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Applied Microbiology and Biotechnology (v.97, #8)

The biotechnological use and potential of plant pathogenic smut fungi by Michael Feldbrügge; Ronny Kellner; Kerstin Schipper (pp. 3253-3265).

Plant pathogens of the family Ustilaginaceae parasitise mainly on grasses and cause smut disease. Among the best characterised members of this family are the covered smut fungus Ustilago hordei colonising barley and oat as well as the head smut Sporisorium reilianum and the corn smut Ustilago maydis, both infecting maize. Over the past years, U. maydis in particular has matured into a model system for diverse topics like plant–pathogen interaction, cellular transport processes or DNA repair. Consequently, a broad set of genetic, molecular and system biological methods has been established. This set currently serves as a strong foundation to improve existing and establish novel biotechnological applications. Here, we review four promising aspects covering different fields of applied science: (1) synthesis of secondary metabolites produced at fermenter level. (2) Lipases and other hydrolytic enzymes with potential roles in biocatalytic processes. (3) Degradation of ligno-cellulosic plant materials for biomass conversion. (4) Protein expression based on unconventional secretion, a novel approach inspired by basic research on mRNA transport. Thus, plant pathogenic Ustilaginaceae offer a great potential for future biotechnological applications by combining basic research and applied science.

Keywords: Smut fungi; Secondary metabolite; Bioconversion; Biofuel; Protein expression

Echinocandins: production and applications by Tamás Emri; László Majoros; Viktória Tóth; István Pócsi (pp. 3267-3284).

The first echinocandin-type antimycotic (echinocandin B) was discovered in the 1970s. It was followed by the isolation of more than 20 natural echinocandins. These cyclic lipo-hexapeptides are biosynthesized on non-ribosomal peptide synthase complexes by different ascomycota fungi. They have a unique mechanism of action; as non-competitive inhibitors of β-1,3-glucan synthase complex they target the fungal cell wall. Results of the structure–activity relationship experiments let us develop semisynthetic derivatives with improved properties. Three cyclic lipohiexapeptides (caspofungin, micafungin and anidulafungin) are currently approved for use in clinics. As they show good fungicidal (Candida spp.) or fungistatic (Aspergillus spp.) activity against the most important human pathogenic fungi including azole-resistant strains, they are an important addition to the antifungal armamentarium. Some evidence of acquired resistance against echinocandins has been detected among Candida glabrata strains in recent years, which enhanced the importance of data collected on the mechanism of acquired resistance developing against the echinocandins. In this review, we show the structural diversity of natural echinocandins, and we summarize the emerging data on their mode of action, biosynthesis and industrial production. Their clinical significance as well as the mechanism of natural and acquired resistance is also discussed.

Keywords: Natural echinocandins; Semisynthetic echinocandins; Non-ribosomal peptide synthase; β-1,3-Glucan synthase; Echinocandin resistance; Paradoxical growth

Biodegradability and biodegradation pathways of endosulfan and endosulfan sulfate by Ryota Kataoka; Kazuhiro Takagi (pp. 3285-3292).

Endosulfan and endosulfan sulfate are persistent organic pollutants that cause serious environmental problems. Although these compounds are already prohibited in many countries, residues can be detected in soils with a history of endosulfan application. Endosulfan is transformed in the environment into endosulfan sulfate, which is a toxic and persistent metabolite. However, some microorganisms can degrade endosulfan without producing endosulfan sulfate, and some can degrade endosulfan sulfate. Therefore, biodegradation has the potential to clean up soil contaminated with endosulfan. In this review, we provide an overview of aerobic endosulfan degradation by bacteria and fungi, and a summary of recent advances and prospects in this research field.

Keywords: Bioavailability; Biodegradation; Endosulfan sulfate; Toxicity; Metabolites

Enzyme-based formulations for decontamination: current state and perspectives by Navdeep Grover; Cerasela Zoica Dinu; Ravi S. Kane; Jonathan S. Dordick (pp. 3293-3300).

Development of noncorrosive, cost-effective, environmentally benign, and broad-spectrum antimicrobial formulations is necessary for clinical, industrial, and domestic purposes. Many current decontaminating formulations are effective, but they require the use of strong oxidizing agents or organic solvents that have deleterious effects on human health and the surrounding environment. The emergence of antibiotic-resistant pathogens has motivated researchers to develop enzyme-based self-decontaminating formulations as alternatives to such chemical decontamination approaches. Hydrolytic and oxidative enzymes can be used to deactivate pathogens, including bacteria, spores, viruses, and fungi. Laccases, haloperoxidases, and perhydrolases catalyze the generation of biocidal oxidants, such as iodine, bromine, hypohalous acid (e.g., HOCl or HOBr), and peracetic acid. These oxidants have broad-spectrum antimicrobial activity. Due to the multi-pathway action of these oxidants, it has proven extremely difficult for microbes to gain resistance. Thus far, few examples have been reported on enzyme-based antimicrobial formulations. For these reasons, various enzyme-containing antimicrobial formulations are highlighted in this review.

Keywords: Decontamination; Antimicrobial; Laccase; Haloperoxidase; Perhydrolase; Nanocomposites

Polyhydroxyalkanoic acids from structurally-unrelated carbon sources in Escherichia coli by Qian Wang; Qianqian Zhuang; Quanfeng Liang; Qingsheng Qi (pp. 3301-3307).

Polyhydroxyalkanoates (PHAs) that contain varied monomers with different chain lengths/structures were normally synthesized when a structurally-related precursor was present. The biosynthesis of PHAs from unrelated carbon sources in microorganisms including Escherichia coli met many challenges in the past. Recently, with the development of metabolic engineering and synthetic biology, the production of PHAs from unrelated carbon sources obtained a breakthrough. Polyesters containing 2-hydroxypropionate, 3-hydroxypropionate, 4-hydroxybutyrate, 3-hydroxyvalarate, and medium-chain-length 3-hydroxyalkanoate monomers can all be synthesized in E. coli by integrating exogenous or endogenous pathways and/or genes. This review will summarize the progresses in this area. In addition, the strategies that lead to the production of PHAs with varied monomers and high polymer content in the cell are discussed.

Keywords: Polyhydroxyalkanoates; Unrelated carbon source; Escherichia coli ; PHA; Metabolic engineering

Recent advances in the metabolic engineering of microorganisms for the production of 3-hydroxypropionic acid as C3 platform chemical by Kris Niño G. Valdehuesa; Huaiwei Liu; Grace M. Nisola; Wook-Jin Chung; Seung Hwan Lee; Si Jae Park (pp. 3309-3321).

Development of sustainable technologies for the production of 3-hydroxypropionic acid (3HP) as a platform chemical has recently been gaining much attention owing to its versatility in applications for the synthesis of other specialty chemicals. Several proposed biological synthesis routes and strategies for producing 3HP from glucose and glycerol are reviewed presently. Ten proposed routes for 3HP production from glucose are described and one of which was recently constructed successfully in Escherichia coli with malonyl–Coenzyme A as a precursor. This resulted in a yield still far from the required level for industrial application. On the other hand, strategies employing engineered E. coli and Klebsiella pneumoniae capable of producing 3HP from glycerol are also evaluated. The titers produced by these recombinant strains reached around 3 %. At its current state, it is evident that a bulk of engineering works is yet to be done to acquire a biosynthesis route for 3HP that is acceptable for industrial-scale production.

Keywords: 3-Hydroxypropionic acid; Metabolic engineering; Escherichia coli ; Klebsiella pneumoniae ; Glycerol; Glucose

Production of long-chain hydroxy fatty acids by microbial conversion by Yujin Cao; Xiao Zhang (pp. 3323-3331).

Hydroxy fatty acids (HFAs) are very important chemicals for versatile applications in biodegradable polymer materials and cosmetic and pharmaceutical industries. They are difficult to be synthesized via chemical routes due to the inertness of the fatty acyl chain. In contrast, these fatty acids make up a major class of natural products widespread among bacteria, yeasts, and fungi. A number of microorganisms capable of producing HFAs from fatty acids or vegetable oils have been reported. Therefore, HFAs could be produced by biotechnological strategies, especially by microbial conversion processes. Microorganisms could oxidize fatty acids either at the terminal carbon or inside the acyl chain to produce various HFAs, including α-HFAs, β-HFAs, mid-position HFAs, ω-HFAs, di-HFAs, and tri-HFAs. The enzymes and their encoded genes responsible for the hydroxylation of the carbon chain have been identified and characterized during the past few years. The involved microbes and catalytic mechanisms for the production of different types of HFAs are systematically demonstrated in this review. It provides a better view of HFA biosynthesis and lays the foundation for further industrial production.

