Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.     

A hybridization assay specific for ribosomal RNA from Escherichia coli

Summary Ribosomal RNA fromEscherichia coli forms specific hybrids with heterologous DNA fromVibrio metschnicovii. The conditions for such hybridization are described, in which the amount of mRNA fromE. coli binding to theVibrio DNA is about 3% of the amount which hybridizes to the same amount of DNA fromE. coli.     

Purification and characterization of NADP+-specific glutamate dehydrogenase fromEscherichia coli

The NADP⁺-specific glutamate dehydrogenase fromEscherichia coli has been purified to electrophoretic homogeneity. The enzyme was purified 40-fold and has a specific activity of 23. Glutamate dehydrogenase fromE. coli is a hexameric enzyme with a native molecular weight of 275 KDa composed of monomers each with a molecular weight of 44.5 KDa. In nondenaturing isoelectric focusing gels, the purified enzyme is resolved into six catalytically active species, each with a molecular weight of 275 KDa and with isoelectric points ranging between pH 5.3 and 5.7. The K_m values for substrates and coenzymes have been determined, and the effect of several divalent ions on catalytic activity has been investigated.     

Cloning and expression analysis of a predicted toxin gene fromPhotorhabdus sp. HB78

The genome of the insect pathogenPhotorhabdus luminescens TT01 strain contains multiple genes predicted to encode toxins. One of these,plu0840, has 55% sequence identity with an enterotoxin fromAeromonas hydrophila. In order to further investigate this gene, we successfully cloned the completeplu0840 fromPhotorhabdus sp. HB78 and expressed it as a GST-Plu0840 fusion protein inEscherichia coli BL21 (DE3) using pGEX-4T-1 as a vector. Most of GST-Plu0840 was insoluble and sequestered into inclusion bodies. The inclusion bodies were harvested and dissolved. The resultant protein was cleaved and purified from the GST-tag. Oral bioassay showed that Plu0840 inhibited growth ofSpodoptera litura andSpodoptera exigua larvae.     

The inhibition of bactericidal activity of sera of newborn germ-free piglets to roughEscherichia coli by yeast mannan

The yeast phosphomannan (PM) derived fromHansenula capsulata strain exerts an inhibitory effect on thein vitro bactericidal activity of fresh sera of newborn, colostrum-deprived germ-free piglets to rough strains ofEscherichia coli (S-16 and Lilly). The experiments presented indicate that the PM function probably takes place at the C1 level. The inhibitory effect of PM does not occur provided bacteria are sensitized by specific antiserum prior to exposure to piglet serum. The antibody which was responsible for removal of PM blockade was of 19S nature, 2-mercaptoethanol-sensitive and can be absorbed by heat inactivated bacteria (roughEscherichia coli) or inhibited by addition of soluble somatic antigen (endotoxin) obtained from the same strain ofEscherichia coli (rough). The possible mechanism of inhibition of bactericidal activity by PM is discussed. This investigation was done in the Laboratory of Dr. M. A. Leon, Pathology Research, St. Luke's Hospital, Cleveland, Ohio, U.S.A.     

Stability of the hybrid plasmid pIM138 and its curing by some eliminating agents

Hybrid plasmid pIM138 was constructed by insertion of a chromosomal fragment with the threonme operon fromEscherichia coli into the pBR322 vector. Molar mass of pIM138 was 2.8 Mg/mol. Heteroduplexes between pBR322 vector and pIM138 hybrid DNA molecules were prepared. The hybrid plasmid shows a high stability against the curing effect of rifampicin and clorobiocm inE. coli SK1590thr host.     

Domain Structure of the Prokaryotic Selenocysteine-specific Elongation Factor SelB

Incorporation of the non-canonical amino acid selenocysteine into proteins requires the activity of the elongation factor SelB which substitutes for the function of EF-Tu. In contrast to EF-Tu, SelB binds selenocystylated tRNA^Secand an mRNA secondary structure adjacent to the UGA selenocysteine codon. To gain information on the domain structure of this specialized translation factor, theselBgenes from two bacteria unrelated toEscherichia coli(Clostridium thermoaceticumandDesulfomicrobium baculatum) were cloned and sequenced. The derived amino acid residue sequences were compared to those of SelB fromE. coliandHaemophilus influenzaeand to EF-Tu sequences. The alignment revealed that SelB contains all three domains characterized for EF-Tu. A fourth, C-terminally located domain shows only limited sequence conservation within the four SelB proteins. To elucidate the function of this C-terminal part a structure-function analysis of SelB fromE. coliwas performed. It showed that a C-terminal 17 kDa subdomain of the translation factor, when expressed separately, specifically binds the mRNA secondary structure. The recognition motif itself could be reduced to a 17 nucleotide minihelix without loss of binding affinity and specificity. A truncated SelB lacking the mRNA binding domain was still able to interact with selenocysteyl-tRNA^Sec. Expression of the mRNA binding domain alone suppressed selenocysteine insertionin vivoby competing with SelB for its binding site at the mRNA. The results indicate that SelB can be considered as an EF-Tu homolog hooked to the mRNAviaits C-terminal domain.     

