scholarly journals Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG

2019 ◽  
Vol 111 (5) ◽  
pp. 1263-1282 ◽  
Author(s):  
Michael Hohl ◽  
Sille Remm ◽  
Haig A. Eskandarian ◽  
Michael Dal Molin ◽  
Fabian M. Arnold ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Drug Permeability ◽  
Mfs Transporter ◽  
In Cells

Formate Dehydrogenase Activity in Cells and Outer Membrane Blebs of Desulfovibrio gigas

Anaerobe ◽  
1995 ◽  
Vol 1 (3) ◽  
pp. 175-182 ◽  
Author(s):  
Tina S. Haynes ◽  
Dwight J. Klemm ◽  
Joseph J. Ruocco ◽  
Larry L. Barton
Keyword(s):  
Outer Membrane ◽  
Dehydrogenase Activity ◽  
Formate Dehydrogenase ◽  
Desulfovibrio Gigas ◽  
Membrane Blebs ◽  
In Cells

Insights into the cell envelope of Paracoccus denitrificans, a member of the α-subdivision of purple bacteria, through studies of its lysozyme susceptibility

1988 ◽  
Vol 34 (8) ◽  
pp. 952-959 ◽  
Author(s):  
Sechan Wee ◽  
Brian J. Wilkinson
Keyword(s):  
Outer Membrane ◽  
Paracoccus Denitrificans ◽  
Cell Envelope ◽  
Divalent Cations ◽  
Purple Bacteria ◽  
Enteric Bacteria ◽  
Complex Medium ◽  
Major Influence ◽  
Outer Membranes ◽  
In Cells

Exponential-phase Paracoccus denitrificans cells grown in a complex medium are known to be lysozyme susceptible without pretreatment with outer membrane perturbing agents. Stationary-phase cells are more lysozyme resistant, but simple washing of these cells with moderate concentrations (154 mM) of monovalent inorganic cations (NaCl, KCl, or LiCl), or 100 mM Tris(hydroxymethyl)aminomethane-HCl (Tris–HCl) rendered them lysozyme susceptible. Subsequent washing with divalent cations reversed the enhanced lysozyme susceptibility. Cells grown in succinate–salts medium were not rendered lysozyme susceptible by NaCl washing. Supplementation of complex medium with various salts used in succinate–salts medium showed that Mg2+ and Ca2+ supplementation resulted in increased growth yields and in cells that were not rendered lysozyme susceptible by NaCl washing. Measurement of the Mg2+ and Ca2+ content of peptone and yeast extract revealed Mg2+ and Ca2+ were present at 50–80 and 15–30 μM concentrations, respectively, in complex medium. Omission of Ca2+ or reduction of Mg2+ from 810 to 50 μM in succinate–salts medium resulted in cells that became lysozyme susceptible after NaCl washing. Incubation of cells grown in succinate–salts medium with Tris–HCl – ethylenediaminetetraacetate – sucrose caused them to become lysozyme susceptible, indicating that their lack of lysozyme susceptibility was due to a more effective outer membrane barrier than that of complex medium grown cells, rather than a lysozyme-resistant peptidoglycan. The protein composition of outer membranes from complex and complex + Mg2+ + Ca2+ grown cells was similar, suggesting that divalent cations did not have a major influence on protein production related to outer membrane stability. The results indicate that certain complex bacteriological media may be deficient in divalent cations for maximum cell envelope stability and growth yield. This might be particularly significant for soil bacteria such as P. denitrificans, which may have evolved to have outer membranes more dependent on divalent cations for structural integrity than animal host adapted bacteria such as enteric bacteria. The results also draw attention to NaCl as an outer membrane perturbing agent.

scholarly journals Membrane Stress Caused by Unprocessed Outer Membrane Lipoprotein Intermediate Pro-Lpp Affects DnaA and Fis-Dependent Growth

2021 ◽  
Vol 12 ◽  
Author(s):  
Digvijay Patil ◽  
Dan Xun ◽  
Markus Schueritz ◽  
Shivani Bansal ◽  
Amrita Cheema ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Physiological State ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Close Link ◽  
Small Regulatory Rna ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Dependent Growth ◽  
In Cells

