, are poorly understood. In this study, we identified unique regions in the O antigen chain length regulators (termed Wzz) of the problematic opportunistic pathogenPseudomonas aeruginosa. We show that these regions are critical for determining O antigen chain length, which provides new insight into the model of the Wzz mechanism. Ultimately, our work adds knowledge toward understanding an important step in the biosynthesis of this virulence factor, which is applicable to a wide range of Gram-negative pathogens.

Bacterial-Mucosal Cell Junctional Complexes

1974 ◽  
Vol 32 ◽  
pp. 144-145
Author(s):  
Roger C. Wagner
Keyword(s):  
Intestinal Wall ◽  
Rat Colon ◽  
Mucosal Cell ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Mucosal Cells ◽  
Mucosal Lining ◽  
Degenerative Alterations ◽  
Junctional Complexes ◽  
Intestinal Mucosal Cells

Bacteria exhibit the ability to adhere to the apical surfaces of intestinal mucosal cells. These attachments either precede invasion of the intestinal wall by the bacteria with accompanying inflammation and degeneration of the mucosa or represent permanent anchoring sites where the bacteria never totally penetrate the mucosal cells.Endemic gram negative bacteria were found attached to the surface of mucosal cells lining the walls of crypts in the rat colon. The bacteria did not intrude deeper than 0.5 urn into the mucosal cells and no degenerative alterations were detectable in the mucosal lining.

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