Bacterial Hemoglobin Research Articles

Effect of Variation in Copper Sources and Growth Conditions on the Copper Uptake by Bacterial Hemoglobin Gene (vgb) Bearing E. coli

Effect of Variation in Copper Sources and Growth Conditions on the Copper Uptake by Bacterial Hemoglobin Gene (vgb) Bearing E. coli

Efficacy of supplementation of α-amylase-producing bacterial culture on the performance, nutrient use, and gut morphology of broiler chickens fed a corn-based diet

A trial was conducted to evaluate the efficacy of an Escherichia coli strain producing α-amylase of Bacillus stearothermophilus on growth performance, nutrient use, and the morphology of the small intestine of broilers fed a corn-based diet. One hundred thirty-five 1-d-old chicks (Cobb 500) were randomly divided into 3 groups and treated as follows: (i) basal diet (control); (ii) basal diet and water supplemented with an E. coli strain that produced amylase, and (iii) basal diet and water supplemented with an E. coli strain that produced amylase plus bacterial hemoglobin. At 21 d of age, supplementation of E. coli improved daily gain (P < 0.05) and feed conversion (P < 0.01). At the end of the trial, birds supplemented with water containing bacteria consumed more and grew faster (P < 0.05) and had better feed conversion (P < 0.10) than broilers given no bacteria. Also, the presence of bacteria improved apparent digestibility of organic matter (P < 0.01). However, no effects were detected for CP or fat digestibility. Supplementation with E. coli reduced relative pancreas weight (P = 0.06) but did not affect the weight of the liver (P > 0.05) and length of duedonum, jejunum, ileum, and cecum (P > 0.05). Length of the villi and crypts were significantly increased with bacterial supplementation. Presence of the bacterial hemoglobin gene did not cause a significant difference in changes observed. The data indicated that supplementation of an E. coli strain capable of producing α-amylase improved digestibility of nutrients and performance of broilers fed a corn-based diet.

Use of Genetically Engineered Microorganisms (GEMs) for the Bioremediation of Contaminants

ABSTRACTThis paper presents a critical review of the literature on the application of genetically engineered microorganisms (GEMs) in bioremediation. The important aspects of using GEMs in bioremediation, such as development of novel strains with desirable properties through pathway construction and the modification of enzyme specificity and affinity, are discussed in detail. Particular attention is given to the genetic engineering of bacteria using bacterial hemoglobin (VHb) for the treatment of aromatic organic compounds under hypoxic conditions. The application of VHb technology may advance treatment of contaminated sites, where oxygen availability limits the growth of aerobic bioremediating bacteria, as well as the functioning of oxygenases required for mineralization of many organic pollutants. Despite the many advantages of GEMs, there are still concerns that their introduction into polluted sites to enhance bioremediation may have adverse environmental effects, such as gene transfer. The extent of horizontal gene transfer from GEMs in the environment, compared to that of native organisms including benefits regarding bacterial bioremediation that may occur as a result of such transfer, is discussed. Recent advances in tracking methods and containment strategies for GEMs, including several biological systems that have been developed to detect the fate of GEMs in the environment, are also summarized in this review. Critical research questions pertaining to the development and implementation of GEMs for enhanced bioremediation have been identified and posed for possible future research.

Correlation Between Bacterial Hemoglobin and Carbon Sources: Their Effect on Copper Uptake by Transformed E. coli Strain αDH5

Escherichia coli alphaDH5 harboring Vitreoscilla hemoglobin gene, vgb (Escherichia:pUC8:16), and control strains (parental and vgb-lacking transformed strains, E. coli:pUC9) were employed for studying the potential ability of copper uptake. The strains were grown on a minimal medium, 5XM9, that contained different carbon (mannitol, fructose, lactose, and maltose) and nitrogen (casein and yeast extract) sources. A clear type-response relationship between the level of copper uptake and the type of carbon and nitrogen sources was observed. Copper uptake ability was better for vgb-bearing cells grown on either mannitol, lactose, fructose, or maltose as carbon source and casein as well as yeast extract as nitrogen source compared with the control medium. The increase in copper uptake was in the following order: parental cells, then pUC9-bearing cells, then VHb-expressing cells.

