Transfer of the chromosomally encoded tetracycline resistance gene <Emphasis Type="Italic">tet</Emphasis>(M) from marine bacteria to <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>

The transferability of the tetracycline (TC) resistance gene tet(M) from marine bacteria to human enteric bacteria was examined by a filter-mating method. Vibrio spp., Lactococcus garvieae, Bacillus spp., Lactobacillus sp., and Paenibacillus sp. were used as donors, and Escherichia coli JM109 and Enterococcus faecalis JH2-2 were used as recipients. The combination of Vibrio spp. and E. coli resulted in 5/68 positive transconjugants with a transfer rate of 10⁻⁷ to 10⁻³; however, no transfer was observed with E. faecalis. In case of L. garvieae and E. faecalis, 6/6 positive transconjugants were obtained with a transfer rate of 10⁻⁶ to 10⁻⁵; however, no transfer was observed with E. coli. The tet(M) gene of Bacillus, Lactobacillus, and Paenibacillus were not transferred to either E. coli or E. faecalis. tet(M) transfer was confirmed in positive E. coli and E. faecalis transconjugants by polymerase chain reaction (PCR) and Southern hybridization. All the donor strains did not harbor plasmids, while they all harbored transposon Tn916. In the transconjugants, the transposon was not detected by PCR, suggesting the possible transfer of tet(M) from the marine bacterial chromosome to the recipient chromosome. This is the first report to show that tet(M) can be transferred from marine bacteria to human enteric bacteria in a species-specific manner.     

Bioaugmented sulfur-oxidizing denitrification system with <Emphasis Type="Italic">Alcaligenes defragrans</Emphasis> B21 for high nitrate containing wastewater treatment

The performance of enriched sludge augmented with the B21 strain of Alcaligenes defragrans was compared with that of enriched sludge, as well as with pure Alcaligenes defragrans B21, in the context of a sulfur-oxidizing denitrification (SOD) process. In synthetic wastewater treatment containing 100–1,000 mg NO₃⁻-N/L, the single strain-seeded system exhibited superior performance, featuring higher efficiency and a shorter startup period, provided nitrate loading rate was less than 0.2 kg NO₃⁻-N/m³ per day. At nitrate loading rate of more than 0.5 kg NO₃⁻-N/m³ per day, the bioaugmented sludge system showed higher resistance to shock loading than two other systems. However, no advantage of the bioaugmented system over the enriched sludge system without B21 strain was observed in overall efficiency of denitrification. Both the bioaugmented sludge and enriched sludge systems obtained stable denitrification performance of more than 80% at nitrate loading rate of up to 2 kg NO₃⁻-N/m³ per day.     

Degradation of trichloroethylene by recombinant E. coli in continuous culture

Trichloroethylene (TCE) degradation by the recombinant E. coli JM109 harboring a TCE-degradative plasmid (pIO720 or pIO72K) in continuous culture was studied. The ampicillin-resistant plasmid, pIO720, contained the cumene dioxygenase genes and the dimethyl sulfide monooxygenase genes. pIO72K was constructed according to replacement of an ampicillin resistance gene on pIO720 by a kanamycin resistance gene. In the case of E. coli JM109 (pIO720) in continuous culture, TCE degradation activity decreased rapidly after continuous culture started, and the remaining number of host cells harboring pIO720 also decreased rapidly. In the case of E. coli JM109 (pIO72K) in continuous culture, TCE degradation activity was stable during continuous culture for at least 300 h and the number of the host cells harboring pIO72K did not decrease. TCE degradation activity of E. coli JM109 (pIO72K) was the highest at a dilution rate of 0.2 h^–1.     

