Quantitation of metal cations bound to membranes and extracted lipopolysaccharide of Escherichia coli

Abstract

Inductively coupled plasma emission spectroscopy was used to quantitate the metal cations bound to outer and cytoplasmic membranes and to extracted lipopolysaccharide from several Escherichia coli K12 strains. The outer membrane was found to be enriched in both calcium and magnesium relative to the cytoplasmic membrane. Both membranes contained significant levels of iron, aluminum, and zinc. The multivalent cation content of the lipopolysaccharide resembled that of the intact outer membrane. Lipopolysaccharide extracted from wild-type k12 strains contained higher levels of Mg than Ca regardless of the growth medium, but the medium used for growth did affect the relative amounts of bound Mg as well as the levels of the minor cations iron, aluminum, and zinc. In contrast, lipopolysaccharide isolated from a deep rough mutant strain, D21f2, contained more Ca than Mg. Electrodialysis of lipopolysaccharide from wild-type k12 strains removed 1 mol of Mg per mol of lipopolysaccharide but did not significantly affect the level of other bound metal ions. Dialysis of lipopolysaccharide against sodium (ethylenedinitrilo)tetraacetate removed most of the Mg and Ca, resulting in a sodium salt. The equimolar replacement of divalent cations with sodium in the sodium salt resulted in a net loss of counterion change. The sodium salt was dialyzed against either tris(hydroxymethyl)aminomethane hydrochloride, CaCl2, MgCl2, or TbCl3, and the resulting lipopolysaccharide salts were analyzed for their ionic composition. It was shown that tris(hydroxymethyl)aminomethane and Ca can replace some but not all of the Na bound to the sodium salt, but all of the other multivalent cations tested replaced Na, resulting in uniform lipopolysaccharide salts. Lipopolysaccharide isolated from the deep rough mutant strain D21f2 was also converted into a sodium salt. Relative to the wild-type lipopolysaccharide, Na was able to neutralize the anionic charge to a greater extent in the mutant lipopolysaccharide. Our results suggest that the loss of specific groups in the core region of the lipopolysaccharide from the mutant strain results in a more open structure that allows the binding of larger cations and of more monovalent cations.

Formation of a Hexagonal Lattice Structure by an R‐Form Lipopolysaccharide of Klebsiella: Effect of Various Divalent Cations on the Lattice Formation

“…From the results they postulated that Mg bound to the LPS was divided into two types by its strength of binding (1). In contrast, we found that, as in the case of the LPS from K. pneumoniae strain LEN-111, Mg bound to the LPS from E. coli K-12 strain JE1011 could be removed almost completely and Ca was significantly decreased with electrodialysis (5).…”

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

“…In Gram-negative bacteria such as E. coli, magnesium is involved in stabilization of membrane phospholipids, lipopolysaccharide, polyphosphate compounds like DNA and RNA, and the ribosome (11)(12)(13). Magnesium is also required to make ATP biologically active and participates in catalysis of certain enzymatic reactions through either direct or indirect mechanisms (14,15).…”

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length

“…The ladderlike banding patterns represent molecules with increasing numbers of 0-antigen repeat units (20,24,45 1, 2, 3, and X were separated on Sephadex G-200, dialyzed, and lyophilized as described in Materials and Methods and suspended in distilled water to a final concentration of 10 mg/ml. In the top row, 10 ,ug of each sample was applied on the nitrocellulose strip, and subsequent rows were spotted with an equal volume of 10-fold serially diluted samples. The sample spots were dried, and the nitrocellulose was either reacted with monoclonal anti-503 antibody and developed as described in Materials and Methods (A) or washed with 10 mM Tris-150 mM NaCl (pH 7.0) and reacted with the silver stain as indicated in Materials and Methods for polyacrylamide gels (B).…”

“…Assays for amino sugars (17), heptose (59), and 2-keto-3-deoxyoctulosonic acid (KDO) (28) were performed as described previously except when noted. Phosphate analyses were performed by using either a colorimetric assay (1) or inductively coupled plasma emission spectroscopy (10). Protein concentrations were estimated by the Pierce BCA protein assay (Pierce Chemical Co., Rockford, Ill.) with bovine serum albumin as the standard.…”