Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency

In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (M_r 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs⁻¹, with a K_m value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs⁻¹ and 0.0766 µMs⁻¹, with a K_m value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII k_cat/K_m ratios were 0.12 µM⁻¹s⁻¹and 0.13 µM⁻¹s⁻¹ and 0.216 µM⁻¹s⁻¹ and 0.094 µM⁻¹s⁻¹ for for cis- and trans-dienelactone, respectively. The k_cat/K_m ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though K_m value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.     

Characterization of the Highly Efficient Sucrose Isomerase from Pantoea dispersa UQ68J and Cloning of the Sucrose Isomerase Gene

Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35°C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50°C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35°C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the K_m was 39.9 mM, the V_max was 638 U mg⁻¹, and the K_cat/K_m was 1.79 × 10⁴ M⁻¹ s⁻¹, compared to a K_m of 76.0 mM, a V_max of 423 U mg⁻¹, and a K_cat/K_m of 0.62 × 10⁴ M⁻¹ s⁻¹ for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.     

AmyM,a Novel Maltohexaose-Forming α-Amylase from Corallococcus sp. Strain EGB

A novel α-amylase, AmyM, was purified from the culture supernatant of Corallococcus sp. strain EGB. AmyM is a maltohexaose-forming exoamylase with an apparent molecular mass of 43 kDa. Based on the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry and peptide mass fingerprinting of AmyM and by comparison to the genome sequence of Corallococcus coralloides DSM 2259, the AmyM gene was identified and cloned into Escherichia coli. amyM encodes a secretory amylase with a predicted signal peptide of 23 amino acid residues, which showed no significant identity with known and functionally verified amylases. amyM was expressed in E. coli BL21(DE3) cells with a hexahistidine tag. The signal peptide efficiently induced the secretion of mature AmyM in E. coli. Recombinant AmyM (rAmyM) was purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography, with a specific activity of up to 14,000 U/mg. rAmyM was optimally active at 50°C in Tris-HCl buffer (50 mM; pH 7.0) and stable at temperatures of <50°C. rAmyM was stable over a wide range of pH values (from pH 5.0 to 10.0) and highly tolerant to high concentrations of salts, detergents, and various organic solvents. Its activity toward starch was independent of calcium ions. The K_m and V_max of recombinant AmyM for soluble starch were 6.61 mg ml⁻¹ and 44,301.5 μmol min⁻¹ mg⁻¹, respectively. End product analysis showed that maltohexaose accounted for 59.4% of the maltooligosaccharides produced. These characteristics indicate that AmyM has great potential in industrial applications.     

Expression of a Temperature-Sensitive Esterase in a Novel Chaperone-Based Escherichia coli Strain

A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4°C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8^T, that allow E. coli to grow at high rates at 4°C (maximum growth rate, 0.28 h⁻¹) (M. Ferrer, T. N. Chernikova, M. Yakimov, P. N. Golyshin, and K. N. Timmis, Nat. Biotechnol. 21:1266-1267, 2003). The expression of a temperature-sensitive esterase in this host at 4 to 10°C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37°C (32,380 versus 190 μmol min⁻¹ g⁻¹). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5°C).     

Identification and Characterization of a Mucilaginibacter sp. Strain QM49 β-Glucosidase and Its Use in the Production of the Pharmaceutically Active Minor Ginsenosides (S)-Rh1 and (S)-Rg2