Phytohormone production by three strains of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and possible physiological and technological implications

The aim of this work was to evaluate phytohormone biosynthesis, siderophores production, and phosphate solubilization in three strains (E109, USDA110, and SEMIA5080) of Bradyrhizobium japonicum, most commonly used for inoculation of soybean and nonlegumes in USA, Canada, and South America. Siderophore production and phosphate solubilization were evaluated in selective culture conditions, which had negative results. Indole-3-acetic acid (IAA), gibberellic acid (GA₃), and abscisic acid (ABA) production were analyzed by gas chromatography–mass spectrometry (GC-MS). Ethylene and zeatin biosynthesis were determined by GS–flame ionization detection and high-performance liquid chromatography (HPLC-UV), respectively. IAA, zeatin, and GA₃ were found in all three strains; however, their levels were significantly higher (p < 0.01) in SEMIA5080 (3.8 μg ml⁻¹), USDA110 (2.5 μg ml⁻¹), and E109 (0.87 μg ml⁻¹), respectively. ABA biosynthesis was detected only in USDA110 (0.019 μg ml⁻¹). Ethylene was found in all three strains, with highest production rate (18.1 ng ml⁻¹ h⁻¹) in E109 cultured in yeast extract mannitol medium plus l-methionine. This is the first report of IAA, GA₃, zeatin, ethylene, and ABA production by B. japonicum in pure cultures, using quantitative physicochemical methodology. The three strains have differential capability to produce the five major phytohormones and this fact may have an important technological implication for inoculant formulation.     

Decolorization of sulfonated azo dyes with two photosynthetic bacterial strains and a genetically engineered Escherichia coli strain

Sulfonated azo dyes were decolorized by two wild type photosynthetic bacterial (PSB) strains (Rhodobacter sphaeroides AS1.1737 and Rhodopseudomonas palustris AS1.2352) and a recombinant strain (Escherichia coli YB). The effects of environmental factors (dissolved oxygen, pH and temperature) on decolorization were investigated. All the strains could decolorize azo dye up to 900 mg l⁻¹, and the correlations between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Repeated batch operations were performed to study the persistence and stability of bacterial decolorization. Mixed azo dyes were also decolorized by the two PSB strains. Azoreductase was overexpressed in E. coli YB; however, the two PSB strains were better decolorizers for sulfonated azo dyes.     

Comparison of microbial hosts and expression systems for mammalian CYP1A1 catalysis

Mammalian cytochrome P450 enzymes are of special interest as biocatalysts for fine chemical and drug metabolite synthesis. In this study, the potential of different recombinant microorganisms expressing rat and human cyp1a1 genes is evaluated for such applications. The maximum specific activity for 7-ethoxyresorufin O-deethylation and gene expression levels were used as parameters to judge biocatalyst performance. Under comparable conditions, E. coli is shown to be superior over the use of S. cerevisiae and P. putida as hosts for biocatalysis. Of all tested E. coli strains, E. coli DH5α and E. coli JM101 harboring rat CYP1A1 showed the highest activities (0.43 and 0.42 U g_CDW⁻¹, respectively). Detection of active CYP1A1 in cell-free E. coli extracts was found to be difficult and only for E. coli DH5α, expression levels could be determined (41 nmol g_CDW⁻¹). The presented results show that efficient expression of mammalian cyp1a1 genes in recombinant microorganisms is troublesome and host-dependent and that enhancing expression levels is crucial in order to obtain more efficient biocatalysts. Specific activities currently obtained are not sufficient yet for fine chemical production, but are sufficient for preparative-scale drug metabolite synthesis.     

Protective action of reactivating factor of <Emphasis Type="Italic">Luteococcus japonicus</Emphasis> subsp. <Emphasis Type="Italic">casei</Emphasis> toward cells of <Emphasis Type="Italic">Escherichia coli</Emphasis> reparation mutants inactivated with UV-light

Reactivating factor (RF) from Luteococcus japonicus subsp. casei had a protective action on UV-irradiated cells of Escherichia coli AB1157 with a native reparation system and on cells of isogenic reparation mutants of E. coli UvrA⁻, RecA⁻, and PolA⁻: the effect resulted in multifold increase of survivability. Defense action of L. casei exometabolite is not connected with stimulating reparation systems in E. coli, and, probably, it is mediated by involvement of the exometabolite in the mechanism of cell division. RF did not provoke the reactivation of E. coli cells inactivated by UV-light.     

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.     