Keywords: Hydroxy fatty acids; Microbial transformation; Catalytic mechanism; Hydroxylase; Monooxygenase

Engineering E. coli for caffeic acid biosynthesis from renewable sugars by Haoran Zhang; Gregory Stephanopoulos (pp. 3333-3341).

Caffeic acid is a valuable aromatic compound that possesses many important pharmacological activities. In structure, caffeic acid belongs to the hydroxycinnamic acid family and can be biosynthesized from the aromatic amino acid tyrosine. In the present paper, the caffeic acid biosynthesis pathway was reconstituted in engineered Escherichia coli to produce caffeic acid from simple biomass sugar glucose and xylose. Different engineering approaches were utilized to optimize the production. Specifically, two parallel biosynthesis routes leading from tyrosine to caffeic acid were studied. The copy number of the intermediate biosynthesis genes was varied to find appropriate gene doses for caffeic acid biosynthesis. Three different media, including a MOPS medium, a synthetic medium, and a rich medium, were also examined to improve the production. The highest specific caffeic acid production achieved was 38 mg/L/OD. Lastly, cultivation of engineered E. coli in a bioreactor resulted in a production of 106 mg/L caffeic acid after 4 days.

Keywords: Escherichia coli ; Caffeic acid; Tyrosine; Heterologous biosynthesis

High yield production of extracellular recombinant levansucrase by Bacillus megaterium by Claudia Korneli; Rebekka Biedendieck; Florian David; Dieter Jahn; Christoph Wittmann (pp. 3343-3353).

In this study, a high yield production bioprocess with recombinant Bacillus megaterium for the production of the extracellular enzyme levansucrase (SacB) was developed. For basic optimization of culture parameters and nutrients, a recombinant B. megaterium reporter strain that produced green fluorescent protein under control of a vector-based xylose-inducible promoter was used. It enabled efficient microtiter plate-based screening via fluorescence analysis. A pH value of pH 6, 20 % of dissolved oxygen, 37 °C, and elevated levels of biotin (100 μg L⁻¹) were found optimal with regard to high protein yield and reduced overflow metabolism. Among the different compounds tested, fructose and glycerol were identified as the preferred source of carbon. Subsequently, the settings were transferred to a B. megaterium strain recombinantly producing levansucrase SacB based on the plasmid-located xylose-inducible expression system. In shake flask culture under the optimized conditions, the novel strain already secreted the target enzyme in high amounts (14 U mL⁻¹ on fructose and 17.2 U mL⁻¹ on glycerol). This was further increased in high cell density fed-batch processes up to 55 U mL⁻¹, reflecting a levansucrase concentration of 0.52 g L⁻¹. This is 100-fold more than previous efforts for this enzyme in B. megaterium and more than 10-fold higher than reported values of other extracellular protein produced in this microorganism so far. The recombinant strain could also handle raw glycerol from biodiesel industry which provided the same amount and quality of the recombinant protein and suggests future implementation into existing biorefinery concepts.

Keywords: Fed-batch; Recombinant protein secretion; Glycosyltransferase; High cell density; Glycerol; Biorefinery

Compensation of the enantioselectivity-activity trade-off in the directed evolution of an esterase from Rhodobacter sphaeroides by site-directed saturation mutagenesis by Fei Guo; Haoming Xu; Hongna Xu; Hongwei Yu (pp. 3355-3362).

Despite directed evolution being a practical and efficient method of improving the properties of enzymes, a trade-off between the targeted property and other essential properties often exists which hinders the efficiency of directed evolution. In our previous work, mutant CVH of an esterase from Rhodobacter sphaeroides with high enantioselectivity was obtained by directed evolution, unfortunately its activity cannot catch another mutant YH. To compensate the trade-off of mutant CVH, site-directed saturation mutagenesis was conducted on four residues, three (Asn62, Met121, and Leu145) were hot spots determined from directed evolution, and one (Tyr27) was introduced to make up the large distance between a mutation (Asn62) and the substrate. A new mutant (HMVY) with high enantioselectivity and comparable activity to YH was obtained. According to the kinetic analysis and molecular dynamics simulations, it was understood that the high enantioselectivity and poor activity of mutant CVH was caused by different decrement of efficiency constants to two isomers, (R)-, (S)-methyl mandelate, and the high enantioselectivity and activity of mutant HWVY was caused by improved activity towards the preferred substrate ((S)-methyl mandelate), which provided the interpretation of the trade-off compensation. This work could provide a way to compensate the trade-off of enantioselectivity and activity in the process of enzyme evolution.

Keywords: Enantioselectivity-activity trade-off; Directed evolution; Molecular dynamics simulation; Site-directed saturation mutagenesis; Kinetic resolution

Morphological changes induced by class III chitin synthase gene silencing could enhance penicillin production of Penicillium chrysogenum by Hui Liu; Zhiming Zheng; Peng Wang; Guohong Gong; Li Wang; Genhai Zhao (pp. 3363-3372).

Chitin synthases catalyze the formation of β-(1,4)-glycosidic bonds between N-acetylglucosamine residues to form the unbranched polysaccharide chitin, which is the major component of cell walls in most filamentous fungi. Several studies have shown that chitin synthases are structurally and functionally divergent and play crucial roles in the growth and morphogenesis of the genus Aspergillus although little research on this topic has been done in Penicillium chrysogenum. We used BLAST to find the genes encoding chitin synthases in P. chrysogenum related to chitin synthase genes in Aspergillus nidulans. Three homologous sequences coding for a class III chitin synthase CHS4 and two hypothetical proteins in P. chrysogenum were found. The gene which product showed the highest identity and encoded the class III chitin synthase CHS4 was studied in detail. To investigate the role of CHS4 in P. chrysogenum morphogenesis, we developed an RNA interference system to silence the class III chitin synthase gene chs4. After transformation, mutants exhibited a slow growth rate and shorter and more branched hyphae, which were distinct from those of the original strain. The results also showed that the conidiation efficiency of all transformants was reduced sharply and indicated that chs4 is essential in conidia development. The morphologies of all transformants and the original strain in penicillin production were investigated by light microscopy, which showed that changes in chs4 expression led to a completely different morphology during fermentation and eventually caused distinct penicillin yields, especially in the transformants PcRNAi1-17 and PcRNAi2-1 where penicillin production rose by 27 % and 41 %, respectively.

Keywords: Chitin synthase; Morphology; Penicillin production; RNA interference

Adaptation of the Spodoptera exigua Se301 insect cell line to grow in serum-free suspended culture. Comparison of SeMNPV productivity in serum-free and serum-containing media by A. Beas-Catena; A. Sánchez-Mirón; F. García-Camacho; A. Contreras-Gómez; E. Molina-Grima (pp. 3373-3381).

Spodoptera exigua Se301 cells have been successfully adapted to two different commercial serum-free media (SFM; Ex-Cell 420 and Serum-Free Insect Medium-1) by gradually reducing the 10 %-added serum-containing medium content from 100 % to 0 % (v/v) in suspended cultures. Both direct adaptation to a serum-free medium and cell growth in the absence of protective additives against fluid dynamic stress [polyvinyl pyrrolidone and polyvinyl alcohol] and disaggregation [dextran sulfate] proved impossible. Cells grew reproducibly in both SFMs once the serum had been completely removed, although the use of Ex-Cell 420 resulted in higher growth rates and cell densities. Turbulence was sufficiently high to reduce growth rates and final cell densities at the highest Reynolds number investigated, although no clear influence of agitation was observed on virus productivity. Both attached and suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Cells adapted to different conditions (attached or suspended culture, serum-containing or serum-free medium) showed different occlusion bodies productivities at the two multiplicities of infection assayed (0.1 and 0.5).

Keywords: Spodoptera exigua ; Baculovirus; Serum-free medium; Suspension culture; Cell damage; Shear stress

Optimization of the pilot-scale production of an ice-binding protein by fed-batch culture of Pichia pastoris by Jun Hyuck Lee; Sung Gu Lee; Hackwon Do; Jong Chan Park; Eunjung Kim; Yong-Hoe Choe; Se Jong Han; Hak Jun Kim (pp. 3383-3393).

Ice-binding proteins (IBPs) can bind to the ice crystal and inhibit its growth. Because this property of IBPs can increase the freeze–thaw survival of cells, IBPs have attracted the attention from industries for their potential use in biotechnological applications. However, their use was largely hampered by the lack of the large-scale recombinant production system. In this study, the codon-optimized IBP from Leucosporidium sp. (LeIBP) was constructed and subjected to high-level expression in methylotrophic Pichia pastoris system. In a laboratory-scale fermentation (7 L), the optimal induction temperature and pH were determined to be 25 °C and 6.0, respectively. Further, employing glycerol fed-batch phase prior to methanol induction phase enhanced the production of recombinant LelBP (rLeIBP) by ∼100 mg/l. The total amount of secreted proteins at these conditions (25 °C, pH 6.0, and glycerol fed-batch phase) was ∼443 mg/l, 60 % of which was rLeIBP, yielding ∼272 mg/l. In the pilot-scale fermentation (700 L) under the same conditions, the yield of rLeIBP was 300 mg/l. To our best knowledge, this result reports the highest production yield of the recombinant IBP. More importantly, the rLeIBP secreted into culture media was stable and active for 6 days of fermentation. The thermal hysteresis (TH) activity of rLeIBP was about 0.42 °C, which is almost the same to those reported previously. The availability of large quantities of rLeIBP may accelerate further application studies.

