Ingrid Hindennach

The Major Proteins of the Escherichia coli Outer Cell Envelope Membrane

A procedure is described that from one batch of cells allows the isolation of all major proteins of the outer cell envelope membrane of Escherichia coli B/r. The method involves differential extraction of cell envelopes with ionic and non-ionic detergents with and without Mg2+ present, and the proteins are finally separated by molecular sieve chromatography in the presence of sodium dodecylsulfate. From 200 g cell paste in ten days (including the five days chromatography) approximately 120 mg protein I (molecular weight approximately 38,000), approximately 110 mg protein II* (molecular weight approximately 33,000), approximately 50 mg protein III (molecular weight approximately 17,000), and approximately 30 mg protein IV (molecular weight approximately 7,000) are obtained in pure state, and these yields are near the expected ones assuming quantitatve recoveries. Protein II* is a heat-modifiable protein (perhaps due to complete unfolding and/or binding of sodium dodecyl-sulfate only at higher temperatures), and the isolated protein is completely in its unmodified form. Protein IV, Brauns lipoprotein, in the cell envelope exists in two forms, one covalently bound to the murein layer and the other not. The isolated protein IV represents the free form of the protein that so far had not been isolated; its protein part dies not differ substantially from that of the bound form.

Correlation of 30S ribosomal proteins ofEscherichia coli isolated in different laboratories

SummaryRibosomal proteins isolated from 30S subunits ofE. coli in four laboratories have been correlated by using two-dimensional gel electrophoresis, immunological techniques, amino acid compositions and molecular weights. The results are given in the Table. A common nomenclature for naming 30 S ribosomal proteins and their genetic loci is proposed.

The Major Proteins of the Escherichia coli Outer Cell Envelope Membrane. Characterization of Proteins II* and III, Comparison of All Proteins

Protein II*, one of the major Escherichia coli outer cell envelope membrane proteins has been characterized. The protein is heat-modifiable and perhaps due to complete unfolding and/or binding of sodium dodecylsulfate only at higher temperatures the modified protein exhibits a higher apparent molecular weight (33,000) than the non-modified form (28,000). Protein-chemical evidence as well as the behavior of two mutant proteins II* very strongly suggest that this protein consists of a single polypeptide chain and that in the strains studied there is no other major protein with similar characteristics. For another outer membrane protein, protein III (molecular weight 17,000), it has not yet been established if it should be classified as a major protein. Protein III consists of one or perhaps two polypeptide chains. The possibility existed that protein III is bound covalently to lipopolysaccharide, and this has been ruled out. Also, the lipopolysaccharide of the E. coli strains studied does not carry covalently bound protein in amounts anywhere near stoichiometry. N-on-protein substituents were neither found in protein II* nor in protein III. It is concluded that in E. coli B/r and the E. coli K12 strains used there are three major proteins: I, II, and IV; protein III may also belong to this class. There are not more major proteins than these. All four proteins are compared and discussed regarding their unknown functions and their relation to E. coli outer membrane proteins studied by other authors.

Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12

The 325-residue outer membrane protein OmpA of Escherichia coli has been proposed to consist of a membrane-embedded moiety (residues 1 to about 170) and a C-terminal periplasmic region. The former is thought to comprise eight transmembrane segments in the form of antiparallel β-strands, forming an amphiphilic β connected by exposed turns. Several questions concerning this model were addressed. Thus no experimental evidence had been presented for the turns at the inner leaflet of the membrane and it was not known whether or not the periplasmic part of the polypeptide plays a role in the process of membrane incorporation. Oligonucleotides encoding trypsin cleavage sites were inserted at the predicted turn sites of the ompA gene and it was shown that the encoded proteins indeed become accessible to trypsin at the modified sites. Together with previous results, these data also show that the turns on both sides of the membrane do not possess specifically topogenic information. In two cases one of the two expected tryptic fragments was lost and could be detected at low concentration in only one case. Therefore, bilateral proteolytic digestion of outer membranes can cause loss of β-strands and does not necessarily produce a reliable picture of protein topology. When ompA genes were constructed coding for proteins ending at residue 228 or 274, the membrane assembly of these proteins was shown to be partially defective with about 20% of the proteins not being assembled. No such defect was observed when, following the introduction of a premature stop codon, a truncated protein was produced ending with residue 171. It is concluded that (1) the proposed β-barrel structure is essentially correct and (2) the periplasmic part of OmpA does not play an active role in, but can, when present in mutant form, interfere with membrane assembly.

