The nucleotide sequence coding for major outer membrane protein OmpA of Shigella dysenteriae.

The nucleotide sequence of the ompA gene from Shigella dysenteriae has been determined and the amino acid sequence of the pro-OmpA protein predicted. Sequence comparison between the ompA genes of S.dysenteriae and Escherichia coli showed that features such as mRNA secondary structure and codon usage, as well as polypeptide function, have been conserved during evolution. The pro-OmpA protein of S.dysenteriae consists of 351 residues, as opposed to the 346 of the E.coli protein and also shows several amino acid changes. These changes have been used to interpret differences in the biological activity of the two proteins.     

Molecular characterization of the gene coding for major outer membrane protein OmpA from Enterobacter aerogenes

The ompA gene from Enterobacter aerogenes was subcloned into a low-copy-number plasmid vector and the resultant plasmid, pTU7En, used to study its expression in Escherichia coli K12. Although the gene was strongly expressed and large amounts of OmpA protein were present in the outer membrane its product was not functionally identical to the E. coli polypeptide. In particular, the E. aerogenes OmpA protein was unable to confer sensitivity to OmpA-specific phages of E. coli. When the primary structure of the protein was deduced from the nucleotide sequence of its gene it was found that three domains differed extensively from the corresponding regions of the E. coli protein. As two of these are known to be exposed on the cell surface we inferred that these alterations are responsible for differences in the biological activity of the two proteins.     

Bacteriophage receptor area of outer membrane protein OmpA of Escherichia coli K-12.

A number of T-even-like bacteriophages use the outer membrane protein OmpA of Escherichia coli as a receptor. We had previously analyzed a series of ompA mutants which are resistant to such phages and which still produce the OmpA protein (R. Morona, M. Klose, and U. Henning, J. Bacteriol. 159:570-578, 1984). Mutational alterations were found near or at residues 70, 110 and 154. Based on these and other results a model was proposed showing the amino-terminal half of the 325-residue protein crossing the outer membrane repeatedly and being cell surface exposed near residues 25, 70, 110, and 154. We characterized, by DNA sequence analysis, an additional 14 independently isolated phage-resistant ompA mutants which still synthesize the protein. Six of the mutants had alterations identical to the ones described before. The other eight mutants possessed seven new alterations: Ile-24----Asn, Gly-28----Val, deletion of Glu-68, Gly-70----Cys, Ser-108----Phe, Ser-108----Pro, and Gly-154----Asp (two isolates). Only the latter alteration resulted in a conjugation-deficient phenotype. The substitutions at Ile-24 and Gly-28 confirmed the expectation that this area of the protein also participates in its phage receptor region. It is unlikely that still other such sites of the protein are involved in the binding of phage, and it appears that the phage receptor area of the protein has now been characterized completely.     

Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12

The 325-residue OmpA protein is one of the major outer membrane proteins of Escherichia coli K-12. A model, in which this protein crosses the membrane eight times in an antiparallel beta-sheet conformation and in which regions around amino acids 25, 70, 110 and 154 are exposed at the cell surface, had been proposed. Linkers were inserted into the ompA gene with the result that OmpA proteins, carrying non-OmpA sequences between residues 153 and 154 or 160 and 162, were synthesized. Intact cells possessing these proteins were treated with proteases. Insertion of 15 residues between residues 153 and 154 made the protein sensitive to proteinase K and the sizes of the two cleavage products were those expected following proteolysis at the area of the insertion. Addition of at least 17 residues between residues 160 and 162 left the protein completely refractory to protease action. Thus, the former area is cell surface exposed while the latter area appears not to be. The insertions did not cause a decrease in the concentration of the hybrid proteins as compared to that of the OmpA protein, and in neither case was synthesis of the protein deleterious to cell growth. It is suggested that this method may serve to carry peptides of practical interest to the cell surface and that it can be used to probe surface-located regions of other membrane proteins.     