Expression of Bacterial Hemoglobin in the Yeast, Pichia pastoris, with a Low O2-Induced Promoter

The PsADH2-promoter of Pichia stipitis alcohol dehydrogenase II (ADH II) gene was employed to control the expression of Vitreoscilla hemoglobin (VHb) gene in Pichia pastoris. As in P. stipitis, the promoter was also induced microaerobically in P. pastoris. The expression level of VHb in P. pastoris at low O2 tension (<5% air saturation) was 16 nmol/g dry cell wt, i.e. about 24-fold higher than that at 60% air saturation. The expressed VHb enhanced growth of P. pastoris under microaerobic conditions. The application of O2-regulated promoter in P. pastoris revealed that induction of high-level expression of heterologous protein is feasible without addition of supplementary compounds.

2‐Chlorobenzoate Biodegradation by Recombinant Burkholderia cepacia under Hypoxic Conditions in a Membrane Bioreactor

The feasibility of applying bacterial hemoglobin technology to degrade 2-chlorobenzoate (2-CBA) through co-metabolism under hypoxic conditions in a membrane bioreactor (MBR) process has been studied in the laboratory. 2-chlorobenzoate removal and chloride release rates in the MBR system varied from 99 to 78% and 98 to 73%, respectively, depending on the operation conditions. Chemical oxygen demand (COD) removal efficiencies were more than 90% at food-to-microorganism ratios ranging from 0.32 to 0.62 g/g/d, and the observed yield was 0.13 to 0.20 g biomass/g COD. The bacterial cell-floc size-distribution analysis showed that there is a significant change in bacterial floc size due to high shear stress during operation of the MBR. To characterize growth kinetics of Burkholderia cepacia strain dinitrotoluene, a mathematical model that describes co-metabolic oxidation of 2-CBA in an MBR has been developed.

ArcA works with Fnr as a positive regulator of Vitreoscilla (bacterial) hemoglobin gene expression in Escherichia coli

Low oxygen induction of the bacterial (Vitreoscilla) hemoglobin gene (vgb) by the Arc system was investigated, as the presumptive vgb Crp site was found to have 73% identity to the Escherichia coli consensus ArcA site. The role of ArcA by itself and with Fnr was examined in E. coli using the wild type vgb promoter and promoter mutants affecting the Fnr and Crp (presumptive ArcA) sites and E. coli strains with all combinations of fnr+/fnr-, arcA+/arcA- genotypes. High-level transcription required both ArcA and Fnr systems to be functional; low oxygen induction required at least one of ArcA and Fnr to be intact. Levels of Vitreoscilla hemoglobin protein (VHb) followed the same trends as seen with mRNA, although the relative decreases in the mutants relative to wild type were less than with transcription. Growth of cells was stimulated by VHb, generally to a greater extent as VHb levels increased.

Highly efficient protein expression and purification using bacterial hemoglobin fusion vector

Recently developed bacterial hemoglobin (VHb) fusion expression vector has been widely used for the production of many target proteins due to its distinctive properties of expressing fusion protein with red color which facilitates visualization of the steps in purification, and increasing solubility of the target proteins. However, after intensive use of the vector, several defects have been found. In this report, we present a modified VHb fusion vector (pPosKJ) with higher efficiency, in which most of the defects were eliminated. First, it was found that thrombin protease often digests target protein as well as inserted thrombin cleavage site, so it was replaced by a TEV cleavage site for more specific cleavage of VHb from target protein. Second, a glycine-rich linker sequence was inserted between 6× his-tag and VHb to improve the affinity of 6× his-tag to Ni–NTA resin, resulting in higher purity of eluted fusion protein. Third, EcoRI and XhoI restriction sites located elsewhere in the vector were removed to make these restriction sites available for the cloning of target protein coding genes. A pPosKJ vector was fully examined with an anti-apoptotic BCL-2 family member of Caenorhabditis elegans, CED-9. A C-terminal VHb fusion expression vector (pPosKJC) was also constructed for stable expression of target proteins that may be difficult to express with an N-terminal fusion. Vaccinia-related kinase 1 (VRK1) was also successfully expressed and purified using the vector with high yield. Taken together, we suggest that the VHb fusion vector may be well suited for high-throughput protein expression and purification.