The complete primary structure of ribosomal protein L1 fromThermus thermophilus

The primary structure of the 23S rRNA binding ribosomal protein L1 from the 50S ribosomal subunit ofThermus thermophilus ribosomes has been elucidated by direct protein sequencing of selected peptides prepared by enzymatic and chemical cleavage of the intact purified protein. The polypeptide chain contains 228 amino acids and has a calculated molecular mass of 24,694 D. A comparison with the primary structures of the corresponding proteins fromEscherichia coli andBacillus stearothermophilus reveals a sequence homology of 49% and 58%, respectively. With respect to both proteins, L1 fromT. thermophilus contains particularly less Ala, Lys, Gln, and Val, whereas its content of Glu, Gly, His, Ile, and Arg is higher. In addition, two fragments obtained by limited proteolysis of the intact, unmodified protein were characterized.     

Effect of metal ions on glucose-6-phosphate dehydrogenase of fungi,bacteria, and rat

We have determined the effects of KCl, ZnCl₂, and MgCl₂ on kinetic parameters of glucose-6-phosphate dehydrogenases from the bacteriaEscherichia coli, Bacillus stearothermophilus, andLeuconostoc mesenteroides, from the fungiAspergillus parasiticus, Alternaria alternata, Aphanomyces astaci, Saccharomyces cerevisiae, andTorula sp., and from the mammalRattus rattus. Each enzyme was stimulated by increasing ionic strength (KCl) and by MgCl₂. One bacterial enzyme, fromE. coli, three fungal enzymes, fromA. parasiticus, S. cerevisiae, andTorula sp., and the rat liver enzyme were inhibited by ZnCl₂. These data are discussed in light of our previous proposal that Zn²⁺ inhibition of this enzyme may stimulate versicolorin synthesis byA. parasiticus.     

The expression of plasmid-determined resistance to ampicillin in colicin K-tolerant mutant of Escherichia coli

A large fraction of colicin K-tolerant mutants of Escherichia coli with R28K plasmid expressed the lowered resistance to ampicillin (Lra phenotype). Transduction experiments have shown that in HC-108 mutant the tolPAB and lra mutations are cotransducible. The tolPAB mutation is responsible specifically for the Lra phenotype due to changes in the cell envelope which result in the elimination of some form of barrier whose effect is to restrict the access of substrate to the antibiotic inactivating β-lactamase. The tolPA mutation has no effect on the stability of R28K. Neither does it affect the susceptibility of the cell to chloramphenicol, kanamycin, and streptomycin nor the resistance to these antibiotics determined by R1drd19.     

Kirromycin-resistant elongation factor Tu from pulvomycin-producing wild-type of streptoverticillium mobaraense

Elongation factor Tu (EF-Tu) ofStreptoverticillium mobaraense, which produces pulvomycin, has been prepared to 90% purity. The purified protein differs significantly from the analogous protein found inEscherichia coli in molecular weight and antibiotic sensitivity. EF-Tu migrates in sodium dodecyl sulfate gel electrophoresis as a 46,000-dalton species. The protein is sensitive to pulvomycin, but highly resistant to kirromycin. EF-Tu fromStv. mobaraense exists in multiple forms as monomer and polymers. By contrast to the monomer, the polymers of EF-Tu are completely resistant to pulvomycin.     

