Histochemical and biochemical urease localization in the periplasm and outer membrane of two Proteus mirabilis strains

1986 ◽  
Vol 32 (10) ◽  
pp. 772-778 ◽  
Author(s):  
Robert J. C. McLeanl ◽  
K.-J. Cheng ◽  
W. Douglas Gould ◽  
J. Curtis Nickel ◽  
J. William Costerton
Keyword(s):  
Outer Membrane ◽  
Proteus Mirabilis ◽  
Urease Activity ◽  
Enzymatic Reaction ◽  
Outer Membranes ◽  
Biochemical Measurements ◽  
Membrane Bound ◽  
Sodium Tetraphenylboron ◽  
Cell Fractions

Proteus mirabilis, a gram-negative bacillus, is often implicated in the formation of infections kidney stones. As ureolytic activity of this organism is thought to play a major role in its pathogenesis, we adapted our recently described urease localization technique to visualize urease activity in vivo. Urease activity was ultra structural] y localized in two clinically isolated P. mirabilis strains by precipitating the enzymatic reaction product (ammonia) with sodium tetraphenylboron. Subsequent silver staining of the cells revealed urease activity to be predominately associated with the periplasm and outer membranes of each strain. Biochemical measurements of urease activity in P. mirabilis cell fractions correlated well with histochemical observations in that the majority of urease activity was associated with the periplasm. Membrane-bound urease activity of these strains was associated mainly with the peptidoglycan in the detergent-insoluble (outer membrane) fraction.

scholarly journals Proteomic analysis of Proteus mirabilis outer membrane proteins reveals differential expression in vivo vs. in vitro conditions

2011 ◽  
Vol 63 (2) ◽  
pp. 174-182 ◽  
Author(s):  
Bruno D'Alessandro ◽  
Leticia M. S. Lery ◽  
Wanda M. A. Krüger ◽  
Analía Lima ◽  
Claudia Piccini ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Differential Expression ◽  
Proteus Mirabilis ◽  
Proteomic Analysis ◽  
Outer Membrane Proteins

scholarly journals An iron-regulated outer-membrane protein of Proteus mirabilis is a haem receptor that plays an important role in urinary tract infection and in in vivo growth

2007 ◽  
Vol 56 (12) ◽  
pp. 1600-1607 ◽  
Author(s):  
Analía Lima ◽  
Pablo Zunino ◽  
Bruno D'Alessandro ◽  
Claudia Piccini
Keyword(s):  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Outer Membrane ◽  
Proteus Mirabilis ◽  
Outer Membrane Proteins ◽  
Mammalian Host ◽  
Tract Infections

Proteus mirabilis, a common cause of urinary tract infections, expresses iron-regulated outer-membrane proteins (OMPs) in response to iron restriction. It has been suggested that a 64 kDa OMP is involved in haemoprotein uptake and that this might have a role in pathogenesis. In order to confirm this hypothesis, this study generated a P. mirabilis mutant strain (P7) that did not express the 64 kDa OMP, by insertion of the TnphoA transposon. The nucleotide sequence of the interrupted gene revealed that it corresponded to a haemin receptor precursor. Moreover, in vitro growth assays showed that the mutant was unable to grow using haemoglobin and haemin as unique iron sources. The authors also carried out in vivo growth and infectivity assays and demonstrated that P7 was not able to survive in an in vivo model and was less efficient than wild-type strain Pr 6515 in colonizing the urinary tract. These results confirmed that the P. mirabilis 64 kDa iron-regulated OMP is a haem receptor that has an important role for survival and multiplication of these bacteria in the mammalian host and in the development of urinary tract infection.

Membrane assembly: movement of phosphatidylserine between the cytoplasmic and outer membranes of Escherichia coli

1982 ◽  
Vol 152 (3) ◽  
pp. 1033-1041
Author(s):  
K E Langley ◽  
E Hawrot ◽  
E P Kennedy
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Sucrose Gradient ◽  
Intracellular Localization ◽  
Temperature Sensitive ◽  
Inner Membrane ◽  
E Coli ◽  
Outer Membranes ◽  
Membrane Bound ◽  
Gradient Fractionation

Phosphatidylserine, normally a trace phospholipid in Escherichia coli, accumulates at high levels in temperature-sensitive phosphatidylserine decarboxylase mutants at nonpermissive temperatures. The intracellular localization of this phospholipid has now been determined. All of the accumulated phosphatidylserine is membrane bound and is distributed about equally between the inner and outer membrane fractions of E. coli as determined by isopycnic sucrose gradient fractionation. Phosphatidylserine is therefore effectively translocated from the inner to the outer membrane. Furthermore, this movement is bidirectional. Outer membrane phosphatidylserine can return to the inner membrane, as shown by the complete conversion of accumulated radioactive phosphatidylserine to phosphatidylethanolamine by inner membrane phosphatidylserine decarboxylase during chase periods. Pulse-chase experiments indicated the newly made phosphatidylserine appears first in the inner membrane and then equilibrates between the inner and outer membranes with a half-time of 12 to 13 min.

