Decolorization kinetics of a recombinant Escherichia coli strain harboring azo-dye-decolorizing determinants from Rhodococcus sp.

A 6.3 kb DNA fragment containing genes responsible for azo-dye decolorization was cloned and expressed in Escherichia coli. The resulting recombinant strain E. coli CY1 decolorized 200 mg azo dye (C.I. Reactive Red 22) l^–1 at 28 °C at 8.2 mg g cell^–1 h^–1, while the host (E. coli DH5) had no color-removal activity. Addition of 0.5 mM isopropyl--d-thiogalacto-pyranoside (IPTG) increased the decolorization rate 3.4-fold. The dependence of the decolorization rate on initial dye concentration essentially followed Monod-type kinetics and the maximal rate occurred with the dye at 600 mg l^–1. The decolorization rate of E. coli CY1 was optimal at 40 °C and pH 11. Aeration (increased dissolved O₂ level) strongly inhibited the decolorization, but decolorization occurred effectively under static incubation conditions (no agitation was employed). The CY1 strain also exhibited excellent stability during repeated-batch operations.     

The isolation and preliminary characterisation of a novel Escherichia coli mutant rorB with enhanced sensitivity to ionising radiation

Summary Escherichia coli K803 cells were mutagenized and screened for the presence of clones sensitive to -rays but not to ultraviolet light. One new mutant of this type, named rorB, was isolated. This mutant is both cross-sensitive to mitomycin C and shows reduced conjugal recombination frequencies, but to a lesser extent than the phenotypically similar mutant recN. Unlike previously reported mutants of E. coli or yeast with an enhanced sensitivity to ionising radiations, rorB appears to be near wild type in ability to rejoin DNA double-strand breaks. The rorB gene maps close to ilvGEDAC at 84.5 min of the E. coli chromosome.     

Textile dye decolorization using cyanobacteria

Cyanobacterial cultures isolated from sites polluted by industrial textile effluents were screened for their ability to decolorize cyclic azo dyes. Gloeocapsa pleurocapsoides and Phormidium ceylanicum decolorized Acid Red 97 and FF Sky Blue dyes by more than 80% after 26 days. Chroococcus minutus was the only culture which decolorized Amido Black 10B (55%). Chlorophyll a synthesis in all cultures was strongly inhibited by the dyes. Visible spectroscopy and TLC confirmed that color removal was due to degradation of the dyes.Revisions requested 10 November 2004/30 November 2004; Revisions received 16 November 2004/ 7 January 2005     

Bioremediation of Textile Azo Dyes by Aerobic Bacterial Consortium Aerobic Degradation of Selected Azo Dyes by Bacterial Consortium

An aerobic bacterial consortium consisting of two isolated strains (BF1, BF2) and a strain of Pseudomonas putida (MTCC1194) was developed for the aerobic degradation of a mixture of textile azodyes and individual azodyes at alkaline pH (9-10.5) and salinity (0.9-3.68 g/l) at ambient temperature (28 +/- 2 degrees C). The degradation efficiency of the strains in different media (mineral media and in the Simulated textile effluent (STE)) and at different dye concentrations were studied. The presence of a H2O2 independent oxidase-laccase (26.5 IU/ml) was found in the culture filtrate of the organism BF2. The analysis of the degraded products by TLC and HPLC, after the microbial treatment of the dyes showed the absence of amines and the presence of low molecular weight oxidative degradation products. The enzymes present in the crude supernatant was found to be reusable for the dye degradation.     

A continuous culture study of an ATPase-negative mutant of Escherichia coli

For anaerobic glucose-limited chemostat cultures of Escherichia coli a value of 8.5 was found for Y _ATP ^max . For anaerobic glucose- or ammoniumlimited chemostat cultures of the ATPase-negative mutant M2-6 of E. coli Y _ATP ^max values of 17.6 and 20.0 were found, respectively. From these data it can be concluded that in the wild type during anaerobic growth 51–58% of the total ATP production is used for energetization of the membrane. Using the Y _ATP values obtained in the anaerobic experiments a P/O ratio of 1.46 could be calculated for aerobic experiments with the wild type. It is concluded that from the energy obtained by respiration in wild type E. coli about 60% is used for membrane energetization and only about 40% for the actual formation of ATP. No dramatic difference in the maintenance requirement for ATP or glucose has been observed between glucose- and ammonium-limited chemostat cultures of the mutant. The large difference in maintenance requirement observed for such cultures of the wild type is therefore supposed to be made possible by ATP hydrolysis by the ATPase.     