TolB protein of Escherichia coli K-12 interacts with the outer membrane peptidoglycan-associated proteins Pal, Lpp and OmpA

The Tol–Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. Transmembrane domains of TolQ, TolR and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. TolB and the central domain of TolA interact in vitro with the outer membrane porins. In this study, both genetic and biochemical analyses were carried out to analyse the links between TolB, Pal and other components of the cell envelope. It was shown that TolB could be cross-linked in vivo with Pal, OmpA and Lpp, while Pal was associated with TolB and OmpA. The isolation of pal and tolB mutants disrupting some interactions between these proteins represents a first approach to characterizing the residues contributing to the interactions. We propose that TolB and Pal are part of a multiprotein complex that links the peptidoglycan to the outer membrane. The Tol–Pal proteins might form transenvelope complexes that bring the two membranes into close proximity and help some outer membrane components to reach their final destination.     

Cloned DNA fragment specifying major outer membrane protein a in Escherichia coli K-12.

Plasmid pMC44 is a recombinant plasmid that contains a 2-megadalton EcoRI fragment of Escherichia coli K-12 DNA joined to the cloning vehicle, pSC101. The polypeptides specified by plasmid pMC44 were identified and compared with those specified by pSC101 to determine those that are unique to pMC44. Three polypeptides specified by plasmid pMC44 were localized in the cell envelope fraction of minicells: a Sarkosyl-insoluble outer membrane polypeptide (designated M2), specified by the cloned 2-megadalton DNA fragment, and two Sarkosyl-soluble membrane polypeptides specified by the cloning plasmid pSC101. Bacteria containing plasmid pMC44 synthesized quantities of M2 approximately equal to the most abundant E. coli K-12 outer membrane protein. Evidence is presented that outer membrane polypeptide M2, specified by the recombinant plasmid pMC44, is the normal E. coli outer membrane protein designated protein a by Lugtenberg and 3b by Schnaitman.     

The signal sequence suffices to direct export of outer membrane protein OmpA of Escherichia coli K-12.

We studied whether information required for export is present within the mature form of the Escherichia coli 325-residue outer membrane protein OmpA. We had previously analyzed overlapping internal deletions in the ompA gene, and the results allowed us to conclude that if such information exists it must be present repeatedly within the membrane part of the protein encompassing amino acid residues 1 to 177 (R. Freudl, H. Schwarz, M. Klose, N. R. Movva, and U. Henning, EMBO J. 4:3593-3598, 1985). A deletion which removed the codons for amino acid residues 1 to 229 of the OmpA protein was constructed. In this construct the signal sequence was fused to the periplasmic part of the protein. The resulting protein, designated Pro-OmpA delta 1-229, was processed, and the mature 95-residue protein accumulated in the periplasm. Hence, information required for export does not exist within the OmpA protein.     

Cloning and expression in Escherichia coli of Serratia marcescens genes encoding prodigiosin biosynthesis

Prodigiosin, the bright red pigment produced by many strains of Serratia marcescens, is synthesized by a bifurcated pathway that terminates in the enzymatic condensation of the two final products, a monopyrrole and a bipyrrole . Sau3A fragments of S. marcescens ( Nima ) DNA were introduced into a strain of Escherichia coli K-12 by use of the cosmid vector pHC79 , and transformed clones were selected based on resistance to ampicillin. Among 879 transformants screened, 2 could be induced to synthesize prodigiosin when supplied with either one or both terminal products of the bifurcated pathway. Data are presented to support the idea that production of prodigiosin is not usually mediated by a plasmid.     

A unique amino acid substitution in the outer membrane protein OmpA causes conjugation deficiency in Escherichia coli K-12

The outer membrane protein OmpA of E. coli K-12 can serve as a receptor for phages and is required for stabilizing mating aggregates during F'-mediated conjugation. Selection for resistance to OmpA-specific phages yields mutants with alterations in the protein at four cell surface exposed sites. It is shown that conjugation deficiency can be caused by apparently only one type of amino acid substitution at one of these sites, the replacement of glycine-154 by aspartic acid. This suggests that, in contrast to binding of phages, a ligand of the donor cell recognizes only a very small area of the protein.     

Escherichia coli K-12 outer membrane protein (OmpA) as a bacteriophage receptor: analysis of mutant genes expressing altered proteins.

