Information within the mature LamB protein necessary for localization to the outer membrane of E coli K12

It has been proposed that the efficient localization of the outer membrane protein LamB requires a functional signal sequence and at least two additional regions contained within the mature protein. We define these regions more precisely by deletion analysis, and we describe methods for cloning deleterious lacZ fusions onto high-copy-number plasmids and generating in-frame deletions. Analysis of the effects of a series of internal lamB deletions on the export of a LamB-LacZ hybrid protein and of the LamB protein itself indicates that necessary informational signal(s) required for localization lie at the amino-terminal end of the protein. In addition, our analysis indicates that there is a region of information close to or within the fusion joint of the largest lamB-lacZ fusion that increases the efficiency of the export process. A unique deletion that removes a protein segment from amino acid 70 to 200 appears to prevent proteolytic removal of the signal sequence. Nevertheless, the mutant protein is exported to the outer membrane.     

Biochemical and genetic studies of recombination proficiency in Escherichia coli K12

Molecular Genetics and Genomics - Residual genetic recombination is carried out by recB - recC - mutants of E. coli. Recombinants (for one gene) formed by a recB - recC - parent were shown to be as...     

Antibodies against synthetic peptides and the topology of LamB, an outer membrane protein from Escherichia coli K12

LamB, an outer membrane protein from Escherichia coli K12, is involved in the transport of maltose and maltodextrins across the outer membrane and constitutes a receptor for a number of bacteriophages. A recent folding model proposes that LamB spans the outer membrane through a number of transmembranous segments separated by regions exposed either to the cell exterior or to the periplasm. This model is essentially based on predictions of structure and genetic arguments relying on the hypothesis that the mutations studied did not alter the folding of the protein. In order to obtain direct evidence with the unaltered protein, we elicited polyclonal antibodies against synthetic peptides corresponding to several LamB sequences. We chose four regions. Three of them [aa 147-161 (peptide 2), aa 371-385 (peptide 3), and aa 399-413 (peptide 4)] are predicted to face the outside of the cell, and the fourth (aa 19-33 (peptide 1)] is predicted to be periplasmic. By immunoblotting against extracts of various mutants, these antibodies were shown to be specific for LamB and targeted to the selected regions. In some cases, the recognition sites for antibodies were narrowed down to parts of a region. In vivo, on intact cells, anti-peptides 2, 3, and 4 reacted with LamB in an ELISA; this confirmed that regions of peptide 2 and 3 are located, at least in part, at the cell exterior and provided the first proof for a similar, situation of the region of peptide 4. Under the same conditions, anti-peptide 1 did not react with LamB.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of paf-acether by E. coli K12

Paf-acether (platelet-activating factor) is one of the most potent mediator of inflammation released from and acting on most cells that participate in inflammatory diseases. Its molecular structure is 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine. Two metabolic steps are involved in its biosynthesis: the action of a phospholipase A2 on choline-containing membrane alkyl-ether lipids results in the production of lyso paf-acether and acetylation of the lyso compound by an acetyltransferase yields the biologically active molecule. Membrane alkyl-ether lipids can therefore be considered as potential precursors of paf-acether and their composition has been studied in various cell types. In this work, we investigated the presence of paf-acether in E. coli. Our results showed that paf-acether can be obtained from E. coli K12 under a variety of bacterial growth conditions. Paf-acether from E. coli exhibited the same physicochemical and biological characteristics as synthetic paf-acether and that from eucaryotic cells. Therefore, it appears that E. coli itself has the ability of producing paf-acether, a result that could be of some importance with respect to the pathogenesis of Enterobacteria and the use of E. coli in the recombinant DNA technology.     

The primary structure of Escherichia coli K12 aspartokinase I-homoserine dehydrogenase I. Site of limited proteolytic cleavage by subtilisin.

The sequence of the first 25 residues of the homoserine dehydrogenase fragment, produced by limited proteolysis of aspartokinase I-homoserine dehydrogenase I with substilisin, has been determined. The sequence of a cyanogen bromide peptide (CB5, 59 residues), isolated from the entire protein, is also presented. Residues 1 to 18 of the subtilisin homoserine dehydrogenase fragment match the sequence 42 to 59 of peptide CB5.     

