Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein.

Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process.     

Interaction between maltose-binding protein and the membrane-associated maltose transporter complex in Escherichia coli

Active transport of maltose in Escherichia coli requires the presence of both maltose-binding protein (MBP) in the periplasm and a complex of MalF, MalG, and MalK proteins (FGK2) located in the cytoplasmic membrane. Earlier, mutants in malF or malG were isolated that are able to grow on maltose in the complete absence of MBP. When the wild-type malE+ allele, coding for MBP, was introduced into these MBP-independent mutants, they frequently lost their ability to grow on maltose. Furthermore, starting from these Mal- strains, Mal+ secondary mutants that contained suppressor mutations in malE were isolated. In this study, we examined the interaction of wild-type and mutant MBPs with wild-type and mutant FGK2 complexes by using right-side-out membrane vesicles. The vesicles from a MBP-independent mutant (malG511) transported maltose in the absence of MBP, with Km and Vmax values similar to those found in intact cells. However, addition of wild-type MBP to these mutant vesicles produced unexpected responses. Although malE+ malG511 cells could not utilize maltose, wild-type MBP at low concentrations stimulated the maltose uptake by malG511 vesicles. At higher concentrations of the wild-type MBP and maltose, however, maltose transport into malG511 vesicles became severely inhibited. This behaviour of the vesicles was also reflected in the phenotype of malE+ malG511 cells, which were found to be capable of transporting maltose from a low external concentration (1 microM), but apparently not from millimolar concentrations present in maltose minimal medium. We found that the mutant FGK2 complex, containing MalG511, had a much higher apparent affinity towards the wild-type MBP than did the wild-type FGK2 complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of the maltose-binding protein with membrane vesicles of Escherichia coli.

The interaction of the radioactively labeled purified maltose-binding protein of Escherichia coli with membrane vesicles was studied. The maltose-binding protein bound specifically to the vesicles, in the presence of maltose, on few sites. Under conditions in which a potential was imposed across the membrane, the specific binding was (i) increased, (ii) dependent on maltose, and (iii) abolished in a mutant defective in the tar gene product, one of the methyl-accepting chemotaxis proteins. At least 1,300 binding sites were present in the membrane fraction of logarithmically growing cells.     

Strain specificity of outer membrane proteins in Escherichia coli.

Outer membrane proteins of various strains of Escherichia coli were compared using three different systems of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The outer membranes of E. coli K-12, E. coli B, and E. coli J-5 had distinctive protein compositions. As regards proteins which interact with peptidoglycan, E. coli K-12 contained O-8 and O-9, while E. coli B possessed one protein which migrated to the position of O-9. Although E. coli J-5 possessed two such proteins, O-8' and O-9', their positions on polyacrylamide gel were different from those of O-8 and O-9. Protein O-7, which migrates slightly more slowly than O-8, was found specifically in E. coli K-12. Proteins O-10 and O-11 were found in all strains tested, although the relative amounts were different depending on the strain. Strains of E. coli K-12 and E. coli J-5 gave three major bands, O-2a, O-2b, and O-3, in the region of high molecular weight. These proteins were repressed by iron in the cultivation media. Strains of E. coli B, on the other hand, gave only O-2b and O-3. E. coli J-5 gave two other major bands in this region, but the amounts were not controlled by iron in the cultivation media.     

Reconstitution of maltose chemotaxis in Escherichia coli by addition of maltose-binding protein to calcium-treated cells of maltose regulon mutants.

Maltose chemotaxis was reconstituted in delta malE cells lacking maltose-binding protein (MBP). Purified MBP was introduced into intact cells during incubation with 250 mM CaCl2 in Tris-hydrochloride buffer at 0 degrees C. After removal of extracellular CaCl2 and MBP, chemotaxis was measured with tethered bacteria in a flow chamber or with free-swimming cells in a capillary assay. About 20% of tethered cells responded to 10(-4) M maltose; the mean response times were about half those of CaCl2-treated wild-type cells (100 s as opposed to 190 s). In capillary tests, the maltose response of reconstituted cells was between 15 and 40% of the aspartate response, about the same percentage as in wild-type cells. The best reconstitution was seen with 0.5 to 1 mM MBP in the reconstitution mixture, which is similar to the periplasmic MBP concentration estimated for maltose-induced wild-type cells. Strains containing large deletions of the malB region and malT mutants lacking the positive regulator gene of the mal regulon also could be reconstituted for maltose chemotaxis, showing that no product of the mal regulon other than MBP is essential for maltose chemotaxis.     

Substrate specificity of the Escherichia coli outer membrane protease OmpP

Escherichia coli OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k(cat)/K(m) = 3.0 x 10(6) M(-1)s(-1)). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1' sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3'. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1', site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F' episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.     