Keywords: Ice-binding protein; Fed-batch culture; LeIBP; Leucosporidium sp.; Pichia pastoris

Human beta-defensin DEFB126 is capable of inhibiting LPS-mediated inflammation by Haiyan Liu; Heguo Yu; Yihua Gu; Aijie Xin; Yonglian Zhang; Hua Diao; Donghai Lin (pp. 3395-3408).

β-Defensins are cationic, antimicrobial peptides that participate in antimicrobial defense as well as the regulation of innate and adaptive immunity. Human β-defensin 126 (DEFB126) is a multifunctional glycoprotein consisting of a conserved β-defensin core and a unique long glycosylated peptide tail. The long glycosylated peptide tail has been proven to be critical for efficient transport of sperm in the female reproductive tract, preventing their immune recognition, and efficient delivery of capacitated sperm to the site of fertilization. However, the functions of the conserved β-defensin core remain to be fully elucidated. In the present work, the conserved β-defensin core of the DEFB126 was expressed to explore its potential antimicrobial and anti-inflammatory activities. The DEFB126 core peptide exhibited both high potency for binding and neutralizing lipopolysaccharide (LPS) in vitro, and potent anti-inflammatory ability by down-regulating the mRNA expression of pro-inflammatory cytokines including IL-α, IL-1β, IL-6 and TNF-α in a murine macrophage cell line RAW264.7. The treatment with the DEFB126 core peptide also led to correspondingly decreased secretion of IL-6 and TNF-α. The blockade of LPS-induced p42/44 and p38 MAPK signal pathway might contribute to the anti-inflammation effects of the DEFB126 core peptide. Furthermore, fluorescence-labeled DEFB126 could enter RAW 264.7 cells and reduce the production of LPS-stimulated inflammatory factors, implying that DEFB126 might also participate in intracellular regulation beyond its direct LPS neutralization. In summary, our results demonstrate that the DEFB 126 core peptide has critical functions in parallel to its C-terminal tail by showing LPS-binding activity, anti-inflammatory effects and intracellular regulatory function.

Keywords: Defensin 126; LPS; Anti-inflammatory; Cytokines; RAW264.7

Biosynthesis of ethylene glycol in Escherichia coli by Huaiwei Liu; Kristine Rose M. Ramos; Kris Niño G. Valdehuesa; Grace M. Nisola; Won-Keun Lee; Wook-Jin Chung (pp. 3409-3417).

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose → d-xylonate → 2-dehydro-3-deoxy-d-pentonate → glycoaldehyde → EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose → d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g L⁻¹ of EG from 40.0 g L⁻¹ of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde → glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.

Keywords: Biosynthesis; d-Xylose; Escherichia coli ; Ethylene glycol

Comparative analysis of the catalytic components in the archaeal dye-linked l-proline dehydrogenase complexes by Ryushi Kawakami; Chiaki Noguchi; Marie Higashi; Haruhiko Sakuraba; Toshihisa Ohshima (pp. 3419-3427).

Two types of hetero-oligomeric dye-linked l-proline dehydrogenases (α₄β₄ and αβγδ types) are expressed in the hyperthermophilic archaea belonging to Thermococcales. In both enzymes, the β subunit (PDHβ) is responsible for catalyzing l-proline dehydrogenation. The genes encoding the two enzyme types form respective clusters that are completely conserved among Pyrococcus and Thermococcus strains. To compare the enzymatic properties of PDHβs from α₄β₄- and αβγδ-type enzyme complexes, eight PDHβs (four of each type) from Pyrococcus furiosus DSM3638, Pyrococcus horikoshii OT-3, Thermococcus kodakaraensis KOD1 JCM12380 and Thermococcus profundus DSM9503 were expressed in Escherichia coli cells and purified to homogeneity using one-step Ni-chelating chromatography. The α₄β₄-type PDHβs showed greater thermostability than most of the αβγδ-type PDHβs: the former retained more than 80 % of their activity after heating at 70 °C for 20 min, while the latter showed different thermostabilities under the same conditions. In addition, the α₄β₄-type PDHβs utilized ferricyanide as the most preferable electron acceptor, whereas αβγδ-type PDHβs preferred 2, 6-dichloroindophenol, with one exception. These results indicate that the differences in the enzymatic properties of the PDHβs likely reflect whether they were from an αβγδ- or α₄β₄-type complex, though the wider divergence observed within αβγδ-type PDHβs based on the phylogenetic analysis may also be responsible for their inconsistent enzymatic properties. By contrast, differences in the kinetic parameters among the PDHβs did not reflect the complex type. Interestingly, the k _cat value for free α₄β₄-type PDHβ from P. horikoshii was much larger than the value for the same subunit within the α₄β₄-complex. This indicates that the isolated PDHβ could be a useful element for an electrochemical system for detection of l-proline.

Keywords: Dye-linked l-proline dehydrogenase; Dehydrogenase complex; Hyperthermophilic archaea; Thermostability; Kinetics

Molecular cloning, overexpression, and characterization of autophosphorylation in calcium-dependent protein kinase 1 (CDPK1) from Cicer arietinum by Ajay Kumar Dixit; Jayabaskaran Chelliah (pp. 3429-3439).

In plants, calcium-dependent protein kinases (CDPKs) are key intermediates in calcium-mediated signaling that couple changes in Ca²⁺ levels to a specific response. In the present study, we report the high-level soluble expression of calcium-dependent protein kinase1 from Cicer arietinum (CaCDPK1) in Escherichia coli. The expression of soluble CaCDPK1 was temperature dependent with a yield of 3–4 mg/l of bacterial culture. CaCDPK1 expressed as histidine-tag fusion protein was purified using Ni–NTA affinity chromatography till homogeneity. The recombinant CaCDPK1 protein exhibited both calcium-dependent autophosphorylation and substrate phosphorylation activities with a V _max and K _m value of 13.2 nmol/min/mg and 34.3 μM, respectively, for histone III-S as substrate. Maximum autophosphorylation was seen only in the presence of calcium. Optimum temperature for autophosphorylation was found to be 37 °C. The recombinant protein showed optimum pH range of 6–9. The role of autophosphorylation in substrate phosphorylation was investigated using histone III-S as exogenous substrate. Our results show that autophosphorylation happens before substrate phosphorylation and it happens via intra-molecular mechanism as the activity linearly depends on enzyme concentrations. Autophosphorylation enhances the kinase activity and reduces the lag phase of activation, and CaCDPK1 can utilize both ATP and GTP as phosphodonor but ATP is preferred than GTP.

Keywords: Calcium; Calcium-dependent protein kinases; Escherichia coli ; Expression; Purification; Ni–NTA affinity; Autophosphorylation; Kinases

Engineering of class I lactate-polymerizing polyhydroxyalkanoate synthases from Ralstonia eutropha that synthesize lactate-based polyester with a block nature by Anna Ochi; Ken’ichiro Matsumoto; Takashi Ooba; Kohei Sakai; Takeharu Tsuge; Seiichi Taguchi (pp. 3441-3447).

Class I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (PhaC_Re) was engineered so as to acquire an unusual lactate (LA)-polymerizing activity. To achieve this, the site-directed saturation mutagenesis of PhaC_Re was conducted at position 510, which corresponds to position 481 in the initially discovered class II LA-polymerizing PHA synthase (PhaC1_PsSTQK), a mutation in which (Gln481Lys) was shown to be essential to its LA-polymerizing activity (Taguchi et al., Proc Natl Acad Sci USA 105(45):17323–17327, 2008). The LA-polymerizing activity of the PhaC_ReA510X mutants was evaluated based on the incorporation of LA units into the P[3-hydroxybutyrate(3HB)] backbone in vivo using recombinant Escherichia coli LS5218. Among 19 PhaC_Re(A510X) mutants, 15 synthesized P (LA-co-3HB), indicating that the 510 residue plays a critical role in LA polymerization. The polymer synthesized by PhaC_ReA510S was fractionated using gel permeation chromatography in order to remove the low molecular weight fractions. The ¹³C and ¹H NMR analyses of the high molecular weight fraction revealed that the polymer was a P(7 mol% LA-co-3HB) copolymer with a weight-averaged molecular weight of 3.2 × 10⁵ Da. Interestingly, the polymer contained an unexpectedly high ratio of an LA-LA*-LA triad sequence, suggesting that the polymer synthesized by PhaC_Re mutant may not be a random copolymer, but presumably had a block sequence.

