Voltage-sensitive ion channel ofEscherichia coli

Summary A voltage-sensitive, cation-selective ion channel ofEscherichia coli has been reconstituted into liposomes and studied with the patch-clamp method. The single channel conductance was 91 pS in symmetric solutions of 150mm KCl. Many channels were open most of the time, with frequent brief transitions to closed levels. Multiple conducting units could close and reopen simultaneously, and this apparent cooperativity in gating was increases with depolarizing voltages. Above a voltage threshold, the channels closed irreversibly, often in groups.     

Export incompatibility of N-terminal basic residues in a mature polypeptide of Escherichia coli can be alleviated by optimising the signal peptide

Summary Export of the outer membrane protein, OmpA, across the cytoplasmic membrane of Escherichia coli was severely inhibited by the presence of two, three, four or six additional basic residues at the N-terminus of the mature polypeptide, but not by three similarily positioned acidic residues. Because a few bacterial proteins do possess basic residues close to the leader peptidase cleavage site and because the type of inhibition described here could pose problems in the construction of hybrid secretory proteins, we also studied means of alleviating this form of export incompatibility. Inhibition was abolished when basic residues were preceded by acidic ones. Also, the processing rates of the mutants with two or six basic residues could be partially restored by increasing the length of the hydrophobic core of the signal peptide. Taking this as a precedent, it is suggested that the structure of the signal peptide is an important feature for maintenance of a reasonable rate of translocation of those exported proteins which possess basic residue(s) at the N-terminus of the mature polypeptide.     

The hydrogenases and formate dehydrogenases ofEscherichia coli

Escherichia coli has the capacity to synthesise three distinct formate dehydrogenase isoenzymes and three hydrogenase isoenzymes. All six are multisubunit, membrane-associated proteins that are functional in the anaerobic metabolism of the organism. One of the formate dehydrogenase isoenzymes is also synthesised in aerobic cells. Two of the formate dehydrogenase enzymes and two hydrogenases have a respiratory function while the formate dehydrogenase and hydrogenase associated with the formate hydrogenlyase pathway are not involved in energy conservation. The three formate dehydrogenases are molybdo-selenoproteins while the three hydrogenases are nickel enzymes; all six enzymes have an abundance of iron-sulfur clusters. These metal requirements alone invoke the necessity for a profusion of ancillary enzymes which are involved in the preparation and incorporation of these cofactors. The characterisation of a large number of pleiotropic mutants unable to synthesise either functionally active formate dehydrogenases or hydrogenases has led to the identification of a number of these enzymes. However, it is apparent that there are many more accessory proteins involved in the biosynthesis of these isoenzymes than originally anticipated. The biochemical function of the vast majority of these enzymes is not understood. Nevertheless, through the construction and study of defined mutants, together with sequence comparisons with homologous proteins from other organisms, it has been possible at least to categorise them with regard to a general requirement for the biosynthesis of all three isoenzymes or whether they have a specific function in the assembly of a particular enzyme. The identification of the structural genes encoding the formate dehydrogenase and hydrogenase isoenzymes has enabled a detailed dissection of how their expression is coordinated to the metabolic requirement for their products. Slowly, a picture is emerging of the extremely complex and involved path of events leading to the regulated synthesis, processing and assembly of catalytically active formate dehydrogenase and hydrogenase isoenzymes. This article aims to review the current state of knowledge regarding the biochemistry, genetics, molecular biology and physiology of these enzymes.     

A temperature-sensitive mutant ofEscherichia coli with an alteration in ribosomal protein L22

A temperature-sensitive, protein synthesis-defective mutant ofEscherichia coli exhibiting an altered ribosomal protein L22 has been investigated. The temperature-sensitive mutation was mapped to therplV gene for protein L22. The genes from the wild type and mutant strains were amplified by the polymerase chain reaction and the products were sequenced. A cytosine to thymine transition at position 22 of the coding sequence was found in the mutant DNA, predicting an arginine to cysteine alteration in the protein. A single cysteine residue was found in the isolated mutant protein. This amino acid change accounts for the altered mobility of the mutant protein in two-dimensional gels and during reversed-phase HPLC. The temperature-sensitive phenotype was fully complemented by a plasmid carrying the wild type L22 gene. Ribosomes from the complemented cells showed only wild type protein L22 by two dimensional gel analysis and were as heat-resistant as control ribosomes in a translation assay. The point mutation in the L22 gene is uniquely responsible for the temperature-sensitivity of this strain.     