Here, we isolated and characterized a new ginsenoside-transforming β-glucosidase (BglQM) from Mucilaginibacter sp. strain QM49 that shows biotransformation activity for various major ginsenosides. The gene responsible for this activity, bglQM, consists of 2,346 bp and is predicted to encode 781 amino acid residues. This enzyme has a molecular mass of 85.6 kDa. Sequence analysis of BglQM revealed that it could be classified into glycoside hydrolase family 3. The enzyme was overexpressed in Escherichia coli BL21(DE3) using a maltose binding protein (MBP)-fused pMAL-c2x vector system containing the tobacco etch virus (TEV) proteolytic cleavage site. Overexpressed recombinant BglQM could efficiently transform the protopanaxatriol-type ginsenosides Re and Rg₁ into (S)-Rg₂ and (S)-Rh₁, respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. The K_m values for p-nitrophenyl-β-d-glucopyranoside, Re, and Rg₁ were 37.0 ± 0.4 μM and 3.22 ± 0.15 and 1.48 ± 0.09 mM, respectively, and the V_max values were 33.4 ± 0.6 μmol min⁻¹ mg⁻¹ of protein and 19.2 ± 0.2 and 28.8 ± 0.27 nmol min⁻¹ mg⁻¹ of protein, respectively. A crude protopanaxatriol-type ginsenoside mixture (PPTGM) was treated with BglQM, followed by silica column purification, to produce (S)-Rh₁ and (S)-Rg₂ at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of (S)-Rh₁ and (S)-Rg₂ from PPTGM using a novel ginsenoside-transforming β-glucosidase of glycoside hydrolase family 3.     

Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.     

Oxygen radical generation by isolated microsomes from soybean seedlings

The generation of active oxygen species by microsomes isolated from soybean seedlings was studied. NADPH-dependent superoxide anion production was 5.0 ± 0.4 nmol · min⁻¹ mg⁻¹ of microsomal protein. Hydrogen peroxide generation by microsomes was 1.40 ± 0.05 nmol · min⁻¹ mg⁻¹ of protein. Hydroxyl radical production, in the presence of ferric EDTA, evaluated through the generation of formaldehyde from dimethyl sulfoxide or tert-butyl alcohol was 0.50 ± 0.04 and 0.44 ± 0.03 nmol · min⁻¹ mg⁻¹, respectively. NADH proved to be suitable as cofactor for oxygen radical generation by microsomes from soybean seedlings. Because transition metals are implicated in radical generation by biological systems, the ability of microsomal membranes to reduce iron complexes was studied. Ferric ATP, ferric citrate, ferric ADP, ferric diethylenetriamine pentaacetic acid, and ferric EDTA were efficiently reduced in the presence of either NADPH or NADH as cofactor. The pattern of effectiveness of the different ferric complexes, on superoxide anion, hydrogen peroxide, and hydroxyl radical production, was similar to that found with animal microsomes. The data presented here indicate that microsomal ability to catalyze oxygen radical generation must be considered as an important contribution to cellular radical steady-state concentrations in cells from soybean seedlings.     

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C_T) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C_T and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10⁻⁵ pg of E. coli O157:H7 DNA ml⁻¹ equivalent to approximately 6.4 × 10³ CFU of E. coli O157:H7 ml⁻¹ based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 10⁴ CFU g⁻¹. E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g⁻¹ with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Application of a Propionyl Coenzyme A Synthetase for Poly(3-Hydroxypropionate-co-3-Hydroxybutyrate) Accumulation in Recombinant Escherichia coli

The genetic operon for propionic acid degradation in Salmonella enterica serovar Typhimurium contains an open reading frame designated prpE which encodes a propionyl coenzyme A (propionyl-CoA) synthetase (A. R. Horswill and J. C. Escalante-Semerena, Microbiology 145:1381–1388, 1999). In this paper we report the cloning of prpE by PCR, its overexpression in Escherichia coli, and the substrate specificity of the enzyme. When propionate was utilized as the substrate for PrpE, a K_m of 50 μM and a specific activity of 120 μmol · min⁻¹ · mg⁻¹ were found at the saturating substrate concentration. PrpE also activated acetate, 3-hydroxypropionate (3HP), and butyrate to their corresponding coenzyme A esters but did so much less efficiently than propionate. When prpE was coexpressed with the polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha in recombinant E. coli, a PHA copolymer containing 3HP units accumulated when 3HP was supplied with the growth medium. To compare the utility of acyl-CoA synthetases to that of an acyl-CoA transferase for PHA production, PHA-producing recombinant strains were constructed to coexpress the PHA biosynthetic genes with prpE, with acoE (an acetyl-CoA synthetase gene from R. eutropha [H. Priefert and A. Steinbüchel, J. Bacteriol. 174:6590–6599, 1992]), or with orfZ (an acetyl-CoA:4-hydroxybutyrate-CoA transferase gene from Clostridium propionicum [H. E. Valentin, S. Reiser, and K. J. Gruys, Biotechnol. Bioeng. 67:291–299, 2000]). Of the three enzymes, PrpE and OrfZ enabled similar levels of 3HP incorporation into PHA, whereas AcoE was significantly less effective in this capacity.     