Expression,purification and characterization of pectate lyase A from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni²⁺-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml⁻¹ medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn²⁺, Ca²⁺, Fe²⁺, Mg²⁺ and Fe³⁺ ions stimulated the pectate lyase activity, but Cu²⁺ and Zn²⁺ inhibited it. The recombinant PelA had a V _max of 77 μmol min⁻¹ mg⁻¹ and an apparent K _m of 0.50 mg ml⁻¹ for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.     

Effect of anaerobic promoters on the microaerobic production of polyhydroxybutyrate (PHB) in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from Ralstonia eutropha for the micro- or anaerobic PHB production in recombinant Escherichia coli. Among the promoters, the one for alcohol dehydrogenase (P _adhE ) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring P _adhE and phbCAB achieved a 48% PHB accumulation in the cell dry weight after 48 h of static culture compared with only 30% PHB production under its native promoter. Sixty-seven percent PHB was produced in the dry weight (CDW) of an acetate pathway deleted (Δpta deletion) E. coli JW2294 harboring the vector pWCY09. In a batch process conducted in a 5.5-l NBS fermentor containing 3 l glucose LB medium, E. coli JW2294 (pWCY09) grew to 7.8 g/l CDW containing 64% PHB after 24 h of microaerobic incubation. In addition, molecular weight of PHB was observed to be much higher under microaerobic culture conditions. The high activity of P _adhE appeared to be the reason for improved micro- or anaerobic cell growth and PHB production while high molecular weight contributed to the static culture condition.     

Impact of oxygen supply on rtPA expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 (DE3): ammonia effects

A phytase with high activity at neutral pH and typical water temperatures (∼25°C) could effectively hydrolyze phytate in aquaculture. In this study, a phytase-producing strain, Pedobacter nyackensis MJ11 CGMCC 2503, was isolated from glacier soil, and the relevant gene, PhyP, was cloned using degenerate PCR and thermal asymmetric interlaced PCR. To our knowledge, this is the first report of detection of phytase activity and cloning of phytase gene from Pedobacter. PhyP belongs to beta-propeller phytase family and shares very low identity (∼28.5%) with Bacillus subtilis phytase. The purified recombinant enzyme (r-PhyP) from Escherichia coli displayed high specific activity for sodium phytate of 24.4 U mg⁻¹. The optimum pH was 7.0, and the optimum temperature was 45°C. The K _m, V _max, and k _cat values were 1.28 mM, 71.9 μmol min⁻¹ mg⁻¹, and 45.1 s⁻¹, respectively. Compared with Bacillus phytases, r-PhyP had higher relative activity at 25°C (r-PhyP (>50%), B. subtilis phytase (<8%)) and hydrolyzed phytate from soybean with greater efficacy at neutral pH. These characteristics suggest that r-PhyP might be a good candidate for an aquatic feed additive in the aquaculture industry.     

Optimization of Sterilization of <Emphasis Type="Italic">Escherichia coli</Emphasis> in Milk by Surfactin and Fengycin Using a Response Surface Method

In this paper, the sensitivity of Escherichia coli to surfactin and fengycin was observed, and the optimization of the antimicrobial activity of surfactin and fengycin to E. coli in milk by a response surface methodology was studied. Results showed that E. coli had high sensitivity to these antibiotics, whose minimal inhibitory concentrations were 15.625 μg·mL⁻¹ and 31.25 μg·mL⁻¹, respectively. The optimization result indicated that E. coli could be sterilized by 5 orders of magnitude when the temperature was 5.5°C, the action time was 15.8 h, and the concentration (surfactin/fengycin weight ratio 1:1) was 14.63 μg·mL⁻¹.     

Cloning,characterization and expression of the chitinase gene of <Emphasis Type="Italic">Enterobacter</Emphasis> sp. NRG4

A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg⁻¹. It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the K_m, k_cat and catalytic efficiency (k_cat/K_m) values of recombinant chitinase were found to be 1.27 mg ml⁻¹, 0.69 s⁻¹ and 0.54 s⁻¹M⁻¹ respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.     