Mutational study of the bacterial hemoglobin distal heme pocket

Ligand binding experiments on three mutants in the distal heme pocket of Vitreoscilla hemoglobin (GlnE7His, ProE8Ala, and GlnE7His,ProE8Ala) were used to probe the role of GlnE7 and ProE8 in the pocket’s unusual structure. The oxygen dissociation constants for the wild type, E8Ala mutant, and E7His mutant proteins were 4.5, 4.7, and 1.7 μM, respectively; the K d for the double mutant was not determinable by our technique. Visible-Soret spectra of the carbonyl and cyanyl forms and FT-IR of the carbonyl form of the E8 mutant were similar to those of the wild type; the opposite was true for the GlnE7His and GlnE7His,ProE8Ala mutants, which also differed from wild type in the visible-Soret spectra of their oxidized forms. Models of the effects of the mutations on distal pocket structure were consistent with the experimental findings, particularly the larger effects of the GlnE7His change.

Comparative production of green fluorescent protein under co-expression of bacterial hemoglobin inEscherichia coli W3110 using different culture scales

Production of green fluorescent protein (GFP) as a model foreign protein using different culture scales under co-expression ofVitreoscilla hemoglobin (VHb) in the industrialEscherichia coli strain W3110 (a K12 derivative), was examined. It was found that the VHb co-expressing W3110, exhibited an exceptional and sustained production ability during cell cultures using different scales, while the VHb non-expressing strain showed variable production levels. This high and sustained production ability indicates that the VHb co-expressingE. coli W3110, could be successfully employed for practical large-scale production cultures without the need for serious consideration of scale-up problems.

Co-expression of bacterial hemoglobin overrides high glucose-induced repression of foreign protein expression in Escherichia coli W3110.

Co-expression of Vitreoscilla hemoglobin (VHb) can enhance production of foreign proteins in several microorganisms, including Escherichia coli. Production of foreign proteins [green fluorescent protein (GFP) and organophosphorous hydrolase (OPH)] has been examined in two typical industrial E. coli strains, W3110 (a K12 derivative) and BL21 (a B derivative). In particular, we investigated the effects of VHb co-expression and media glucose concentration on target protein production. We employed the nar O(2)-dependent promoter for self-tuning of VHb expression based on the natural changes in dissolved O(2) levels over the duration of culture. Foreign protein production in strain BL21 was decreased by a high glucose concentration but co-expression of VHb had no effect on this. In contrast, co-expression of VHb in strain W3110 overrode the glucose-induced repression and resulted in steady expression of foreign proteins.

Enhancement of 2,4-dinitrotoluene biodegradation by Burkholderia sp. in sand bioreactors using bacterial hemoglobin technology.

Continuous flow sand column bioreactor experiments were conducted to investigate the effect of 2,4-dinitrotoluene (DNT) concentration (i.e. DNT loading rate) and influent dissolved oxygen (DO) concentration on aerobic biodegradation of DNT by wild type (DNT) and recombinant (YV1) Burkholderia sp., the latter containing plasmid pSC160 which carries the gene (vgb) encoding the hemoglobin (VHb) from the bacterium Vitreoscilla. The experiments were conducted in two continuous flow packed bed sand column bioreactors, one growing the wild type strain and the other growing YV1. Under oxygen-rich feed conditions (6.8 mg DO/L in the feed) with an influent DNT concentration of 99.6 mg/L (DNT loading rate approximately = 9.2 mg/m2/day), the effluent DNT concentration from the wild type bioreactor reached 0.7 mg DNT/L in 40 days whereas it was less than 0.2 mg DNT/L for the YV1 bioreactor in about 25 days. When influent DNT concentration was increased to 214 mg/L (DNT loading rate approximately = 20.3 mg/m2/day) while maintaining the same influent DO level of 6.8 mg/L, the effluent DNT concentration increased to about 5 mg/L for the wild type bioreactor whereas it was maintained at less than 0.2 mg/L for the YV1 bioreactor. Additionally, when influent DO was reduced from 6.8 mg/L to 3.1 mg/L while the influent DNT concentration remained at 214 mg/L, the effluent DNT concentration increased to more than 20 mg/L for the wild type bioreactor but up to only 1.7 mg/L for the YV1 bioreactor. A subsequent increase of influent DO back to 6.6 mg/L reduced the effluent DNT concentration to about 5 mg/L for the wild type bioreactor and to 0.10-0.19 mg/L for the YV1 bioreactor. These results confirm the utility of vgb technology to enhance biodegradation of aromatic compounds under hypoxic conditions and also that this enhancement can be maintained over extended periods of time as evidenced by plasmid stability in Burkholderia YV1.