Correlation of Intracellular Trehalose Concentration with Desiccation Resistance of Soil Escherichia coli Populations

Naturalized soil Escherichia coli populations need to resist common soil desiccation stress in order to inhabit soil environments. In this study, four representative soil E. coli strains and one lab strain, MG1655, were tested for desiccation resistance via die-off experiments in sterile quartz sand under a potassium acetate-induced desiccation condition. The desiccation stress caused significantly lower die-off rates of the four soil strains (0.17 to 0.40 day⁻¹) than that of MG1655 (0.85 day⁻¹). Cellular responses, including extracellular polymeric substance (EPS) production, exogenous glycine betaine (GB) uptake, and intracellular compatible organic solute synthesis, were quantified and compared under the desiccation and hydrated control conditions. GB uptake appeared not to be a specific desiccation response, while EPS production showed considerable variability among the E. coli strains. All E. coli strains produced more intracellular trehalose, proline, and glutamine under the desiccation condition than the hydrated control, and only the trehalose concentration exhibited a significant correlation with the desiccation-contributed die-off coefficients (Spearman''s ρ = −1.0; P = 0.02). De novo trehalose synthesis was further determined for 15 E. coli strains from both soil and nonsoil sources to determine its prevalence as a specific desiccation response. Most E. coli strains (14/15) synthesized significantly more trehalose under the desiccation condition, and the soil E. coli strains produced more trehalose (106.5 ± 44.9 μmol/mg of protein [mean ± standard deviation]) than the nonsoil reference strains (32.5 ± 10.5 μmol/mg of protein).     

Analysis of Escherichia coli O157:H7 Survival in Ovine or Bovine Manure and Manure Slurry

Farm animal manure or manure slurry may disseminate, transmit, or propagate Escherichia coli O157:H7. In this study, the survival and growth of E. coli O157:H7 in ovine or bovine feces under various experimental and environmental conditions were determined. A manure pile collected from experimentally inoculated sheep was incubated outside under fluctuating environmental conditions. E. coli O157:H7 survived in the manure for 21 months, and the concentrations of bacteria recovered ranged from <10² to 10⁶ CFU/g at different times over the course of the experiment. The DNA fingerprints of E. coli O157:H7 isolated at month 1 and month 12 were identical or very similar. A second E. coli O157:H7-positive ovine manure pile, which was periodically aerated by mixing, remained culture positive for 4 months. An E. coli O157:H7-positive bovine manure pile was culture positive for 47 days. In the laboratory, E. coli O157:H7 was inoculated into feces, untreated slurry, or treated slurry and incubated at −20, 4, 23, 37, 45, and 70°C. E. coli O157:H7 survived best in manure incubated without aeration at temperatures below 23°C, but it usually survived for shorter periods of time than it survived in manure held in the environment. The bacterium survived at least 100 days in bovine manure frozen at −20°C or in ovine manure incubated at 4 or 10°C for 100 days, but under all other conditions the length of time that it survived ranged from 24 h to 40 days. In addition, we found that the Shiga toxin type 1 and 2 genes in E. coli O157:H7 had little or no influence on bacterial survival in manure or manure slurry. The long-term survival of E. coli O157:H7 in manure emphasizes the need for appropriate farm waste management to curtail environmental spread of this bacterium. This study also highlights the difficulties in extrapolating laboratory data to on-farm conditions.     

Characterization of the Recombinant Exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 Important for Food Protein Hydrolysis

The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkat_{H-Ala-Pro-pNA} L⁻¹, which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 µkat_H-Ala-pNA L⁻¹. After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn²⁺-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co²⁺>Zn²⁺>Mn²⁺>Ca²⁺>Mg²⁺. PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.     

