Localization of the dicarboxylate binding protein in the cell envelope of Escherichia coli K12

1980 ◽  
Vol 58 (10) ◽  
pp. 885-897 ◽  
Author(s):  
Mary A. Bewick ◽  
Theodore C. Y. Lo
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Cell Envelope ◽  
Binding Protein ◽  
Osmotic Shock ◽  
Outer Region ◽  
Periplasmic Space ◽  
Whole Cells ◽  
Escherichia Coli K12 ◽  
The Moment

Examination of the localization of the dicarboxylate binding protein (DBP) in the cell envelope of Escherichia coli K12 reveals that this protein is present on the cell surface, and also in the inner and outer regions of the periplasmic space. The cell surface DBP is released by treating the cells with EDTA. This protein can be surface labeled by lactoperoxidase radio-iodination, and by diazo[125I]iodosulfanilic acid in whole cells. It also binds tightly, but not covalently, to lipopolysaccharide. The DBP located in the outer region of the periplasmic space is released when the outer membrane is dissociated by EDTA – osmotic shock treatment. The DBP located in the inner region of the periplasmic space is released only when the EDTA – osmotic shocked cells are subjected to lysozyme treatment. At the moment, it is not certain whether this protein is bound to or trapped by the peptidoglycan network. This protein cannot be surface labeled in whole cells or in EDTA – osmotic shock treated cells; and it is not associated with lipopolysaccharide. Analysis of transport mutants indicates that these DBP are coded by the same gene.

Dicarboxylic acid transport in Escherichia coli K12: involvement of a binding protein in the translocation of dicarboxylic acids across the outer membrane of the cell envelope

1979 ◽  
Vol 57 (6) ◽  
pp. 653-661 ◽  
Author(s):  
Mary A. Bewick ◽  
Theodore C. Y. Lo
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Outer Membrane ◽  
Transport System ◽  
Cell Envelope ◽  
Binding Protein ◽  
Dicarboxylic Acids ◽  
Escherichia Coli K12 ◽  
Dicarboxylate Transport ◽  
Active Transport System

We have previously found that the dicarboxylate transport system in Escherichia coli K12 is an active transport system and that at least one binding protein and two cytoplasmic membrane transport components are involved in the uptake of dicarboxylic acids. Recently, through surface labelling studies, some dicarboxylate binding proteins were found to be exposed on the cell surface. In the present paper, we demonstrate that the dicarboxylate transport component located in the outer membrane can be inactivated by two different kinds of nonpenetrating inhibitors, viz. proteases, and diazosulfanilic acid. These inhibitors seem to act on the dicarboxylate binding protein. By adding this protein to inactivated cells or to transport-negative mutants, we have succeeded in reconstituting the dicarboxylate transport system. These findings suggest that the dicarboxylate binding protein found on the cell surface plays an essential role in the translocation of dicarboxylic acids across the outer membrane.

scholarly journals Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A

2020 ◽  
Vol 8 (6) ◽  
pp. 826
Author(s):  
Federica A. Falchi ◽  
Flaviana Di Lorenzo ◽  
Roberto Pizzoccheri ◽  
Gianluca Casino ◽  
Moira Paroni ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Lipid A ◽  
Cell Envelope ◽  
Osmotic Shock ◽  
Ectopic Expression ◽  
Phenotypic Effect ◽  
Osmotic Lysis ◽  
Membrane Defects ◽  
Inner Membrane Protein

LpxT is an inner membrane protein that transfers a phosphate group from the essential lipid undecaprenyl pyrophosphate (C-55PP) to the lipid A moiety of lipopolysaccharide, generating a lipid A tris-phosphorylated species. The protein is encoded by the non-essential lpxT gene, which is conserved in distantly related Gram-negative bacteria. In this work, we investigated the phenotypic effect of lpxT ectopic expression from a plasmid in Escherichia coli. We found that lpxT induction inhibited cell division and led to the formation of elongated cells, mostly with absent or altered septa. Moreover, the cells became sensitive to detergents and to hypo-osmotic shock, indicating that they had cell envelope defects. These effects were not due to lipid A hyperphosphorylation or C-55PP sequestering, but most likely to defective lipopolysaccharide transport. Indeed, lpxT overexpression in mutants lacking the L,D-transpeptidase LdtD and LdtE, which protect cells with outer membrane defects from osmotic lysis, caused cell envelope defects. Moreover, we found that pyrophosphorylated lipid A was also produced in a lpxT deletion mutant, indicating that LpxT is not the only protein able to perform such lipid A modification in E. coli.

scholarly journals Initial Steps of Colicin E1 Import across the Outer Membrane of Escherichia coli

2007 ◽  
Vol 189 (7) ◽  
pp. 2667-2676 ◽  
Author(s):  
Muriel Masi ◽  
Phu Vuong ◽  
Matthew Humbard ◽  
Karen Malone ◽  
Rajeev Misra
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Outer Membrane ◽  
Vitamin B ◽  
Whole Cells ◽  
E Coli ◽  
Colicin E1 ◽  
Unfolded State ◽  
Translocation Domain

ABSTRACT Data suggest a two-receptor model for colicin E1 (ColE1) translocation across the outer membrane of Escherichia coli. ColE1 initially binds to the vitamin B12 receptor BtuB and then translocates through the TolC channel-tunnel, presumably in a mostly unfolded state. Here, we studied the early events in the import of ColE1. Using in vivo approaches, we show that ColE1 is cleaved when added to whole cells. This cleavage requires the presence of the receptor BtuB and the protease OmpT, but not that of TolC. Strains expressing OmpT cleaved ColE1 at K84 and K95 in the N-terminal translocation domain, leading to the removal of the TolQA box, which is essential for ColE1's cytotoxicity. Supported by additional in vivo data, this suggests that a function of OmpT is to degrade colicin at the cell surface and thus protect sensitive E. coli cells from infection by E colicins. A genetic strategy for isolating tolC mutations that confer resistance to ColE1, without affecting other TolC functions, is also described. We provide further in vivo evidence of the multistep interaction between TolC and ColE1 by using cross-linking followed by copurification via histidine-tagged TolC. First, secondary binding of ColE1 to TolC is dependent on primary binding to BtuB. Second, alterations to a residue in the TolC channel interfere with the translocation of ColE1 across the TolC pore rather than with the binding of ColE1 to TolC. In contrast, a substitution at a residue exposed on the cell surface abolishes both binding and translocation of ColE1.

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