Design of a secondary alcohol degradation pathway from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> DSM 50106 in an engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

The genes encoding an alcohol dehydrogenase, Baeyer–Villiger monooxygenase and an esterase from P. fluorescens DSM 50106, which seemed to be metabolically connected based on the sequence of the corresponding open reading frames, were cloned into one vector (pABE) and functionally expressed in Escherichia coli. Overall expression levels were quite low, however, using whole cells of E. coli JM109 pABE expressing the three recombinant enzymes, conversion of secondary alcohols (C_n) to the corresponding primary alcohols (C_n−2) and acetic acid via ketone and ester was possible. In this way, 2-decanol was almost completely converted within 20 h at 30°C. Thus, it could be shown that the three enzymes are metabolically connected and that they are most probably involved in alkane degradation via sub-terminal oxidation of the acyclic aliphatic hydrocarbons.     

Biokinetic parameters and behavior of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> during anaerobic growth

The biokinetics of glucose metabolism were evaluated in Aeromonas hydrophila during growth in an anaerobic biosystem. After approx 34 h growth, A. hydrophila metabolized 5,000 mg glucose l⁻¹ into the end-products ethanol, acetate, succinate and formate. The maximum growth rate, μ _m, half saturation coefficients, K _s, microbial yield coefficient, Y, cell mass decay rate coefficient, k _d, and substrate inhibition coefficient, K _si were 0.25 ± 0.03 h⁻¹, 118 ± 31 mg glucose l⁻¹, 0.12 μg DNA mg glucose⁻¹, 0.01 h⁻¹, and 3,108 ± 1,152 mg glucose l⁻¹, respectively. These data were used to predict the performance of a continuous growth system with an influent glucose concentration of 5,000 mg l⁻¹. Results of the analysis suggest that A. hydrophila will metabolize glucose at greater than 95% efficiency when hydraulic retention times (HRTs) exceed 7 h, whereas the culture is at risk of washing out at an HRT of 6.7 h.     

Decolorization of azo dyes by <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1 in the presence of humic acids

The effects of humic acid (HA) on azo dye decolorization by Shewanella oneidensis MR-1 were studied. It was found that HA species isolated from different sources could all accelerate the decolorization of Acid Red 27 (AR27). Anoxic and anaerobic conditions were required for the enhancement of azo dye decolorization by HA. In the presence of 50 mg DOC L⁻¹ Aldrich HA, 15–29% increases in decolorization efficiencies of azo dyes with different structures were achieved in 11 h. The enhancing effects increased with the increase of HA concentrations ranging from 25 to 150 mg DOC L⁻¹, and the decolorization rates were directly proportional to the HA concentrations when they were below 100 mg DOC L⁻¹. Lactate and formate were good electron donors for AR27 decolorization in the presence of HA. Both nitrate (0.1–3.0 mM) and nitrite (0.3–1.2 mM) inhibited AR27 decolorization in the presence of HA, and negligible decolorization was observed before their removal. Soluble FeCl₃ could accelerate the decolorization process in the presence of HA, whereas insoluble hematite could not. These findings may affect the understanding of bioremediation of azo dye-polluted environments and help improve the treatment of azo dye wastewaters.     

Motility influences biofilm architecture in Escherichia coli

Eight Escherichia coli strains were studied in minimal medium with a continuous flow system using confocal microscopy. K12 wild-type strains ATCC 25404 and MG1655 formed the best biofilms (∼43 μm thick, 21 to 34% surface coverage). JM109, DH5α, and MG1655 motA formed intermediate biofilms (∼13 μm thick, 41 to 58% surface coverage). BW25113, MG1655 qseB, and MG1655 fliA had poor biofilms (surface coverage less than 5%). The best biofilm-formers, ATCC 25404 and MG1655, displayed the highest motility, whereas the worst biofilm former, BW25113, was motility-impaired. The differences in motility were due to differences in expression of the motility loci qseB, flhD, fliA, fliC, and motA (e.g., qseB expression in MG1655 was 139-fold higher than BW25113 and 209-fold higher than JM109). Motility affected the biofilm architecture as those strains which had poor motility (E. coli JM109, E. coli MG1655 motA, and DH5α) formed flatter microcolonies compared with MG1655 and ATCC 25404, which had more dramatic vertical structures as a result of their enhanced motility. The presence of flagella was also found to be important as qseB and fliA mutants (which lack flagella) had less biofilm than the isogenic paralyzed motA strain (threefold less thickness and 15-fold less surface coverage).     