Identification of an Na+-Dependent Malonate Transporter of Malonomonas rubra and Its Dependence on Two Separate Genes

Two membrane proteins encoded by the malonate fermentation gene cluster of Malonomonas rubra, MadL and MadM, have been synthesized in Escherichia coli. MadL and MadM were shown to function together as a malonate transport system, whereas each protein alone was unable to catalyze malonate transport. Malonate transport by MadLM is Na⁺ dependent, and imposition of a ΔpNa⁺ markedly enhanced the rate of malonate uptake. The kinetics of malonate uptake into E. coli BL21(DE3) cells synthesizing MadLM at different pH values indicated that Hmalonate⁻ is the transported malonate species. The stimulation of malonate uptake by Na⁺ ions showed Michaelis-Menten kinetics, and a K_m for Na⁺ of 1.2 mM was determined. These results suggest that MadLM is an electroneutral Na⁺/Hmalonate⁻ symporter and that it is dependent on two separate genes.     

Rectal Carriage of Enterohemorrhagic Escherichia coli O157 in Slaughtered Cattle

Escherichia coli O157:H7 is an important cause of diarrhea, hemorrhagic colitis, and potentially fatal human illness. Cattle are considered a primary reservoir of infection, and recent experimental evidence has indicated that the terminal rectum is the principal site of bacterial carriage. To test this finding in naturally colonized animals, intact rectum samples from 267 cattle in 24 separate lots were obtained immediately after slaughter, and fecal material and mucosal surfaces were cultured for E. coli O157 by direct and enrichment methods. Two locations, 1 and 15 cm proximal to the recto-anal junction, were tested. In total, 35 animals were positive for E. coli O157 at at least one of the sites and 232 animals were negative as determined by all tests. The frequency of isolation and the numbers of E. coli O157 cells were higher at the site closer to the recto-anal junction, confirming our previous experimental findings. We defined low- and high-level carriers as animals with E. coli O157 levels of <1 × 10³ CFU g⁻¹ or <1 × 10³ CFU ml⁻¹ and animals with E. coli O157 levels of ≥1 × 10³ CFU g⁻¹ or ≥1 × 10³ CFU ml⁻¹ in feces or tissues, respectively. High-level carriage was detected in 3.7% of the animals (95% confidence interval, 1.8 to 6.8%), and carriage on the mucosal surface of the terminal rectum was associated with high-level fecal excretion. In summary, our results support previous work demonstrating that the mucosal epithelium in the bovine terminal rectum is an important site for E. coli O157 carriage in cattle. The data also support the hypothesis that high-level fecal shedding (≥1 × 10³ CFU g of feces⁻¹) of enterohemorrhagic E. coli O157 results from colonization of this site.     

Identification, Cloning, and Characterization of a Novel Ketoreductase from the Cyanobacterium Synechococcus sp. Strain PCC 7942

A new ketoreductase useful for asymmetric synthesis of chiral alcohols was identified in the cyanobacterium Synechococcus sp. strain PCC 7942. Mass spectrometry of trypsin-digested peptides identified the protein as 3-ketoacyl-[acyl-carrier-protein] reductase (KR) (EC 1.1.1.100). The gene, referred to as fabG, was cloned, functionally expressed in Escherichia coli, and subsequently purified to homogeneity. The enzyme displayed a temperature optimum at 44°C and a broad pH optimum between pH 7 and pH 9. The NADPH-dependent KR was able to asymmetrically reduce a variety of prochiral ketones with good to excellent enantioselectivities (>99.8%). The KR showed particular high specific activity for asymmetric reduction of ethyl 4-chloroacetoacetate (38.29 ± 2.15 U mg⁻¹) and 2′,3′,4′,5′,6′-pentafluoroacetophenone (8.57 ± 0.49 U mg⁻¹) to the corresponding (S)-alcohols. In comparison with an established industrial enzyme like the alcohol dehydrogenase from Lactobacillus brevis, the KR showed seven-times-higher activity toward 2′,3′,4′,5′,6′-pentafluoroacetophenone, with a remarkably higher enantiomeric excess (>99.8% [S] versus 43.3% [S]).     