Major proteins of the outer cell envelope membrane ofEscherichia coli K-12: Multiple species of protein I

SummaryProtein I, one of the major outer membrane proteins ofE. coli, in a number of strains exists as two electrophoretically separable species Ia and Ib. Two phages, TuIa and TuIb, have been found which use, as receptors, proteins Ia and Ib, respectively. Selection for resistance to phage TuIb yielded mutants still possessing protein Ia and missing protein Ib (Ia+ Ib-). Selection in this background, for resistance to phage TuIa yielded one class of mutants missing both species of protein I and another synthesizing a new species of protein I, polypeptide Ic.Tryptic fingerprints of Ia and Ic are very similar and the sequence of 8 N-terminal amino acids is identical for Ia and Ic. Yet, Ic showed an entirely different pattern of cyanogen bromide fragments than that of protein Ia. With another example (cyanogen bromide fragments of protein II*, with and without performic acid oxidation) it is shown that protein modification can lead to gross changes of the electrophoretic mobility of cyanogen bromide fragments. It is not unlikely that all protein I species observed so far represent in vivo modifications of one and the same polypeptide chain.A genetic analysis together with data from other laboratories revealed that at least 4 widely separated chromosomal loci are involved in the expression of the protein I species known to date.

Major proteins of the Escherichia coli outer cell envelope membrane preliminary characterization of the phage λ receptor protein

The outer cell envelope membrane of Gram-negative bacteria harbors a set of a few major or abundant proteins. For some of those from Salmonella typhimurium and Escherichiu coli it has been shown that they function as non-specific hydrophilic pores for various low molecular weight hydrophilic solutes [ 141, and such proteins have therefore been called porins [l] . In E. coli K-l 2, these porins are represented by the group of proteins I, polypeptides Ia and Ib ([5,6] ; identical with proteins la/lb [7,8] ; b/c [9] ; O-9/0-8 [lo,1 l] ; Al/AZ [12]). Such a porin of E. coli BE is the matrix protein [ 131 , probably corresponding to protein Ia in K-l 2 strains. In agreement with porin function it has been shown that these polypeptides are trans-membrane proteins [13-l 51. In maltose-induced cells the receptor for phage X (lamB protein) also constitutes a major outer membrane protein [ 161. It is known to much increase the outer membrane’s permeability for maltose and maltotriose, and is thought to form a pore for these sugars [ 17 ] and, to a certain degree, also for glucose and lactose [ 181. There are striking similarities between E. coli’s proteins I and lamB. Both are tightly but noncovalently bound to the murein layer of the cell envelope [13] (for lamB: Schnaitman, personal communication). In the murein-protein complexes obtained as in [ 131 both are completely resistant to proteolytic degradation ([ 13,191 and see below). Similarity concerning functions and the behavior just mentioned may allow to expect a similar structure of

Cloned structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12

SummarypTU 100 is a hybrid plasmid constructed by cloning a 7.5 Kb EcoRI fragment (carrying the wildtype ompA gene) onto pSC 101 (Henning et al., 1979). This plasmid confers sensitivity to phages Tull* and K3h1 when present in an ompA host strain, due to the expression of the phage receptor protein II* from the plasmid ompA+ gene. Plasmid mutants have been isolated that have become resistant to one or both of these phages. Restriction endonuclease analysis and DNA-sequencing studies in these plasmids demonstrate that a BamHI site and two PvuII sites are located within the ompA gene. BamHI cuts the gene at a site corresponding to residue 227 within a total of 325 amino acid residues.Neither the wildtype ompA gene nor the BamHI fragment encoding the NH2-terminal part of the protein (residues 1–227) could be transferred to a high copy number plasmid, presumably due to lethal overproduction of the protein or its NH2-terminal fragment. However, the NH2-terminal fragment derived from one of the ompA mutants of pTU100 could be transferred to the high copy number plasmid pBR322, and was expressed in the presence of the amber suppressors supD or supF. Under these conditions two new envelope proteins with apparent molecular weights of 30,000 and 24,000 were synthesized, and the cells became sensitive to phage TuII*, indicating the presence of phage receptor activity in the outer membrane. The major, 24,000 dalton protein has the molecular weight expected of a protein comprising residues 1–227 of protein II*. DNA-sequencing studies demonstrated that no termination codons are present in the DNA region immediately downstream from the BamHI site at residue 227 in this hybrid plasmid, and it is therefore likely that the 24,000-dalton protein arises from the posttranslational proteolytic cleavage of a larger polypeptide. The 30,000-dalton protein is a likely candidate for such a larger polypeptide. These results also demonstrate that the 98 CO2H-terminal residues of wildtype protein II* (resisdues 228–325) are not required either for the activity of the protein as a phage receptor or for its incorporation into the outer membrane.

