The effect of the cationic proteins of human blood cells on the growth of Escherichia coli

The antibacterial effect of cationic proteins (CP) on donor leukocytes and thrombocytes with respect to the growth of E. coli has been demonstrated in vitro, the maximum recorded inhibition being caused by the action of leukocytic CP. Differences in the inhibitory action may be linked with the presence of anomalies in the amino acid composition of leukocytic CP and thrombocytic CP, manifested by the deterioration of the basic properties of the latter, as well as by the fractional composition whose characteristic features for thrombocytic CP are the appearance of high-molecular components and a decrease in the proportion of low-molecular fractions. In patients with different forms of leukosis (chronic myeloleukemia, chronic lympholeukemia, acute myelomonoblastic leukosis) leukocytic CP retain their antibacterial activity. Under the action of blood cell CP changes in the outer and cytoplasmic membranes have been noted.     

Voltage-dependent cationic channel of Escherichia coli

Verapamil and dimethylcurine are Ca²⁺ entry blockers of essentially different chemical structures which presumably bind to the same arylalkylamine receptor of the L-type Ca channel. A systematic conformational analysis of methoxyverapamil (D-600) and dimethylcurine has been carried out using a molecular mechanics method. The lowest minimum-energy conformations of D-600 are predisposed to chelate Ca²⁺ by four oxygen atoms of the stacked methoxyphenyl moieties. Comparison of the lowest energy conformations of D-600-Ca²⁺ and dimethylcurine revealed a similar spatial disposition of cationic groups and methoxyphenyl moieties in the two compounds. A three-dimensional model of arylalkylamine receptor was suggested which incorporates two nucleophilic areas of the Ca channel. Dimethylcurine binds to these areas by its quaternary amine functions, whereas D-600 does so by amine function and via coordinated Ca²⁺. The results support the hypotheses on ternary complex formation between the ligands of Ca channel, their receptors, and Ca²⁺.     

Binding of plasminogen to Escherichia coli adhesion proteins

Immobilization of plasminogen via its lysine-binding sites is regarded as a prerequisite for its activation and function in fibrinolysis and pericellular proteolysis. In the present study, the interaction of plasminogen with fimbriae found on Escherichia coli strains causing invasive human infections was studied. Plasminogen displayed concentration-dependent and saturable binding to immobilized type 1 fimbriae and, several fold lower binding to P and S fimbriae. The binding to fimbriae was effectively inhibited by -aminocaproic acid indicating that it was mediated by the lysine-binding sites of plasminogen. Binding studies with mutated fimbriae and inhibition tests indicated that the interaction was not dependent on the lectin subunit of the fimbriae. These results indicate the existence of a novel type of host-microbe interaction which may be important in the invasion by bacteria of host tissues.     

Acid shock proteins of Escherichia coli

Synthesis of total cellular proteins of Escherichia coli was studied after transfer of cultures from pH 6.9 to pH 4.3. Proteins induced by such an external pH shift down were identified by mono- and bi-dimensional electrophoresis. 30 to 45 min after an acid shift, a group of at least sixteen polypeptides was markedly induced. Four of these polypeptides corresponded to the well known heat shock proteins GroEL, DnaK, HtpG and HtpM. Their pH induction was RpoH-dependent. Three other pH-induced proteins were previously identified as stress proteins induced either by osmolarity or aerobiosis or low temperature (proteins 32 (defined in this paper), C70.0 and C62.7). Seven other proteins were specifically induced after an acid shift and were called acid shock proteins (ASP). The induction of one of these proteins was RpoH-dependent, whereas that of others was RpoH-independent.     

Membrane potential in Escherichia coli bacteria subjected to the action of low temperature freezing and blood serum complement

The action of a blood serum complement on Escherichia coli cells or their freezing does not cause cell destruction visible in the electron microscope, but the permeability barrier is disordered and exogenous substrates can penetrate into the cell. When these exogenous respiration substrates are oxidised, the energy-dependent uptake of phenyl dicarboundecarborane (PCB-), a lipophilic anion and an indicator of the membrane potential, is observed. Apparently, the uptake of PCB- is associated with the generation of local membrane potentials when the permeability barrier of cells is damaged.     

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.     

The effect of cationic surface-active agents on the Escherichia coli ATPase

The authors studied the effect of cationic surfactants (CS), such as alkyl(C8H17-C18H37)dimethylbenzylammonium (I), alkyl(C8H17-C16H33)benzyltrimethylammonium (II), alkyl(C8H17-C16H33)di-beta-hydroxyethylbenzylammonium (III) chlorides and chlorhydrate of glycine decyl ester (IV) on the ATPase activity of E. coli 1257 cell, spheroplasts, and isolated membranes. Changes in the ATPase activity of the E. coli cells and spheroplasts were found to depends on the concentration and the structure of the cationic surfactants. The removal of the cell wall increased the destroying effect of CS on the cytoplasmic membranes and enhanced the ATPase inhibition. The compounds with 16 and 18 carbon atom radical had the highest inhibitory effect. The action of cationic surfactants on the membrane is accompanied by changes in the protein and phospholipid composition and by significant solubilization of ATPase with pronounced inactivation of the enzyme. The kinetics of inhibition of E. coli membrane ATPase was studied to the presence of the homological series I and IV. The cationic surfactants under study inhibited the ATP hydrolysis catalysed by E. coli ATPase by a mixed type mechanism. Ki = 58.21.10(-4) M for IC10H21; 10.67.10(-4) M for IC12H25; 0.58.10(-4) M for IC16H33; 0.16.10(-4) M for IC18H37, and 5.93.10(-4) M for IV.     

