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Applied Microbiology and Biotechnology (v.90, #2)

Enzymatic deglycation of Amadori products in bacteria: mechanisms, occurrence and physiological functions by Veronika Maria Deppe; Johannes Bongaerts; Timothy O’Connell; Karl-Heinz Maurer; Friedhelm Meinhardt (pp. 399-406).

Amadori products (fructosamines)—ubiquitously occurring in nature—are precursors of the toxic and cell damaging ‘advanced glycation endproducts’; thus, it is not surprising that numerous organisms have developed systems to degrade such compounds. The deglycating enzymes differ with respect to their mechanisms as well as to their substrate specificities. Furthermore, different physiological functions are proposed for the different enzymes. The fructosamine 3-kinases of mammals and homologous proteins (fructosamine 3-kinase related proteins), which are common to all taxa, are thought to focus on intracellular repair functions. In contrast, in Bacillus subtilis and Escherichia coli, the cooperative action of a kinase and a deglycase facilitates Amadori degradation. As genes encoding these enzymes are co-transcribed with ABC transporter genes, it is thought that these genes facilitate the utilisation of extracellular Amadori products. Indeed, it has been shown that fructosamines can serve as the sole carbon and nitrogen sources. Here, we provide an overview of known deglycating systems with the emphasis on Amadori product degradation in bacteria.

Keywords: Maillard reaction; Deglycation; Fructosamines

Utility of gel-free, label-free shotgun proteomics approaches to investigate microorganisms by Bianca Porteus; Chawalit Kocharunchitt; Rolf E. Nilsson; Tom Ross; John P. Bowman (pp. 407-416).

This review will examine the current situation with label-free, quantitative, shotgun-oriented proteomics technology and discuss the advantages and limitations associated with its capability in capturing and quantifying large portions of proteomes of microorganisms. Such an approach allows (1) comparisons between physiological or genetic states of organisms at the protein level, (2) ‘painting’ of proteomic data onto genome data-based metabolic maps, (3) enhancement of the utility of genomic data and finally (4) surveying of non-genome sequenced microorganisms by taking advantage of available inferred protein data in order to gain new insights into strain-dependent metabolic or physiological capacities. The technology essentially is a powerful addition to systems biology with a capacity to be used to ask hypothesis-driven ‘top-down’ questions or for more empirical ‘bottom-up’ exploration.

Keywords: Proteomics; MudPIT; Protein quantification; LTQ-orbitrap; Quantification; Spectral counting

Perspectives for production and application of resveratrol by Konstantin V. Kiselev (pp. 417-425).

The polyphenol trans-resveratrol (3,5,4′-trihydroxy-trans-stilbene) is one of the best known plant secondary metabolites. The number of articles devoted to trans-resveratrol has been steadily increasing. Trans-resveratrol is a molecule that is beneficial to human health; this explains the high level of interest in trans-resveratrol among different research groups. Therefore, it is important to develop an effective method to produce this compound commercially. The applicability of biotechnology for trans-resveratrol extraction is still uncertain. This review describes and compares the available biotechnological methods of trans-resveratrol production, focusing on their advantages and disadvantages.

Keywords: Cell cultures; Resveratrol; Microorganisms

Polyrotaxanes for applications in life science and biotechnology by Jia Jing Li; Feng Zhao; Jun Li (pp. 427-443).

Due to their low cytotoxicity, controllable size, and unique architecture, cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes have inspired interesting exploitation as novel biomaterials. This review will update the recent progress in the studies on the structures of polyrotaxanes and polypseudorotaxanes based on different CDs and polymers, followed by summarizing their potential applications in life science and biotechnology, such as drug delivery, gene delivery, and tissue engineering. CD-based biodegradable polypseudorotaxane hydrogels could be used as promising injectable drug delivery systems for sustained and controlled drug release. Polyrotaxanes with drug or ligand-conjugated CDs threaded on polymer chain with biodegradable end group could be useful for controlled and multivalent targeting delivery. Cationic polyrotaxanes consisting of multiple oligoethylenimine-grafted CDs threaded on a block copolymer chain were attractive non-viral gene carries due to the strong DNA-binding ability, low cytotoxicity, and high gene transfection efficiency. Cytocleavable end caps were also introduced in the polyrotaxane systems in order to ensure efficient endosomal escape for intracellular trafficking of DNA. Finally, hydrolyzable polyrotaxane hydrogels with cross-linked α-CDs could be a desirable scaffold for cartilage and bone tissue engineering.

Keywords: Polyrotaxane; Polypseudorotaxane; Cyclodextrin; Drug delivery; Gene delivery; Tissue engineering

Wound dressings for a proteolytic-rich environment by Andreia Vasconcelos; Artur Cavaco-Paulo (pp. 445-460).

Wound dressings have experienced continuous and significant changes over the years based on the knowledge of the biochemical events associated with chronic wounds. The development goes from natural materials used to just cover and conceal the wound to interactive materials that can facilitate the healing process, addressing specific issues in non-healing wounds. These new types of dressings often relate with the proteolytic wound environment and the bacteria load to enhance the healing. Recently, the wound dressing research is focusing on the replacement of synthetic polymers by natural protein materials to delivery bioactive agents to the wounds. This article provides an overview on the novel protein-based wound dressings such as silk fibroin keratin and elastin. The improved properties of these dressings, like the release of antibiotics and growth factors, are discussed. The different types of wounds and the effective parameters of healing process will be reviewed.

Keywords: Wound healing; Silk fibroin; Collagen; Elastin; Keratin

Elicitation of Streptomyces coelicolor with dead cells of Bacillus subtilis and Staphylococcus aureus in a bioreactor increases production of undecylprodigiosin by Khalid Jaber Kadhum Luti; Ferda Mavituna (pp. 461-466).

Since microorganisms normally co-exist with other species in nature, they have developed complex metabolic and physiological responses as a result of such interspecies interactions. We utilized some of these interactions by introducing heat-killed cells of Bacillus subtilis and Staphylococcus aureus to Streptomyces coelicolor cultures and, as a result, stimulated undecylprodigiosin production. Undecylprodigiosin is not only an antibiotic; it has also been attributed with antitumor activities, but, in a defined medium, pure cultures of S. coelicolor produced only low concentrations. Elicitation with B. subtilis increased the maximum undecylprodigiosin concentration by threefold and S. aureus by fivefold compared with the pure culture of S. coelicolor. Growth and glucose consumption of elicited S. coelicolor, however, remained similar to those observed in the pure culture. Furthermore, another positive outcome of the elicitation with both B. subtilis and S. aureus was the earlier onset of undecylprodigiosin production by 24 h compared with the pure culture of S. coelicolor. This is the first time that such a phenomenon has been seen in 2L bioreactors. Our work supports the use of biotic elicitation in order to enhance the production of secondary metabolites for industrial-scale applications.

Keywords: Streptomyces coelicolor ; Bacillus subtilis ; Staphylococcus aureus ; Elicitation; Undecylprodigiosin; Bioreactor

Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis by Hisashi Harada; Kazutoshi Shindo; Kanoko Iki; Ayuko Teraoka; Sho Okamoto; Fengnian Yu; Jun-ichiro Hattan; Ryutaro Utsumi; Norihiko Misawa (pp. 467-476).

Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB and NsFER), which are derived from Pseudomonas putida and cyanobacterium Nostoc sp. strain PCC 7120, respectively, as well as three higher-plant NADPH-P450 reductases, the Arabidopsis thaliana ATR2 and two corresponding enzymes derived from ginger (Zingiber officinale), named ZoRED1 and ZoRED2. We also constructed plasmids for functional analysis of two P450s, α-humulene-8-hydroxylase (CYP71BA1) from shampoo ginger (Zingiber zerumbet) and germacrene A hydroxylase (P450NS; CYP110C1) from Nostoc sp. PCC 7120, and co-transformed E. coli with each of the pAC-Mv-based plasmids. Production levels of 8-hydroxy-α-humulene with recombinant E. coli cells (for CYP71BA1) were 1.5- to 2.3-fold higher than that of a control strain without the mevalonate-pathway genes. Level of the P450NS product with the combination of NsRED and NsFER was 2.9-fold higher than that of the CamA and CamB. The predominant product of P450NS was identified as 1,2,3,5,6,7,8,8a-octahydro-6-isopropenyl-4,8a-dimethylnaphth-1-ol with NMR analyses.

Keywords: Cytochrome P450; NADPH-P450 reductase; Sesquiterpene; Germacrene A; Ginger

Continuous ethanol production from wheat straw hydrolysate by recombinant ethanologenic Escherichia coli strain FBR5 by Badal C. Saha; Michael A. Cotta (pp. 477-487).

Continuous production of ethanol from alkaline peroxide pretreated and enzymatically saccharified wheat straw hydrolysate by ethanologenic recombinant Escherichia coli strain FBR5 was investigated under various conditions at controlled pH 6.5 and 35°C. The strain FBR5 was chosen because of its ability to ferment both hexose and pentose sugars under semi-anaerobic conditions without using antibiotics. The average ethanol produced from the available sugars (21.9–47.8 g/L) ranged from 8.8 to 17.3 g/L (0.28–0.45 g/g available sugars, 0.31–0.48 g/g sugar consumed) with ethanol productivity of 0.27–0.78 g l⁻¹ h⁻¹ in a set of 14 continuous culture (CC) runs (16–105 days). During these CC runs, no loss of ethanol productivity was observed. This is the first report on the continuous production of ethanol by the recombinant bacterium from a lignocellulosic hydrolysate.

Keywords: Continuous culture; Ethanol; Recombinant ethanologenic Escherichia coli ; Wheat straw hydrolysate; Alkaline peroxide pretreatment; Enzymatic hydrolysis

Increase of lycopene production by supplementing auxiliary carbon sources in metabolically engineered Escherichia coli by Yeong-Su Kim; Jae-Hee Lee; Nam-Hee Kim; Soo-Jin Yeom; Seon-Won Kim; Deok-Kun Oh (pp. 489-497).

In the fed-batch culture of glycerol using a metabolically engineered strain of Escherichia coli, supplementation with glucose as an auxiliary carbon source increased lycopene production due to a significant increase in cell mass, despite a reduction in specific lycopene content. l-Arabinose supplementation increased lycopene production due to increases in cell mass and specific lycopene content. Supplementation with both glucose and l-arabinose increased lycopene production significantly due to the synergistic effect of the two sugars. Cell growth by the consumption of carbon sources was related to endogenous metabolism in the host E. coli. Supplementation with l-arabinose stimulated only the mevalonate pathway for lycopene biosynthesis and supplementation with both glucose and l-arabinose stimulated synergistically only the mevalonate pathway. In the fed-batch culture of glycerol with 10 g l⁻¹ glucose and 7.5 g l⁻¹ l-arabinose, the cell mass, lycopene concentration, specific lycopene content, and lycopene productivity after 34 h were 42 g l⁻¹, 1,350 mg l⁻¹, 32 mg g cells⁻¹, and 40 mg l⁻¹ h⁻¹, respectively. These values were 3.9-, 7.1-, 1.9-, and 11.7-fold higher than those without the auxiliary carbon sources, respectively. This is the highest reported concentration and productivity of lycopene.

Keywords: Lycopene; Metabolically engineered Escherichia coli ; Auxiliary carbon source; Fed-batch culture

Covalent immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase onto modified silica nanoparticles by Ye-Wang Zhang; Manish Kumar Tiwari; Marimuthu Jeya; Jung-Kul Lee (pp. 499-507).

Rare sugars have many applications in food industry, as well as pharmaceutical and nutrition industries. Xylitol dehydrogenase (XDH) can be used to synthesize various rare sugars enzymatically. However, the immobilization of XDH has not been performed to improve the industrial production of rare sugars. In this study, silica nanoparticles which have high immobilization efficiency were selected from among several carriers for immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase (ReXDH) and subjected to characterization. Among four different chemical modification methods to give different functional groups, the silica nanoparticle derivatized with epoxy groups showed the highest immobilization efficiency (92%). The thermostability of ReXDH was improved more than tenfold by immobilization on epoxy-silica nanoparticles; the t 1/2 of the ReXDH was enhanced from 120 min to 1,410 min at 40 °C and from 30 min to 450 min at 50 °C. The K _m of ReXDH was slightly altered from 17.9 to only 19.2 mM by immobilization. The immobilized ReXDH had significant reusability, as it retained 81% activity after eight cycles of batch conversion of xylitol into l-xylulose. A ∼ 71% conversion and a productivity of 10.7 g h^-1 l^-1 were achieved when the immobilized ReXDH was employed to catalyze the biotransformation of xylitol to l-xylulose, a sugar that has been used in medicine and in the diagnosis of hepatitis. These results suggest that immobilization of ReXDH onto epoxy-silica nanoparticles has potential industrial application in rare sugar production.

Keywords: Covalent immobilization; Functional groups; Nanoparticle; Silica; Stability; Xylitol dehydrogenase

Improvement of the quality of wheat bread by addition of glycoside hydrolase family 10 xylanases by Han Zheng; Bing Guo; Xiu-Lan Chen; Sou-Jin Fan; Yu-Zhong Zhang (pp. 509-515).

Although many xylanases are widely used in the baking industry, only one glycoside hydrolase family 10 (GH 10) xylanase has previously been reported to be effective in baking. In this study, we compared the effectiveness of two GH 10 xylanases, psychrophilic XynA from Glaciecola mesophila and mesophilic EX1 from Trichoderma pseudokoningii, in bread making. The optimal dosages needed to improve wheat flour dough and bread quality were 270-U/kg flour for EX1 and 0.9-U/kg flour for XynA. At their optimal dosage, both XynA and EX1 had significant dough-softening ability, resulting in a 50% reduction in Brabender units. XynA was more effective than EX1 in reducing the time to reach maximum consistency. XynA and EX1 showed similar effects in improving the bread volume (~30% increase). EX1 was more effective in reducing the initial crumb firmness. Although both enzymes exhibited similar anti-staling effects on the bread, based on a decrease in the bread firmness, XynA had a greater effect on reducing the firming rate, and EX1 showed an enhanced reduction in the initial firmness. These results show that these two GH 10 xylanases have unique advantages in improving dough and bread quality and indicate their potential in bread making.

