scholarly journals Envelope Disorder of Escherichia coli Cells Lacking Phosphatidylglycerol

2002 ◽  
Vol 184 (19) ◽  
pp. 5418-5425 ◽  
Author(s):  
Motoo Suzuki ◽  
Hiroshi Hara ◽  
Kouji Matsumoto
Keyword(s):  
Escherichia Coli ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Inner Membrane ◽  
Cellular Processes ◽  
Escherichia Coli Cells ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Null Mutants

ABSTRACT Phosphatidylglycerol, the most abundant acidic phospholipid in Escherichia coli, is considered to play specific roles in various cellular processes that are essential for cell viability. A null mutation of pgsA, which encodes phosphatidylglycerophosphate synthase, does indeed confer lethality. However, pgsA null mutants are viable if they lack the major outer membrane lipoprotein (Lpp) (lpp mutant) (S. Kikuchi, I. Shibuya, and K. Matsumoto, J. Bacteriol. 182:371-376, 2000). Here we show that Lpp expressed from a plasmid causes cell lysis in a pgsA lpp double mutant. The envelopes of cells harvested just before lysis could not be separated into outer and inner membrane fractions by sucrose density gradient centrifugation. In contrast, expression of a mutant Lpp (LppΔK) lacking the COOH-terminal lysine residue (required for covalent linking to peptidoglycan) did not cause lysis and allowed for the clear separation of the outer and inner membranes. We propose that in pgsA mutants LppΔK could not be modified by the addition of a diacylglyceryl moiety normally provided by phosphatidylglycerol and that this defect caused unmodified LppΔK to accumulate in the inner membrane. Although LppΔK accumulation did not lead to lysis, the accumulation of unmodified wild-type Lpp apparently led to the covalent linking to peptidoglycan, causing the inner membrane to be anomalously anchored to peptidoglycan and eventually leading to lysis. We suggest that this anomalous anchoring largely explains a major portion of the nonviable phenotypes of pgsA null mutants.

THE TERMINAL GROUPS OF RIBONUCLEATE CHAINS IN EACH OF THE 16S AND 23S COMPONENTS OF ESCHERICHIA COLI RIBOSOMAL RNA

1967 ◽  
Vol 45 (6) ◽  
pp. 937-948 ◽  
Author(s):  
J. L. Nichols ◽  
B. G. Lane
Keyword(s):  
Escherichia Coli ◽  
Chain Length ◽  
Ribosomal Rna ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Quantitative Estimates ◽  
16S Rna ◽  
The Mean ◽  
End Group

Ribosomal ribonucleates from Escherichia coli have been resolved into 16S and 28S components by sucrose density-gradient centrifugation, and the chain termini in each of the 16S and 23S RNA components have been analyzed by hydrolysis with alkali. The principal 5′-linked end group of 16S RNA was found to be adenosine, and the principal 5′-linked end group of 23S RNA was found to be uridine. The principal 3′-linked end group of 16S RNA was also found to be adenosine, whereas the principal 3′-linked end group of 23S RNA was found to be guanosine. Quantitative estimates of chain length based on analyses for 5′-iinked terminals indicate that the mean chain length for 16S RNA is about 1.3 × 103nucleotide residues and the mean chain length for 23S RNA is about 2.1 × 103nucleotide residues.

scholarly journals Viability of an Escherichia coli pgsANull Mutant Lacking Detectable Phosphatidylglycerol and Cardiolipin

2000 ◽  
Vol 182 (2) ◽  
pp. 371-376 ◽  
Author(s):  
Shin Kikuchi ◽  
Isao Shibuya ◽  
Kouji Matsumoto
Keyword(s):  
Escherichia Coli ◽  
Cell Viability ◽  
Total Phospholipid ◽  
E Coli ◽  
Cellular Processes ◽  
Acidic Phospholipid ◽  
Outer Membrane Lipoprotein ◽  
Rich Media ◽  
Acidic Phospholipids ◽  
Membrane Lipoprotein

ABSTRACT Phosphatidylglycerol, the most abundant acidic phospholipid inEscherichia coli, has been considered to play specific roles in various cellular processes and is believed to be essential for cell viability. It is functionally replaced in some cases by cardiolipin, another abundant acidic phospholipid derived from phosphatidylglycerol. However, we now show that a null pgsAmutant is viable, if the major outer membrane lipoprotein is deficient. The pgsA gene normally encodes phosphatidylglycerophosphate synthase that catalyzes the committed step in the biosynthesis of these acidic phospholipids. In the mutant, the activity of this enzyme and both phosphatidylglycerol and cardiolipin were not detected (less than 0.01% of total phospholipid, both below the detection limit), although phosphatidic acid, an acidic biosynthetic precursor, accumulated (4.0%). Nonetheless, the null mutant grew almost normally in rich media. In low-osmolarity media and minimal media, however, it could not grow. It did not grow at temperatures over 40°C, explaining the previous inability to construct a null pgsA mutant (W. Xia and W. Dowhan, Proc. Natl. Acad. Sci. USA 92:783–787, 1995). Phosphatidylglycerol and cardiolipin are therefore nonessential for cell viability or basic life functions. This notion allows us to formulate a working model that defines the physiological functions of acidic phospholipids in E. coli and explains the suppressing effect of lipoprotein deficiency.