Substrate specificity of the Escherichia coli outer membrane protease OmpT

OmpT is a surface protease of gram-negative bacteria that has been shown to cleave antimicrobial peptides, activate human plasminogen, and degrade some recombinant heterologous proteins. We have analyzed the substrate specificity of OmpT by two complementary substrate filamentous phage display methods: (i) in situ cleavage of phage that display protease-susceptible peptides by Escherichia coli expressing OmpT and (ii) in vitro cleavage of phage-displayed peptides using purified enzyme. Consistent with previous reports, OmpT was found to exhibit a virtual requirement for Arg in the P1 position and a slightly less stringent preference for this residue in the P1' position (P1 and P1' are the residues immediately prior to and following the scissile bond). Lys, Gly, and Val were also found in the P1' position. The most common residues in the P2' position were Val or Ala, and the P3 and P4 positions exhibited a preference for Trp or Arg. Synthetic peptides based upon sequences selected by bacteriophage display were cleaved very efficiently, with kcat/Km values up to 7.3 x 10(6) M(-1) s(-1). In contrast, a peptide corresponding to the cleavage site of human plasminogen was hydrolyzed with a kcat/Km almost 10(6)-fold lower. Overall, the results presented in this work indicate that in addition to the P1 and P1' positions, additional amino acids within a six-residue window (between P4 and P2') contribute to the binding of substrate polypeptides to the OmpT binding site.     

Chemical heterogeneity of major outer membrane pore proteins of Escherichia coli.

Peptide mapping and isoelectric focusing were used to compare the major outer membrane pore proteins from various strains of Escherichia coli K-12, including strains carrying mutations in the nmpA, nmpB, and nmpC genes which result in the production of new membrane proteins. Proteins 1a, 1b, and 2 and the NmpA proteins each gave unique peptide and isoelectric focusing profiles, indicating that these are different polypeptides. The NmpA protein and the NmpB protein appeared to be identical by these criteria. The NmpC protein and protein 2 were nearly identical, although one different peptide was observed in comparing the proteolytic peptide maps of these proteins and there were slight differences in their isoelectric focusing profiles. Antiserum against protein 2 showed partial cross-reactivity with the NmpC protein. These results indicate that the various pore proteins of E. coli K-12 fall into four different classes.     

Cloning of phoE, the structural gene for the Escherichia coli phosphate limitation-inducible outer membrane pore protein.

The hybrid plasmid pLC44-11 from the Clarke and Carbon collection, which was known to carry the proA gene, was shown also to contain the phoE gene. In vitro recombination techniques were used to subclone a 4.9-kilobase-pair DNA fragment of pLC44-11 into the plasmid vectors pACYC184 and pBR322. Expression of this fragment in a minicell system showed that it codes for the PhoE protein and for polypeptides with apparent molecular weights of 47,000 and 17,000. These results supply definite proof for the earlier supposition that the phoE gene is the structural gene for the outer membrane PhoE protein. Overproduction of the PhoE protein in a phoS strain resulted in reduced amounts of OmpF and LamB proteins.     

Silent and functional changes in the periplasmic maltose-binding protein of Escherichia coli K12. I. Transport of maltose

The malE gene encodes the periplasmic maltose-binding protein (MBP). Nineteen mutations that still permit synthesis of stable MBP were generated by random insertion of a BamHI octanucleotide into malE and six additional mutations by in-vitro recombinations between mutant genes. The sequence changes were determined; in most cases the linker insertion is accompanied by a small deletion (30 base-pairs on average). The mutant MBP were studied for export, growth on maltose and maltodextrins, maltose transport and binding, and maltose-induced fluorescence changes. Sixteen mutant MBP (out of 21 studied in detail) were found in the periplasmic space: 12 of them retained a high affinity for maltose, and 10 activity for growth on maltose. The results show that several regions of MBP are dispensable for stability, substrate binding and export. Three regions (residues 207 to 220, 297 to 303 and 364 to 370) may be involved in interactions with the MalF or MalG proteins. A region near the C-terminal end is important for maltose binding. Two regions of the mature protein (residues 18 to 42 and 280 to 296) are required for export to, or solubility in, the periplasm.     

Effects of linker insertions on the biogenesis and functioning of the Escherichia coli outer membrane pore protein PhoE

Summary The swi1 ⁺ gene is necessary for effective mating-type (MT) switching in Schizosaccharomyces pombe. It was cloned on a 4.2 kb genomic DNA fragment. By site-directed integration into the genome and gene disruption experiments it was proved that the swi1 ⁺ gene itself and not a suppressor had been isolated. Disruption of the swi1 ⁺ gene causes a phenotype identical to that of the original swi1 mutant, i.e. the strain still shows some MT switching. The swi1 gene is unique in the genome and gives rise to a 3 kb mRNA.     

Escherichia coli outer membrane protein K is a porin.

Protein K is an outer membrane protein found in pathogenic encapsulated strains of Escherichia coli. We present evidence here that protein K is structurally and functionally related to the E. coli K-12 porin proteins (OmpF, OmpC, and PhoE). Protein K was found to cross-react with antibody to OmpF protein and to share 8 out of 17 peptides in common with the OmpF protein. Strains that are OmpC porin- and OmpF porin- and contain protein K as their major outer membrane protein have increased rates of uptake of nutrients and a faster growth rate relative to the parental porin- strain. The protein K-containing strains are at least 1,000-fold more sensitive to colicins E2 and E3 than is the porin -deficient strain. These data suggest that protein K is a functional porin in E. coli. The porin function of protein K was also demonstrated in vitro, using black lipid membranes. Protein K increased the conductance in these membranes in discrete, uniform steps characteristic of channels with a size of about 2 nS.     