Keywords: Bread quality; Farinograph characteristics; Glaciecola mesophila KMM 241; Xylanase; Trichoderma pseudokoningii K9301

Crystal structure of Clostridium thermocellum ribose-5-phosphate isomerase B reveals properties critical for fast enzyme kinetics by Junho Jung; Jin-Kwang Kim; Soo-Jin Yeom; Yeh-Jin Ahn; Deok-Kun Oh; Lin-Woo Kang (pp. 517-527).

Ribose-5-phosphate isomerase (Rpi) catalyzes the conversion of d-ribose 5-phosphate (R5P) to d-ribulose 5-phosphate, which is an important step in the non-oxidative pathway of the pentose phosphate pathway and the Calvin cycle of photosynthesis. Recently, Rpis have been used to produce valuable rare sugars for industrial purposes. Of the Rpis, d-ribose-5-phosphate isomerase B from Clostridium thermocellum (CtRpi) has the fastest reactions kinetics. While Thermotoga maritime Rpi (TmRpi) has the same substrate specificity as CtRpi, the overall activity of CtRpi is approximately 200-fold higher than that of TmRpi. To understand the structural basis of these kinetic differences, we determined the crystal structures, at 2.1-Å resolution or higher, of CtRpi alone and bound to its substrates, R5P, d-ribose, and d-allose. Structural comparisons of CtRpi and TmRpi showed overall conservation of their structures with two notable differences. First, the volume of the CtRpi substrate binding pocket (SBP) was 20% less than that of the TmRpi SBP. Second, the residues next to the sugar-ring opening catalytic residue (His98) were different. We switched the key residues, involved in SBP shaping or catalysis, between CtRpi and TmRpi by site-directed mutagenesis, and studied the enzyme kinetics of the mutants. We found that tight interactions between the two monomers, narrow SBP width, and the residues near the catalytic residue are all critical for the fast enzyme kinetics of CtRpi.

Keywords: D-ribose phosphate isomerase; Clostridium thermocellum ; Rare sugar; Crystal structure

Antibacterial activity of Acinetobacter baumannii phage ϕAB2 endolysin (LysAB2) against both Gram-positive and Gram-negative bacteria by Meng-Jiun Lai; Nien-Tsung Lin; Anren Hu; Po-Chi Soo; Li-Kuang Chen; Long-Hui Chen; Kai-Chih Chang (pp. 529-539).

To investigate the nature and origin of the antibacterial activity of the lytic phage ϕAB2 toward Acinetobacter baumannii, we successfully isolated and characterized a novel phage lysozyme (endolysin) from ϕAB2 and named it LysAB2. To analyze antibacterial activity of LysAB2, the complete LysAB2 and two deletion derivatives were constructed, purified and characterized. Zymographic assays showed that only the intact LysAB2 could lyse the peptidoglycan of A. baumannii and the Staphylococcus aureus cell wall. Antibacterial analysis also showed that only the intact LysAB2 retained the complete bactericidal activity. When applied exogenously, LysAB2 exhibited a broad bacteriolytic activity against a number of Gram-negative and Gram-positive bacteria. Thermostability assays indicated that LysAB2 was stable at 20∼40°C. Its optimal pH was 6.0, and it was active from pH 4 to 8. Scanning electron microscopy revealed that exposure to 500 μg ml⁻¹ LysAB2 for up to 60 min caused a remarkable modification of the cell shape of the bacteria. Treating bacteria with LysAB2 clearly enhanced permeation of the bacterial cytoplasmic membrane. These results indicate that LysAB2 is an effective lysozyme against bacteria, and they suggest that it is a good candidate for a therapeutic/disinfectant agent to control nosocomial infections caused by multiple drug-resistant bacteria.

Keywords: Antibacterial activity; Acinetobacter baumannii ; Phage; Endolysin; Zymographic assays

A thermostable feruloyl-esterase from the hemicellulolytic bacterium Thermobacillus xylanilyticus releases phenolic acids from non-pretreated plant cell walls by Harivony Rakotoarivonina; Beatrice Hermant; Brigitte Chabbert; Jean-Pierre Touzel; Caroline Remond (pp. 541-552).

A gene (Tx-est1) encoding a thermostable feruloyl-esterase was isolated from the genome of the Gram-positive hemicellulolytic thermophilic bacterium Thermobacillus xylanilyticus. This gene contains an open reading frame of 1,020 bp encoding a protein with molecular mass of 37.4 kDa, similar to feruloyl-esterases from cellulolytic bacteria and fungi. The recombinant enzyme Tx-Est1 was expressed and produced in Escherichia coli. Tx-Est1 contains the conserved putative lipase residues Ser 202, Asp 287, and His 322 which act as catalytic triad in its C-terminus part. Purified Tx-Est1 was active against phenolic acid derivatives and stable at high temperatures. Optimal activity was observed at 65 °C and the optimal pH was around 8.5. The kinetic parameters of the esterase were determined on various substrates. The enzyme displayed activity against methyl esters of hydrocinnamic acids and feruloylated arabino-xylotetraose, exhibiting high specificity and affinity for the latter. Our results showed that Tx-Est1 is a thermostable feruloyl-esterase which could be useful to hydrolyze arabinoxylans from graminaceous plant cell walls as the enzyme is able to release phenolic acids from a lignocellulose biomass.

Keywords: Feruloyl-esterase; Thermobacillus xylanilyticus ; Hemicelluloses; Phenolic acids; Lignocellulosic biomass

An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent by M. Enamul Kabir; Senthilkumar Krishnaswamy; Masahiko Miyamoto; Yasuhiro Furuichi; Tadazumi Komiyama (pp. 553-564).

Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data showed that the binding affinity for this single-domain antibody was 272-fold higher (K _d = 1.07 × 10⁻¹⁰ M) and antifungal activity was three- to fivefold more efficient (IC₅₀ = 0.46 × 10⁻⁶ to 1.17 × 10⁻⁶ M) than that for the conventional antibody (K _d = 2.91 × 10⁻⁸ M and IC₅₀ = 2.14 × 10⁻⁶ to 3.78 × 10⁻⁶ M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain synthetic antibodies will find their way into a number of biotechnological or medical applications.

Keywords: Phage-display panning; Single-domain antibody fragment; Antifungal activity; HM-1 killer toxin; Killer toxin (HM-1) neutralizing monoclonal antibody

A celluloytic complex from Clostridium cellulovorans consisting of mannanase B and endoglucanase E has synergistic effects on galactomannan degradation by Sang Duck Jeon; Kyung Ok Yu; Seung Wook Kim; Sung Ok Han (pp. 565-572).

In our previous study using a fluorescently labeled cohesin biomarker, we detected and identified a putative cellulosomal mannanase belonging to the glycosyl hydrolase family 26 from Clostridium cellulovorans in xylan-containing cultures. In this study, a mannanase gene, manB from C. cellulovorans, was expressed in Escherichia coli. The optimal pH of a purified enzyme was around pH 7.0 and the optimal temperature was 40°C. The purified mannanase B (ManB) showed high hydrolytic activity toward galactomannan. An assembly of ManB with mini-CbpA, which contains a carbohydrate-binding module that provides proximity to insoluble substrates, increased the activity toward galactomannan [locust bean gum (LBG) and guar gum] 1.7- and 2.0-fold over those without mini-CbpA. We tested the synergistic effects on galactomannan (LBG and guar gum) degradation using cellulosomal mannanase ManB with cellulosomal endoglucanase E, which was predicted to have mannanase activity in C. cellulovorans as a cellulolytic complex. When assembled with the mini-CbpA, the mixture of endoglucanase E (EngE) and ManB at a molar ratio of 1:2 showed the highest synergistic effect (2.4-fold) on LBG. The mixture at a ratio of 1:3 showed the highest synergistic effect (2.8-fold) on guar gum. These synergistic actions indicated that ManB assembled with mini-CbpA hydrolyzed insoluble galactomannan, which in turn promoted soluble galactomannan degradation by EngE.