Keywords: Lactate-polymerizing enzyme; Site-directed mutagenesis; Polyhydroxybutyrate polymerase; Block copolymer; Cupriavidus necator

Staphylococcal Phage 2638A endolysin is lytic for Staphylococcus aureus and harbors an inter-lytic-domain secondary translational start site by Igor Abaev; Juli Foster-Frey; Olga Korobova; Nina Shishkova; Natalia Kiseleva; Pavel Kopylov; Sergey Pryamchuk; Mathias Schmelcher; Stephen C. Becker; David M. Donovan (pp. 3449-3456).

Staphylococcus aureus is a notorious pathogen highly successful at developing resistance to virtually all antibiotics to which it is exposed. Staphylococcal phage 2638A endolysin is a peptidoglycan hydrolase that is lytic for S. aureus when exposed externally, making it a new candidate antimicrobial. It shares a common protein organization with more than 40 other reported staphylococcal peptidoglycan hydrolases. There is an N-terminal M23 peptidase domain, a mid-protein amidase 2 domain (N-acetylmuramoyl-L-alanine amidase), and a C-terminal SH3b cell wall-binding domain. It is the first phage endolysin reported with a secondary translational start site in the inter-lytic-domain region between the peptidase and amidase domains. Deletion analysis indicates that the amidase domain confers most of the lytic activity and requires the full SH3b domain for maximal activity. Although it is common for one domain to demonstrate a dominant activity over the other, the 2638A endolysin is the first in this class of proteins to have a high-activity amidase domain (dominant over the N-terminal peptidase domain). The high activity amidase domain is an important finding in the quest for high-activity staphylolytic domains targeting novel peptidoglycan bonds.

Keywords: Bacteriophage; Endolysin; M23 peptidase domain; SH3b cell wall-binding domain; Staphylococcus aureus

Effects of membrane-bound glucose dehydrogenase overproduction on the respiratory chain of Gluconobacter oxydans by Maria Meyer; Paul Schweiger; Uwe Deppenmeier (pp. 3457-3466).

The acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources as a natural part of its metabolism, and this feature has been exploited for many biotechnological applications. The most important enzymes used to harness the biocatalytic oxidative capacity of G. oxydans are the pyrroloquinoline quinone (PQQ)-dependent dehydrogenases. The membrane-bound PQQ-dependent glucose dehydrogenase (mGDH), encoded by gox0265, was used as model protein for homologous membrane protein production using the previously described Gluconobacter expression vector pBBR1p452. The mgdh gene had ninefold higher expression in the overproduction strain compared to the parental strain. Furthermore, membranes from the overexpression strain had a five- and threefold increase of mGDH activity and oxygen consumption rates, respectively. Oxygen consumption rate of the membrane fraction could not be increased by the addition of a substrate combination of glucose and ethanol in the overproduction strain, indicating that the terminal quinol oxidases of the respiratory chain were rate limiting. In contrast, addition of glucose and ethanol to membranes of the control strain increased oxygen consumption rates approaching the observed rates with G. oxydans overproducing mGDH. The higher glucose oxidation rates of the mGDH overproduction strain corresponded to a 70 % increase of the gluconate production rate compared to the control strain. The high rate of glucose oxidation may be useful in the industrial production of gluconates and ketogluconates, or as whole-cell biosensors. Furthermore, mGDH was purified to homogeneity by one-step strep-tactin affinity chromatography and characterized. To our knowledge, this is the first report of a membrane integral quinoprotein being purified by affinity chromatography and serves as a proof-of-principle for using G. oxydans as a host for membrane protein expression and purification.

Keywords: Acetic acid bacteria; Glucose dehydrogenase; Membrane protein; Biotransformation; Overexpression; Protein purification; Gluconate

Enhancing the cyclodextrin production by synchronous utilization of isoamylase and α-CGTase by Xuguo Duan; Sheng Chen; Jian Chen; Jing Wu (pp. 3467-3474).

Cyclodextrins (CD) are cyclic α-1,4-glucans composed of glucose units, and they have multiple applications in food, pharmaceuticals, cosmetics, agriculture, chemicals, etc. CD are usually produced by cyclodextrin glycosyltransferase (CGTase) from starch. In the present study, a simultaneous conversion approach was developed to improve the yield of CD from starch by conjunction use of isoamylase with α-CGTase. The isoamylase of Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The biochemical characterization of the enzyme showed that the optimum temperature and pH of the recombinant enzyme was 50 °C and 5.5, respectively, and it maintained 60 %, 85 % and 78 % relative activity at 30 °C, 40 °C and 60 °C, respectively. When the recombinant isoamylase and α-CGTase were used simultaneously to convert potato starch (15 %, w/v) into CD, the optimum conditions were found to be: 10 U of α-CGTase and 48 U of isoamylase per gram of substrate, with reaction temperature of 30 °C and pH 5.6. On the optimum condition, the total yield of CD reached 84.6 % (w/w) after 24 h, which was 31.2 % higher than transformation with α-CGTase alone. This is the first report of synchronous bioconversion of CD by both α-CGTase and isoamylase, and represents the highest efficiency of CD production reported so far.

Keywords: Isoamylase; Thermobifida fusca ; Characterization; Cyclodextrin; Synchronous production

Asymmetric reduction of diketones by two Gluconobacter oxydans oxidoreductases by Paul Schweiger; Harald Gross; Jessica Zeiser; Uwe Deppenmeier (pp. 3475-3484).

Two genes encoding recombinant cytosolic oxidoreductases from Gluconobacter oxydans, gox0313 and gox0646, were heterologously expressed in Escherichia coli and the resulting proteins were purified and characterized. GOX0313 was identified as a medium-chain alcohol dehydrogenase, whereas GOX0646 was classified as a ketocarbonyl reductase. GOX0313 had a broad substrate spectrum and oxidized various primary alcohols. However, GOX0313 had a preference for substrate reduction, reducing many aldehydes and α-diketones. In contrast, GOX0646 had a narrow substrate spectrum and reduced α-diketones, preferring short-chain ketocarbonyls. Both enzymes regio- and stereospecifically reduced α-diketones to the corresponding (S)-hydroxy ketone, as shown by NMR. These products are difficult to produce chemically, requiring complicated protecting group chemistry. Furthermore, hydroxy ketones find industrial application in the production of pheromones, fragrances, flavors, and pharmaceuticals. Hence, these enzymes are interesting biocatalysts for the production of enantiomerically pure building blocks that are difficult to prepare chemically.

Keywords: Acetic acid bacteria; Biotransformation; Chiral; Stereospecific; Ketone reduction

Two novel Physcomitrella patens fatty acid elongases (ELOs): identification and functional characterization by Pradinunt Eiamsa-ard; Akkharawit Kanjana-Opas; Edgar B. Cahoon; Pichit Chodok; Sireewan Kaewsuwan (pp. 3485-3497).

The lower plant Physcomitrella patens synthesizes several long-chain polyunsaturated fatty acids (LC-PUFAs) by a series of desaturation and elongation reactions. In the present study, the full-length cDNAs for two novel fatty acid elongases designated PpELO1 and PpELO2 were isolated from P. patens using a PCR-based cloning strategy. These cDNAs encoding proteins of 335 and 280 amino acids with predicted molecular masses of 38.7 and 32.9 kDa, respectively, are predicted to contain seven transmembrane domains with a possible localization in the subcellular endoplasmic reticulum. Sequence comparisons and phylogenetic analysis revealed that they are closely related to other LC-PUFA elongases of the lower eukaryotes such as the Δ⁵- and Δ⁶-elongases of Marchantia polymorpha as well as the Δ⁶-elongase of P. patens. Heterologous expression of the PpELO1 in Saccharomyces cerevisiae led to the elongation of Δ⁹-, Δ⁶-C₁₈, and Δ⁵-C₂₀ LC-PUFAs, whereas only Δ⁹- and Δ⁶-C₁₈ LC-PUFA substrates were used by PpELO2. Chimeric proteins were constructed to identify the amino acid regions most likely to be involved in the determination of the fatty acid substrate specificity. The expression of eight chimeric proteins in yeast revealed that substitution of the C-terminal 50 amino acids from PpELO1 into PpELO2 resulted in a high specificity for C₂₀ fatty acid substrates. As a result, we suggest that the C-terminal region of PpELO1 is sufficient for C₂₀ substrate elongation. Overall, these results provide important insights into the structural basis for substrate specificity of PUFA-generating ELO enzymes.