Silent and functional changes in the periplasmic maltose-binding protein of Escherichia coli K12. II. Chemotaxis towards maltose

We examined the chemotactic behavior of ten Escherichia coli mutants able to synthesize a modified periplasmic maltose-binding protein (MBP) retaining high affinity for maltose. Eight were able to grow on maltose (Mal+), two were not (Mal-). In the capillary assay six out of eight of the Mal+ strains showed an optimal response at the same concentration of maltose as the wild-type strain; the amplitude of the response was strongly reduced in two Mal+ mutants and partially affected in one. The amplitude of the chemotactic response of the two Mal- strains was at least equal to that of the wild type, so that the chemotactic and transport functions of MBP were dissociated in these two cases. We define two regions of the protein (residues 297 to 303 and 364 to 369), that are important both for the chemotactic response and for transport, and one region (residues 207 to 220) that is essential for transport but dispensable for chemotaxis. Interestingly, some regions that were found to be inessential for transport are also dispensable for chemotaxis.     

Gene encoding a hybrid OmpF-PhoE pore protein in the outer membrane of Escherichia coli K12

Summary To study the structure-function relationship of outer membrane pore proteins of E. coli K12, a hybrid gene was constructed in which the DNA encoding amino acid residues 2–73 of the mature PhoE protein is replaced by the homologous part of the related ompF gene. The product of this gene is incorporated normally into the outer membrane. It was characterized with respect to its pore activity and its phage receptor and colicin receptor properties. It is concluded (i) that the preference of the PhoE protein pore for negatively charged solutes is partly determined by the amino terminal 73 amino acids, (ii) that part of the receptor site of PhoE protein for phage TC45 is located in this part of the protein, (iii) that colicin N uses OmpF protein as (part of) its receptor, (iv) that the specificity of OmpF protein as a colicin N receptor is completely located within the 80 amino terminal amino acid residues, whereas the specificity of this protein as a colicin A receptor is completely located within the 260 carboxy terminal amino acid residues, and (v) that the amino terminal 73 amino acid residues of PhoE protein span the membrane at least once.     

Interspecific reconstitution of maltose transport and chemotaxis in Escherichia coli with maltose-binding protein from various enteric bacteria.

In Escherichia coli, the periplasmic maltose-binding protein (MBP), the product of the malE gene, is the primary recognition component of the transport system for maltose and maltodextrins. It is also the maltose chemoreceptor, in which capacity it interacts with the signal transducer Tar (taxis to aspartate and some repellents). In studies of the maltose system in other members of the family Enterobacteriaceae, we found that MBP is produced by Salmonella typhimurium, Klebsiella pneumoniae, Enterobacter aerogenes, and Serratia marcescens. MBP from all of these species cross-reacted with antibody against the E. coli protein and had a similar molecular weight (about 40,000). The Shigella flexneri and Proteus mirabilis strains we examined did not synthesize MBP. The isoelectric points of MBP from different species varied from the acid extreme of E. coli (4.8) to the basic extreme of E. aerogenes (8.9). All species with MBP transported maltose with high affinity, although the Vmax for K. pneumoniae was severalfold lower than that for the other species. Maltose chemotaxis was observed only in E. coli and E. aerogenes. In S. typhimurium LT2, Tar was completely inactive in maltose taxis, although it signaled normally in response to aspartate. MBP isolated from all five species could be used to reconstitute maltose transport and taxis in a delta malE strain of E. coli after permeabilization of the outer membrane with calcium.     

Role of a major outer membrane protein in Escherichia coli.

Mutants of Escherichia coli B/r lacking a major outer membrane protein, protein b, were obtained by selecting for resistance to copper. These mutants showed a decreased ability to utilize a variety of metabolites when the metabolites were present at low concentrations. Also, mutants of E. coli K-12 lacking proteins b and c from the outer membrane were shown to have an identical defect in the uptake of various metabolites. These results are discussed with regard to their implications as to the role of these proteins in permeability of the outer membrane,     

Escherichia coli outer membrane protease OmpT confers resistance to urinary cationic peptides

Escherichia coli OmpT, located in the outer membrane, has been characterized as a plasminogen activator, with the ability to hydrolyze protamine and block its entry. In this investigation, a complex of low molecular weight cationic peptides purified from human urine by a combination of membrane ultrafiltration and weak cation exchange chromatography was characterized. The impact of OmpT on E. coli resistance to urinary cationic peptides was investigated by testing ompT knockout strains. The ompT mutants were more susceptible to urinary cationic peptides than ompT⁺ strains, and this difference was abolished by complementation of the mutants with pUC19 carrying the ompT gene. The urinary protease inhibitor ulinastatin greatly decreased the resistance of the ompT⁺ strains. Overall, the data indicate that OmpT may help E. coli persist longer in the urinary tract by enabling it to resist the antimicrobial activity of urinary cationic peptides.