Keywords: Mannanase; Cellulosome; Clostridium cellulovorans ; Synergistic effect; Galactomannan

Isolation and characterization of cold-active family VIII esterases from an arctic soil metagenome by Eun Young Yu; Min-A Kwon; Miae Lee; Joon Young Oh; Ji-Eun Choi; Ji Young Lee; Bong-Keun Song; Dae-Hyun Hahm; Jae Kwang Song (pp. 573-581).

Functional screening for lipolytic enzymes at low temperatures resulted in the isolation of the novel cold-active esterases, EstM-N1 and EstM-N2, from a metagenomic DNA library of arctic soil samples. EstM-N1 and EstM-N2 were 395 and 407 amino acids in length, respectively, and showed the highest similarity to class C β-lactamases. However, they shared a relatively low level of sequence similarity (30%) with each other. Phylogenetic analysis of bacterial lipolytic enzymes confirmed that EstM-N1 and EstM-N2 belonged to family VIII of bacterial esterases/lipases. The (His)₆-tagged esterases were purified to about 99% homogeneity from the soluble fraction of recombinant Escherichia coli cultures. The purified EstM-N1 and EstM-N2 retained more than 50% of maximal activity in the temperature range of 0–35°C, with optimal temperatures of 20°C and 30°C, respectively. Both enzymes preferred the short acyl chains of p-nitrophenyl esters and exhibited very narrow substrate specificity, indicating that they are typical esterases. The β-lactamase activity of EstM-N1 and EstM-N2 was also detected and reached about 31% and 13% of the positive control enzyme, Bacillus cereus β-lactamase, respectively. These first cold-active esterases belonging to family VIII are expected to be useful for potential biotechnological applications as interesting biocatalysts.

Keywords: Esterase; Metagenome; Family VIII; Cold-active enzyme

The sense strand pre-cleaved RNA duplex mediates an efficient RNA interference with less off-target and immune response effects by Xiaozhao Lu; Guodong Yang; Jie Zhang; Haiyan Fu; Liang Jin; Mengying Wei; Li Wang; Zifan Lu (pp. 583-589).

RNA interference is an appealing and promising therapeutic approach in cancer and other diseases. Designing novel strategies aiming to increase the efficiency, duration, and reduce the off-target silencing by sense strand is of great significance for its future application clinically. Here, we report that RNA duplex with the sense strand pre-cleaved at the base between base 10 and 11 relative to the 5′ end of the antisense strand induced a target-specific RNA silencing effectively. Furthermore, different from the canonical RNA duplex, this novel RNA duplex rarely inhibits the luciferase activity in the reporter, bearing the target sequence corresponding to the sense strand, suggesting a less off-target effects of this novel strategy. Furthermore, the immune response of the novel RNA duplex induced a much milder immune response as seen from the NFkappaB activity. In addition, our newly designed RNA duplex should be easier for preservation than the asymmetric RNA duplex. Our results establish a novel method to design a new class of RNA duplex for improved RNA interference.

Keywords: RNAi; Off-target effects; Immune response

Molecular analysis of ∆6 desaturase and ∆6 elongase from Conidiobolus obscurus in the biosynthesis of eicosatetraenoic acid, a ω3 fatty acid with nutraceutical potentials by Li Tan; Dauenpen Meesapyodsuk; Xiao Qiu (pp. 591-601).

Conidiobolus obscurus, an entomopathogenic fungus able to infect aphids, was previously reported to produce substantial amounts of very long chain polyunsaturated fatty acids (VLCPUFAs) that may mediate the insect infection. However, the genes involved in the biosynthesis of these VLCPUFAs from the order Entomophthorales have yet to be identified. Using degenerate reverse transcriptase-polymerase chain reaction and rapid amplification of the cDNA end methods, we cloned a ∆6 desaturase cDNA (CoD6) and a ∆6 elongase cDNA (CoE6) from C. obscurus. Expression of CoD6 and CoE6 in Saccharomyces cerevisiae revealed CoD6 could introduce a Δ6 double bond into α-linolenic acid (18:3n-3), and CoE6 preferentially elongated 18-carbon Δ6 desaturated fatty acid stearidonic acid (18:4n-3). When the fungus was grown under a temperature shift from 20°C to 10°C, the transcript level of CoD6 and CoE6 increased, whereas when the fungal culture was shifted from 20°C to 30°C, the transcript level of both genes decreased. The entire eicosatetraenoic acid biosynthetic pathway was reconstituted in yeast using four genes, CoD6 and CoE6 from C. obscurus, CpDes12 (a Δ12 desaturase) and CpDesX (a ω3 desaturase) from Claviceps purpurea. Yeast transformants expressing the four genes produced ten new fatty acids including the final product eicosatetraenoic acid (ETA). This represents the reconstitution of the entire ETA pathway in yeast without supplementation of any exogenous fatty acids.

Keywords: VLCPUFAs; Eicosatetraenoic acid; Δ6 desaturase; Δ6 elongase; Conidiobolus obscurus

Tailor-made type II Pseudomonas PHA synthases and their use for the biosynthesis of polylactic acid and its copolymer in recombinant Escherichia coli by Taek Ho Yang; Yu Kyung Jung; Hye Ok Kang; Tae Wan Kim; Si Jae Park; Sang Yup Lee (pp. 603-614).

Previously, we have developed metabolically engineered Escherichia coli strains capable of producing polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] by employing evolved Clostridium propionicum propionate CoA transferase (Pct Cp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 Ps_6-19). Introduction of mutations four sites (E130, S325, S477, and Q481) of PhaC1 Ps_6-19 have been found to affect the polymer content, lactate mole fraction, and molecular weight of P(3HB-co-LA). In this study, we have further engineered type II Pseudomonas PHA synthases 1 (PhaC1s) from Pseudomonas chlororaphis, Pseudomonas sp. 61-3, Pseudomonas putida KT2440, Pseudomonas resinovorans, and Pseudomonas aeruginosa PAO1 to accept short-chain-length hydroxyacyl-CoAs including lactyl-CoA and 3-hydroxybutyryl-CoA as substrates by site-directed mutagenesis of four sites (E130, S325, S477, and Q481). All PhaC1s having mutations in these four sites were able to accept lactyl-CoA as a substrate and supported the synthesis of P(3HB-co-LA) in recombinant E. coli, whereas the wild-type PhaC1s could not accumulate polymers in detectable levels. The contents, lactate mole fractions, and the molecular weights of P(3HB-co-LA) synthesized by recombinant E. coli varied depending upon the source of the PHA synthase and the mutants used. PLA homopolymer could also be produced at ca. 7 wt.% by employing the several PhaC1 variants containing E130D/S325T/S477G/Q481K quadruple mutations in wild-type E. coli XL1-Blue.