Keywords: Physcomitrella patens ; Polyunsaturated fatty acid (LC-PUFA); Fatty acid elongase (ELO); Chimeric protein

Stable integration and expression of heterologous genes in several lactobacilli using an integration vector constructed from the integrase and attP sequences of phage ΦAT3 isolated from Lactobacillus casei ATCC 393 by Chao-Fen Lin; Ta-Chun Lo; Yang-Cheng Kuo; Thy-Hou Lin (pp. 3499-3507).

An integration vector capable of stably integrating and maintaining in the chromosomes of several lactobacilli over hundreds of generations has been constructed. The major integration machinery used is based on the ΦAT3 integrase (int) and attP sequences determined previously. A novel core sequence located at the 3′ end of the tRNA^leu gene is identified in Lactobacillus fermentum ATCC 14931 as the integration target by the integration vector though most of such sequences found in other lactobacilli are similar to that determined previously. Due to the lack of an appropriate attB site in Lactococcus lactis MG1363, the integration vector is found to be unable to integrate into the chromosome of the strain. However, such integration can be successfully restored by cotransforming the integration vector with a replicative one harboring both attB and erythromycin resistance sequences into the strain. Furthermore, the integration vector constructed carries a promoter region of placT from the chromosome of Lactobacillus rhamnosus TCELL-1 which is used to express green fluorescence and luminance protein genes in the lactobacilli studied.

Keywords: Lactic acid bacteria; ΦAT3 integrase; Integrative vectors; Site-specific integration; Heterologous expression

Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010 by Xing Wan; Ruiqing Li; Per E. J. Saris; Timo M. Takala (pp. 3509-3518).

Leuconostoc carnosum 4010 is a protective culture for meat products. It kills the foodborne pathogen Listeria monocytogenes by producing two class IIa (pediocin-like) bacteriocins, leucocin A and leucocin C. The genes for leucocin A production have previously been characterised from Leuconostoc gelidum UAL 187, whereas no genetic studies about leucocin C has been published. Here, we characterised the genes for the production of leucocins A and C in L. carnosum 4010. In this strain, leucocin A and leucocin C operons were localised in different plasmids. Unlike in L. gelidum, leucocin A operon in L. carnosum 4010 only contained the structural and the immunity genes lcaAB without transporter genes lcaECD. On the contrary, leucocin C cluster included two intact operons. Novel genes lecCI encode the leucocin C precursor and the 97-aa immunity protein LecI, respectively. LecI shares 48 % homology with the immunity proteins of sakacin P and listeriocin. Another leucocin C operon lecXTS, encoding an ABC transporter and an accessory protein, was 97 % identical with the leucocin A transporter operon lcaECD of L. gelidum. For heterologous expression of leucocin C in Lactococcus lactis, the mature part of the lecC gene was fused with the signal sequence of usp45 in the secretion vector pLEB690. L. lactis secreted leucocin C efficiently, as shown by large halos on lawns of L. monocytogenes and Leuconostoc mesenteroides indicators. The function of LecI was then demonstrated by expressing the gene lecI in L. monocytogenes. LecI-producing Listeria was less sensitive to leucocin C than the vector strain, thus corroborating the immunity function of LecI.

Keywords: Leuconostoc ; Class IIa bacteriocin; Leucocin C; Heterologous expression

Expression of salt-induced 2-Cys peroxiredoxin from Oryza sativa increases stress tolerance and fermentation capacity in genetically engineered yeast Saccharomyces cerevisiae by Il-Sup Kim; Young-Saeng Kim; Ho-Sung Yoon (pp. 3519-3533).

Peroxiredoxins (Prxs), also termed thioredoxin peroxidases (TPXs), are a family of thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative chloroplastic 2-Cys thioredoxin peroxidase (OsTPX) was identified by proteome analysis from leaf tissue samples of rice (Oryza sativa) seedlings exposed to 0.1 M NaCl for 3 days. To investigate the relationship between the OsTPX gene and the stress response, OsTPX was cloned into the yeast expression vector p426GPD under the control of the glyceraldehyde-3-phosphate dehydrogenase (GPD1) promoter, and the construct was transformed into Saccharomyces cerevisiae cells. OsTPX expression was confirmed by semi-quantitative reverse transcription-polymerase chain reaction and western blot analyses. OsTPX contained two highly conserved cysteine residues (Cys114 and Cys236) and an active site region (FTFVCPT), and it is structurally very similar to human 2-Cys Prx. Heterologous OsTPX expression increased the ability of the transgenic yeast cells to adapt and recover from reactive oxygen species (ROS)-induced oxidative stresses, such as a reduction of cellular hydroperoxide levels in the presence of hydrogen peroxide and menadione, by improving redox homeostasis. OsTPX expression also conferred enhanced tolerance to tert-butylhydroperoxide, heat shock, and high ethanol concentrations. Furthermore, high OsTPX expression improved the fermentation capacity of the yeast during glucose-based batch fermentation at a high temperature (40 °C) and at the general cultivation temperature (30 °C). The alcohol yield in OsTPX-expressing transgenic yeast increased by approximately 29 % (0.14 g g⁻¹) and 21 % (0.12 g g⁻¹) during fermentation at 40 and 30 °C, respectively, compared to the wild-type yeast. Accordingly, OsTPX-expressing transgenic yeast showed prolonged cell survival during the environmental stresses produced during fermentation. These results suggest that heterologous OsTPX expression increases acquired tolerance to ROS-induced oxidative stress by improving cellular redox homeostasis and improves fermentation capacity due to improved cell survival during fermentation, especially at a high temperature.

Keywords: Thioredoxin peroxidase; Stress response; Fermentation; Transgenic yeast

Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities by Rasha Abou Aleinein; Razan Hamoud; Holger Schäfer; Michael Wink (pp. 3535-3543).

The coding sequence, which corresponds to the mature antimicrobial peptide ranalexin from the frog Rana catesbeiana, was chemically synthesized with preferred codons for expression in Escherichia coli. It was cloned into the vector pET32c (+) to express a thioredoxin-ranalexin fusion protein which was produced in soluble form in E. coli BL21 (DE3) induced under optimized conditions. After two purification steps through affinity chromatography, about 1 mg of the recombinant ranalexin was obtained from 1 L of culture. Mass spectrometrical analysis of the purified recombinant ranalexin demonstrated its identity with ranalexin. The purified recombinant ranalexin is biologically active. It showed antibacterial activities similar to those of the native peptide against Staphylococcus aureus, Streptococcus pyogenes, E. coli, and multidrug-resistant strains of S. aureus with minimum inhibitory concentration values between 8 and 128 μg/ml. The recombinant ranalexin is also cytotoxic in HeLa and COS7 human cancer cells (IC₅₀ = 13–15 μg/ml).

Keywords: Ranalexin; Fusion protein; Antimicrobial peptide; Escherichia coli ; Recombinant protein

Functional characterization of Edwardsiella tarda twin-arginine translocation system and its potential use as biological containment in live attenuated vaccine of marine fish by Yamin Wang; Qiyao Wang; Weizheng Yang; Bing Liu; Yuanxing Zhang (pp. 3545-3557).

Bacterial twin-arginine translocation (Tat) system contributes to translocate folded proteins to the periplasm and plays pleiotropic roles in physiological fitness. Here, we showed that the fish pathogen Edwardsiella tarda Tat pathway was functional and was essential for H₂S production and hemolytic activity. E. tarda Tat mutant was more susceptible to diverse stresses such as high temperature, SDS, ethanol, and high-salt conditions. However, E. tarda Tat mutant displayed marginal in vivo virulence attenuation in fish models. Comparative proteomics analysis using two-dimensional gel electrophoresis (2-DGE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry were performed to identify proteins undergoing changes in expression levels under high-salt conditons when the Tat pathway was mutilated. Of the 96 differently expressed proteins on the 2-DGE map, 15 proteins were successfully identified with a MASCOT score >45 (p < 0.05) and fold change higher than 2. These significantly differentially expressed proteins were functionally related to basal metabolism and the biosynthesis of proteins and macromolecules. The results of plate counting further confirmed that the Tat mutant was high-salt-sensitive, indicating that Tat mutant merits as a novel salt-sensitive biological containment system for live attenuated vaccine (LAV) in marine fish vaccinology. To test this, we deleted the type III secretion system genes and cured endogenous plasmid pEIB202 to construct a LAV candidate in the context of Tat abrogation in E. tarda. The results indicated that the LAV candidate was highly attenuated when injected intraperitoneally and elicited significant protection against challenge of wild-type E. tarda in turbot while being rapidly eliminated in seawater.

Keywords: Tat system; Biological containment; Marine fish vaccinology; Edwardsiella tarda

Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae by Jin Hou; Tobias Österlund; Zihe Liu; Dina Petranovic; Jens Nielsen (pp. 3559-3568).

The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high level over-expression of HSF1-R206S increased heterologous α-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously.