In Escherichia coli, repression of phosphatidylglycerol synthase A gene (pgsA) lowers the levels of membrane acidic phospholipids, particularly phosphatidylglycerol (PG), causing growth-arrested phenotype. The interrupted synthesis of PG is known to be associated with concomitant reduction of chromosomal content and cell mass, in addition to accumulation of unprocessed outer membrane lipoprotein intermediate, pro-Lpp, at the inner membrane. However, whether a linkage exists between the two altered-membrane outcomes remains unknown. Previously, it has been shown that pgsA+ cells overexpressing mutant Lpp(C21G) protein have growth defects similar to those caused by the unprocessed pro-Lpp intermediate in cells lacking PG. Here, we found that the ectopic expression of DnaA(L366K) or deletion of fis (encoding Factor for Inversion Stimulation) permits growth of cells that otherwise would be arrested for growth due to accumulated Lpp(C21G). The DnaA(L366K)-mediated restoration of growth occurs by reduced expression of Lpp(C21G) via a σE-dependent small-regulatory RNA (sRNA), MicL-S. In contrast, restoration of growth via fis deletion is only partially dependent on the MicL-S pathway; deletion of fis also rescues Lpp(C21G) growth arrest in cells lacking physiological levels of PG and cardiolipin (CL), independently of MicL-S. Our results suggest a close link between the physiological state of the bacterial cell membrane and DnaA- and Fis-dependent growth.

scholarly journals The SecA Subunit of Escherichia coliPreprotein Translocase Is Exposed to the Periplasm

1998 ◽  
Vol 180 (21) ◽  
pp. 5776-5779 ◽  
Author(s):  
Jerry Eichler ◽  
William Wickner
Keyword(s):  
Plasma Membrane ◽  
Outer Membrane ◽  
Conformational Changes ◽  
Protease Resistance ◽  
In Cells

ABSTRACT SecA undergoes conformational changes during translocation, inserting domains into and across the membrane or enhancing the protease resistance of these domains. We now show that some SecA bound at SecYEG is accessible from the periplasm to a membrane-impermeant probe in cells with a permeabilized outer membrane but an intact plasma membrane.

scholarly journals A Multiprotein Complex Anchors Adhesive Holdfast at the Outer Membrane ofCaulobacter crescentus

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Nina I. Sulkowski ◽  
Gail G. Hardy ◽  
Yves V. Brun ◽  
Tanmay A. M. Bharat
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Biofilm Formation ◽  
Caulobacter Crescentus ◽  
Cell Envelope ◽  
Bacterial Colonization ◽  
Bacterial Species ◽  
Specific Cell ◽  
Content Type ◽  
In Cells

ABSTRACTAdhesion allows microbes to colonize surfaces and is the first stage in biofilm formation. Stable attachment of the freshwater alphaproteobacteriumCaulobacter crescentusto surfaces requires an adhesive polysaccharide called holdfast, which is synthesized at a specific cell pole and ultimately found at the tip of cylindrical extensions of the cell envelope called stalks. Secretion and anchoring of holdfast to the cell surface are governed by proteins HfsDAB and HfaABD, respectively. The arrangement and organization of these proteins with respect to each other and the cell envelope, and the mechanism by which the holdfast is anchored on cells, are unknown. In this study, we have imaged a series ofC. crescentusmutants using electron cryotomography, revealing the architecture and arrangement of the molecular machinery involved in holdfast anchoring in cells. We found that the holdfast is anchored to cells by a defined complex made up of the HfaABD proteins and that the HfsDAB secretion proteins are essential for proper assembly and localization of the HfaABD anchor. Subtomogram averaging of cell stalk tips showed that the HfaABD complex spans the outer membrane. The anchor protein HfaB is the major component of the anchor complex located on the periplasmic side of the outer membrane, while HfaA and HfaD are located on the cell surface. HfaB is the critical component of the complex, without which no HfaABD complex was observed in cells. These results allow us to propose a working model of holdfast anchoring, laying the groundwork for further structural and cell biological investigations.IMPORTANCEAdhesion and biofilm formation are fundamental processes that accompany bacterial colonization of surfaces, which are of critical importance in many infections.Caulobacter crescentusbiofilm formation proceeds via irreversible adhesion mediated by a polar polysaccharide called holdfast. Mechanistic and structural details of how the holdfast is secreted and anchored on cells are still lacking. Here, we have assigned the location and described the arrangement of the holdfast anchor complex. This work increases our knowledge of the relatively underexplored field of polysaccharide-mediated adhesion by identifying structural elements that anchor polysaccharides to the cell envelope, which is important in a variety of bacterial species.