Microscale process evaluation of recombinant biocatalyst libraries: application to Baeyer–Villiger monooxygenase catalysed lactone synthesis

Microscale processing techniques are rapidly emerging as a cost- effective means for parallel experimentation and hence the evaluation of large libraries of recombinant biocatalysts. In this work, the potential of an automated microscale process is demonstrated in a linked sequence of operations comprising fermentation, enzyme induction and bioconversion using three whole-cell biocatalysts each expressing cyclohexanone monoxygenase (CHMO). The biocatalysts, Escherichia coli TOP 10 [pQR239], E. coli JM107 and Acinetobacter calcoaceticus NCIMB 9871, were first produced in 96-deep square well fermentations at various carbon source concentrations (10 and 20 g L⁻¹ glycerol). Following induction of CHMO activity biomass concentrations of up to 6 g_DCW L⁻¹ were obtained. Cells from each fermentation were subsequently used for the Baeyer–Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one, cyclohexanone and cyclopentanone. Each bioconversion was performed at two initial substrate concentrations (0.5 and 1.0 g L⁻¹) in order to simultaneously explore both substrate specificity and inhibition. The microscale process sequences yielded quantitative and reproducible data for each biocatalyst on maximum growth rate, biomass yield, initial rate of lactone formation, specific biocatalyst activity and bioconversion yield. E. coli TOP 10 [pQR239] was demonstrated to be an efficient biocatalyst showing substrate specificities and substrate inhibition effects in line with previous studies. Finally, in order to show that the data obtained with E. coli TOP 10 [pQR239] at microwell scale (1,000 μL) could be related to larger scales of operation, the process was performed in a 2-L stirred-tank bioreactor. Using conditions designed to enable microwell kinetic measurements under none oxygen-limited conditions, the fermentation and bioconversion data obtained at the two scales showed good quantitative agreement. This study therefore confirms the potential of automated microscale experimentation for the whole-process evaluation of recombinant biocatalyst libraries and the specification of pilot and process scale operating conditions.     

Quantitative Structure-Activity Relationships for the Pre-Steady State of <Emphasis Type="Italic">Pseudomonas Species</Emphasis> Lipase Inhibitions by <Emphasis Type="Italic">p</Emphasis>-Nirophenyl-<Emphasis Type="Italic">N</Emphasis>-Substituted Carbamates

The pre-steady states of Pseudomonas species lipase inhibitions by p-nitrophenyl-N-substituted carbamates (1–6) are composed of two steps: (1) formation of the non-covalent enzyme–inhibitor complex (E:I) from the inhibitor and the enzyme and (2) formation of the tetrahedral enzyme–inhibitor adduct (E–I) from the E:I complex. From a stopped-flow apparatus, the dissociation constant for the E:I complex, K_S, and the rate constant for formation of the tetrahedral E–I adduct from the E:I complex, k₂ are obtained from the non-linear least-squares of curve fittings of first-order rate constant (k_obs) versus inhibition concentration ([I]) plot against k_obs=k₂+k₂[I]/(K_S+[I]). Values of pK_S, and log k₂ are linearly correlated with the σ^* values with the ρ^* values of −2.0 and 0.36, respectively. Therefore, the E:I complexes are more positive charges than the inhibitors due to the ρ^* value of −2.0. The tetrahedral E–I adducts on the other hand are more negative charges than the E:I complexes due to the ρ^* value of 0.36. Formation of the E:I complex from the inhibitor and the enzyme are further divided into two steps: (1) the pre-equilibrium protonation of the inhibitor and (2) formation of the E:I complex from the protonated inhibitor and the enzyme.