Keywords: Heterologous protein production; Heat shock response; HSF1; Chaperones; Saccharomyces cerevisiae

Evaluation of control mechanisms for Saccharomyces cerevisiae central metabolic reactions using metabolome data of eight single-gene deletion mutants by Tomokazu Shirai; Fumio Matsuda; Mami Okamoto; Akihiko Kondo (pp. 3569-3577).

We performed metabolome and metabolite–metabolite correlation analyses for eight single-gene deletion mutants of Saccharomyces cerevisiae to evaluate the physiology of glucose metabolism. The irreversible enzyme reactions can become bottlenecks when intracellular metabolism is perturbed by direct interference from the central metabolic pathway by gene deletions or by a deletion of transcriptional regulator. Metabolome data reveal that transcriptional factor, gcr2, regulates the reaction that converts 3-phosphoglycerate into phosphoenolpyruvate. Metabolome data also suggest that the reaction catalyzed by pyruvate kinase makes one of the rate-limiting reactions throughout the glycolytic pathway.

Keywords: Metabolome analysis; Saccharomyces cerevisiae ; Single-gene mutant; Correlation analysis

Investigations on three genes in Ralstonia eutropha H16 encoding putative cyanophycin metabolizing enzymes by Katja Adames; Karina Euting; Anna Bröker; Alexander Steinbüchel (pp. 3579-3591).

The genome sequence of the facultative chemolithoautotrophic bacterium Ralstonia eutropha H16 exhibited two coding sequences with high homologies to cyanophycin synthetases (CphA) as well as one gene coding for a putative cyanophycinase (CphB). To investigate whether or not the genes cphA _H16 (H16_A0774), cphA′_H16 (H16_A0775) and cphB _H16 (H16_B1013) encode active cyanophycin (CGP) metabolism proteins, several functional analyses were performed. Extensive in silico analysis revealed that all characteristic motifs are conserved within CphA_H16, whereas CphA′_H16 misses a large part of the so-called J-loop present in other active cyanophycin synthetases. Although transcription of both genes was demonstrated by RT-PCR, and heterologously expressed cphA genes led to light-scattering inclusions in recombinant cells of Escherichia coli, no CGP could be isolated from the cells or detected by HPLC analysis. For all enzyme assay experiments carried out, significant enzyme activities were determined for CphA and CphA′ in recombinant E. coli cells if crude cell extracts were applied. Homologous expression of cphA genes in cells of R. eutropha H16∆phaC1 did not result in the formation of light-scattering inclusions, and no CGP could be isolated from the cells or detected by HPLC analysis. No transcription of cphB encoding a putative cyanophycinase could be detected by RT-PCR analysis and no overexpression was achieved in several strains of E. coli. Furthermore, no enzyme activity was detected by using CGP overlay agar plates.

Keywords: Ralstonia eutropha ; Poly(amino acids); Cyanophycin synthetase; Cyanophycinase; cphA ; cphA′; cphB

Evaluation of anti-quorum sensing activity of silver nanowires by Mohini S. Wagh (nee Jagtap); Rajendra H. Patil; Deepali K. Thombre; Milind V. Kulkarni; Wasudev N. Gade; Bharat B. Kale (pp. 3593-3601).

A menace of antimicrobial resistance with growing difficulties in eradicating clinical pathogens owing to the biofilm has prompted us to take up a facile aqueous-phase approach for the synthesis of silver nanowires (SNWs) by using ethylene glycol as a reducing agent and polyvinylpyrrolidone (PVP) as a capping agent. This synthesis is a reflux reaction seedless process. The obtained SNWs were about 200–250 nm in diameter and up to 3–4 μm in length. The SNWs were characterized by field emission scanning electron microscopy, transmission electron microscopy, UV–Vis spectroscopy, and X-Ray powder diffraction, and the chemical composition of the sample was examined by energy dispersive X-ray spectrum. The SNWs did not show an antibacterial activity against test organisms, Bacillus subtilis NCIM 2063 and Escherichia coli NCIM 2931; however, it showed a promising property of a quorum sensing-mediated inhibition of biofilm in Pseudomonas aeruginosa NCIM 2948 and violacein synthesis in Chromobacterium violaceum ATCC 12472, which is hitherto unattempted, by polyol approach.

Keywords: Silver nanowires; Polyol; Anti-quorum; Pseudomonas; CLSM

Expression of the immunoreactive buckwheat major allergenic storage protein in Lactococcus lactis by Suguru Shigemori; Shinichi Yonekura; Takashi Sato; Hajime Otani; Takeshi Shimosato (pp. 3603-3611).

Proteins from buckwheat (Fagopyrum esculentum) are strong allergens that can cause serious symptoms, including anaphylaxis, in patients with hypersensitivity. In this study, we successfully developed a modified lactic acid bacterial vector (pNSH) and a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced a major allergenic storage protein of buckwheat, Fagag1 (61.2 kDa, GenBank accession number AF152003), with or without a green fluorescent protein (GFP) tag. GFP fluorescence allows for rapid, simple, and accurate measurement of target protein expression by microscopy or fluorimetry. We describe a convenient method for production of rGFP-Fagag1 fusion and rFagag1 proteins with a good yield in an advantageous probiotic host. We found that in vitro treatment of splenocytes isolated from buckwheat crude protein-immunized mice with rFagag1 increased the expression of allergic inflammation cytokines such as IL-4, IL-13, and IL-17 F. Because it was less antigenic, rGFP-Fagag1 protein from NZ9000 might be of limited use; however, rFagag1 from NZ9000 evoked a robust response as measured by induction of IL-4 and IL-17 F expression levels. The observed allergic activity is indicative of a Th2 cell-mediated immune response and is similar to the effects induced by exposure to buckwheat crude protein. Our results suggest that expression of rFagag1 in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to buckwheat allergens.

Keywords: Buckwheat; Fagopyrum esculentum ; Allergen; Fagag1; Lactococcus lactis ; Th2

Aerobic transformation of zinc into metal sulfide by photosynthetic microorganisms by Chad D. Edwards; Joseph C. Beatty; Jacqueline B. R. Loiselle; Katya A. Vlassov; Daniel D. Lefebvre (pp. 3613-3623).

Industrial activity over the last two centuries has increased heavy metal contamination worldwide, leading to greater human exposure. Zinc is particularly common in industrial effluents and although an essential nutrient, it is highly toxic at elevated concentrations. Photoautotrophic microbes hold promise for heavy metal bioremediation applications because of their ease of culture and their ability to produce sulfide through metabolic processes that in turn are known to complex with the metal ion, Hg(II). The green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae, and the cyanobacterium Synechococcus leopoliensis were all able to synthesize sulfide and form zinc sulfide when exposed to Zn(II). Supplementation of their respective media with sulfite and cysteine had deleterious effects on growth, although ZnS still formed in Cyanidioschyzon cells to the same extent as in unsupplemented cells. The simultaneous addition of sulfate and Zn(II) had similar effects to that of Zn(II) alone in all three species, whereas supplying sulfate prior to exposure to Zn(II) enhanced metal sulfide production. The coupled activities of serine acetyltransferase and O-acetylserine(thiol)lyase (SAT/OASTL) did not increase significantly in response to conditions in which enhanced ZnS formation occurred; sulfate added prior to and simultaneously with Zn(II). However, even low activity could provide sufficient sulfate assimilation over this relatively long-term study. Because the extractable activity of cysteine desulfhydrase was elevated in cells that produced higher amounts of zinc sulfide, cysteine is the probable source of the sulfide in this aerobic process.

Keywords: Heavy metals; Metal transformation; Cyanobacteria; Algae; Zinc sulfide; Sulfate assimilation

Physiological and molecular analysis of carbon source supplementation and pH stress-induced lipid accumulation in the marine diatom Phaeodactylum tricornutum by Florence Mus; Jean-Paul Toussaint; Keith E. Cooksey; Matthew W. Fields; Robin Gerlach; Brent M. Peyton; Ross P. Carlson (pp. 3625-3642).

A detailed physiological and molecular analysis of lipid accumulation under a suite of conditions including nitrogen limitation, alkaline pH stress, bicarbonate supplementation, and organic acid supplementation was performed on the marine diatom Phaeodactylum tricornutum. For all tested conditions, nitrogen limitation was a prerequisite for lipid accumulation and the other culturing strategies only enhanced accumulation highlighting the importance of compounded stresses on lipid metabolism. Volumetric lipid levels varied depending on condition; the observed rankings from highest to lowest were for inorganic carbon addition (15 mM bicarbonate), organic acid addition (15 carbon mM acetate), and alkaline pH stress (pH 9.0). For all lipid-accumulating cultures except acetate supplementation, a common series of physiological steps were observed. Upon extracellular nitrogen exhaustion, culture growth continued for approximately 1.5 cell doublings with decreases in specific protein and photosynthetic pigment content. As nitrogen limitation arrested cell growth, carbohydrate content decreased with a corresponding increase in lipid content. Addition of the organic carbon source acetate appeared to activate alternative metabolic pathways for lipid accumulation. Molecular level data on more than 50 central metabolism transcripts were measured using real-time PCR. Analysis of transcripts suggested the central metabolism pathways associated with bicarbonate transport, carbonic anhydrases, and C4 carbon fixations were important for lipid accumulation. Transcriptomic data also suggested that repurposing of phospholipids may play a role in lipid accumulation. This study provides a detailed physiological and molecular-level foundation for improved understanding of diatom nutrient cycling and contributes to a metabolic blueprint for controlling lipid accumulation in diatoms.

