Novel properties of Escherichia coli exonuclease III.

The specificity of hydrolysis of polynucleotide termini by Escherichia coli exonuclease III was studied with the use of oligothymidylate annealed to polydeoxyadenylate. The size of the products after 3' leads to 5'-hydrolysis of 5'-labeled substrate is temperature-dependent. At 25 degrees the enzyme can hydrolyze a polynucleotide chain up to the last 5'-terminal dinucleotide. A gradation of higher 5'-terminal oligonucleotides of defined chain lengths is produced after limit digestion by the enzyme when the temperature is raised between 25 degrees to 60 degrees. When the oligothymidylate was labeled at the 3'-ends with ribonucleotides, it was observed that exonuclease III can cleave a single or two consecutive ribonucleotides regardless of whether the ribonucleotides are base-paired or mismatched.     

Purification and properties of recombinant rat catalase produced in Escherichia coli

Catalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.     

Purification and properties of two truncated endoglucanases produced in Escherichia coli harbouring Clostridium cellulolyticum endoglucanase gene celCCD

The endoglucanase gene, celCCD, of Clostridium cellulolyticum has been expressed in Escherichia coli. Multiple active polypeptides were detected in the E. coli cells. The relative molecular mass (M_r) of two major active polypeptides were 56 000 (D56) and 38 000 (D38), which were smaller than the deduced M_r of the mature protein (63 401). D56 and D38 were purified from the periplasmic fraction. The N-terminal sequences of the two purified polypeptides were identical to that of the mature endoglucanase (Ala-Ile-Asn-Ser-Gln-Asp-Met-Val---) deduced from the nucleotide sequence. These data indicated that these polypeptides were produced by processing the original mature protein in the C-terminal region. The enzymatic properties of these two polypeptides were very similar, except that the specific activity of D38 was 2–3.5-fold higher than that of D56, and D38 was more heat stable than D56.Correspondence to: T. Kodama     

Identification of indigo-related pigments produced by Escherichia coli containing a cloned Rhodococcus gene.

Pigments produced by Escherichia coli containing a cloned piece of DNA from Rhodococcus sp. ATCC 21145 were extracted in chloroform and separated into blue and pink components. Evidence from TLC, NMR spectroscopy, absorption spectrum analysis and solubility behaviour suggested that the blue pigment was indigo and the pink pigment was indirubin, a structural isomer of indigo. The proposed pathway for pigment production on LB agar involves the conversion of tryptophan to indole by tryptophanase of E. coli and the oxidation of indole to indigo by the product of the cloned Rhodococcus DNA insert.     

The biotechnological potential and design of novel carotenoids by gene combination in Escherichia coli.

Carotenoids are antioxidants with considerable pharmaceutical potential. More than 600 carotenoid structures are known but their availability is limited owing to practical difficulties associated with chemical synthesis and isolation from microorganisms or plant tissue. To overcome some of these problems, heterologous expression of carotenoid genes in Escherichia coli can be used for the synthesis of rare derivatives or even of novel carotenoids. Novel and rare carotenoids can be obtained by combining carotenoid genes from different host species in E. coli.     

Microwave improved Escherichia coli transformation

Aims: The calcium chloride chemical transformation of Escherichia coli is still the most widley used cloning method in small laboratories. Therefore, any practicable improvement in its transformation efficiency seems to be of general interest. Methods and Results: We found that giving calcium chloride competent cells a 1 min microwave pulse at the lowest power setting (180 W), instead of the classic 1–2 min 42°C heat‐shock step, increases the transformation efficiency around threefold (3.3 ± 0.5). Moreover, when both treatments were given in a 2‐min 42°C ? 5 min on ice ? 1 min microwave pluse sequence, an additional improvement of 1.6 was obtained, resulting in an overall increase in efficiency of approximately 5.3‐fold compared to classical heat shock. Conclusions: This transformation method significantly improves the classical heat shock treatment. Significance and Impact of the Study: This method might be useful to those laboratories that cannot afford an electroporation apparatus.     

Novel chitosan-based films cross-linked by genipin with improved physical properties

Novel cross-linked chitosan-based films were prepared using the solution casting technique. A naturally occurring and nontoxic cross-linking agent, genipin, was used to form the chitosan and chitosan/poly(ethylene oxide) (PEO) blend networks, where two types of PEO were used, one with a molecular weight of 20 000 g/mol (HPEO) and the other of 600 g/mol (LPEO). Genipin is used in traditional Chinese medicine and extracted from gardenia fruit. Importantly, it overcomes the problem of physiological toxicity inherent in the use of some common synthetic chemicals as cross-linking agents. The mechanical properties and the stability in water of cross-linked and un-crosslinked chitosan and chitosan/PEO blend films were investigated. It was shown that, compared to the transparent yellow, un-cross-linked chitosan/PEO blend films, the genipin-cross-linked chitosan-based film, blue in color, was more elastic, was more stable, and had better mechanical properties. Genipin-cross-linking produced chitosan networks that were insoluble in acidic and alkaline solutions but were able to swell in these aqueous media. The swelling characteristics of the films exhibit sensitivity to the environmental pH and temperature. The surface properties of the films were also examined by contact angle measurements using water and mixtures of water/ethanol. The results showed that, with the one exception of cross-linked pure chitosan in 100% water, the cross-linked chitosan and chitosan/PEO blends were more hydrophobic than un-crosslinked ones.     

Characterization of fimbriae produced by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J. A. Girón, A. S. Y. Ho, and G. K. Schoolnik, Science 254:710-713, 1991). Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid. In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains. Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E. coli and F1845 of diffuse-adhering E. coli, respectively. The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E. coli, which suggests a different receptor specificity. Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin. Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane. We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC.     