scholarly journals TonB Interacts with Nonreceptor Proteins in the Outer Membrane of Escherichia coli

2002 ◽  
Vol 184 (6) ◽  
pp. 1640-1648 ◽  
Author(s):  
Penelope I. Higgs ◽  
Tracy E. Letain ◽  
Kelley K. Merriam ◽  
Neal S. Burke ◽  
HaJeung Park ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Receptors ◽  
Strong Interactions ◽  
Cross Linking ◽  
Outer Membrane Receptor ◽  
Outer Membranes

ABSTRACT The Escherichia coli TonB protein serves to couple the cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes and vitamin B12 across the outer membrane. Consistent with this role, TonB has been demonstrated to participate in strong interactions with both the cytoplasmic and outer membranes. The cytoplasmic membrane determinants for that interaction have been previously characterized in some detail. Here we begin to examine the nature of TonB interactions with the outer membrane. Although the presence of the siderophore enterochelin (also known as enterobactin) greatly enhanced detectable cross-linking between TonB and the outer membrane receptor, FepA, the absence of enterochelin did not prevent the localization of TonB to the outer membrane. Furthermore, the absence of FepA or indeed of all the iron-responsive outer membrane receptors did not alter this association of TonB with the outer membrane. This suggested that TonB interactions with the outer membrane were not limited to the TonB-dependent outer membrane receptors. Hydrolysis of the murein layer with lysozyme did not alter the distribution of TonB, suggesting that peptidoglycan was not responsible for the outer membrane association of TonB. Conversely, the interaction of TonB with the outer membrane was disrupted by the addition of 4 M NaCl, suggesting that these interactions were proteinaceous. Subsequently, two additional contacts of TonB with the outer membrane proteins Lpp and, putatively, OmpA were identified by in vivo cross-linking. These contacts corresponded to the 43-kDa and part of the 77-kDa TonB-specific complexes described previously. Surprisingly, mutations in these proteins individually did not appear to affect TonB phenotypes. These results suggest that there may be multiple redundant sites where TonB can interact with the outer membrane prior to transducing energy to the outer membrane receptors.

scholarly journals Structural insight into outer membrane asymmetry maintenance of Gram-negative bacteria by the phospholipid transporter MlaFEDB

2020 ◽  
Author(s):  
Xiaodi Tang ◽  
Shenghai Chang ◽  
Wen Qiao ◽  
Qinghua Luo ◽  
Yuejia Chen ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Bound State ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Membrane Asymmetry ◽  
Outer Membranes ◽  
Transport Direction ◽  
Insight Into

The asymmetric phospholipid outer membrane (OM) of Gram-negative bacteria serves as the first line of defense against cytotoxic substances such as antibiotics. The Mla pathway is known to maintain the lipid asymmetry of the OM by transporting phospholipids between the inner and outer membranes. Six Mla proteins MlaFEDBCA are involved, with the ABC transporter MlaFEDB acts through a mechanism yet to be elucidated. Here we determine cryo-EM structures of MlaFEDB in apo, phospholipid-, ADP- or AMP-PNP-bound state to 3.3-3.75 Å resolution and establish a proteoliposome-based transport system containing MlaFEDB, MlaC and MlaA/OmpF to reveal the transport direction of phospholipids. Mutagenetic in vitro transport assays and in vivo sensitivity assays reveal functional residues which recognize and transport phospholipids as well as regulate the activity and structural stability of the MlaFEDB complex. Our work provides molecular basis for understanding the mechanism of the Mla pathway which could be targeted for antimicrobial drug development.

Binding of mutagenic pyrolyzates to fractions of intestinal bacterial cells

1992 ◽  
Vol 38 (7) ◽  
pp. 614-617 ◽  
Author(s):  
Xue Bin Zhang ◽  
Yoshiyuki Ohta
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Outer Membrane ◽  
Bacterial Strain ◽  
Cytoplasmic Membrane ◽  
Intestinal Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
Membrane Bound ◽  
Cell Fractions

The binding of mutagenic pyrolyzates to cell fractions from some gram-negative intestinal bacteria and to thermally treated bacterial cells was investigated. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) were effectively bound by several of the bacterial cells. The cell wall skeletons of all bacteria effectively bound Trp-P-1 and Trp-P-2. Their cytoplasmic fractions retained Trp-P-1 and Trp-P-2, but to a lesser extent than the cell wall skeletons. 2-Amino-3-methylimidazo [4,5-f]quinoline (IQ) was not found in their cytoplasmic fractions. These cell wall skeletons also bound 2-amino-6-methyldipyrido[1,2-a:3′2′-d] imidazole (Glu-P-1), 2-amino-5-phenylpyridine (Phe-P-1), IQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQX). The amount of each mutagen bound differed with the type of mutagen and the bacterial strain used. The outer membrane of Escherichia coli IFO 14249 showed binding of about 123.7 μg/mg of Trp-P-2, and its cytoplasmic membrane bound 57.14 μg/mg. Trp-P-2 bound to the bacterial cells was extracted with ammonia (5%), methanol, and ethanol but not with water. Key words: cell wall skeletons, outer membrane, cytoplasmic membrane, binding of mutagenic pyrolyzates.

