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Applied Microbiology and Biotechnology (v.96, #4)

The immunomodulatory effects of lactic acid bacteria for improving immune functions and benefits by Yueh-Ting Tsai; Po-Ching Cheng; Tzu-Ming Pan (pp. 853-862).

Probiotics have a number of beneficial health effects in humans and animals, such as reducing lactose intolerance symptoms and enhancing the bioavailability of nutrients. Probiotics help regulate intestinal microflora and immunomodulatory properties. Probiotics also decrease the prevalence of allergies in susceptible individuals, inhibit the inflammatory responses in the gut, and have antagonistic effects against intestinal and food-borne pathogens. Bacteria typically colonize the intestinal tract first and then reinforce the host defense systems by inducing generalized mucosal immune responses, including modulation of DC/NK interaction, a balanced T-helper cell response, self-limited inflammatory response, and the secretion of polymeric IgA. A lot of reports showed that lactic acid bacteria (LAB) as Lactobacillus and Bifidobacterium and their fermented products are effective at enhancing innate and adaptive immunity, prevent gastric mucosal lesion development, alleviate allergies, and put up defense against intestinal pathogen infection. In this review paper, we compared the influence of immunomodulatory effects on the function and efficacy of lactobacillus products with different strains. We also discuss the beneficial effects of several LAB strain and its derivative products for human immunity and related diseases.

Keywords: Lactobacillus paracasei subsp. paracasei NTU 101; Innate immune response; Mucosal immunity; Allergy; Escherichia coli O157:H7

Potential uses of spent mushroom substrate and its associated lignocellulosic enzymes by Chia-Wei Phan; Vikineswary Sabaratnam (pp. 863-873).

Mushroom industries generate a virtually in-exhaustible supply of a co-product called spent mushroom substrate (SMS). This is the unutilised substrate and the mushroom mycelium left after harvesting of mushrooms. As the mushroom industry is steadily growing, the volume of SMS generated annually is increasing. In recent years, the mushroom industry has faced challenges in storing and disposing the SMS. The obvious solution is to explore new applications of SMS. There has been considerable discussion recently about the potentials of using SMS for production of value-added products. One of them is production of lignocellulosic enzymes such as laccase, xylanase, lignin peroxidase, cellulase and hemicellulase. This paper reviews scientific research and practical applications of SMS as a readily available and cheap source of enzymes for bioremediation, animal feed and energy feedstock.

Keywords: Spent mushroom substrate; Spent mushroom compost; Lignocellulosic enzyme; Bioremediation; Animal feed; Energy feedstock

Paracetamol in the environment and its degradation by microorganisms by Shijin Wu; Lili Zhang; Jianmeng Chen (pp. 875-884).

Paracetamol (4′-hydroxyacetanilide, N-acetyl-p-aminophenol, acetaminophen, and paracetamol) is a widely used over-the-counter analgesic and antipyretic drug. Paracetamol and structural analogs are ubiquitous in the natural environment and easily accumulate in aquatic environment, which have been detected in surface waters, wastewater, and drinking water throughout the world. Paracetamol wastewater is mainly treated by chemical oxidation processes. Although these chemical methods may be available for treating these pollutants, the harsh reaction conditions, the generation of secondary pollutants, and the high operational cost associated with these methods have often made them not a desirable choice. Biodegradation of paracetamol is being considered as an environmentally friendly and low-cost option. The goal of this review is to provide an outline of the current knowledge of biodegradation of paracetamol in the occurrence, degrading bacteria, and proposed metabolic/biodegrading pathways, enzymes and possible intermediates. The comprehensive understanding of the metabolic pathways and enzyme systems involved in the utilization of paracetamol means will be helpful for optimizing and allowing rational design of biodegradation systems for paracetamol-contaminated wastewater.

Keywords: Paracetamol (acetaminophen); Structural analogs; Metabolic/biodegrading pathway

Recent advances in technology supporting biopharmaceutical production from mammalian cells by M. Butler; A. Meneses-Acosta (pp. 885-894).