High-affinity calcium-binding proteins in Escherichia coli

Crude extracts of Escherichia coli contain at least three heat stable proteins of Mr, 33,000, 47,000, and 60,000, which bind 45Ca2+ in buffers containing micromolar calcium and physiological salt concentrations. Fractions containing these proteins neither activated the calmodulin-dependent enzyme, NAD kinase, nor inhibited the activity of this enzyme in the presence of brain calmodulin. Radioimmunoassay of crude extracts for calmodulin indicated the presence of a calmodulin-like antigen. Crude extracts also contain proteins that interact with 2-trifluoromethyl-10H-(3'-aminopropyl)phenothiazine-Sepharose in a calcium-dependent manner, but proteins eluted from this resin did not bind calcium with high affinity.     

Expression of N-formylated proteins in Escherichia coli

In bacteria, protein expression initiates with a formyl-methionine group. Addition of the antibiotic actinonin, a known peptide deformylase inhibitor, at the time of induction of protein expression results in the retention of the formyl group by the overexpressed protein. In addition, because deformylation is a prerequisite for removal of the initiating methionine, this post-translational processing step is also prevented by actinonin, and the N-formyl methionine residue is retained by proteins from which it is normally removed. We have demonstrated the applicability of this system for obtaining N-modified forms of several different proteins and use one of these modified molecules to show that the N-terminal amino group is not required for ClpXP degradation of proteins bearing an N-terminal recognition signal.     

Penicillin-binding proteins of pathogenic Escherichia coli strains

The penicillin-binding proteins of 11 pathogenic Escherichia coli strains, including enteropathogenic, enterotoxigenic, enteroinvasive, enteroaggregative, and enterohemorrhagic E. coli, were detected in gels following the labeling of isolated cell envelopes with [³H]benzylpenicillin. The electrophoretic profiles, sensitivities to and morphological changes induced by β-lactam antibiotics showed that the penicillin-binding proteins of most pathogenic E. coli possess structural and physiological functions similar to those of E. coli K12.     

Histone-like proteins in the purified Escherichia coli deoxyribonucleoprotein

Analysis of E.coli chromosomes isolated under conditions similar to those used for isolation of eukaryotic chromatin has shown that: 1) The proteins of highly purified E.coli deoxyribonucleoprotein are mainly in addition to RNA polymerase two specific histone-like proteins of apparent molecular weight of 17,000 and 9,000 (proteins 1 and 2, respectively). 2) Proteins 1 and 2 occur in approximately equal molar amounts in the isolated E.coli chromosome, and their relative content corresponds to one molecule of protein 1 plus one molecule of protein 2 per 150-200 base pairs of DNA. 3) There are no long stretches of naked DNA in the purified E.coli deoxyribonucleoprotein suggesting a fairly uniform distribution of the proteins 1 and 2 along DNA. 4) The protein 2 is apparently identical to the DNA-binding protein HU which was isolated previously /1/ from extracts of E.coli cells. 5) Digestion of the isolated E.coli chromosomes with staphylococcal nuclease proceeds through discrete deoxyribonucleoprotein intermediates (in particular, at approximately 120 base pairs) which contain both proteins 1 and 2. However, since no repeating multimer structure was observed so far in nuclease digests of the E.coli chromosome, it seems premature to draw definite conclusions about possible similarities between the nucleosomal organization of the eukaryotic chromatin and the E.coli chromatin structure.Images     

Production of disulfide-bonded proteins in Escherichia coli

Disulfide bonds are covalent bonds formed post-translationally by the oxidation of a pair of cysteines. A disulfide bond can serve structural, catalytic, and signaling roles. However, there is an inherent problem to the process of disulfide bond formation: mis-pairing of cysteines can cause misfolding, aggregation and ultimately result in low yields during protein production. Recent developments in the understanding of the mechanisms involved in the formation of disulfide bonds have allowed the research community to engineer and develop methods to produce multi-disulfide-bonded proteins to high yields. This review attempts to highlight the mechanisms responsible for disulfide bond formation in Escherichia coli, both in its native periplasmic compartment in wild-type strains and in the genetically modified cytoplasm of engineered strains. The purpose of this review is to familiarize the researcher with the biological principles involved in the formation of disulfide-bonded proteins with the hope of guiding the scientist in choosing the optimum expression system.