scholarly journals VirB7 Lipoprotein Is Exocellular and Associates with the Agrobacterium tumefaciens T Pilus

2001 ◽  
Vol 183 (12) ◽  
pp. 3642-3651 ◽  
Author(s):  
Vitaliya Sagulenko ◽  
Evgeniy Sagulenko ◽  
Simon Jakubowski ◽  
Elena Spudich ◽  
Peter J. Christie
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Cell Surface ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Effector Proteins ◽  
Cross Linking ◽  
Sucrose Density Gradient Centrifugation ◽  
Type Iv ◽  
Outer Membrane Lipoprotein

ABSTRACT Agrobacterium tumefaciens transfers oncogenic T-DNA and effector proteins to plant cells via a type IV secretion pathway. This transfer system, assembled from the products of the virBoperon, is thought to consist of a transenvelope mating channel and the T pilus. When screened for the presence of VirB and VirE proteins, material sheared from the cell surface of octopine strain A348 was seen to possess detectable levels of VirB2 pilin, VirB5, and the VirB7 outer membrane lipoprotein. Material sheared from the cell surface of mostvirB gene deletion mutants also possessed VirB7, but not VirB2 or VirB5. During purification of the T pilus from wild-type cells, VirB2, VirB5, and VirB7 cofractionated through successive steps of gel filtration chromatography and sucrose density gradient centrifugation. A complex containing VirB2 and VirB7 was precipitated from a gel filtration fraction enriched for T pilus with both anti-VirB2 and anti-VirB7 antiserum. Both the exocellular and cellular forms of VirB7 migrated as disulfide-cross-linked dimers and monomers when samples were electrophoresed under nonreducing conditions. A mutant synthesizing VirB7 with a Ser substitution of the lipid-modified Cys15 residue failed to elaborate the T pilus, whereas a mutant synthesizing VirB7 with a Ser substitution for the disulfide-reactive Cys24 residue produced very low levels of T pilus. Together, these findings establish that the VirB7 lipoprotein localizes exocellularly, it associates with the T pilus, and both VirB7 lipid modification and disulfide cross-linking are important for T-pilus assembly. T-pilus-associated VirB2 migrated in nonreducing gels as a monomer and a disulfide-cross-linked homodimer, whereas cellular VirB2 migrated as a monomer. A strain synthesizing a VirB2 mutant with a Ser substitution for the reactive Cys64 residue elaborated T pilus but exhibited an attenuated virulence phenotype. Dithiothreitol-treated T pilus composed of native VirB2 pilin and untreated T pilus composed of the VirB2C64S mutant pilin distributed in sucrose gradients more predominantly in regions of lower sucrose density than untreated, native T pili. These findings indicate that intermolecular cross-linking of pilin monomers is not required for T-pilus production, but cross-linking does contribute to T-pilus stabilization.

scholarly journals Deletion of lolB, Encoding an Outer Membrane Lipoprotein, Is Lethal for Escherichia coli and Causes Accumulation of Lipoprotein Localization Intermediates in the Periplasm

2001 ◽  
Vol 183 (22) ◽  
pp. 6538-6542 ◽  
Author(s):  
Kimie Tanaka ◽  
Shin-Ichi Matsuyama ◽  
Hajime Tokuda
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Temperature Sensitive ◽  
Helper Plasmid ◽  
Inner Membrane ◽  
Outer Membrane Lipoprotein ◽  
Lipoprotein Complex ◽  
Membrane Lipoprotein

ABSTRACT Outer membrane lipoproteins of Escherichia coli are released from the inner membrane upon the formation of a complex with a periplasmic chaperone, LolA, followed by localization to the outer membrane. In vitro biochemical analyses revealed that the localization of lipoproteins to the outer membrane generally requires an outer membrane lipoprotein, LolB, and occurs via transient formation of a LolB-lipoprotein complex. On the other hand, a mutant carrying the chromosomal lolB gene under the control of thelac promoter-operator grew normally in the absence of LolB induction if the mutant did not possess the major outer membrane lipoprotein Lpp, suggesting that LolB is only important for the localization of Lpp in vivo. To examine the in vivo function of LolB, we constructed a chromosomal lolB null mutant harboring a temperature-sensitive helper plasmid carrying the lolBgene. At a nonpermissive temperature, depletion of the LolB protein due to loss of the lolB gene caused cessation of growth and a decrease in the number of viable cells irrespective of the presence or absence of Lpp. LolB-depleted cells accumulated the LolA-lipoprotein complex in the periplasm and the mature form of lipoproteins in the inner membrane. Taken together, these results indicate that LolB is the first example of an essential lipoprotein for E. coliand that its depletion inhibits the upstream reactions of lipoprotein trafficking.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

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