HPLC separation of enterobactin and linear 2,3-dihydroxybenzoylserine derivatives: a study on mutants ofEscherichia coli defective in regulation (fur), esterase (fes) and transport (fepA)

Reversed-phase HPLC separation of enterobactin and its 2,3-dihydroxybenzoylserine derivatives was used for a comparative analysis of mutants of Escherichia coli, defective in the regulation of enterobactin biosynthesis (fur), enterobactin transport (fepA) and enterobactin esterase (fes). A complete separation of all 2,3-dihydroxybenzoylserine compounds was achieved: the monomer (DHBS), the linear dimer (DHBS)₂ and trimer (DHBS)₃, the cyclic trimer, enterobactin, as well as 2,3-dihydroxybenzoic acid. The production of all these compounds was followed after ethylacetate extraction from acidified culture fluids. Enterobactin was found to be the predominant product in all mutant strains. The mutant strains behaved differently with regard to the breakdown products. All degradation products, such as DHBS, (DHBS)₂ and (DHBS)₃, were detected in the overproducing fur mutant where both transport and esterase are still functioning, while only the monomer, DHBS, was detected in the fepA mutant and no degradation was found in the esterase-deficient fes mutant. From the pattern of breakdown products it may be inferred that the esterase acts in two different ways, depending on whether transport is functioning or not. Thus, esterolytic cleavage of ferric enterobactin after entering the cells results in a mixture of all three hydrolysis products, i.e. DHBS, (DHBS)₂ and (DHBS)₃, while cleavage of iron-free enterobactin subsequent to its biosynthesis yields only the monomer. Thus, the results of quantitative HPLC analysis of enterobactin and its breakdown products show that different enterobactin esterase products arise, depending on whether iron is bound to enterobactin or not.     

Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli

Adenoregulin is a 33 amino acid antimicrobial peptide isolated from the skin of the arboreal frog Phyllomedusa bicolor. Natural adenoregulin is synthesized with an amidated valine residue at C-terminus and shows lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. A synthetic gene for adenoregulin (ADR) with an additional amino acid glutamine at C-terminus was cloned into pET32a vector to allow expression of ADR as a Trx fusion protein in Escherichia coli BL21(DE3). The resulting expression level of the fusion protein could reach up to 20% of the total cell proteins. The fusion protein could be purified effectively by Ni2+-chelating chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant ADR displayed antimicrobial activity similar to that of the synthetic ADR reported earlier. Comparing the antimicrobial activities of the recombinant adenoregulin with C-amidated terminus to that without an amidated C-terminus, we found that the amide of glutamine at C-terminus of ADR improved its potency on certain microorganisms such as Tritirachium album and Saccharomyces cerevisiae.     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

The close proximity ofEscherichia coli genes: Consequences for stop codon and synonymous codon use

It is shown that synonymous codon usage is less biased in favor of those codons preferred by highly expressed genes at the end ofEscherichia coli genes than in the middle. This appears to be due to the close proximity of manyE. coli genes. It is shown that a substantial number of genes overlap either the Shine-Dalgarno sequence or the coding sequence of the next gene on the chromosome and that the codons that overlap have lower synonymous codon bias than those which do not. It is also shown that there is an increase in the frequency of A-ending codons, and a decrease in the frequency of G-ending codons at the end ofE. coli genes that lie close to another gene. It is suggested that these trends in composition could be associated with selection against the formation of mRNA secondary structure near the start of the next gene on the chromosome. Stop codon use is also affected by the close proximity of genes; many genes are forced to use TGA and TAG stop codons because they terminate either within the Shine-Dalgarno or coding sequence of the next gene on the chromosome. The implications these results have for the evolution of synonymous codon use are discussed.     