The outer membrane protein OmpA of Escherichia coli K-12 serves as a receptor for a number of T-even-like phages. We have isolated a series of ompA mutants which are resistant to such phages but which still produce the OmpA protein. None of the mutants was able to either irreversibly or reversibly bind the phage with which they had been selected. Also, the OmpA protein is required for the action of colicins K and L and for the stabilization of mating aggregates in conjugation. Conjugal proficiency was unaltered in all cases. Various degrees of colicin resistance was found; however, the resistance pattern did not correlate with the phage resistance pattern. DNA sequence analyses revealed that, in the mutants, the 325-residue OmpA protein had suffered the following alterations: Gly-65----Asp, Gly-65----Arg, Glu-68----Gly, Glu-68----Lys (two isolates), Gly-70----Asp (four isolates), Gly-70----Val, Ala-Asp-Thr-Lys-107----Ala-Lys (caused by a 6-base-pair deletion), Val-110----Asp, and Gly-154----Ser. These mutants exhibited a complex pattern of resistance-sensitivity to 14 different OmpA-specific phages, suggesting that they recognize different areas of the protein. In addition to the three clusters of mutational alterations around residues 68, 110, and 154, a site around residue 25 has been predicted to be involved in conjugation and in binding of a phage and a bacteriocin (R. Freudl, and S. T. Cole, Eur. J. Biochem, 134:497-502, 1983; G. Braun and S. T. Cole, Mol. Gen. Genet, in press). These four areas are regularly spaced, being about 40 residues apart from each other. A model is suggested in which the OmpA polypeptide repeatedly traverses the outer membrane in cross-beta structure, exposing the four areas to the outside.     

Promoter exchange between ompF and ompC, genes for osmoregulated major outer membrane proteins of Escherichia coli K-12.

Expression of the ompF and ompC genes coding for major outer membrane proteins OmpF and OmpC is regulated in opposite directions by medium osmolarity. Chimera genes were constructed by a reciprocal exchange of the promoter-signal sequence region between the two genes. The chimera gene construction was designed so that the proteins synthesized by these genes were essentially the same as the OmpC and OmpF proteins. Studies with the chimera genes demonstrated that the osmoregulation of the OmpF-OmpC synthesis was promoter dependent. They also showed that cells grew normally even when the osmoregulation took place in opposite directions. The effects of the ompR2 and envZ mutations, which suppress ompC and ompF expression, respectively, also became reversed. The reduced expression was still subject to the promoter-controlled osmoregulation. Based on these observations, the mechanism of regulation of the ompF-ompC gene expression and its physiological importance are discussed.     

The nature of information, required for export and sorting, present within the outer membrane protein OmpA of Escherichia coli K-12.

Information, in addition to that provided by signal sequences, for translocation across the plasma membrane is thought to be present in exported proteins of Escherichia coli. Such information must also exist for the localization of such proteins. To determine the nature of this information, overlapping inframe deletions have been constructed in the ompA gene which codes for a 325-residue major outer membrane protein. In addition, one deletion, encoding only the NH2-terminal part of the protein up to residue 160, was prepared. The location of each product was determined by immunoelectron microscopy. Proteins missing residues 4-45, 43-84, 46-227, 86-227 or 160-325 of the mature protein were all efficiently translocated across the plasma membrane. The first two proteins were found in the outer membrane, the others in the periplasmic space. It has been proposed that export and sorting signals consist of relatively small amino acid sequences near the NH2 terminus of an outer membrane protein. On the basis of sequence homologies it has also been suggested that such proteins possess a common sorting signal. The locations of the partially deleted proteins described here show that a unique export signal does not exist in the OmpA protein. The proposed common sorting signal spans residues 1-14 of OmpA. Since this region is not essential for routing the protein, the existence of a common sorting signal is doubtful. It is suggested that information both for export (if existent) and localization lies within protein conformation which for the former process should be present repeatedly in the polypeptide.     

