Alterations in the Outer Membrane of the Cell Envelope of Heptose-Deficient Mutants of Escherichia coli

Abstract

The composition of the cell envelope of a heptose-deficient lipopolysaccharide mutant of Escherichia coli, GR467, was studied after fractionation into its outer and cytoplasmic membrane components by means of sucrose density gradient centrifugation. The outer membrane of GR467 had a lower density than that of its parent strain, CR34. Analysis of the fractionated membranes of GR467 indicated that the phospholipid-to-protein ratio had increased 2.4-fold in the outer membrane. The ratio in the mutant cytoplasmic membrane was also increased, although to a lesser extent. By employing a third parameter, the lipid A content of the outer membrane, it was found that the observed phospholipidto-protein change in the outer membrane was due predominantly to a decrease in the relative amount of protein. This decrease in protein was particularly significant, since it was concomitant with a 68% decrease in the lipid A recovered in the outer membrane of GR467 relative to the lipid A recovered in the outer membrane of CR34. Similar findings were observed in a second heptose-deficient mutant of E. coli, RC-59. The apparent protein deficiency in GR467 was further studied by subjecting solubilized envelope proteins to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. It was found that major envelope proteins which were localized in the outer membrane were greatly diminished in GR467. Two revertants of GR467 with the wild-type amounts of heptose had wild-type relative levels of protein in their outer membranes. A partial heptose revertant had a relative level of protein in its outer membrane between those of the mutant and wild type. The organization of the gram-negative bacterial cell envelope may be considered on a number of levels: (i) the chemical composition and linear structure of the major macromolecules, e.g., lipopolysaccharide, lipoprotein, protein, phospholipid, and peptidoglycan; (ii) the stereochemistry of these macromolecules; (iii) the cross-linking of repeating units of a given macromolecule; and (iv) the bonds linking different macromolecules. These factors all contribute to the formation of a multilayered structure which supports such phenomena as active transport of small molecules, enzymatic reactions, and, indeed, the growth of existing envelope structures involving translocation of preformed subunits or in situ synthesis. The cell envelope is generally thought to consist of three distinct layers-the outermost layer containing lipopolysaccharide (LPS), protein, and phospholipid, a rigid peptidoglycan layer, and the cytoplasmic membrane consisting mainly of protein and phospholipid (7, 22, 24, 28, 37, 44). Extensive literature exists describing the chemical composition and linear structure of the LPS in gram-negative bacteria. A large portion of the information available has been derived from analyses of Salmonella strains (19). These analyses indicate that the LPS structure can be divided into three regions, designated as 0-antigen polysaccharide, core polysaccharide, and lipid A. The 0-antigen sugars, wh...

Mutants of Escherichia coli K12 lacking all ‘major’ proteins of the outer cell envelope membrane

“…1-6, envelopes; 7-9 whole cells; 1, wild type; 2, P692; 3-5, three different TulI* resistant mutants of P692; 6,7, wild type; 8, P692; 9, Tull* resistant mutant of P692. Photographs 6-9 were exposed shorter than 1-5 for a clearer presentation of missing bands, and many minor bands are not visible in [6][7][8][9]. Both bands at I in 1 and 6 belong to protein I (see table 1).…”

“…One could therefore have been led to suspect that lack of one major protein is compensated for by increased production of another one, possibly in order to maintain integrity of the outer membrane. Doubts to this view came from the studies of Koplow and Goldfine [7] as well as from those of Ames [ 14] ; lipopolysaccharide defective mutants of L: coli K12 and Salmonella typhimurium showed rather significant deficiencies of all outer membrane proteins in the 38 000-33 000 daltons range. The former authors showed that in the outer membrane of such a mutant the phospholipid-to-protein ratio had increased 2.4-fold.…”

“…Recently, a number of mutants have been isolated exhibiting various types of abnormal protein composition of the outer membrane. Such mutants miss one or another major protein [8,10,13] or harbor decreased amounts of several of these proteins [7,14]. Also, it appears that the relative amounts of these proteins can differ substantially depending on growth conditions [ 10].…”

Characterization of a coronavirus

“…The author is not aware of any other reported example of similar behavior by a virus structural protein. However, the electrophoretic mobility in SDS of one of the outer membrane proteins from E. coli, as well as flagellin, and some proteins of the human erythrocyte membrane are also reduced after boiling (Bragg and Hou, 1972;Fairbanks et al, 1972;Garten et al, S. STURMAN 1975;Inouye and Yee, 1973;Kondoh and incompletely denatured by SDS at 25" or Hotani, 1974;Koplow and Goldfine, 1974;37". Reithmeier and Bragg, 1974; The role of P-MSH and DTT at elevated 1973aDTT at elevated ,b, 1974.…”

Identification by Tn10 transposon mutagenesis of host factors involved in the biosynthesis of K99 fimbriae of Escherichia coli: Effect of LPS core mutations

“…In so-called deep-rough LPS mutants (Rdl, Rd 2 or Re LPS), the incorporation of a number of outer membrane proteins ~s strongly reduced [16] Therefore, the effects of the transposon insertion m the rfaQGP gene cluster could result in a strongly reduced incorporation of FanD m the outer membrane and/or m a hampered transport of K99 major subumts across the outer membrane Experiments carned out by Wfllemsen m the laboratory of Dr Jann and in cooperation w~th Hemtz Hoschutzky gave some mformat~on on the possible role of LPS in the formation of K99 flmbrlae K99 and K88ab fimbrlae were purlfled [17] [22] and special color filters were used for photography Lane 5, lmmunoblottlng of K99 fimbnal subumts following nondenaturmg gel electrophoresls (m the absence of SDS) Blots were developed with LPS antiserum obtained from Hoschutzky M, marker proteins, molecular masses an kDa antigen These fimbnae preparations were subjected to denaturing SDS polyacrylamlde gel electrophoresls and the gels were stained for protein and LPS Furthermore, K99 subumts were ISOlated from the penplasm and subjected to nondenaturmg polyacrylamlde gel electrophoresls and ~mmunoblottmg with specific antl-LPS antiserum The result showed that the K99 major subumt stained with the LPS staining, whereas the K88ab subunlts did not Furthermore, K99 subunlts reacted specifically with the LPS antiserum (Fig 4) This strongly suggested that the purified K99 major fimbnal subumt contained covalently bound LPS Perhaps LPS molecules play a crucial, so far unknown role m the process of K99 biogenesis Attempts to isolate K99 fimbrme from the mutant strain EP14 and to analyze these flmbrme for LPS binding were not successful, simply because cells of strata EP14 do not synthesis enough K99 fimbrme (they are practically K99-negatwe) to allow their Isolation LPS as produced by mutant EP14 may not be able to bound to K99 subunlts, which might result in a lack of fimbrlae production Recently, Stanley et al [18], described an effect of an rfaP lesion on the loss of actwlty in the secreted form of E cohhaemolysm Besides, E coh rfaH and rfaG mutants are mable to produce surface flagella and sex plh [19,20] These findlngs and our results point to a more general reqmrement of functional LPS m the secreuon of proteins across the outer membrane, especially of proteins involved in virulence and fertlhty 206…”