An Escherichia coli mutant defective in the NAD-dependent succinate semialdehyde dehydrogenase

Escherichia coli mutants, unable to grown on 4-hydroxyphenylacetate, have been isolated and found to be defective in the NAD-dependent succinate semialdehyde dehydrogenase. When the mutants are grown with 4-aminobutyrate as sole nitrogen source an NAD-dependent succinate semialdehyde dehydrogenase seen in the parental strain is absent but, as in the parental strain, an NADP-dependent enzyme is induced. Growth of the mutants is inhibited by 4-hydroxyphenylacetate due to the accumulation of succinate semialdehyde. The mutants are more sensitive to inhibition by exogenous succinate semialdehyde than is the parental strain. Secondary mutants able to grow in the presence of 4-hydroxyphenylacetate but still unable to use it as sole carbon source were defective in early steps of 4-hydroxyphenylacetate catabolism and so did not form succinate semialdehyde from 4-hydroxyphenylacetate. The gene encoding the NAD-dependent succinate semialdehyde dehydrogenase of Escherichia coli K-12 was located at min 34.1 on the genetic map.     

Microbial decolorization of azo dyes and dye industry effluent by Fomes lividus

The white rot fungus, Fomes lividus, was isolated from the logs of Shorea robusta in the Western Ghats region of Tamil Nadu, India. The fungus was tested for decolorization of azo dyes such as orange G (50 M) congo red (50 M) amido black 10B (25 M) and also for colour removal from dye industry effluents. The results revealed that the fungus could remove only 30.8% of orange G in the synthetic solution, whereas congo red and amido black 10B were removed by 74.0 and 98.9% respectively. A dye industry effluent was treated by the fungus in batch and continuous mode. In batch mode treatment, a maximum decolorization of 84.4% was achieved on day 4, and in continuous mode a maximum decolorization of 37.5% was obtained on day 5. The colour removal by the basidiomycete fungus might be due to adsorption of the dyes to the mycelial surface and metabolic breakdown. These results suggested that the batch mode treatment of Fomes lividus is one of the most efficient ways for colour removal in dye industry effluents.     

The complete sequence and segregational stability analysis of a new cryptic plasmid pIGWZ12 from a clinical strain of Escherichia coli

A new cryptic plasmid from a multi-resistant, multi-plasmid clinical strain of Escherichia coli has been isolated. The sequence of the 4072-base-pair pIGWZ12 (GenBank Accession No. DQ311641) was determined and analyzed. Two open-reading frames that code for proteins involved in plasmid mobilization and initiation of replication were identified. The putative origin of replication possesses all characteristic features of the theta mechanism for replicating plasmids. pIGWZ12 is stably maintained without selective pressure in bacterial cultures (for up to 80 generations), making it a good candidate for engineering a new cloning vector.     

Escherichia coli strains with defined mutations which alter cellular permeability

Summary We describe the construction and analysis of an isogenic series ofEscherichia coli K12 strains that vary in their outer membrane permeability. They carry mutations that alter the amount and the type of porin present in the outer membrane. The permeability profiles of these strains suggest that both the amount and the type of porin present in the outer membrane affects permeability. Several of the strains carry mutations that extend the permeability of the outer membrane to include a variety of compounds that are normally excluded from entering the cell.     

Mechanism of 2-aminopurine-stimulated mutagenesis in Escherichia coli

2-Aminopurine (2AP), a base analog, causes both transition and frameshift mutations in Escherichia coli. The analog is thought to cause mutations by two mechanisms: directly, by mispairing with cytosine, and indirectly, by saturation of mismatch repair (MMR). The goal of this work was to measure the relative contribution of these two mechanisms to the occurrence of transition mutations. Our data suggest that, in contrast to 2-aminopurine-stimulated frameshift mutations, the majority of transition mutations are a direct effect of base mispairing.     