Fimbrial adhesins produced by atypical enteropathogenic Escherichia coli strains

Atypical enteropathogenic Escherichia coli (aEPEC) has emerged as a significant cause of pediatric diarrhea worldwide; however, information regarding its adherence mechanisms to the human gut mucosa is lacking. In this study, we investigated the prevalence of several (fimA, ecpA, csgA, elfA, and hcpA) fimbrial genes in 71 aEPEC strains isolated from children with diarrhea (54 strains) and healthy individuals (17 strains) in Brazil and Australia by PCR. These genes are associated with adhesion and/or biofilm formation of pathogenic and commensal E. coli. Here, the most prevalent fimbrial genes found, in descending order, were hcpA (98.6%), ecpA (86%), fimA (76%), elfA (72%), and csgA (19.7%). Phenotypic expression of pili in aEPEC strains was assessed by several approaches. We were not able to detect the hemorrhagic coli pilus (HCP) or the E. coli laminin-binding fimbriae (ELF) in these strains by using immunofluorescence. Type 1 pili and curli were detected in 59% (by yeast agglutination) and 2.8% (by Congo red binding and immunofluorescence) of the strains, respectively. The E. coli common pilus (ECP) was evidenced in 36.6% of the strains on bacteria adhering to HeLa cells by immunofluorescence, suggesting that ECP could play an important role in cell adherence for some aEPEC strains. This study highlights the complex nature of the adherence mechanisms of aEPEC strains involving the coordinated function of fimbrial (e.g., ECP) and nonfimbrial (e.g., intimin) adhesins and indicates that these strains bear several pilus operons that could potentially be expressed in different niches favoring colonization and survival in and outside the host.     

Novel SOS phenotypes caused by second-site mutations in the recA430 gene of Escherichia coli

E coli recA430 mutants are recombination-proficient, extremely UV sensitive, UV nonmutable and partially deficient in RecA-mediated proteolysis and in RecA-dependent 'induced replisome reactivation' (IRR), the ability to recover DNA replication activity after UV irradiation. To determine how this pleiotropic phenotype can be altered by mutation, we isolated 10 independent derivatives of a recA430 strain, selecting for increased UV resistance. Eight of the 10 owed their resistance to altered recA alleles. We here describe the phenotypes conferred by two of the new recA alleles (recA720 and recA727), each of which contains the original recA430 mutation (G662 to A) and a second-site transition: T167 to C in recA720, and G103 to A in recA727. The second-site change in recA720 suppresses all the defects caused by recA430, and causes RecA720 to exhibit greater activity than RecA+ in some respects. Some, but not all, of the recA430 defects are partially corrected by the second-site mutation in recA727.     

Guanine nucleotide binding properties of the mammalian RalA protein produced in Escherichia coli

The simian ralA cDNA was inserted in a ptac expression vector, and high amounts of soluble ral protein were expressed in Escherichia coli. The purified p24ral contains 1 mol of bound nucleotide/mol of protein that can be exchanged against external nucleotide. The ral protein exchanges GDP with a t 1/2 of 90 min at 37 degrees C in the presence of Mg2+, and has a low GTPase activity (0.07 min-1 at 37 degrees C). We have also studied its affinity for various guanine nucleotides and analogs. NMR measurements show that the three-dimensional environment around the nucleotide is similar in p21ras and p24ral. In addition to these studies on the wild-type ral protein, we used in vitro mutagenesis to introduce substitutions corresponding to the Val12, Val12 + Thr59, and Leu61 substitutions of p21ras. These mutant ral proteins display altered nucleotide exchange kinetics and GTPase activities, however, the effects of the substitutions are less pronounced than in the ras proteins. p24ralVal12 + Thr59 autophosphorylates on the substituted Thr, as a side reaction of the GTP hydrolysis, but the rate is much lower than those of the Thr59 mutants of p21ras. These results show that ras and ral proteins have similar structures and biochemical properties. Significant differences are found, however, in the contribution of the Mg2+ ion to GDP binding, in the rate of the GTPase reaction and in the sensitivity of these two proteins to substitutions around the phosphate-binding site, suggesting that the various "small G-proteins" of the ras family perform different functions.     

Protocol for preparing proteins with improved solubility by co-expressing with molecular chaperones in Escherichia coli

Eight combinations of molecular chaperones (e.g., DnaK/DnaJ/GrpE/ClpB) are co-expressed with the target recombinant protein to compare their effectiveness in improving its soluble yield. This system allows the most complete and rational approach proposed so far to use the chaperone activity for optimizing the host cell folding machinery. Furthermore, a two-step protocol is presented, in which protein synthesis and protein refolding are uncoupled. Molecular chaperones and target protein accumulate in the first growth phase and target protein aggregates are then disaggregated in vivo after the block of protein synthesis. The optimal chaperone combination to maximize the soluble yield of a specific protein remains unpredictable. Therefore, a small-scale purification selection step is useful for screening among expression combinations before scaling-up production. Applying such a strategy, we could increase the solubility of 70% of the tested constructs with yields of up to 42-fold more protein than in controls. The procedure takes 2 d.     

Novel Escherichia coli hybrids with enhanced butanol tolerance

Hybrids between Escherichia coli and Lactobacillus brevis were generated via protoplast fusion. Growth kinetics of five hybrid strains and E. coli were used to evaluate the butanol tolerance of the novel strains under different conditions. The hybrid strains tolerated up to 2% (v/v) butanol compared to the 1% (v/v) maximum for E. coli. The growth inhibitory effects of butanol were also significantly less in several of the hybrids compared to E. coli. These results demonstrate the potential use of protoplast fusion to generate butanol-tolerant strains.