The demand for production of glycoproteins from mammalian cell culture continues with an increased number of approvals as biopharmaceuticals for the treatment of unmet medical needs. This is particularly the case for humanized monoclonal antibodies which are the largest and fastest growing class of therapeutic pharmaceuticals. This demand has fostered efforts to improve the efficiency of production as well as to address the quality of the final product. Chinese hamster ovary cells are the predominant hosts for stable transfection and high efficiency production on a large scale. Specific productivity of recombinant glycoproteins from these cells can be expected to be above 50 pg/cell/day giving rise to culture systems with titers of around 5 g/L if appropriate fed-batch systems are employed. Cell engineering can delay the onset of programmed cell death to ensure prolonged maintenance of productive viable cells. The clinical efficacy and quality of the final product can be improved by strategic metabolic engineering. The best example of this is the targeted production of afucosylated antibodies with enhanced antibody-dependent cell cytotoxicity, an important function for use in cancer therapies. The development of culture media from non-animal sources continues and is important to ensure products of consistent quality and without the potential danger of contamination. Process efficiencies may also be improved by employing disposable bioreactors with the associated minimization of downtime. Finally, advances in downstream processing are needed to handle the increased supply of product from the bioreactor but maintaining the high purity demanded of these biopharmaceuticals.

Keywords: Biopharmaceuticals; CHO cells; Glycosylation; Apoptosis; Antibodies; Vaccines

Fluorescence resonance energy transfer (FRET)-based biosensors: visualizing cellular dynamics and bioenergetics by Sohila Zadran; Steve Standley; Kaylee Wong; Erick Otiniano; Arash Amighi; Michel Baudry (pp. 895-902).

Förster (or fluorescence) resonance energy transfer (FRET) is a process involving the radiation-less transfer of energy from a “donor” fluorophore to an “acceptor” fluorophore. FRET technology enables the quantitative analysis of molecular dynamics in biophysics and in molecular biology, such as the monitoring of protein–protein interactions, protein–DNA interactions, and protein conformational changes. FRET-based biosensors have been utilized to monitor cellular dynamics not only in heterogeneous cellular populations, but also at the single-cell level in real time. Lately, applications of FRET-based biosensors range from basic biological to biomedical disciplines. Despite the diverse applications of FRET, FRET-based sensors still face many challenges. There is an increasing need for higher fluorescence resolution and improved specificity of FRET biosensors. Additionally, as more FRET-based technologies extend to medical diagnostics, the affordability of FRET reagents becomes a significant concern. Here, we will review current advances and limitations of FRET-based biosensor technology and discuss future FRET applications.

Keywords: Förster resonance energy transfer (FRET); Biosensors; Imaging; Fluorescence; Medical diagnostics

Recombinant production of hyperthermostable CelB from Pyrococcus furiosus in Lactobacillus sp. by N. Böhmer; S. Lutz-Wahl; L. Fischer (pp. 903-912).

Lactic acid bacteria (LAB) are used widespread in the food industry as traditional starters for various fermented foods. For recombinant protein production, LAB would be superior with view from the food safety demands since most of them are Generally Recognized As Safe organisms. We investigated the two pSIP expression systems, pSIP403 and pSIP409 (Sørvig et al. 2005), to produce a hyper-thermophilic β-glycosidase (CelB) from Pyrococcus furiosus in Lactobacillus plantarum NC8 and Lactobacillus casei as hosts, respectively. Both lactobacilli harboring the pSIP409-celB vector produced active CelB in batch bioreactor cultivations (MRS medium) while the specific CelB activity of the cell free extract was about 44 % higher with L. plantarum (1,590 ± 90 nkat/mg_protein) than with L. casei (1,070 ± 66 nkat/mg_protein) using p-nitrophenyl-β-galactoside (pNPGal) as the substrate. A fed-batch bioreactor cultivation of L. plantarum NC8 pSIP409-celB resulted in a specific CelB activity of 2,500 ± 120 nkat p_NPGal/mg_protein after 28 h. A repeated dosage of the inducer spp-IP did not increase the enzyme expression further. As alternative for the cost intensive MRS medium, a basal whey medium with supplements (yeast extract, Tween 80, NH₄-citrate) was developed. In bioreactor cultivations using this medium, about 556 ± 29 nkat p_NPGal/mg_protein of CelB activity was achieved. It was shown that both LAB were potential expression hosts for recombinant enzyme production. The pSIP expression system can be applied in L. casei.