Genetic analysis of the structure and function of RNase P fromE. coli

A brief review of the genetic studies on ribonuclease P (RNase P) fromEscherichia coli is presented. Temperature-sensitive mutants ofE. coli defective in tRNA processing were isolated by screening cells which were unable to synthesize a suppressor tRNA at restrictive temperature. Structural analysis of accumulated tRNA precursors showed that the isolated mutants were defective in RNase P activity. Analyses of the mutants revealed that the enzyme is essential for the synthesis of all tRNA molecules in cells and that the enzymes consists of two subunits. Analyses of the isolated mutants revealed a possible domain structure of the RNA subunit of the enzyme.Abbreviations E. coli Escherichia coli - RNase P ribonuclease P     

Effect of Overexpression of Actinobacillus succinogenes Phosphoenolpyruvate Carboxykinase on Succinate Production in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation. In contrast, in the phosphoenolpyruvate carboxylase mutant E. coli strain K-12 ppc::kan, PEPCK overexpression increased succinate production 6.5-fold.     

Electrophoretic resolution of microheterogeneity inVibrio cholerae lipopolysaccharide

Lipopolysaccharide (LPS) fromVibrio cholerae has been analysed by sodium dodecyl sulfate-potyacrylamide gel electrophoresis. Under normal conditions of electrophoresis which resolveEscherichia coli LPS, V.cholerae LPS shows two diffuse and unresolved bands. However, on long gels at low concentration it can be resolved into two major band types. There are at least 10 slow moving, discrete bands of regular periodicity and three fast moving bands. Comparison with LPS fromE. coli indicates that the heterogeneity occurs over a much smaller range of molecular weight in V.cholerae LPS, with the entire spectrum of discrete bands being contained within the space of fourE. coli repeating units.     

Effect of Phosphorus on Survival of Escherichia coli in Drinking Water Biofilms

The effect of phosphorus addition on survival of Escherichia coli in an experimental drinking water distribution system was investigated. Higher phosphorus concentrations prolonged the survival of culturable E. coli in water and biofilms. Although phosphorus addition did not affect viable but not culturable (VBNC) E. coli in biofilms, these structures could act as a reservoir of VBNC forms of E. coli in drinking water distribution systems.     

Possibilities of the conjugation process in mycobacteria

Results obtained when studying conjugation in mycobacteria by means of different methods are summarized. The method of conjugation on surface of a solid complete medium was tested with different auxotrophic mutants of different strains ofMycobacterium smegmatis. It was not possible to obtain positive results even by means of the above method. This was probably due to unsuitability of the chosen strains ofMycobacterium smegmatis. Preparation of the donor strain by transfer of the F factor fromEscherichia coli F’ORF 1ade ⁺ lac⁺ pro⁺ toMycobacterium phlei PA adeStm ^r by means of sexduction is described. Frequency of the phenotype PAade ⁺ Stm^r increased in the average by two and a half orders of magnitude with respect to the control, however, a further transfer from cultures of the cellsade ⁺ Stm^r to cells ade could not be demonstrated. Experiments aimed at transferring the R factor from strainsEscherichia coli K-12 toMycobacterium phlei were unsuccessful.     

Polarity induced by chloramphenicol and relief by suA

The suA allele, known to relieve polarity in Escherichia coli, also relieves a unique polar effect on E. coli tryptophan operon messenger RNA produced by chloramphenicol.     

Heavy meromyosin decoration of filaments fromEscherichia coli

A fibrous protein complex extracted fromEscherichia coli B/r by the method of Minkoff and Damadian [2] demonstrates arrowhead complexes when reacted with heavy meromyosin.     

Further purification,characterization and salt activation of acyl-CoA synthetase fromEscherichia coli

Acyl-CoA synthetase was further purified fromEscherichia coli in good yield and fold purification by affinity chromatography on CoA-Sepharose 4B. The molecular weight of the active form of the purified enzyme was estimated as 45 000 by Sephadex G-100 and 47 000 by Sephadex G-200. Sedimentation equilibrium ultracentrifugation analysis revealed a molecular weight of 50 000. The sedimentation coefficient was calculated as 4.4. S. An absorption maximum at 276 nm was observed in the ultraviolet light absorption spectrum. The molar extinction coefficient was 9.2 · 10⁴. Kinetic constants were determined fortrans fatty acids. All ions tested, including chaotropic and lyotropic ions, stimulated or inhibited acyl-CoA synthetase activity depending on their concentrations in the assay system. In a series of chaotropes, the lower concentration required to maximally activate acyl-CoA synthetase in increasing order of potency of chaotropic ions. The inhibitory effect of chaotrope on the enzyme activity was reversible. These data suggest that salts have a common mode of action and influence acyl-CoA synthetase activity primarily through their effect on the solution structure.