A single amino acid substitution alters conductance and gating of OmpC porin ofEscherichia coli

Summary We have reconstituted into liposomes outer-membrane fractions fromEscherichia coil strains which express OmpC porins with altered pore properties. Single-channel experiments were performed with the patch-clamp technique on blisters generated from the reconstituted liposomes. Our goal was to identify positively the activity pattern of OmpC in our reconstituted system. The properties of the parent strain were compared to those of a strain whose OmpC porin has a single amino acid substitution in a postulated transmembrane segment. The parent and the mutant strain each exhibit a cation-selective channel of high open probability and gating to closed levels of various amplitudes. However, the mutant channel appeared to be 9 to 30% larger in unit conductance. It tended to close and reopen most often in groups of three units, as opposed to two units in the parent channel. The results are discussed in terms of the observed phenotype and of their implication as to the structure-function relationship of the porin channels.     

RNA polymerase sigma-related proteins in Escherichia coli: detection by antibodies against a synthetic peptide

Summary Antibodies were raised against a synthetic tetradecameric peptide with an amino acid sequence, DLIQEGNIGLMKAV, which corresponds to the most highly conserved region of bacterial RNA polymerase factors. In a Western-blot analysis of total Escherichia coli proteins, the antiserum reacted specifically with at least three proteins with apparent molecular weights of 75 kDa, 27 kDa and 23 kDa, in addition to the known factors (⁷⁰ and ³²). The majorities of ⁷⁰ and ³² were recovered as associated forms with the RNA polymerase on glycerol gradient centrifugation, while the other cross-reacting proteins were not. Unambiguous evidence was obtained which indicated that the intracellular level of ³² increased rapidly upon heatshock, at least in the strain containing high copy numbers of the rpoH gene.     

Escherichia coli formate dehydrogenase mutants with altered selenopolymer profiles

Four classes of Escherichia coli mutants deficient in either or both of their anaerobic selenium-containing formate dehydrogenases (FDH) were isolated. A class I mutant devoid of FDH_H activity specifically linked to benzyl viologen (BV) produced a small amount of the FDH_H 80,000 dalton selenopeptide. Three class II mutants were deficient in FDH_N activity specifically linked to phenazine methosulfate (PMS) and exhibited a selenopeptide doublet rather than the FDH_N 110,000 dalton selenosubunit. Three class III mutants were selenium incorporation deficient and did not exhibit either FDH activity or ⁷⁵Selabeled selenopolymers. A class IV mutant was devoid of PMS-linked FDH_N activity; neither its FDH_N 110,000 dalton selenosubunit nor its BV-linked FDH_H activity was fully regulated by nitrate.Abbreviations FDH formate dehydrogenase - BV benzyl viologen - MV methyl viologen - PMS phenazine methosulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Genetics of pentose-phosphate pathway enzymes ofEscherichia coli K-12

The pentose-phosphate pathway ofEscherichia coli K-12, in addition to its role as a route for the breakdown of sugars such as glucose or pentoses, provides the cell with intermediates for the anabolism of amino acids, vitamins, nucleotides, and cell wall constituents. Through its oxidative branch, it is a major source of NADPH. The expression of the gene for NADP-dependent 6-phospho-gluconate dehydrogenase (gnd) is regulated by the growth rate inE. coli. The recently identified gene for ribulose-5-phosphate 3-epimerase (rpe) is part of a large operon that comprises among others genes for the biosynthesis of aromatic amino acids. In recent years, genes for all enzymes of the pathway have been cloned and sequenced. Isoenzymes have been found for transketolase (genestktA andtktB), ribose-5-phosphate isomerase (rpiA andrpiB) and transaldolase (talA andtalB).     