Isolation of different bacteriophages using the LamB protein for adsorption on Escherichia coli K-12.

Ten phages which use the LamB protein for adsorption have been isolated from sewage waters. Nine have a shape similar to lambda and require only the LamB protein for adsorption. One has a shape similar to T phages and can use either the LamB or the OmpC protein. Preliminary characterization by a number of criteria showed that at least nine of these phages were different and also differed from other known phages which use the LamB protein, such as lambda, 21, and K10.     

Coliphage which requires either the LamB protein or the OmpC protein for adsorption to Escherichia coli K-12.

Either of two different proteins in the outer membrane of Escherichia coli K-12 (LamB and OmpC) can function in the constitution of receptor activity for a newly isolated T-even bacteriophage. This bacteriophage (SSI) differs from other T-even phages which use the OmpC protein as their receptors. The simple procedure used to isolate phage SSI may be suitable for the detection of bacteriophages with novel outer membrane receptor requirements.     

In vitro proteolytic cleavage of the Escherichia coli Ada protein by the ompT gene product.

Down regulation of the adaptive response to alkylation damage in Escherichia coli has been proposed to occur by proteolytic cleavage of the regulatory Ada protein. In this paper, it is shown that proteolysis of the Ada protein as observed in cell extracts is caused by the ompT gene product. This protease, however, was not involved in switching off the adaptive response in vivo.     

Topography of the insertion of LamB protein into the outer membrane of Escherichia coli wild-type and lac-lamB cells

The appearance of newly induced LamB protein at the cell surface of Escherichia coli was followed topographically by immuno-electron microscopy. LamB protein was induced in E. coli wild-type or lac-lamB cells for a short period of time (4 to 6 min), such that the overall level of LamB protein in induced cells was at least twofold higher than that in uninduced cells. Antibodies bound to LamB protein exposed at the cell surface were labeled with a protein A-gold probe, and the probe distribution in briefly induced cells was compared to that in uninduced cells. Analysis of large numbers of cells showed that newly inserted LamB protein appeared homogeneously over the entire cell surface, both in wild-type cells and in lac-lamB cells. A peak of insertion which was observed at the division site of the cell was also observed in the absence of induction and in control experiments in which a nonspecific probe was used. It is concluded therefore that insertion of LamB protein into the cell envelope of E. coli occurs at multiple sites over the entire cell surface. The average amount of LamB protein which appeared at the cell surface after induction was determined for various cell size classes. It was found that cells of various size classes all synthesized LamB protein after induction, indicating that synthesis of the protein was not restricted to cells in a particular stage of the cell cycle. However, the rate of LamB synthesis was found to vary during the cell cycle: this rate was constant regardless of cell size in nondividing cells, whereas it increased in dividing cells. It is concluded that the accumulation of newly induced LamB protein follows a linear pattern.     

Mutagenesis by random linker insertion into the lamB gene of Escherichia coli K12

Summary Gene lamB encodes an outer membrane protein involved in maltose and maltodextrin transport as well as phage adsorption. The active form is a trimer. We characterized 11 mutations in lamB, obtained after random insertion of a BamH1 linker and screening for stable immunodetectable mutant proteins. Six mutations resulted in the loss of the distal part of the LamB protein either by deletion (five cases) or frameshift (one case). The six corresponding proteins had all lost the ability to confer phage sensitivity and the capacity to grow on dextrins, and to yield immunnodetectable oligomers. Induction of a high level of the four longest of these proteins was toxic to the cell. Five other mutations were due to in-frame insertions. In four cases, the corresponding proteins still had the ability to yield immunodetectable oligomers, to confer phage sensitivity and the capacity to grow on dextrins and were not toxic on induction. In one case (AJC73), the mutant protein had lost the first three properties and was toxic on induction. Deletions and duplications between some of the inserts were also constructed and studied. To account for our results we present a hypothetical scheme in which trimerization would not only be needed for phage sensitivity and growth on dextrins but also for proper insertion into the outer membrane. The C-terminus of the protein, as well as other regions such as the site of mutation AJC73, would be required for the formation of stable trimers. We tentatively interpret toxicity as due to improper insertion into the outer membrane. Our results also show that it is possible to insert several amino acids (up to 11 in one case) at a number of positions in LamB without appreciably affecting its export and activities.     