Preliminary characterisation of an Escherichia coli K5 lyase-deficient strain producing the K5 polysaccharide

Escherichia coli K5 polysaccharide has structural analogies with N-acetylheparosan, a non-sulphated precursor of heparin and, for this reason, can be considered an attractive precursor for the production of semi-synthesis heparin analogues. This polysaccharide has two components: a high molecular weight (HMW) one and a low molecular weight (LMW) one, whose ratio varies depending on the action of a lyase enzyme synthesized by the same K5 producer strain. The present paper reports the production of the K5 polysaccharide by a spontaneous E. coli mutant strain lacking the lyase activity. Similar K5 polysaccharide yields, 180 mg l(-1) after 16 h fermentation, were obtained by both the wild and mutant strains, though K5 lyase activity was only observed in the culture filtrates from the wild strain. The time course of the specific filtrate volume (1 m(-2)) and of the specific filtrate flux rate (1 m(-2) h(-1)) during ultrafiltration (UF) of culture filtrates where the lyase enzyme acted on the K5 chain, showed a decrease of UF performance, probably because of membrane fouling by the LMW K5 fraction. In particular, the specific filtrate volume and specific filtrate flux rate of wild strain samples reached respectively 13 l m(-2) and 4 l m(-2) h(-1), compared to 25 l m(-2) and 15 l m(-2) h(-1) obtained from the mutant strain samples. PCR molecular analysis of the DNA region encoding for the lyase enzyme showed that, in the mutant strain, molecular rearrangements occurred in both regulatory and structural regions.     

Toroidal nucleoids in Escherichia coli exposed to chloramphenicol

The DNA of growing cells of Escherichia coli occurs in one or a few lobular bodies known as nucleoids. Upon exposure to chloramphenicol, the nucleoids assume compact, rounded forms ("cm-nucleoids") that have been described as ring- or sphere-shaped. Multiple views of single cells or spheroplasts, however, support a different, curved toroid shape for cm-nucleoids. The multiple views were obtained either by DNA fluorescence imaging as the cells or spheroplasts reoriented in liquid medium or by optical sectioning using phase-contrast or fluorescence imaging of immobilized cells. The curved toroid shape is consistent with electron microscope images of thin sections of chloramphenicol-treated cells. The relationship of this structure to active and inactive nucleoids and to the smaller toroidal forms made by in vitro DNA condensation is discussed.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 522/C1 and the international type strain from Escherichia coli O 178

The structure of the O-antigenic polysaccharide (PS) from the enteroaggregative Escherichia coli strain 522/C1 has been determined. Component analysis and (1)H and (13)C NMR spectroscopy techniques were used to elucidate the structure. Inter-residue correlations were determined by (1)H,(1)H-NOESY and (1)H,(13)C-heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [ structure: see text]. Analysis of NMR data reveals that on average the PS consists of four repeating units and indicates that the biological repeating unit contains an N-acetylgalactosamine residue at its reducing end. Serotyping of the E. coli strain 522/C1 showed it to be E. coli O 178:H7. Determination of the structure of the O-antigen PS of the international type strain from E. coli O 178:H7 showed that the two polysaccharides have identical repeating units. In addition, this pentasaccharide repeating unit is identical to that of the capsular polysaccharide from E. coli O9:K 38, which also contains O-acetyl groups.     

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     

Construction of Escherichia coli dnaK-deletion mutant infected by lambdaDE3 for overexpression and purification of recombinant GrpE proteins

Escherichia coli is widely employed to produce recombinant proteins because this microorganism is simple to manipulate, inexpensive to culture, and of short duration to produce a recombinant protein. However, contamination of molecular chaperone DnaK during purification of the recombinant protein is sometimes a problem, since DnaK sometimes has a negative effect on subsequent experiments. Previously, several efforts have been done to remove the DnaK contaminants by several sequential chromatography or washing with some expensive chemicals such as ATP. Here, we developed a simple and inexpensive method to express and purify recombinant proteins based on an E. coli dnaK-deletion mutant. The E. coli ΔdnaK52 mutant was infected by λDE3 phage to overexpress desired recombinant proteins under the control of T7 promoter. Using this host cell, recombinant hexa histidine-tag fused GrpE, which is well known as a co-chaperone for DnaK and to strongly interact with DnaK, was overexpressed and purified by one-step nickel affinity chromatography. As a result, highly purified recombinant GrpE was obtained without washing with ATP. The purified recombinant GrpE showed a folded secondary structure and a dimeric structure as previous findings. In vitro ATPase activity assay and luciferase-refolding activity assay demonstrated that the recombinant GrpE worked together with DnaK. Thus, this developed method would be rapid and useful for expression and purification of recombinant proteins which is difficult to remove DnaK contaminants.     