Effects of iron-limitation ofEscherichia coli on growth,the respiratory chains and gallium uptake

The effects of iron limitation on growth, the composition and function of the respiratory chains, and gallium uptake inEscherichia coli have been studied. Decreasing the iron concentration in a defined medium using Chelex resin gave lower growth yields in both continuous culture and prolonged batch culture. In the former, ironlimited (entering [Fe]2.0 M) cells exhibited diminished respiration rates, respiration-driven proton translocation quotients, and levels of non-haem iron and cytochromes. The cellular concentration of haemoproteinb-590 (a cytochromea ₁-like hydroperoxidase) decreased 20-fold on iron limitation, whilst a CO-binding pigment with an absorption maximum in the dithionite-treated form near 500 nm appeared. Gallium(III) (9 M) added to iron-limited, but not iron-sufficient, cultures diminished growth yields further; cells grown with low entering concentrations of iron took up less gallium than iron-sufficient cells. These results are attributed to the interference by gallium(III) with siderophore-mediated metal uptake. Gallium also stimulated iron uptake and was itself accumulated by iron-sufficient cells, suggesting that gallium(III) also affects the iron transport system(s) of low affinity.     

Biochemical analysis of galactose induced bacteriostasis ingalT mutants ofescherichia coli K 12

Escherichia coli mutants completely defective in galactose-1-phosphate uridyl transferase (EC 2.7.7.10) and growing in glycerol medium undergo rapid cessation of growth when exposed to galactose. Toxicity due to galactose is equally pronounced when glycerol is replaced by other carbon sources, like succinate and proline. Gas chromatographic analysis failed to detect even trace amounts of galactitol. Moreover, galactose-1-phosphate had no inhibitory role on some of the critical enzymes of cellular metabolism. General loss of energy (ATP) due to futile phosphorylation of galactose is probably the cause of bacteriostasis. ThegalT mutants can serve as models of human transferaseless galactosemia only to a limited extent     

Physicochemical characterization ofEscherichia coli

EightEscherichia coli strains were characterized by determining their adhesion to xylene, surface free energy, zeta potential, relative surface charge, and their chemical composition. The latter was done by applying X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (IR). No relationship between the adhesion to xylene and the water contact angles of these strains was found. Three strains had significantly lower surface free energies than the other strains. Surface free energies were either obtained from polar and dispersion parts or from Lifshitz-van der Waals and acid/base parts of the surface free energy. A correlation (r=0.97) between the polar parts and the electron-donor contributions to the acid/base part of the surface free energy was found. The zeta potentials of all strains, measured as a function of pH (2–11), were negative. Depending on the zeta potential as a function of pH, three groups were recognized among the strains tested. A relationship (r=0.84) was found between the acid/base component of the surface free energy and the zeta potential measured at pH=7.4. There was no correlation between results of XPS and IR studies. Data from the literature of XPS and IR studies of the gram-positive staphylococci and streptococci were compared with data from the gram-negativeE. coli used in this study. It appeared that in these three groups of bacteria, the polysaccharide content detected by IR corresponded well with the oxygen-to-carbon ratio detected by XPS.     

Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners

It has been reported that it is difficult to express cationic antibacterial peptides in engineered bacteria because such peptides are highly toxic to the host bacteria cells and sensitive to intracellular proteases. Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi and tumor cells, which may possibly be used as an antimicrobial agent. Here we tried to express ABP-CM4 in Escherichia coli cells using either the GST fusion system or the intein-mediated fusion expression system. In order to investigate the possible use of these two fusion partners in cationic small peptide expression and purification, a mutant ABP-CMt, which is a highly positively charged peptide with +9 charges at neutral pH, was designed. In the present study, we have shown that both ABP-CM4 and ABP-CMt peptides can be expressed and purified by the intein-mediated expression system but not by the GST fusion expression system. Thus the intein-mediated peptide expression and purification system potentially could be employed for the production of recombinant protease-sensitive and cytotoxic peptides.     

The density distribution of gene loci over the genetic map ofEscherichia coli: Its structural,functional and evolutionary implications

Summary A quantitative analysis was carried out on the dispersion of gene loci over theE. coli genetic map. Therefore, the map was divided into regions characterized by an homogeneous gene density. This created a distribution pattern of gene loci that contained a symmetry axis located near to the origin of DNA replication. The pattern could be subdivided into a set of 22 functional domains containing gene loci whose products revealed a biochemical or functional relatedness. A correlation was found between the boundary positions of these domains and the distribution of F plasmid- and DNA insertion sites over theE. coli chromosome. The structural, functional and evolutionary implications of these findings are discussed.