Keywords: Diatom; Phaeodactylum tricornutum ; Alkaline pH stress; Nitrogen limitation; Bicarbonate addition; Lipids; Biodiesel

Improved PCR for identification of Pseudomonas aeruginosa by Hyeon Jin Choi; Myeong Ho Kim; Min Seok Cho; Byoung Kyu Kim; Joo Young Kim; ChangKug Kim; Dong Suk Park (pp. 3643-3651).

The aim of the present study was to develop a noble and specific marker for a quantitative polymerase chain reaction (PCR) assay for the species-specific detection of Pseudomonas aeruginosa based on the O-antigen acetylase gene. It is an important challenge to characterize populations of the bacterium P. aeruginosa, an opportunist by virtue of its physiological and genetic adaptability. However, molecular and serological methods currently available for sensitive and specific detection of P. aeruginosa are by no means satisfactory because there have been critical defects in the diagnosis and identification of P. aeruginosa strains in that these assays also detect other Pseudomonas species, or do not obtain amplified products from P. aeruginosa strains. Therefore, a primer set was designed based on the O-antigen acetylase gene of P. aeruginosa PA01 because it has been known that this gene is structurally diverse among species. The specificity of the primer set was evaluated using genomic DNA from six isolates of P. aeruginosa, 18 different species of Pseudomonas, and 23 other reference pathogenic bacteria. The primer set used in the PCR assay amplified a 232-bp amplicon for only six P. aeruginosa strains. The assay was also able to detect at least 1.41 × 10³ copies/μl of cloned amplified target DNA using purified DNA, or 2.7 × 10² colony-forming unit per reaction when using calibrated cell suspension. In conclusion, this assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of water with a low level or latent infection of P. aeruginosa.

Keywords: Pseudomonas aeruginosa ; O-antigen acetylase gene; Detection; Real-time PCR

More refined diversity of anammox bacteria recovered and distribution in different ecosystems by Ping Han; Ji-Dong Gu (pp. 3653-3663).

A newly reported 16S rRNA gene-based PCR primer set was successfully applied to detect anammox bacteria from four ecosystem samples, including sediments from marine, reservoir, mangrove wetland, and wastewater treatment plant sludge. This primer set showed ability to amplify a much wider coverage of all reported anammox bacterial genera. Based on the phylogenetic analyses of 16S rRNA gene of anammox bacteria, two new clusters were obtained, one closely related to Candidatus Scalindua, and the other in a previously reported novel genus related to Candidatus Brocadia. In the Scalindua cluster, four new subclusters were also found in this study, mainly by sequences of the South China Sea sediments, presenting a higher diversity of Candidatus Scalindua in marine environment. Community structure analyses indicated that samples were grouped together based on ecosystems, showing a niche-specific distribution. Phylogenetic analyses of anammox bacteria in samples from the South China Sea also indicated distinguished community structure along the depth. Pearson correlation analysis showed that the amount of anammox bacteria in the detected samples was positively correlated with the nitrate concentration. According to Canonical Correspondence Analysis, pH, temperature, nitrite, and nitrate concentration strongly affected the diversity and distribution of anammox bacteria in South China Sea sediments. Results collectively indicated a promising application of this new primer set and higher anammox bacteria diversity in the marine environment.

Keywords: Anammox; Diversity; Distribution; Primer, 16S rRNA gene

Detection and validation of volatile metabolic patterns over different strains of two human pathogenic bacteria during their growth in a complex medium using multi-capillary column-ion mobility spectrometry (MCC-IMS) by Nils Kunze; Julia Göpel; Martin Kuhns; Melanie Jünger; Michael Quintel; Thorsten Perl (pp. 3665-3676).

Headspace analyses over microbial cultures using multi-capillary column-ion mobility spectrometry (MCC-IMS) could lead to a faster, safe and cost-effective method for the identification of pathogens. Recent studies have shown that MCC-IMS allows identification of bacteria and fungi, but no information is available from when on during their growth a differentiation between bacteria is possible. Therefore, we analysed the headspace over human pathogenic reference strains of Escherichia coli and Pseudomonas aeruginosa at four time points during their growth in a complex fluid medium. In order to validate our findings and to answer the question if the results of one bacterial strain can be transferred to other strains of the same species, we also analysed the headspace over cultures from isolates of random clinical origin. We detected 19 different volatile organic compounds (VOCs) that appeared or changed their signal intensity during bacterial growth. These included six VOCs exclusively changing over E. coli cultures and seven exclusively changing over P. aeruginosa cultures. Most changes occurred in the late logarithmic or static growth phases. We did not find differences in timing or trends in signal intensity between VOC patterns of different strains of one species. Our results show that differentiation of human pathogenic bacteria by headspace analyses using MCC-IMS technology is best possible during the late phases of bacterial growth. Our findings also show that VOC patterns of a bacterial strain can be transferred to other strains of the same species.

Keywords: Escherichia coli ; Pseudomonas aeruginosa ; Bacteria; Identification; Headspace analyses

Selection of DNA aptamers for capture and detection of Salmonella Typhimurium using a whole-cell SELEX approach in conjunction with cell sorting by Hari P. Dwivedi; R. Derike Smiley; Lee-Ann Jaykus (pp. 3677-3686).

Alternative ligands such as nucleic acid aptamers can be used for pathogen capture and detection and offer advantages over antibodies, including reduced cost, ease of production and modification, and improved stability. DNA aptamers demonstrating binding specificity to Salmonella enterica serovar Typhimurium were identified by whole-cell-systematic evolution of ligands by exponential enrichment (SELEX) beginning with a combinatorial library of biotin-labeled single stranded DNA molecules. Aptamer specificity was achieved using whole-cell counter-SELEX against select non-Salmonella genera. Aptamers binding to Salmonella were sorted, cloned, sequenced, and characterized for binding efficiency. Out of 18 candidate aptamers screened, aptamer S8-7 showed relatively high binding affinity with an apparent dissociation constant (K _d value) of 1.73 ± 0.54 μM and was selected for further characterization. Binding exclusivity analysis of S8-7 showed low apparent cross-reactivity with other foodborne bacteria including Escherichia coli O157: H7 and Citrobacter braakii and moderate cross-reactivity with Bacillus cereus. Aptamer S8-7 was successfully used as a ligand for magnetic capture of serially diluted Salmonella Typhimurium cells, followed by downstream detection using qPCR. The lower limit of detection of the aptamer magnetic capture-qPCR assay was 10²–10³ CFU equivalents of Salmonella Typhimurium in a 290-μl sample volume. Mean capture efficiency ranged from 3.6 to 12.6 %. Unique aspects of the study included (a) the use of SELEX targeting whole cells; (b) the application of flow cytometry for aptamer pool selection, thereby favoring purification of ligands with both high binding affinity and targeting abundant cell surface moieties; and (c) the use of pre-labeled primers that circumvented the need for post-selection ligand labeling. Taken together, this study provides proof-of-concept that biotinylated aptamers selected by whole-cell SELEX can be used in a qPCR-based capture-detection platform for Salmonella Typhimurium.

Keywords: Salmonella ; DNA aptamers; Real-time PCR; Whole-cell SELEX

Degradation of paracetamol by pure bacterial cultures and their microbial consortium by Lili Zhang; Jun Hu; Runye Zhu; Qingwei Zhou; Jianmeng Chen (pp. 3687-3698).

Three bacterial strains utilizing paracetamol as the sole carbon, nitrogen, and energy source were isolated from a paracetamol-degrading aerobic aggregate, and assigned to species of the genera Stenotrophomonas and Pseudomonas. The Stenotrophomonas species have not included any known paracetamol degraders until now. In batch cultures, the organisms f1, f2, and fg-2 could perform complete degradation of paracetamol at concentrations of 400, 2,500, and 2,000 mg/L or below, respectively. A combination of three microbial strains resulted in significantly improved degradation and mineralization of paracetamol. The co-culture was able to use paracetamol up to concentrations of 4,000 mg/L, and mineralized 87.1 % of the added paracetamol at the initial of 2,000 mg/L. Two key metabolites of the biodegradation pathway of paracetamol, 4-aminophenol, and hydroquinone were detected. Paracetamol was degraded predominantly via 4-aminophenol to hydroquinone with subsequent ring fission, suggesting new pathways for paracetamol-degrading bacteria. The degradation of paracetamol could thus be performed by the single isolates, but is stimulated by a synergistic interaction of the three-member consortium, suggesting a possible complementary interaction among the various isolates. The exact roles of each of the strains in the consortium need to be further elucidated.