A Zea mays GTP-binding protein of the ARF family complements an Escherichia coli mutant with a temperature-sensitive malonyl-coenzyme A:acyl carrier protein transacylase

In an attempt to isolate a plant malonyl-coenzyme A:acyl carrier protein transacylase cDNA clone, by direct genetic selection in an Escherichia coli fabD mutant (LA2-89) with a maize cDNA expression library, a Zea mays cDNA clone encoding a GTP-binding protein of the ARF family was isolated. Complementation of a mutation affecting bacterial membrane lipid biosynthesis by a plant ARF protein, could indicate the existence of as yet unidentified bacterial equivalents of this ubiquitous eucaryotic GTP-binding protein.     

Purification and properties of dipeptidase from Escherichia coli AJ005

Summary A Co²⁺-dependent dipeptidase from E. coli strain AJ005, a peptidase-deficient mutant, was purified with streptomycin sulfate, ammonium sulfate and DEAE-cellulose. The purified dipeptidase increased by about 106-fold in specific activity, with dilysine as a substrate. The dipeptidase cleaved dilysine to two lysines among the lysine homopolymers, the possibility remaining that it is active toward peptides other than dilysine, since it was investigated in the present study only for activity toward lysine homopolymers. Activity was inhibited 54% by 10^–3 M KCN and completely by 10^–3 M PCMB, EDTA and benzethonium chloride, but not at all by soybean trypsin inhibitors. 78% and 95% of its activity was lost with 30 minutes' treatment at 45°C and 50°C, respectively. The apparent Km value was 6.7 × 10^–4 M for dilysine. It is probable that the dipeptidase differs from dipeptidase DP.Abbreviations EDTA Ethylenediaminetetraacetate - PCMB pchloromercuribenzoate     

Chromosome replication in Escherichia coli induced by oversupply of DnaA

Summary Overexpression of DnaA protein from a multicopy plasmid accompanied by a shift to 42°C causes initiation of one extra round of replication in a dnaA ⁺ strain grown in glycerol minimal medium. This extra round of replication does not lead to an extra cell division, such that cells contain twice the normal number of chromosomes.     

Two streptokinase genes are expressed with different solubility in Escherichia coli W3110

The streptokinase (SK) gene from S. equisimilis H46A (ATCC 12449) was cloned in E. coli W3110 under the control of the tryptophan promoter. The recombinant SK, which represented 15% of total cell protein content, was found in the soluble fraction of disrupted cells. The solubility of this SK notably differed from that of the product of the SK gene from S. equisimilis (ATCC 9542) which had been cloned in E. coli W3110 by using similar expression vector and cell growth conditions, and occurred in the form of inclusion bodies.     

Evidence for Escherichia coli polymerase II mutagenic bypass of intrastrand DNA crosslinks

The mutagenic potentials of DNAs containing site- and stereospecific intrastrand DNA crosslinks were evaluated in Escherichia coli cells that contained a full complement of DNA polymerases or were deficient in either polymerases II, IV, or V. Crosslinks were made between adjacent N(6)-N(6) adenines and consisted of R,R- and S,S-butadiene crosslinks and unfunctionalized 2-, 3-, and 4-carbon tethers. Although replication of single-stranded DNAs containing the unfunctionalized 3- and 4-carbon tethers were non-mutagenic in all strains tested, replication past all the other intrastrand crosslinks was mutagenic in all E. coli strains, except the one deficient in polymerase II in which no mutations were ever detected. However, when mutagenesis was analyzed in cells induced for SOS, mutations were not detected, suggesting a possible change in the overall fidelity of polymerase II under SOS conditions. These data suggest that DNA polymerase II is responsible for the in vivo mutagenic bypass of these lesions in wild-type E. coli.