An outer membrane protein (OmpA) of Escherichia coli K-12 undergoes a conformational change during export

Pulse-chase experiments were performed to follow the export of the Escherichia coli outer membrane protein OmpA. Besides the pro-OmpA protein, which carries a 21-residue signal sequence, three species of ompA gene products were distinguishable. One probably represented an incomplete nascent chain, another the mature protein in the outer membrane, and the third, designated imp-OmpA (immature processed), a protein which was already processed but apparently was still associated with the plasma membrane. The pro- and imp-OmpA proteins could be characterized more fully by using a strain overproducing the ompA gene products; pro- and imp-OmpA accumulated in large amounts. It could be shown that the imp- and pro-OmpA proteins differ markedly in conformation from the OmpA protein. The imp-OmpA, but not the pro-OmpA, underwent a conformational change and gained phage receptor activity upon addition of lipopolysaccharide. Utilizing a difference in detergent solubility between the two polypeptides and employing immunoelectron microscopy, it could be demonstrated that the pro-OmpA protein accumulated in the cytoplasm while the imp-OmpA was present in the periplasmic space. The results suggest that the pro-OmpA protein, bound to the plasma membrane, is processed, and the resulting imp-OmpA, still associated with the plasma membrane, recognizes the lipid A moiety of the lipopolysaccharide. The resulting conformational change may then force the protein into the outer membrane.     

Action of a major outer cell envelope membrane protein in conjugation of Escherichia coli K-12.

Protein II, a major outer cell envelope membrane protein, was found together with lipopolysaccharide to stoichiometrically inhibit conjugation in Escherichia coli K12.     

Nucleotide sequences of the trpG regions of Escherichia coli,Shigella dysenteriae,Salmonella typhimurium and Serratia marcescens

The nucleotide sequences of Serratia marcescens trpG and the corresponding regions of Escherichia coli, Shigella dysenteriae and Salmonella typhimurium trpD have been determined. Analysis of the nucleotide sequence divergence suggests the following evolutionary relationships: Serratia-[Salmonella, (Escherichia, Shigella)]. Partial reconstruction of ancestral nucleotide sequences and subsequent analysis of nucleotide substitutions show that the majority of nucleotide substitutions in the evolution of trp(G)D are transitions that result in a reduction of G + C content. Since most of the nucleotide substitutions are in the third position of codons, bias in synonymous codon usage also reflects G + C content. The trpE-trp(G)D junction in the four organisms is characterized by overlapping translation termination and initiation codons. The relative positions of trpE and trp(G)D thus became fixed in evolution before the fusion of trpG and trpD. Nucleotide sequences representing the fusion of trpG and trpD in Escherichia, Shigella and Salmonella are not more nor less divergent than other portions of the trp(G)D coding sequences.     

Comparative analysis of the structure and function of recA genes from Serratia marcescens Sb and Escherichia coli K-12

Nucleotide sequence of the 1276 bp fragment of Serratia marcescens DNA coding for the recASM gene has been determined. This structure was shown to contain an ORF corresponding to a protein with molecular weight of 37766 D. Comparative analysis of the regulatory part of recASM and recAEC (Escherichia coli) demonstrated identity of "-35" and "-10" boxes for these genes and similarity of the SOS box and the enhancer sequences. A comparison of the amino acids sequences of RecASM, RecAEC and RecAPA (Pseudomonas aeruginosa) proteins revealed a great conservatism in the N-terminus and in some structural patches (alpha-helices and beta-sheets) of the RecA proteins predicted by the model of Blanar et al. In contrast, a strong variability of the C-terminus (for the last 25 amino acids, in particular) was revealed. A necessity for definite amino acids composition of the carboxy-terminal end is discussed.     

Diploidy for a structural gene specifying a major protein of the outer cell envelope membrane from Escherichia coli K-12.

Homogenotes, heterogenotes, and intergeneric hybrids have been studied that are diploid for the structural gene of a major outer cell envelope membrane protein (protein II) from Escherichia coli. This protein can act as a phage receptor. In wild-type homogenotes, diploidy for the gene did not cause a gene dosage effect. It could be shown with two heterogenotes that both the chromosomal mutant and the episomal wild-type genes are expressed, and in each case more of the mutant than the wild-type protein species was found in the cell envelope. In on case of 21 phage-resistant mutants missing protein II was a trans effect observed of the mutant gene on the expression of the episomal wild type gene. Transfer of E. coli episomes carrying the protein II structural gene into Salmonella typhimurium and Proteus mirabilis resulted in intergeneric hybrids that became sensitive to the relevant phage and harbored the E. coli protein II in their cell envelopes. The results may be taken as suggestive evidence for a simple feedback mechanism for the regulation of synthesis of protein II, and they show that there are no highly specific requirements on protein primary structure for incorporation into an outer cell envelope membrane.