Single mutations in a gene for a tail fiber component of an Escherichia coli phage can cause an extension from a protein to a carbohydrate as a receptor

The T-even type Escherichia coli phage Ox2 recognizes the outer membrane protein OmpA as a receptor. This recognition is accomplished by the 266 residue protein 38, which is located at the free ends of the virions long tail fibers. Host-range mutants had been isolated in three consecutive steps: Ox2----Ox2h5----Ox2h10----Ox2h12, with Ox2h12 recognizing the outer membrane protein OmpC efficiently and having lost some affinity for OmpA. Protein 38 consists, in comparison with these proteins of other phages, of two constant and one contiguous array of four hypervariable regions; the alterations leading to Ox2h12 were all found within the latter area. Starting with Ox2h12, further host-range mutants could be isolated on strains resistant to the respective phage: Ox2h12----h12h1----h12h1.1----h12h1.11----h12 h1.111. It was found that Ox2h12h1.1 (and a derivative of Ox2h10, h10h4) probably uses, instead of OmpA or OmpC, yet another outer membrane protein, designated OmpX. Ox2h12h1.11 was obtained on a strain lacking OmpA, -C and -X. This phage could not grow on a mutant of E. coli B, possessing a lipopolysaccharide (LPS) with a defective core oligosaccharide; Ox2h12h1.111 was obtained from this strain. It turned out that the latter two mutants used LPS as a receptor, most likely via its glucose residues. Selection for resistance to them in E. coli B (ompA+, ompC-, ompX-) yielded exclusively LPS mutants, and in another strain, possessing OmpA, C and X, the majority of resistant mutants were of this type. Isolated LPS inactivated the mutant phages very well and was inactive towards Ox2h12. By recombining the genes of mutant phages into the genome of parental phages it could be shown that the phenotypes were associated with gene 38. All mutant alterations (mostly single amino acid substitutions) were found within the hypervariable regions of protein 38. In particular, a substitution leading to Ox2h12h1.11 (Arg170----Ser) had occurred at the same site that led to Ox2h10 (His170----Arg), which binds to OmpC in addition to OmpA. It is concluded that not only can protein 38 gain the ability to switch from a protein to a carbohydrate as a receptor but can do so using the same domain of the polypeptide.

Lethal mutations in the structural gene of an outer membrane protein (OmpA) of Escherichia coli K12

SummaryThe gene ompA encodes a major outer membrane protein of Escherichia coli. Localized mutagenesis of the part of the gene corresponding to the 21-residue signal sequence and the first 45 residues of the protein resulted in alterations which caused cell lysis when expressed. DNA sequence analyses revealed that in one mutant type the last CO2H-terminal residue of the signal sequence, alanine, was replaced by valine. The proteolytic removal of the signal peptide was much delayed and most of the unprocessed precursor protein was fractioned with the outer membrane. However, this precursor was completely soluble in sodium lauryl sarcosinate which does not solubilize the OmpA protein or fragments thereof present in the outer membrane. Synthesis of the mutant protein did not inhibit processing of the OmpA or OmpF proteins. In the other mutant type, multiple mutational alterations had occurred leading to four amino acid substitutions in the signal sequence and two affecting the first two residues of the mature protein. A reduced rate of processing could not be clearly demonstrated. Membrane fractionation suggested that small amounts of this precursor were associated with the plasma membrane but synthesis of this mutant protein also did not inhibit processing of the wild-type OmpA or OmpF proteins. Several lines of evidence left no doubt that the mature, mutant protein is stably incorporated into the outer membrane. It is suggested that the presence, in the outer membrane, of the mutant precursor protein in the former case, or of the mutant protein in the latter case perturbs the membrane architecture enough to cause cell death.

Characterisation of the promoters for the ompA gene which encodes a major outer membrane protein of Escherichia coli

SummaryThe regulatory region of the ompA gene from Escherichia coli has been characterized by biochemical and genetic approaches. Two overlapping promoters, P1 and P2, organized in that order with respect to the ompA coding sequence, were identified and it was found that ompA possesses an unusually long leader region. Both P1 and P2 were active in an in vitro transcription system although S1 mapping analysis of the ompA mRNA made in vivo showed that P2 was mainly responsible for transcription of the gene. Confirmation of this was obtained by studying down-promoter mutants of ompA cloned in pSC101. These mutants were classified into two groups, deletions and insertions. The deletions, which were caused by the IS102 insertion element found in pSC101 removed the-35 regions of both P1 and P2. However, since P2 was distally situated with respect to the IS element it was less extensively damaged and it is proposed that the residual P2 sequence is responsible for the low level of expression observed. In addition to an IS102 insertion in the promoter region four IS1 insertion mutants were characterized. These had integrated at different positions in the ompA leader region and were all incompletely polar.