Keywords: Biodegradation; Paracetamol; Metabolite; Pathway

Anaerobic bioremediation of RDX by ovine whole rumen fluid and pure culture isolates by H. L. Eaton; J. M. Duringer; L. D. Murty; A. M. Craig (pp. 3699-3710).

The ability of ruminal microbes to degrade the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in ovine whole rumen fluid (WRF) and as 24 bacterial isolates was examined under anaerobic conditions. Compound degradation was monitored by high-performance liquid chromatography analysis, followed by liquid chromatography–tandem mass spectrometry identification of metabolites. Organisms in WRF microcosms degraded 180 μM RDX within 4 h. Nitroso-intermediates hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) were present as early as 0.25 h and were detected throughout the 24-h incubation period, representing one reductive pathway of ring cleavage. Following reduction to MNX, peaks consistent with m/z 193 and 174 were also produced, which were unstable and resulted in rapid ring cleavage to a common metabolite consistent with an m/z of 149. These represent two additional reductive pathways for RDX degradation in ovine WRF, which have not been previously reported. The 24 ruminal isolates degraded RDX with varying efficiencies (0–96 %) over 120 h. Of the most efficient degraders identified, Clostridium polysaccharolyticum and Desulfovibrio desulfuricans subsp. desulfuricans degraded RDX when medium was supplemented with both nitrogen and carbon, while Anaerovibrio lipolyticus, Prevotella ruminicola, and Streptococcus bovis IFO utilized RDX as a sole source of nitrogen. This study showed that organisms in whole rumen fluid, as well as several ruminal isolates, have the ability to degrade RDX in vitro and, for the first time, delineated the metabolic pathway for its biodegradation.

Keywords: Bioremediation; RDX; Rumen; Ovine; Anaerobic

Understanding the degradation of Congo red and bacterial diversity in an air–cathode microbial fuel cell being evaluated for simultaneous azo dye removal from wastewater and bioelectricity generation by Jian Sun; Youming Li; Yongyou Hu; Bin Hou; Yaping Zhang; Sizhe Li (pp. 3711-3719).

We investigated the mechanism of Congo red degradation and bacterial diversity in a single-chambered microbial fuel cell (MFC) incorporating a microfiltration membrane and air–cathode. The MFC was operated continuously for more than 4 months using a mixture of Congo red and glucose as fuel. We demonstrated that the Congo red azo bonds were reduced at the anode to form aromatic amines. This is consistent with the known mechanism of anaerobic biodegradation of azo dyes. The MFC developed a less dense biofilm at the anode in the presence of Congo red compared to its absence indicating that Congo red degradation negatively affected biofilm formation. Denaturing gradient gel electrophoresis and direct 16S ribosomal DNA gene nucleotide sequencing revealed that the microbial communities differed depending on whether Congo red was present in the MFC. Geobacter-like species known to generate electricity were detected in the presence or absence of Congo red. In contrast, Azospirillum, Methylobacterium, Rhodobacter, Desulfovibrio, Trichococcus, and Bacteroides species were only detected in its presence. These species were most likely responsible for degrading Congo red.

Keywords: Microbial fuel cell; Azo dye; Wastewater treatment; Bioelectricity; Microbial diversity

Adsorption of rare earth ions onto the cell walls of wild-type and lipoteichoic acid-defective strains of Bacillus subtilis by Hiroshi Moriwaki; Remi Koide; Ritsuko Yoshikawa; Yuya Warabino; Hiroki Yamamoto (pp. 3721-3728).

The aim of this study is to investigate the potential of cell walls of wild-type and lipoteichoic acid-defective strains of Bacillus subtilis 168 to adsorb rare earth ions. Freeze-dried cell powders prepared from both strains were used for the evaluation of adsorption ability for the rare earth ions, namely, La(III), Eu(III), and Tm(III). The rare earth ions were efficiently adsorbed onto powders of both wild-type strain (WT powder) and lipoteichoic acid-defective strain (∆LTA powder) at pH 3. The maximum adsorption capacities for Tm(III) by WT and ∆LTA powders were 43 and 37 mg g⁻¹, respectively. Removal (in percent) of Tm(III), La(III), and Eu(III) from aqueous solution by WT powder was greater than by ∆LTA powder. These results indicate that rare earth ions are adsorbed to functional groups, such as phosphate and carboxyl groups, of lipoteichoic acid. We observed coagulated ∆LTA powder in the removal of rare earth ions (1–20 mg L⁻¹) from aqueous solution. In contrast, sedimentation of WT powder did not occur under the same conditions. This unique feature of ∆LTA powder may be caused by the difference of the distribution between lipoteichoic acid and wall teichoic acid. It appears that ∆LTA powder is useful for removal of rare earth ions by adsorption, because aggregation allows for rapid separation of the adsorbent by filtration.

Keywords: Rare earth ion; Bacillus subtilis 168; Lipoteichoic acid; Lipoteichoic acid-defective strain; Adsorbent

AHL signaling molecules with a large acyl chain enhance biofilm formation on sulfur and metal sulfides by the bioleaching bacterium Acidithiobacillus ferrooxidans by Alex González; Sören Bellenberg; Sigde Mamani; Lina Ruiz; Alex Echeverría; Laurent Soulère; Alain Doutheau; Cecilia Demergasso; Wolfgang Sand; Yves Queneau; Mario Vera; Nicolas Guiliani (pp. 3729-3737).

Biofilm formation plays a pivotal role in bioleaching activities of bacteria in both industrial and natural environments. Here, by visualizing attached bacterial cells on energetic substrates with different microscopy techniques, we obtained the first direct evidence that it is possible to positively modulate biofilm formation of the extremophilic bacterium Acidithiobacillus ferrooxidans on sulfur and pyrite surfaces by using Quorum Sensing molecules of the N-acylhomoserine lactone type (AHLs). Our results revealed that AHL-signaling molecules with a long acyl chain (12 or 14 carbons) increased the adhesion of A. ferrooxidans cells to these substrates. In addition, Card-Fish experiments demonstrated that C14-AHL improved the adhesion of indigenous A. ferrooxidans cells from a mixed bioleaching community to pyrite. Finally, we demonstrated that this improvement of cell adhesion is correlated with an increased production of extracellular polymeric substances. Our results open up a promising means to develop new strategies for the improvement of bioleaching efficiency and metal recovery, which could also be used to control environmental damage caused by acid mine/rock drainage.

Keywords: Biomining; Biofilm; Acidithiobacillus ferrooxidans ; Quorum sensing; Homoserine lactone; Atomic force microscopy

Hollow fiber membrane based H₂ diffusion for efficient in situ biogas upgrading in an anaerobic reactor by Gang Luo; Irini Angelidaki (pp. 3739-3744).

Bubbleless gas transfer through a hollow fiber membrane (HFM) module was used to supply H₂ to an anaerobic reactor for in situ biogas upgrading, and it creates a novel system that could achieve a CH₄ content higher than 90 % in the biogas. The increase of CH₄ content and pH, and the decrease of bicarbonate concentration were related with the increase of the H₂ flow rate. The CH₄ content increased from 78.4 % to 90.2 % with the increase of the H₂ flow rate from 930 to 1,440 ml/(l day), while the pH in the reactor remained below 8.0. An even higher CH₄ content (96.1 %) was achieved when the H₂ flow rate was increased to 1,760 ml/(l day); however, the pH increased to around 8.3 due to bicarbonate consumption which hampered the anaerobic process. The biofilm formed on the HFM was found not to be beneficial for the process since it increased the resistance of H₂ diffusion to the liquid. The study also demonstrated that the biofilm formed on the membrane only contributed 22–36 % to the H₂ consumption, while most of the H₂ was consumed by the microorganisms in the liquid phase.

Keywords: In situ biogas upgrading; Hollow fiber membrane; H₂ ; Anaerobic digestion

Erratum to: Purification and characterization of a nitrilase from Aspergillus niger K10 by Ondřej Kaplan; Vojtěch Vejvoda; Ondřej Plíhal; Petr Pompach; Daniel Kavan; Pavla Bojarová; Karel Bezouška; Martina Macková; Maria Cantarella; Vladimír Jirků; Vladimír Křen; Ludmila Martínková (pp. 3745-3746).

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Applied Microbiology and Biotechnology (v.97, #8)

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Applied Microbiology and Biotechnology (v.97, #8)