Expression of vitreoscilla hemoglobin improves growth and levels of extracellular enzyme in yarrowia lipolytica

Enhancement in oxygen uptake by high-cell-density cultivations has been achieved previously by expression of the bacterial hemoglobin gene from Vitreoscilla. The Vitreoscilla hemoglobin (VHb) gene was expressed in the yeast Yarrowia lipolytica to study the effect of expression in this commercially important yeast. The expression of VHb in this yeast was found to enhance growth, contrary to reported observations in wild-type Saccharomyces cerevisiae in which there was no significant growth enhancement. VHb-expressing Y. lipolytica exhibited higher specific growth rate, enhanced oxygen uptake rate, and higher respiratory activity. We report the beneficial effects of VHb expression on growth under microaerobic as well as under nonlimiting dissolved oxygen conditions. Earlier studies in Y. lipolytica have demonstrated inhibition of mycelia formation by respiratory inhibitors and poor nitrogen source, conditions poor for growth. VHb(+) Y. lipolytica cells were more efficient at forming mycelia, indicating better utilization of available oxygen as compared with the VHb(-) cells. Expression of VHb was also found to increase the levels of enzyme ribonuclease secreted into the medium, a property that may be beneficial for producing heterologous proteins in Y. lipolytica.

Biodegradation of 2-chlorobenzoate by recombinant Burkholderia cepacia expressing Vitreoscilla hemoglobin under variable levels of oxygen availability.

The influence of bacterial hemoglobin, VHb, on dechlorination and degradation of 2-chlorobenzoate (2-CBA) by recombinant Burkholderia sp. under variable oxygen availability with an initial dissolved oxygen concentration of 0.27 mM-0.72 mM was investigated in batch and continuous culture. Ability to express VHb was provided to recombinant Burkholderia by transformation with the VHb gene, vgb, on plasmid pSC160. 100% of 0.5 mM CBA was degraded in cultures with 85% and 70% of total volume as headspace air in closed reactors by both wild type and recombinant Burkholderia. The recombinant cultures were able to dechlorinate and degrade 100% of the 2-CBA in less than 48 hours at 30 degrees C compared to more than 120 hours for wild type cultures. The rate and extent of CBA degradation by recombinant cultures with 40% of total volume as headspace air was higher than those achieved by wild type cells at the end of the 168 hours of incubation period, 98 and 73%, respectively. The chloride released: CBA degraded molar ratio for cultures with 40% of total volume headspace air was nearly stoichiometric (molar ratio = 1.0) for recombinant strains, whereas it was non-stoichiometric (molar ratio = 0.24) for wild type cells. The results suggest a suicidal meta-pathway for wild type cells and a complete dechlorination and degradation pathway for recombinant cells under hypoxic conditions. The degradation and dechlorination ability of both types of cells was also investigated in continuous reactor studies by varying the dilution rate under hypoxic conditions. Regarding potential of the recombinant strain for 2-CBA degradation in either open ecosystems or closed bioreactor bioremediation systems, the stability of the plasmid containing vgb in the recombinant cells was also studied; the plasmid was 100% stable at 0.025 h(-1) dilution rate (approximately 1.7 d hydraulic retention time), even after one month.

Bacterial Hemoglobins and Flavohemoglobins for Alleviation of Nitrosative Stress in Escherichia coli

Escherichia coli MG1655 cells expressing novel bacterial hemoglobin and flavohemoglobin genes from a medium-copy-number plasmid were grown in shake flask cultures under nitrosative and oxidative stress. E. coli cells expressing these proteins display enhanced resistance against the NO(.) releaser sodium nitroprusside (SNP) relative to that of the control strain bearing the parental plasmid. Expression of bacterial hemoglobins originating from Campylobacter jejuni (CHb) and Vitreoscilla sp. (VHb) conferred resistance on SNP-challenged cells. In addition, it has been shown that NO(.) detoxification is also a common feature of flavohemoglobins originating from different taxonomic groups and can be transferred to a heterologous host. These observations have been confirmed in a specific in vitro NO(.) consumption assay. Protein extracts isolated from E. coli strains overexpressing flavohemoglobins consumed authentic NO(.) more readily than protein extracts from the wild-type strain. Oxidative challenge to the cells evoked nonuniform responses from the various cell cultures. Improved oxidative-stress-sustaining properties had also been observed when the flavohemoglobins from E. coli, Klebsiella pneumoniae, Deinococcus radiodurans, and Pseudomonas aeruginosa were expressed in E. coli.