Keywords: PLA; P(3HB-co-LA); Pseudomonas PhaC1 variants; Site-directed mutagenesis

The construction of a library of synthetic promoters revealed some specific features of strong Streptomyces promoters by Nicolas Seghezzi; Patrick Amar; Brian Koebmann; Peter R. Jensen; Marie-Joëlle Virolle (pp. 615-623).

Streptomyces are bacteria of industrial interest whose genome contains more than 73% of bases GC. In order to define, in these GC-rich bacteria, specific sequence features of strong promoters, a library of synthetic promoters of various sequence composition was constructed in Streptomyces. To do so, the sequences located upstream, between and downstream of the −35 and −10 consensus promoter sequences were completely randomized and some variability was introduced in the −35 (position 6) and −10 (positions 3, 4 and 5) hexamers recognized by the major vegetative sigma factor HrdB. The synthetic promoters were cloned into the promoter-probe plasmid pIJ487 just upstream of the promoter-less aphII gene that confers resistance to neomycin. This synthetic promoter library was transformed into Streptomyces lividans, and the resulting transformants were screened for their ability to grow in the presence of different concentrations of neomycin (20, 50, and 100 μg ml⁻¹). Promoter strengths varied up to 12-fold, in small increments of activity increase, as determined by reverse transcriptase-PCR. This collection of promoters of various strengths can be useful for the fine-tuning of gene expression in genetic engineering projects. Thirty-eight promoters were sequenced, and the sequences of the 14 weakest and 14 strongest promoters were compared using the WebLogo software with small sample correction. This comparison revealed that the −10 box, the −10 extended motif as well as the spacer of the strong Streptomyces promoters are more G rich than those of the weak promoters.

Keywords: Streptomyces ; Synthetic promoters; Promoter strength; aphII ; Neomycin

Co-transcription of the celC gene cluster in Clostridium thermocellum by Michael Newcomb; Jonathan Millen; Chun-Yu Chen; J. H. David Wu (pp. 625-634).

Clostridium thermocellum, an anaerobic, thermophilic, and ethanogenic bacterium produces a large cellulase complex termed the cellulosome and many free glycosyl hydrolases. Most cellulase genes scatter around the genome. We mapped the transcripts of the six-gene cluster celC–glyR3–licA–orf4–manB–celT and determined their transcription initiation sites by primer extension. Northern blot showed that celC–glyR3–licA were co-transcribed into a polycistronic messenger with the transcription initiation site at −20 bp. Furthermore, RT-PCR mapping showed that manB and celT, two cellulosomal genes immediately downstream, were co-transcribed into a bicistronic messenger with the initiation site at −233 bp. In contrast, rf4 was transcribed alone with the two initiation sites at −130 and −138 bp, respectively. Finally, quantitative RT-PCR analysis showed that celC, glyR3, and licA were coordinately induced by growing on laminarin, a β-1,3 glucan. Gene expression peaked at the late exponential phase. Taking together with our previous report that GlyR3 binds to the celC promoter in the absence of laminaribiose, a β-1,3 glucose dimer, these results indicate that celC, glyR3, and licA form an operon repressible by GlyR3 and inducible by laminaribiose, signaling the availability of β-1,3 glucan. The celC operon is the first glycosyl hydrolase operon reported in this bacterium.

Keywords: Cellulase; Operon; Cellulosome; Gene regulation; Cellulolytic enzyme; Cellulase operon

Conversion of 11-hydroxy-O-methylsterigmatocystin to aflatoxin G₁ in Aspergillus parasiticus by Hongmei Zeng; Hidemi Hatabayashi; Hiroyuki Nakagawa; Jingjing Cai; Ryoya Suzuki; Emi Sakuno; Toshitsugu Tanaka; Yasuhiro Ito; Kenneth C. Ehrlich; Hiromitsu Nakajima; Kimiko Yabe (pp. 635-650).

In aflatoxin biosynthesis, aflatoxins G₁ (AFG₁) and B₁ (AFB₁) are independently produced from a common precursor, O-methylsterigmatocystin (OMST). Recently, 11-hydroxy-O-methylsterigmatocystin (HOMST) was suggested to be a later precursor involved in the conversion of OMST to AFB₁, and conversion of HOMST to AFB₁ was catalyzed by OrdA enzyme. However, the involvement of HOMST in AFG₁ formation has not been determined. In this work, HOMST was prepared by incubating OrdA-expressing yeast with OMST. Feeding Aspergillus parasiticus with HOMST allowed production of AFG₁ as well as AFB₁. In cell-free systems, HOMST was converted to AFG₁ when the microsomal fraction, the cytosolic fraction from A. parasiticus, and yeast expressing A. parasiticus OrdA were added. These results demonstrated (1) HOMST is produced from OMST by OrdA, (2) HOMST is a precursor of AFG₁ as well as AFB₁, and (3) three enzymes, OrdA, CypA, and NadA, and possibly other unknown enzymes are involved in conversion of HOMST to AFG₁.

Keywords: Aflatoxin G₁ formation; HOMST; OrdA; NadA; CypA

GGDEF proteins YeaI, YedQ, and YfiN reduce early biofilm formation and swimming motility in Escherichia coli by Viviana Sanchez-Torres; Hongbo Hu; Thomas K. Wood (pp. 651-658).

The second messenger 3′–5′-cyclic diguanylic acid (c-di-GMP) promotes biofilm formation, and c-di-GMP is synthesized by diguanylate cyclases (characterized by a GGDEF domain) and degraded by phosphodiesterases. Here, we evaluated the effect of the 12 E. coli GGDEF-only proteins on biofilm formation and motility. Deletions of the genes encoding the GGDEF proteins YeaI, YedQ, YfiN, YeaJ, and YneF increased swimming motility as expected for strains with reduced c-di-GMP. Alanine substitution in the EGEVF motif of YeaI abolished its impact on swimming motility. In addition, extracellular DNA (eDNA) was increased as expected for the deletions of yeaI (tenfold), yedQ (1.8-fold), and yfiN (3.2-fold). As a result of the significantly enhanced motility, but contrary to current models of decreased biofilm formation with decreased diguanylate cyclase activity, early biofilm formation increased dramatically for the deletions of yeaI (30-fold), yedQ (12-fold), and yfiN (18-fold). Our results indicate that YeaI, YedQ, and YfiN are active diguanylate cyclases that reduce motility, eDNA, and early biofilm formation and contrary to the current paradigm, the results indicate that c-di-GMP levels should be reduced, not increased, for initial biofilm formation so c-di-GMP levels must be regulated in a temporal fashion in biofilms.

Keywords: Diguanylate cyclase; Cyclic-di-GMP; Biofilm; GGDEF; Motility

Microbial production of polyhydroxyalkanoate block copolymer by recombinant Pseudomonas putida by Shi Yan Li; Cui Ling Dong; Shen Yu Wang; Hai Mu Ye; Guo-Qiang Chen (pp. 659-669).