scholarly journals A Protein Complex Containing Mdm10p, Mdm12p, and Mmm1p Links Mitochondrial Membranes and DNA to the Cytoskeleton-based Segregation Machinery

2003 ◽  
Vol 14 (11) ◽  
pp. 4618-4627 ◽  
Author(s):  
Istvan R. Boldogh ◽  
Dan W. Nowakowski ◽  
Hyeong-Cheol Yang ◽  
Haesung Chung ◽  
Sharon Karmon ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Reciprocal Relationship ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Mitochondrial Movement ◽  
Close Proximity ◽  
Mtdna Deletion ◽  
Mitochondrial Motility ◽  
In Cells

Previous studies indicate that two proteins, Mmm1p and Mdm10p, are required to link mitochondria to the actin cytoskeleton of yeast and for actin-based control of mitochondrial movement, inheritance and morphology. Both proteins are integral mitochondrial outer membrane proteins. Mmm1p localizes to punctate structures in close proximity to mitochondrial DNA (mtDNA) nucleoids. We found that Mmm1p and Mdm10p exist in a complex with Mdm12p, another integral mitochondrial outer membrane protein required for mitochondrial morphology and inheritance. This interpretation is based on observations that 1) Mdm10p and Mdm12p showed the same localization as Mmm1p; 2) Mdm12p, like Mdm10p and Mmm1p, was required for mitochondrial motility; and 3) all three proteins coimmunoprecipitated with each other. Moreover, Mdm10p localized to mitochondria in the absence of the other subunits. In contrast, deletion of MMM1 resulted in mislocalization of Mdm12p, and deletion of MDM12 caused mislocalization of Mmm1p. Finally, we observed a reciprocal relationship between the Mdm10p/Mdm12p/Mmm1p complex and mtDNA. Deletion of any one of the subunits resulted in loss of mtDNA or defects in mtDNA nucleoid maintenance. Conversely, deletion of mtDNA affected mitochondrial motility: mitochondria in cells without mtDNA move 2–3 times faster than mitochondria in cells with mtDNA. These observations support a model in which the Mdm10p/Mdm12p/Mmm1p complex links the minimum heritable unit of mitochondria (mtDNA and mitochondrial outer and inner membranes) to the cytoskeletal system that drives transfer of that unit from mother to daughter cells.

Cytoplasmic Tubules in Cells Infected with Laryngotracheitis Virus

1969 ◽  
Vol 27 ◽  
pp. 376-377
Author(s):  
A. M. Watrach
Keyword(s):  
Tissue Culture ◽  
Small Proportion ◽  
Chicken Embryo ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Morphologic Characteristics ◽  
Infectious Laryngotracheitis ◽  
Laryngotracheitis Virus ◽  
In Cells

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.

Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

1971 ◽  
Vol 29 ◽  
pp. 548-549
Author(s):  
Awtar Krishan ◽  
Dora Hsu
Keyword(s):  
Granular Material ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Metabolic Inhibitors ◽  
Protein And Rna Synthesis ◽  
Apparent Reduction ◽  
Plant Alkaloids ◽  
In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

Molecular Structure of the Outermost Cell Wall Component of Spirillum Serpens

1977 ◽  
Vol 35 ◽  
pp. 342-343
Author(s):  
Wah Chiu ◽  
David Grano
Keyword(s):  
Molecular Structure ◽  
Cell Wall ◽  
Outer Membrane ◽  
Gel Electrophoresis ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Cell Wall Component ◽  
Protein Components ◽  
Exposure Technique ◽  
Structural Aspects

The periodic structure external to the outer membrane of Spirillum serpens VHA has been isolated by similar procedures to those used by Buckmire and Murray (1). From SDS gel electrophoresis, we have found that the isolated fragments contain several protein components, and that the crystalline structure is composed of a glycoprotein component with a molecular weight of ∽ 140,000 daltons (2). Under an electron microscopic examination, we have visualized the hexagonally-packed glycoprotein subunits, as well as the bilayer profile of the outer membrane. In this paper, we will discuss some structural aspects of the crystalline glycoproteins, based on computer-reconstructed images of the external cell wall fragments.The specimens were prepared for electron microscopy in two ways: negatively stained with 1% PTA, and maintained in a frozen-hydrated state (3). The micrographs were taken with a JEM-100B electron microscope with a field emission gun. The minimum exposure technique was essential for imaging the frozen- hydrated specimens.

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

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