Enhanced detoxification of organophosphates using recombinant Escherichia coli with co-expression of organophosphorus hydrolase and bacterial hemoglobin

A significantly improved, recombinant Escherichia coli has been developed to degrade the toxic organophosphorus compound, Paraoxon, to non-toxic materials by co-expression of organophosphorus hydrolase (OPH) under trc promoter and Vitreoscilla hemoglobin (VHb) under O2dependent nar promoter. VHb-expressing whole cells had significant enhancement of OPH activity (48%, 18.7 vs. 27.8 unit l−1) and bioconversion efficiency Vmax/Km (44%, 0.14 vs. 0.2 min−1) compared to VHb-free system.

Effect of the Bacterial Hemoglobin vhbGene on the Efficiency of Intergeneric Conjugation Escherichia coli–Streptomyces and Biosynthesis of Antibiotics in Streptomyces

The bacterial hemoglobin vhbgene was cloned from sliding bacterium Vitreoscillasp. as an element of the system ensuring survival of this microorganism in an environment that contains insufficient amount of oxygen. The vhbgene was transferred fromEscherichia colito some Streptomycesstrains, producers of antibiotics, by the method of intergeneric conjugation using conjugative–integrative plasmid vectors pIH1 and pCH2. The stability of plasmid DNA inheritance was analyzed in the genomes of exconjugants. A positive effect of the vhbgene on processes of conjugation and antibiotic production in a number of examined strains was shown.

Bacterial Hemoglobin gene (vgb) Transformed into Escherichia coli enhances Lead-Uptake and Minimizes it’s Adsorption

Bacterial Hemoglobin gene (vgb) Transformed into Escherichia coli enhances Lead-Uptake and Minimizes it’s Adsorption

Proton NMR investigation of heme and surrounding proton in low-spin cyanide-ligated bacterial hemoglobin fromVitreoscilla

(1)H NMR spectra of low-spin cyanide-ligated bacterial hemoglobin from Vitreoscilla (VtHb-CN) are reported. The assignments of the(1)H NMR spectra of VtHb-CN have been made through MCOSY, NOESY, 1D TOE and SUPERWEFT experiments. Almost all resonance peaks of heme and ligated His85 are identified. The spin-lattice relaxation time T (1)'s and the variation relationships of chemical shifts of these peaks with temperature have been acquired, from which the distances between the measured protons and Fe(3+), and the diamagnetic chemical shifts have been acquired, respectively. The ionization constants of pK (a)'s of ligated His85 are determined through pH titration of chemical shift, which is 4.95 for ligated His85 C(2)H proton. The lower pK (a) is attributed to the influence of the Fe(3+) of carrying positive charge and the coordination of His85 and Fe(3+) of heme.

Error-prone PCR ofVitreoscilla hemoglobin (VHb) to support the growth of microaerobicEscherichia coli

Expression of the gene encoding bacterial hemoglobin (VHb) from Vitreoscilla has been previously used to improve recombinant cell growth and enhance product formation under microaerobic conditions. It is very likely that the properties of VHb are not optimized for foreign hosts; therefore, we used error-prone PCR to generate a number of randomly mutated vhb genes to be expressed and studied in Escherichia coli. In addition, the mutated VHb proteins also contained an extension of eight residues (MTMITPSF) at the amino terminus. VHb mutants were screened for improved growth properties under microaerobic conditions and 15 clones expressing mutated hemoglobin protein were selected for further characterization and cultivated in a microaerobic bioreactor to analyze the physiological effects of novel VHb proteins on cell growth. The expression of four VHb mutants, carried by pVM20, pVM50, pVM104, and pVM134, were able to enhance microaerobic growth of E. coli by approximately 22%, 155%, 50%, and 90%, respectively, with a concomitant decrease of acetate excretion into the culture medium. The vhb gene in pVM20 contains two mutations substituting residues Glu19(A17) and Glu137(H23) to Gly. pVM50 expresses a VHb protein carrying two mutations: His36(C1) to Arg36 and Gln66(E20) to Arg66. pVM104 and pVM134 express VHb proteins carrying the mutations Ala56(E10) to Gly and Ile24(B5) to Thr, respectively. Our experiments also indicate that the positive effects elicited by mutant VHb-expression from pVM20 and pVM50 are linked to the peptide tail. Removal of the N-terminal sequence reduced cell growth approximately 23% and 53%, respectively, relative to wild-type controls. These results clearly demonstrate that it is possible to obtain mutated VHb proteins with improved characteristics for improving microaerobic growth of E. coli by using combined mutation techniques, addition of a peptide tail, and random error-prone PCR.