Polyhydroxyalkanoate (PHA) synthesis genes phaPCJ Ac cloned from Aeromonas caviae were transformed into Pseudomonas putida KTOY06ΔC, a mutant of P. putida KT2442, resulting in the ability of the recombinant P. putida KTOY06ΔC (phaPCJ A.c) to produce a short-chain-length and medium-chain-length PHA block copolymer consisting of poly-3-hydroxybutyrate (PHB) as one block and random copolymer of 3-hydroxyvalerate (3HV) and 3-hydroxyheptanoate (3HHp) as another block. The novel block polymer was studied by differential scanning calorimetry (DSC), nuclear magnetic resonance, and rheology measurements. DSC studies showed the polymer to possess two glass transition temperatures (T _g), one melting temperature (T _m) and one cool crystallization temperature (T _c). Rheology studies clearly indicated a polymer chain re-arrangement in the copolymer; these studies confirmed the polymer to be a block copolymer, with over 70 mol% homopolymer (PHB) of 3-hydroxybutyrate (3HB) as one block and around 30 mol% random copolymers of 3HV and 3HHp as the second block. The block copolymer was shown to have the highest tensile strength and Young’s modulus compared with a random copolymer with similar ratio and a blend of homopolymers PHB and PHVHHp with similar ratio. Compared with other commercially available PHA including PHB, PHBV, PHBHHx, and P3HB4HB, the short-chain- and medium-chain-length block copolymer PHB-b-PHVHHp showed differences in terms of mechanical properties and should draw more attentions from the PHA research community.

Keywords: PHB; Polyhydroxyalkanoates; Block copolymer; Pseudomonas putida ; 3-Hydroxybutyrate; 3-Hydroxyvalerate; 3-Hydroxyheptanoate

Functional characterization of a gene cluster involved in gentisate catabolism in Rhodococcus sp. strain NCIMB 12038 by Ting-Ting Liu; Ying Xu; Hong Liu; Sha Luo; Ya-Jie Yin; Shuang-Jiang Liu; Ning-Yi Zhou (pp. 671-678).

Rhodococcus sp. strain NCIMB 12038 utilizes naphthalene as a sole source of carbon and energy, and degrades naphthalene via salicylate and gentisate. To identify the genes involved in this pathway, we cloned and sequenced a 12-kb DNA fragment containing a gentisate catabolic gene cluster. Among the 13 complete open reading frames deduced from this fragment, three (narIKL) have been shown to encode the enzymes involved in the reactions of gentisate catabolism. NarI is gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate, NarL is a mycothiol-dependent maleylpyruvate isomerase which catalyzes the isomerization of maleylpyruvate to fumarylpyruvate, and NarK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. The narX gene, which is divergently transcribed with narIKL, has been shown to encode a functional 3-hydroxybenzoate 6-monooxygenase. This led us to discover that this strain is also capable of utilizing 3-hydroxybenzoate as its sole source of carbon and energy. Both NarL and NarX were purified to homogeneity as His-tagged proteins, and they were determined by gel filtration to exist as a trimer and a monomer, respectively. Our study suggested that the gentisate degradation pathway was shared by both naphthalene and 3-hydroxybenzoate catabolism in this strain.

Keywords: Catabolism; Gene cluster; Gentisate; Rhodococcus sp. strain NCIMB 12038

Lead induces oxidative stress and phenotypic markers of apoptosis in Saccharomyces cerevisiae by Jurrian Vanden Bussche; Eduardo V. Soares (pp. 679-687).

In the present work, the mode of cell death induced by Pb in Saccharomyces cerevisiae was studied. Yeast cells Pb-exposed, up to 6 h, loss progressively the capacity to proliferate and maintained the membrane integrity evaluated by the fluorescent probes bis(1,3-dibutylbarbituric acid trimethine oxonol) and propidium iodide. Pb-induced death is an active process, requiring the participation of cellular metabolism, since the simultaneous addition of cycloheximide attenuated the loss of cell proliferation capacity. Cells exposed to Pb accumulated intracelullarly reactive oxygen species (ROS), evaluated by 2′,7′-dichlorodihydrofluorescein diacetate. The addition of ascorbic acid (a ROS scavenger) strongly reduced the oxidative stress and impaired the loss of proliferation capacity in Pb-treated cells. Pb-exposed cells displayed nuclear morphological alterations, like chromatin fragmentation, as revealed by diaminophenylindole staining. Together, the data obtained indicate that yeast cells exposition to 1 mmol/l Pb results in severe oxidative stress which can be the trigger of programmed cell death by apoptosis.

Keywords: Apoptosis; Ascorbic acid; Lead; Heavy metals toxicity; Oxidative stress; ROS production

Biodegradation and metabolic pathway of β-chlorinated aliphatic acid in Bacillus sp. CGMCC no. 4196 by Chunjiao Lin; Lirong Yang; Gang Xu; Jianping Wu (pp. 689-696).

In this study, a bacterial Bacillus sp. CGMCC no. 4196 was isolated from mud. This strain exhibited the ability to degrade high concentration of 3-chloropropionate (3-CPA, 120 mM) or 3-chlorobutyrate (30 mM), but not chloroacetate or 2-chloropropionate (2-CPA). The growing cells, resting cells, and cell-free extracts from this bacterium had the capability of 3-CPA degradation. The results indicated that the optimum biocatalyst for 3-CPA biodegradation was the resting cells. The 3-CPA biodegradation pathway was further studied through the metabolites and critical enzymes analysis by HPLC, LC-MS, and colorimetric method. The results demonstrated that the metabolites of 3-CPA were 3-hydroxypropionic acid (3-HP) and malonic acid semialdehyde, and the critical enzymes were 3-CPA dehalogenase and 3-HP dehydroxygenase. Thus, the mechanism of the dehalogenase-catalyzed reaction was inferred as hydrolytic dehalogenation which was coenzyme A-independent and oxygen-independent. Finally, the pathway of β-chlorinated aliphatic acid biodegradation could be concluded as follows: the β-chlorinated acid is first hydrolytically dehalogenated to the β-hydroxyl aliphatic acid, and the hydroxyl aliphatic acid is oxidized to β-carbonyl aliphatic acid by β-hydroxy aliphatic acid dehydroxygenase. It is the first report that 3-HP was produced from 3-CPA by β-chlorinated aliphatic acid dehalogenase.

Keywords: Dehalogenase; β-chlorinated aliphatic acid; 3-Chloropropionate; 3-Chlorobutyrate; Bacillus sp.

The effect of trehalose on the fermentation performance of aged cells of Saccharomyces cerevisiae by Eduardo T. V. Trevisol; Anita D. Panek; Sergio Cantu Mannarino; Elis C. A. Eleutherio (pp. 697-704).

The fermentation process offers a wide variety of stressors for yeast, such as temperature, aging, and ethanol. To evaluate a possible beneficial effect of trehalose on ethanol production, we used mutant strains of Saccharomyces cerevisiae possessing different deficiencies in the metabolism of this disaccharide: in synthesis, tps1; in transport, agt1; and in degradation, ath1 and nth1. According to our results, the tps1 mutant, the only strain tested unable to synthesize trehalose, showed the lowest fermentation yield, indicating that this sugar is important to improve ethanol production. At the end of the first fermentation cycle, only the strains deficient in transport and degradation maintained a significant level of the initial trehalose. The agt1, ath1, and nth1 strains showed the highest survival rates and the highest proportions of non-petites. Accumulation of petites during fermentation has been correlated to low ethanol production. When recycled back for a subsequent fermentation, those mutant strains produced the highest ethanol yields, suggesting that trehalose is required for improving fermentative capacity and longevity of yeasts, as well as their ability to withstand stressful industrial conditions. Finally, according to our results, the mechanism by which trehalose improves ethanol production seems to involve mainly protection against protein oxidation.