Keywords: Lactobacillus plantarum ; Lactobacillus casei ; Recombinant CelB; Food grade; pSIP

Efficient secreted production of (R)-3-hydroxybutyric acid from living Halomonas sp. KM-1 under successive aerobic and microaerobic conditions by Yoshikazu Kawata; Kazunori Kawasaki; Yasushi Shigeri (pp. 913-920).

Production of (R)-3-hydroxybutyric acid [(R)-3-HB] by strain Halomonas sp. KM-1 under successive aeration conditions was investigated. The first aerobic condition allowed both cell growth and intracellular storage of poly-(R)-3-hydroxybutyric acid (PHB). The second microaerobic condition, achieved by reducing the culture agitation rate, lead to the degradation of PHB to (R)-3-HB. The amount of PHB stored in KM-1 cells after 48-h cultivation under aerobic conditions was 16.4 g/l. In contrast, after a shift from aerobic to microaerobic conditions and a further 18-h cultivation, PHB content in KM-1 cells decreased to 0.9 g/l. Numerous intracellular PHB-containing granules were observed in cells under aerobic conditions by electron microscopy. After the shift to microaerobic conditions, the number and size of granules were significantly reduced, in agreement with the degradation of prestored PHB. On the other hand, under microaerobic conditions, the concentration of (R)-3-HB in the medium reached a maximum of 15.2 g/l, indicating the production and extracellular secretion of (R)-3-HB as a result of PHB digestion. Notably, cell lysis was not observed during the successive aeration conditions as assessed by elution of genomic DNA to the culture supernatant, cell morphology observed by electron microscopy and counts of colony formation. In this simple system utilizing a change of aeration during cultivation of strain Halomonas sp. KM-1, we obtained one of the highest levels of microbiological production of (R)-3-HB reported to date.

Keywords: Halomonas ; Poly-(R)-3-hydroxybutyric acid (PHB); (R)-3-hydroxybutyric acid; Microaerobic; Glycerol

Fusing the vegetative insecticidal protein Vip3Aa7 and the N terminus of Cry9Ca improves toxicity against Plutella xylostella larvae by Fang Dong; Ruiping Shi; Shanshan Zhang; Tao Zhan; Gaobing Wu; Jie Shen; Ziduo Liu (pp. 921-929).

Bacillus thuringiensis insecticidal crystal proteins (ICPs) and vegetative insecticidal proteins (VIPs) have been widely used as a kind of safe bio-insecticides. A problem that has been of concern worldwide is how to improve their insecticidal activities. In this study, to determine the synergism between VIPs and ICPs effect on insecticidal activity, a construct that produces a chimeric protein of the Vip3Aa7 and the N terminus ofCry9Ca, named V3AC9C, was expressed in Escherichia coli BL21 cells. In additional experiments, the V3AC9C chimeric protein, the single Vip3Aa7, and the single N terminus of Cry9Ca were treated with trypsin. SDS–PAGE showed that the V3AC9C could be processed into two single toxins. Bioassays tested on third instar larvae of Plutella xylostella showed that the toxicity of the chimeric protein was markedly better than either of the single toxins. Interestingly, the toxicity of the chimeric protein was 3.2-fold higher than a mixture of the Vip3Aa7 and Cry9Ca toxins (mass ratio of 1:1). The synergism factor (SF) of chimeric protein containing Vip3Aa7 and Cry9Ca was calculated to be 4.79. The SF in mixture of toxins is only 1.46. Hence, the effect was more than the sum of the Vip3Aa7 and Cry9C activities. Analysis of the protein’s solubility showed that the Vip3Aa7 helped the N terminus of Cry9Ca to dissolve in an alkaline buffer. It was concluded that the increase in the toxicity of the V3AC9C chimeric protein over the constituent proteins mainly resulted from this increase in solubility. These results lay a foundation for the development of a new generation of bio-insecticides and multi-gene transgenic plants.