scholarly journals In Vitro and In Vivo Functional Activity of Chlamydia MurA, a UDP-N-Acetylglucosamine Enolpyruvyl Transferase Involved in Peptidoglycan Synthesis and Fosfomycin Resistance

2003 ◽  
Vol 185 (4) ◽  
pp. 1218-1228 ◽  
Author(s):  
Andrea J. McCoy ◽  
Robin C. Sandlin ◽  
Anthony T. Maurelli
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Enzymatic Reaction ◽  
Developmental Cycle ◽  
E Coli ◽  
Obligate Intracellular ◽  
Membrane Bound ◽  
Fosfomycin Resistance

ABSTRACT Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal ΔmurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.

scholarly journals An ExbD Disordered Domain Peptide Inhibits TonB System Activity

2020 ◽  
Author(s):  
Dale R. Kopp ◽  
Kathleen Postle
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Protonmotive Force ◽  
Gram Negative Bacteria ◽  
System Function ◽  
Gram Negative ◽  
Conserved Motif ◽  
Outer Membranes ◽  
Tonb System

ABSTRACTThe TonB system energizes transport of essential nutrients, such as iron siderophores, across unenergized outer membranes of Gram-negative bacteria. The integral cytoplasmic membrane proteins of the TonB system--ExbB, ExbD, and TonB--transduce the protonmotive force of the cytoplasmic membrane to TonB-dependent outer membrane transporters for active transport. ExbD protein is anchored in the cytoplasmic membrane, with the majority of it occupying the periplasm. We previously identified a conserved motif within a periplasmic disordered domain that is essential for TonB system function. Here we demonstrated that export of a peptide derived from that motif into the periplasm prevented TonB system function and inhibited all known ExbD interactions in vivo. Formaldehyde crosslinking captured the ExbD peptide in multiple ExbD and TonB complexes. Furthermore, peptides with mutations in the conserved motif not only had significantly reduced ability to inhibit TonB system activity, but they also altered interactions with ExbD and TonB, indicating the specificity of the interaction. Conserved motif peptide interactions with ExbD and TonB mostly occurred between Stage II and Stage III of the TonB energy transduction cycle, a transition that is characterized by the use of protonmotive force. Taken together, the data suggest that the ExbD disordered domain motif has multiple interactions with TonB and ExbD during between Stage II and III of the TonB energization cycle. Because of the essentiality of the motif, it may be a potential template for design of novel antibiotics that target the TonB system.IMPORTANCEGram-negative bacteria are intrinsically antibiotic-resistant due to the diffusion barrier posed by their outer membranes. The TonB system allows them to circumvent this barrier for their own nutritional needs, including iron. The ability of bacteria to acquire iron is a virulence factor for many Gram-negative pathogens. However, no antibiotics currently target the TonB system. Because TonB and ExbD must interact productively in the periplasm for transport across the outer membrane, they constitute attractive targets for potential antibiotic development where chemical characteristics need not accommodate the need to cross the hydrophobic cytoplasmic membrane. Here we show that a small ExbD-derived peptide can interfere with the TonB-ExbD interaction to inhibit the TonB system in vivo.

Régulation de la synthèse de la catalase chez Proteus mirabilis

1979 ◽  
Vol 25 (3) ◽  
pp. 302-311 ◽  
Author(s):  
H. Jouve ◽  
G. Sauret ◽  
A. M. Labouré ◽  
J. Pelmont
Keyword(s):  
Proteus Mirabilis ◽  
Culture Medium ◽  
Specific Activity ◽  
Enzyme Synthesis ◽  
Appreciable Effect ◽  
Outer Membranes ◽  
Order Of Magnitude ◽  
Mechanical Separation

During the log-phase growth of Proteus mirabilis the specific activity of catalase decreases, while at the beginning of or during the stationary phase an increase takes place which is abolished by inhibitors of nucleic acid or protein synthesis. Glucose in the culture medium has no appreciable effect on the level of enzyme synthesis nor does the passage of bacteria to anaerobiosis bring any noticeable change. Successive additions of hydrogen peroxide up to weak final concentrations (0.2–0.5 mM) stimulate catalase synthesis. Determination of the enzyme in vivo reveals but a weak proportion of the total catalase which can only be titrated after the breakdown of cells. The titrable enzyme in vivo represents, as an order of magnitude, the activity found associated with the cell wall, in an easily released form after the mechanical separation of the inner and outer membranes. Thus, bacteria can act upon exogenous peroxide only through a peripheral catalase while they possess in a masked form an important reserve of cytoplasmic enzyme.

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