Keywords: Trehalose; Saccharomyces cerevisiae ; Alcoholic fermentation; Ethanol stress; Oxidative stress

Menthol diminishes Staphylococcus aureus virulence-associated extracellular proteins expression by Jiazhang Qiu; Mingjing Luo; Jing Dong; Jianfeng Wang; Hongen Li; Xiaoliang Wang; Yanhong Deng; Haihua Feng; Xuming Deng (pp. 705-712).

Staphylococcus aureus is a significant human pathogen that is the major cause of a broad spectrum of illnesses, ranging from minor skin infections to life-threatening deep tissue infections and toxinosis. The ability of the organism to cause such a broad range of infections is, to a great extent, attributed to the secretion of a myriad of virulence-related extracellular proteins. Therefore, virulence as a target for antimicrobial chemotherapy has gained great interest. Menthol is a monocyclic terpene alcohol that occurs naturally in plants of the Mentha species lacking anti-S. aureus activity. In this paper, we demonstrate via hemolytic activity assays, tumor necrosis factor release assays, Western blot assays, and real-time reverse transcription-PCR assays that low concentrations of menthol can markedly inhibit the expression of α-hemolysin, enterotoxins A and B, and toxic shock syndrome toxin 1 in S. aureus. Our results indicate that menthol may be useful in managing S. aureus infections when used in combination with β-lactam antibiotics, which can often increase S. aureus toxin secretion when used at subinhibitory concentrations. In addition, the menthol basic structure has potential applications in the development of new anti-virulence drugs.

Keywords: Staphylococcus aureus ; Menthol; α-Hemolysin; Toxic shock syndrome toxin 1; Staphylococcal enterotoxins

Use of soybean vinasses as a germinant medium for a Geobacillus stearothermophilus ATCC 7953 sterilization biological indicator by Regina E. F. Dlugokenski; Sandra R. B. R. Sella; Belquis P. Guizelini; Luciana P. S. Vandenberghe; Adenise L. Woiciechowski; Carlos R. Soccol; João C. Minozzo (pp. 713-719).

A novel low-cost medium was developed from by-products and wastes from the ethanol agro-industry to replace commercial media in the production of a steam sterilization biological indicator (BI). Various recovery media were developed using soybean or sugarcane molasses and vinasse to prepare a self-contained BI. Media performance was evaluated by viability and heat resistance (D _121 °C value) according to regulatory standards. A medium produced with a soybean vinasse ratio of 1:70 (1.4%) (w/v) produced the results, with D _121 °C = 2.9 ± 0.5 min and Usk = 12.7 ± 2.1 min. The addition of 0.8% (w/v) yeast extract improved the germination of heat-damaged spores. The pH variation from 6.0 to 7.3 resulted in a gradual increase in the D _121 °C value. The absence of calcium chloride resulted in a decrease in germination, while no significant differences were observed with starch addition. Soybean vinasses may thus be used as the main component of a culture medium to substitute for commercial media in the production of self-contained biological indicators. The use of ethanol production waste in this biotechnological process realized a reliable performance, minimized the environmental impact, and decreased BI production costs while producing a high quality product.

Keywords: Biological indicator; Ethanol by-products; G. stearothermophilus ; Germination medium; Soybean vinasse

Construction of a reference plasmid molecule containing eight targets for the detection of genetically modified crops by Xiumin Wang; Da Teng; Yalin Yang; Fang Tian; Qingfeng Guan; Jianhua Wang (pp. 721-731).

A standard plasmid containing eight targets was developed for quantitative detection of genetically modified (GM) soybeans and cotton. These eight targets were joined in tandem to form the pTLE8 plasmid with a length of 3,680 bp. This plasmid contains part of the endogenous soybean Lec1 gene, the Cauliflower mosaic virus (CaMV) 35S promoter, the Agrobacterium tumefaciens nopaline synthase (NOS) terminator, the PAT gene of the soybean line A2704-12, the event-specific 5′-junction region of Roundup-Ready Soya (RRS, 35SG), the Cry1A(c) gene from Bacillus thuringiensis (Bt), the endogenous cotton Sad1 gene, and a part of RRS EPSPS gene. The PCR efficiencies with pTLE8 as a calibrator ranged from 99.4% to 100.2% for the standard curves of the RRS EPSPS gene and the taxon-specific Lec1 gene (R ² ≥ 0.996). The limits of detection and quantification were nine and 15 copies, respectively. The standard deviation (SD) and relative standard deviation (RSD) values of repeatability were from 0.09 to 0.52 and from 0.28% to 2.11%, and those for reproducibility were from 0.12 to 1.15 and 0.42% to 3.85%, respectively. The average conversion factor (Cf) for the CRMs RRS quantification was 0.91. The RSD of the mean values for known samples ranged from 3.09% to 18.53%, and the biases were from 0.5% to 40%. These results show that our method using the pTLE8 plasmid as a reference material (RM) is reliable and feasible in the identification of GM soybeans, thus paving the way for the establishment of identification management systems for various products containing GMO components.

Keywords: Calibrator; Construction; Genetically modified crops; Overlap PCR; Real-time PCR; Reference molecules

Surface-modified sulfur nanoparticles: an effective antifungal agent against Aspergillus niger and Fusarium oxysporum by Samrat Roy Choudhury; Mahua Ghosh; Amrita Mandal; Dipankar Chakravorty; Moumita Pal; Saheli Pradhan; Arunava Goswami (pp. 733-743).

Surface-modified sulfur nanoparticles (SNPs) of two different sizes were prepared via a modified liquid-phase precipitation method, using sodium polysulfide and ammonium polysulfide as starting material and polyethylene glycol-400 (PEG-400) as the surface stabilizing agent. Surface topology, size distribution, surface modification of SNPs with PEG-400, quantitative analysis for the presence of sulfur in nanoformulations, and thermal stability of SNPs were determined by atomic force microscopy (AFM), dynamic light scattering (DLS) plus high-resolution transmission electron microscopy (HR-TEM), fourier transform infrared (FT-IR) spectroscopy, energy dispersive X-ray (EDX) spectroscopy, and thermogravimetric analysis (TGA), respectively. A simultaneous study with micron-sized sulfur (S⁰) and SNPs was carried out to evaluate their fungicidal efficacy against Aspergillus niger and Fusarium oxysporum in terms of radial growth, sporulation, ultrastructural modifications, and phospholipid content of the fungal strains using a modified poisoned food technique, spore-germination slide bioassay, environmental scanning electron microscopy (ESEM), and spectrometry. SNPs expressed promising inhibitory effect on fungal growth and sporulation and also significantly reduced phospholipid content.

Keywords: Elemental sulfur; Sulfur nanoparticles; Antifungal; Aspergillus niger ; Fusarium oxysporum

Seasonal variability of bacteria in fine and coarse urban air particulate matter by Andrea Franzetti; Isabella Gandolfi; Eleonora Gaspari; Roberto Ambrosini; Giuseppina Bestetti (pp. 745-753).