Keywords: Bacillus thuringiensis ; Vegetative insecticidal proteins; Insecticidal crystal proteins; Fusing expression; Synergism factor

Formation of the two novel glycolipid biosurfactants, mannosylribitol lipid and mannosylarabitol lipid, by Pseudozyma parantarctica JCM 11752^T by Tomotake Morita; Tokuma Fukuoka; Tomohiro Imura; Dai Kitamoto (pp. 931-938).

In order to develop novel glycolipid biosurfactants, Pseudozyma parantarctica JCM 11752^T, which is known as a producer of mannosylerythritol lipids (MEL), was cultivated using different sugar alcohols with the presence of vegetable oil. When cultivated in a medium containing 4 % (w/v) olive oil and 4 % d-ribitol or d-arabitol, the yeast strain provided different glycolipids, compared to the case of no sugar alcohol. On TLC, both of the extracted glycolipid fractions gave two major spots corresponding to MEL-A (di-acetylated MEL) and MEL-B (mono-acetylated MEL). Based on ¹H NMR analysis, one glycolipid was identified as MEL-A, but the other was not MEL-B. On high-performance liquid chromatography after acid hydrolysis, the unknown glycolipid from the d-ribitol culture provided mainly two peaks identical to d-mannose and d-ribitol, and the other unknown glycolipid from the d-arabitol culture did two peaks identical to d-mannose and d-arabitol. Accordingly, the two unknown glycolipids were identified as mannosylribitol lipid (MRL) and mannosylarabitol lipid (MAL), respectively. The observed critical micelle concentration (CMC) and surface tension at CMC of MRL were 1.6 × 10⁻⁶ M and 23.7 mN/m, and those of MAL were 1.5 × 10⁻⁶ M and 24.2 mN/m, respectively. These surface-tension-lowering activities were significantly higher compared to conventional MEL. Furthermore, on a water-penetration scan, MRL and MAL efficiently formed not only the lamella phase (L_α) but also the myelins at a wide range of concentrations, indicating their excellent self-assembling properties and high hydrophilicity. The present two glycolipids should thus facilitate the application of biosurfactants as new functional materials.

Keywords: Mannosylarabitol lipid; Mannosylribitol lipid; Biosurfactant; Glycolipid; Yeast; Pseudozyma

Molecular cloning and functional analysis of a recombinant ribosome-inactivating protein (alpha-momorcharin) from Momordica charantia by Shuzhen Wang; Yubo Zhang; Honggao Liu; Ying He; Junjie Yan; Zhihua Wu; Yi Ding (pp. 939-950).