Escherichia coli K12 mutants defective in the glycine cleavage enzyme system

Two routes of one-carbon biosynthesis have been described in Escherichia coli K12. One is from serine via the serine hydroxymethyltransferase (SHMT) reaction, and the other is from glycine via the glycine cleavage (GCV) enzyme system. To isolate mutants deficient in the GCV pathway, we used a selection procedure that is based on the assumption that loss of this enzyme system in strains blocked in serine biosynthesis results in their inability to use glycine as a serine source. Mutants were accordingly isolated that grow with a serine supplement, but not with a glycine supplement. Enzyme assays demonstrated that three independently isolated mutants have no detectable GCV enzyme activity. The absence of a functional GCV pathway results in the excretion of glycine, but has no affect on the cell's primary source of one-carbon units, the SHMT reaction. The new mutations, designated gcv, were mapped between the serA and lysA genes on the E. coli chromosome.     

Membrane assembly: Posttranslational insertion of M13 procoat protein into E. coli membranes and its proteolytic conversion to coat protein in vitro

The major coat protein (gene 8 product) of bacteriophage M13 is an integral membrane protein during infection of host cells. It is synthesized as a larger precursor (procoat) with a leader sequence of 23 amino acids at its amino terminus. In vivo studies have shown that procoat only inserts into the host-cell plasma membrane after its synthesis is completed. We now demonstrate that procoat can post-translationally insert into inverted cytoplasmic membrane vesicles from E. coli and can be processed proteolytically to yield coat protein. Procoat changes from an assembly-competent substrate to an incompetent (denatured) form within minutes after its synthesis; much of the procoat that accumulates during an hour of in vitro synthesis is therefore denatured. These studies emphasize the importance of stringent criteria for the demonstration of obligate cotranslational assembly.     

Mapping of insertion element IS30 in the Escherichia coli K12 chromosome

Summary We identified seven phage clones containing the insertion element IS30 in a phage library mini-set, which includes 476 clones carrying chromosomal segments that cover almost the entire chromosome ofEscherichia coli K12 W3110 (Kohara et al. 1987). We could assign locations and orientations to four copies of IS30 (namedis30A tois30D) on the W3110 chromosome by restriction analysis of phage DNAs containing them. These IS30s were present at the same locations in chromosomes of both W3110 and anotherE. coli K12 strain JE5519, and thus are assumed to be present in otherE. coli K12 derivatives, including early isolates. Among the IS30 copies found, one (is30B) contained a large deletion and possessed only a 181 by stretch of the right terminal region of IS30.EMBL Accession Number: The EMBL accession number of the sequence reported in this paper is X17345     

Effect of charged residue substitutions on the membrane-interactive properties of signal sequences of the Escherichia coli LamB protein.

Although the central role of the signal sequence in protein export is well established, the molecular details underlying signal sequence in vivo function remain unclear. As part of our continuing effort to relate signal sequence phenotypes to specific biophysical properties, we have carried out an extensive characterization of the secondary structure and lipid interactions for a family of peptides corresponding to the wild-type E. coli LamB signal sequence, and mutants that harbor charged residue point mutations in the hydrophobic core region. We used membrane-resident fluorescence quenching according to the parallax method to determine the relative depth of insertion of tryptophan-labeled analogs of these peptides into the acyl chain region of bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. Also, restriction of acyl chain motion upon peptide binding was evaluated using steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Each of these peptides showed evidence of insertion into the acyl chain region, although most likely not in a transmembrane orientation. The mutant peptides were shown to have a reduced insertion potential relative to the wild-type peptide. Furthermore, tryptophan spectral properties indicated that insertion of the wild-type and mutant peptides enhances bilayer hydration. This effect was particularly pronounced with peptides harboring negatively charged aspartate point substitutions. The results are discussed in relation to the potential roles of signal sequences in mediating protein translocation.     

Multiple IS insertion sequences near the replication terminus in Escherichia coli K-12.

In order to assess the feasibility of semi-automatic procedures for large genome sequencing, a fragment of 9.4 kb of Escherichia coli chromosomal DNA isolated at random was sequenced. It was found to map at 30 min on the chromosome map and to harbour two insertion sequences (IS2 and IS30) as well as several putative coding sequences which had no feature in common with known proteins.