The current knowledge about the microbial communities associated with airborne particulate matter, particularly in urban areas, is limited. This study aims to fill this gap by describing the microbial community associated with coarse (PM10) and fine (PM2.5) particulate matter using pyrosequencing. Particulate matter was sampled on Teflon filters over 3 months in summer and 3 months in winter in Milan (Italy), and the hypervariable V3 region of the gene 16S rRNA amplified from the DNA extracted from the filters. The results showed large seasonal variations in the microbial communities, with plant-associated bacteria dominating in summer and spore-forming bacteria in winter. Bacterial communities from PM10 and PM2.5 were also found to differ from each other by season. In all samples, a high species richness, comparable with that of soils, but a low evenness was found. The results suggest that not only can the sources of the particulate influence the presence of specific bacterial groups but also that environmental factors and stresses can shape the bacterial community.

Keywords: Particulate matter; Airborne bacteria

Biodegradation of fenvalerate and 3-phenoxybenzoic acid by a novel Stenotrophomonas sp. strain ZS-S-01 and its use in bioremediation of contaminated soils by Shaohua Chen; Liu Yang; Meiying Hu; Jingjing Liu (pp. 755-767).

A bacterial strain ZS-S-01, newly isolated from activated sludge, could effectively degrade fenvalerate and its hydrolysis product 3-phenoxybenzoic acid (3-PBA). Based on the morphology, physiological biochemical characteristics, and 16 S rDNA sequence, strain ZS-S-01 was identified as Stenotrophomonas sp. Strain ZS-S-01 could also degrade and utilize deltamethrin, beta-cypermethrin, beta-cyfluthrin, and cyhalothrin as substrates for growth. Strain ZS-S-01 was capable of degrading fenvalerate rapidly without a lag phase over a wide range of pH and temperature, even in the presence of other carbon sources, and metabolized it to yield 3-PBA, then completely degraded it. No persistent accumulative product was detected by HPLC and GC/MS analysis. Studies on biodegradation in various soils showed that strain ZS-S-01 demonstrated efficient degradation of fenvalerate and 3-PBA (both 50 mg·kg⁻¹) with a rate constant of 0.1418–0.3073 d⁻¹, and half-lives ranged from 2.3 to 4.9 days. Compared with the controls, the half-lives for fenvalerate and 3-PBA reduced by 16.9–156.3 days. These results highlight strain ZS-S-01 may have potential for use in bioremediation of pyrethroid-contaminated environment.

Keywords: Biodegradation; Fenvalerate; 3-Phenoxybenzoic acid; Stenotrophomonas sp. strain ZS-S-01; Bioremediation; Kinetics

Functional genes based analysis of sulfur-oxidizing bacteria community in sulfide removing bioreactor by Jian-Fei Luo; Wei-Tie Lin; Yong Guo (pp. 769-778).

Sulfur-oxidizing bacteria (SOB) are the main microorganisms that participate in the bioremediation of sulfide-rich wastewater. To reveal the SOB community structure and determine which members of SOB contribute to the sulfide oxidation in a sulfide-rich cloth printing and dyeing wastewater treatment plant, specific primer pairs dsrA 625F/877R, soxB 704F/1199R, and sqr 473F/982R based on the SOB functional genes encoding dissimilatory sulfite reductase, sulfate thioesterase/thiohydrolase, and sulfide: quinone oxidoreductase were designed. The restriction fragment length polymorphism analysis showed that the diversity indices and the abundance of each OTU have no significant changes after time, which suggested the SOB community in the sulfide removing bioreactor have high steady phylogenetic analysis of functional gene-based clone libraries detected the SOB from Chlorobia, α-proteobacteria, β-proteobacteria, and γ-proteobacteria. The combined clone library showed the presence of dominant members of the SOB species closely related to families Halothiobacillaceae (17%), Hydrogenophilaceae (14%), and Rhodocyclaceae (13%), which may contribute to the sulfide oxidation in wastewater treatment process. This work provides a precise understanding of SOB microbial community within sulfide removing bioreactor, and the result gives assistance for the optimization of the treatment systems for sulfide biological degradation.

Keywords: Sulfur-oxidizing bacteria (SOB); Microbial community; Functional gene; Clone library; Sulfide-rich wastewater

Quantitative analyses of ammonia-oxidizing Archaea and bacteria in the sediments of four nitrogen-rich wetlands in China by Shanyun Wang; Yu Wang; Xiaojuan Feng; Liming Zhai; Guibing Zhu (pp. 779-787).

With the rapid development of ammonia-synthesizing industry, the ammonia-nitrogen pollution in wetlands acting as the sink of point and diffuse pollution has been increased dramatically. Most of ammonia-nitrogen is oxidized at least once by ammonia-oxidizing prokaryotes to complete the nitrogen cycle. Current research findings have expanded the known ammonia-oxidizing prokaryotes from the domain Bacteria to Archaea. However, in the complex wetlands environment, it remains unclear whether ammonia oxidation is exclusively or predominantly linked to Archaea or Bacteria as implied by specific high abundance. In this research, the abundance and composition of Archaea and Bacteria in sediments of four kinds of wetlands with different nitrogen concentration were investigated by using quantitative real-time polymerase chain reaction, cloning, and sequencing approaches based on amoA genes. The results indicated that AOA distributed widely in wetland sediments, and the phylogenetic tree revealed that archaeal amoA functional gene sequences from wetlands sediments cluster as two major evolutionary branches: soil/sediment and sediment/water. The bacteria functionally dominated microbial ammonia oxidation in different wetlands sediments on the basis of molecule analysis, potential nitrification rate, and soil chemistry. Moreover, the factors influencing AOA and AOB abundances with environmental indicator were also analyzed, and the results addressed the copy numbers of archaeal and bacterial amoA functional gene having the higher correlation with pH and ammonia concentration. The pH had relatively great negative impact on the abundance of AOA and AOB, while ammonia concentration showed positive impact on AOB abundance only. These findings could be fundamental to improve understanding of the importance of AOB and AOA in nitrogen and other nutrients cycle in wetland ecosystems.

Keywords: Wetland; Sediment; Abundance; Biodiversity; amoA functional gene

Evaluation of hydrolysis and fermentation rates in microbial fuel cells by Sharon B. Velasquez-Orta; Eileen Yu; Krishna P. Katuri; Ian M. Head; Tom P. Curtis; Keith Scott (pp. 789-798).

This study determined the influence of substrate degradation on power generation in microbial fuel cells (MFCs) and microbial community selection on the anode. Air cathode MFCs were fed synthetic medium containing different substrates (acetate, glucose and starch) using primary clarifier sewage as source of electroactive bacteria. The complexity of the substrate affected the MFC performance both for power generation and COD removal. Power output decreased with an increase in substrate complexity from 99 ± 2 mW m⁻² for acetate to 4 ± 2 mW m⁻² for starch. The organic matter removal and coulombic efficiency (CE) of MFCs with acetate and glucose (82% of COD removal and 26% CE) were greater than MFCs using starch (60% of COD removal and 19% of CE). The combined hydrolysis–fermentation rate obtained (0.0024 h⁻¹) was considerably lower than the fermentation rate (0.018 h⁻¹), indicating that hydrolysis of complex compounds limits current output over fermentation. Statistical analysis of microbial community fingerprints, developed on the anode, showed that microbial communities were enriched according to the type of substrate used. Microbial communities producing high power outputs (fed acetate) clustered separately from bacterial communities producing low power outputs (fed complex compounds).

Keywords: Microbial fuel cell; Fermentation; Hydrolysis; Organic substrates

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Applied Microbiology and Biotechnology (v.90, #2)

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Applied Microbiology and Biotechnology (v.90, #2)