Alpha-momorcharin (α-MC), a member of the ribosome-inactivating protein (RIP) family, has been used not only as antiviral, antimicrobial, and antitumor agents, but also as toxicant to protozoa, insects, and fungi. In this study, we expressed the protein in Escherichia coli Rosetta (DE3) pLysS strain and purified it by nickel–nitrilotriacetic acid affinity chromatography. A total of 85 mg of homogeneous protein was obtained from 1 l culture supernatant of Rosetta (DE3) pLysS, showing a high recovery rate of 73.9%. Protein activity assay indicated that α-MC had both N-glycosidase activity and DNA-nuclease activity, the former releasing RIP diagnostic RNA fragment (Endo’s fragment) from rice rRNAs and the latter converting supercoiled circular DNA of plasmid pET-32a(+) into linear conformations in a concentration-dependent manner. Specially, we found that α-MC could inhibit the mycelial growth of Fusarium solani and Fusarium oxysporum with IC₅₀ values of 6.23 and 4.15 μM, respectively. Results of optical microscopy and transmission electron microscopy demonstrated that α-MC caused extensive septum formation, loss of integrity of the cell wall, separation of the cytoplasm from the cell wall, deformation of cells with irregular budding sites, and apoptosis in F. solani. Moreover, α-MC was active against Pseudomonas aeruginosa with an IC₅₀ value of 0.59 μM. The α-MC protein carries a high potential for the design of new antifungal drugs or the development of transgenic crops resistant to pathogens.

Keywords: Ribosome-inactivating proteins; Alpha-momorcharin; Heterologous expression; N-glycosidase; Antifungal activity

Expression and characterization of a novel metagenome-derived cellulase Exo2b and its application to improve cellulase activity in Trichoderma reesei by Alei Geng; Gen Zou; Xing Yan; Qianfu Wang; Jun Zhang; Fanghua Liu; Baoli Zhu; Zhihua Zhou (pp. 951-962).

A metagenomic fosmid library containing 1 × 10⁵ clones was constructed from a biogas digester fed with pig ordure and rice straw. In total, 121 clones with activity of 4-methylumbelliferyl-cellobiosidase were screened from the metagenomic library. A novel GH5 cellulase gene exo2b was identified from a sequenced clone EXO02C10 and expressed in Escherichia coli BL21. The corresponding recombinant Exo2b protein showed high specific activity toward both carboxymethylcellulose (CMC; 260 U/mg protein) and β-d-glucan from barley (849 U/mg), with an optimal pH and temperature of 7.5 and 58 °C, respectively. Exo2b showed stable activity at a wide pH range from 5.5 to 9.0 and was highly thermostable at 60 °C in the presence of 60 mM cysteine. Residual activity was maintained at nearly 100% when Exo2b was incubated at 60 °C for 15 h. A thin-layer chromatography analysis of the hydrolysis products confirmed that Exo2b was an endo-β-1,4-glucanase and it could also produce oligosaccharide smaller than cellotetraose. The fragment encoding the Exo2b catalytic domain was then fused with the cbh1 gene from Trichoderma reesei, and the fused gene was successfully expressed in T. reesei Rut-C30. Compared to that of the parent strain, the filter paper activity and CMCase activity of the secreted proteins of a selected transformant A1 increased by 24% and 18%, respectively. Besides, the glucose concentration from the hydrolysis of pretreated corn stover by the A1 secreted proteins increased by 19.8%. The present study demonstrated the potential application of metagenome originated cellulase genes to modify cellulase producing fungi.

Keywords: Metagenome; Cellulase; Gene fusion; Fungi; Pretreated corn stover

Cloning, expression, and characterization of an adenylate cyclase from Arthrobacter sp. CGMCC 3584 by Ying He; Nan Li; Yong Chen; Xiaochun Chen; Jianxin Bai; Jinglan Wu; Jingjing Xie; Hanjie Ying (pp. 963-970).

The cya gene encoding adenylate cyclase was cloned from Arthrobacter sp. CGMCC 3584 by thermal asymmetric interlaced PCR for the first time. It exhibited an open reading frame containing 1,125 bp and encoding 374 amino acids. Amino acid sequence analysis showed that this enzyme was a class III adenylate cyclase. Expression of the cya gene was carried out in Escherichia coli Rosetta, and purification was performed via Ni²⁺-NTA agarose gel column. SDS-PAGE indicated that the molecular mass of the recombinant adenylate cyclase was 45 kDa. The V _max and K _m were determined to be 5.06 μmol/min/mg and 7.56 mM, respectively. The optimum pH and temperature were 8.0 and 35 °C. Several divalent metal ions were found to activate the enzyme to different extents, and the maximal specific activity reached 3.04 μmol/min/mg when 50 mM Mg²⁺ was added. This was the first report of the cloning of an adenylate cyclase gene from Arthrobacter sp.