Ultrasound-mediated DNA transfer for bacteria

In environmental microbiology, the most commonly used methods of bacterial DNA transfer are conjugation and electroporation. However, conjugation requires physical contact and cell–pilus–cell interactions; electroporation requires low-ionic strength medium and high voltage. These limitations have hampered broad applications of bacterial DNA delivery. We have employed a standard low frequency 40 kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5α and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl₂, temperature of 22°C, plasmid concentration of 0.8 ng/µl, P. putida UWC1 cell concentration of 2.5 × 10⁹ CFU (colony forming unit)/ml and reaction volume of 500 µl, the efficiency of ultrasound DNA delivery (UDD) was 9.8 ± 2.3 × 10⁻⁶ transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5α and P. fluorescens SBW25 with efficiencies of 1.16 ± 0.13 × 10⁻⁶ and 4.33 ± 0.78 × 10⁻⁶ transformants per cell, respectively. Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale.     

Size-Dependent Antimicrobial Effects of Novel Palladium Nanoparticles

Investigating the interactions between nanoscale materials and microorganisms is crucial to provide a comprehensive, proactive understanding of nanomaterial toxicity and explore the potential for novel applications. It is well known that nanomaterial behavior is governed by the size and composition of the particles, though the effects of small differences in size toward biological cells have not been well investigated. Palladium nanoparticles (Pd NPs) have gained significant interest as catalysts for important carbon-carbon and carbon-heteroatom reactions and are increasingly used in the chemical industry, however, few other applications of Pd NPs have been investigated. In the present study, we examined the antimicrobial capacity of Pd NPs, which provides both an indication of their usefulness as target antimicrobial compounds, as well as their potency as potential environmental pollutants. We synthesized Pd NPs of three different well-constrained sizes, 2.0±0.1 nm, 2.5±0.2 nm and 3.1±0.2 nm. We examined the inhibitory effects of the Pd NPs and Pd²⁺ ions toward gram negative Escherichia coli (E. coli) and gram positive Staphylococcus aureus (S. aureus) bacterial cultures throughout a 24 hour period. Inhibitory growth effects of six concentrations of Pd NPs and Pd²⁺ ions (2.5×10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, and 10⁻⁹ M) were examined. Our results indicate that Pd NPs are generally much more inhibitory toward S. aureus than toward E. coli, though all sizes are toxic at ≥10⁻⁵ M to both organisms. We observed a significant difference in size-dependence of antimicrobial activity, which differed based on the microorganism tested. Our work shows that Pd NPs are highly antimicrobial, and that fine-scale (<1 nm) differences in size can alter antimicrobial activity.     

Escherichia coli Behavior in the Presence of Organic Matter Released by Algae Exposed to Water Treatment Chemicals

When exposed to oxidation, algae release dissolved organic matter with significant carbohydrate (52%) and biodegradable (55 to 74%) fractions. This study examined whether algal organic matter (AOM) added in drinking water can compromise water biological stability by supporting bacterial survival. Escherichia coli (1.3 × 10⁵ cells ml⁻¹) was inoculated in sterile dechlorinated tap water supplemented with various qualities of organic substrate, such as the organic matter coming from chlorinated algae, ozonated algae, and acetate (model molecule) to add 0.2 ± 0.1 mg of biodegradable dissolved organic carbon (BDOC) liter⁻¹. Despite equivalent levels of BDOC, E. coli behavior depended on the source of the added organic matter. The addition of AOM from chlorinated algae led to an E. coli growth equivalent to that in nonsupplemented tap water; the addition of AOM from ozonated algae allowed a 4- to 12-fold increase in E. coli proliferation compared to nonsupplemented tap water. Under our experimental conditions, 0.1 mg of algal BDOC was sufficient to support E. coli growth, whereas the 0.7 mg of BDOC liter⁻¹ initially present in drinking water and an additional 0.2 mg of BDOC acetate liter⁻¹ were not sufficient. Better maintenance of E. coli cultivability was also observed when AOM was added; cultivability was even increased after addition of AOM from ozonated algae. AOM, likely to be present in treatment plants during algal blooms, and thus potentially in the treated water may compromise water biological stability.