Keywords: Adenylate cyclase; Arthrobacter ; Cyclic adenosine monophosphate; Expression; Characterization

Identification of antibody-interacting proteins that contribute to the production of recombinant antibody in mammalian cells by Daisuke Nishimiya; Yuji Ogura; Hidetaka Sakurai; Tohru Takahashi (pp. 971-979).

Protein folding and assembly processes are essential for antibody secretion; however, the endogenous proteins involved in these processes remain largely unknown. Therefore, except for some well-known endoplasmic reticulum (ER) chaperones such as GRP78/Bip and protein disulfide isomerase, enhancement of recombinant antibody expression by co-expression of interacting proteins has been largely elusive. Here, in addition to known ER chaperones, we identified additional endogenous proteins that interact with recombinant antibody in mammalian cells by immunoprecipitation coupled with liquid chromatography–tandem mass spectrometry. Most of our identified proteins enhanced antibody production, and furthermore, some of their combinations resulted in greater enhancement. In particular, eukaryotic initiation factor 4A combined with other proteins had approximately fourfold higher effect on antibody production. Identified proteins that could improve antibody expression contain not only ER-resident proteins like GRP78/Bip but also non-ER-resident proteins. These results suggest that this method could be effective in the investigation of novel proteins that are involved in enhancing recombinant antibody production because immunoprecipitation coupled with mass spectroscopy could identify proteins which directly interact with the antibody.

Keywords: Antibody production; Chaperone; Mammalian cell; LC–MS/MS; IP

Characterization of triglyceride lipase genes of fission yeast Schizosaccharomyces pombe by Hisashi Yazawa; Hiromichi Kumagai; Hiroshi Uemura (pp. 981-991).

Triglycerides (TG) are major storage lipids for eukaryotic cells. In this study, we characterized three genes of fission yeast Schizosaccharomyces pombe, SPCC1450.16c, SPAC1786.01c, and SPAC1A6.05c, that show high homology to Saccharomyces cerevisiae TG lipase genes, TGL3, TGL4, and TGL5. Deletion of each gene increased TG content by approximately 1.7-fold compared to the parental wild-type strain, and their triple deletion mutant further increased TG content to 2.7-fold of the wild-type strain, suggesting that all three genes encode TG lipase and are functioning in S. pombe. The triple deletion mutant showed no growth defect in rich and synthetic medium, but its growth was sensitive to cerulenin, an inhibitor of fatty acid synthesis. This growth defect by cerulenin was restored by adding oleic acid in media, suggesting that these genes were involved in the mobilization of TG in S. pombe. When ricinoleic acid was produced in the triple mutant by introducing CpFAH12 fatty acid hydroxylase gene from Claviceps purpurea, percent composition of ricinoleic acid increased by 1.1-fold compared to the wild-type strain, in addition to a 1.6-fold increase in total fatty acid content per dry cell weight (DCW). In total, the ricinoleic acid production per DCW increased by 1.8-fold in the triple deletion mutant.

Keywords: Schizosaccharomyces pombe ; Yeast; Triglyceride lipase; Triglyceride; Fatty acid

Cloning, sequencing, and characterization of lipase genes from a polyhydroxyalkanoate (PHA)-synthesizing Pseudomonas resinovorans by Jeung Hee Lee; Richard D. Ashby; David S. Needleman; Ki-Teak Lee; Daniel K. Y. Solaiman (pp. 993-1005).

Lipase (lip) and lipase-specific foldase (lif) genes of a biodegradable polyhydroxyalkanoate (PHA)-synthesizing Pseudomonas resinovorans NRRL B-2649 were cloned using primers based on consensus sequences, followed by polymerase chain reaction-based genome walking. Sequence analyses showed a putative Lip gene product (314 amino acids, a.a.) with its catalytic active site (Ser₁₁₁, Asp₂₅₈, and His₂₈₀) identified. The foldase lif gene that is located 55 bp downstream of lip codes for a putative Lif (345 a.a.). To verify the biological function of the cloned lip gene for lipase expression in P. resinovorans, we constructed a lip knock-out mutant (lip::Tn5) by transposon insertion. Complementation of the lip knock-out P. resinovorans mutant with a lipase expression plasmid (pBS29-P2-lip) was performed, and its effect on lipase expression was investigated. The wild-type P. resinovorans and the lip::Tn5[pBS29-P2-lip] recombinant (but not the lip::Tn5 mutant) showed fluorescence on rhodamine B plates indicative of lipase activity. The wild type exhibited extracellular lipase activity when grown on medium containing triacylglycerol substrates (tallow, olive oil, and tributyrin) as sole carbon sources, but the lip::Tn5 mutant did not show such activity. Lipase activity of various strains was also confirmed by TLC analysis of the composition of acylglycerols and free fatty acid in the extracts of the spent culture medium. We further found that tributyrin was more effective than olive oil in inducing lipase expression in P. resinovorans.

Keywords: Lipase; Lipase-specific foldase; Pseudomonas resinovorans ; Rhodamine B; Transposon

Comparative analysis of temperature-dependent transcriptome of Pseudomonas aeruginosa strains from rhizosphere and human habitats by Da-Qiang Wu; Yaqian Li; Yuquan Xu (pp. 1007-1019).

In this study, we investigated the effects of a change in growth temperature on the transcriptome of two strains of Pseudomonas aeruginosa. The chosen P. aeruginosa strains were M18 and PAO1, which are adapted to two different niches, rhizosphere and human, respectively. To assess the changes induced by a change in temperature, we used a newly designed microarray covering the complete genome of four P. aeruginosa strains: PAO1, M18, PA14 and LESB58, which proved informative and reliable for the transcriptome study. Using the microarray, we analysed the transcriptome profile changes of two P. aeruginosa strains of M18 and PAO1 at their originating and non-originating temperatures: 28 °C for the rhizosphere and 37 °C for the human. The transcriptome profiles showed significant temperature-dependent differences (64.8 % in M18 and 66.8 % in PAO1) compared with the genome structure (6 % in M18 and 4.1 % in PAO1). Furthermore, we found that the specific induced genes at the non-originating growth temperature of the each strain (207 genes in M18 and 229 genes in PAO1) were evidently more than those induced at the originating growth temperature (158 genes in M18 and 169 genes in PAO1). The functional analysis of several newly found specific regulated operons (such as phh, liu, hmg) in the two strains indicated possible strategies implemented to respond to the non-originating temperature. This study provides new insight into how P. aeruginosa species responds to temperature change and a microarray platform covering the complete genomes of four widely studied P. aeruginosa strains.

Keywords: Pseudomonas aeruginosa ; Transcriptome; Microarray; Temperature-dependent

Extracellular glutathione fermentation using engineered Saccharomyces cerevisiae expressing a novel glutathione exporter by Kentaro Kiriyama; Kiyotaka Y. Hara; Akihiko Kondo (pp. 1021-1027).

A novel extracellular glutathione fermentation method using engineered Saccharomyces cerevisiae was developed by following three steps. First, a platform host strain lacking the glutathione degradation protein and glutathione uptake protein was constructed. This strain improved the extracellular glutathione productivity by up to 3.2-fold compared to the parental strain. Second, the ATP-dependent permease Adp1 was identified as a novel glutathione export ABC protein (Gxa1) in S. cerevisiae based on the homology of the protein sequence with that of the known human glutathione export ABC protein (ABCG2). Overexpression of this GXA1 gene improved the extracellular glutathione production by up to 2.3-fold compared to the platform host strain. Finally, combinatorial overexpression of the GXA1 gene and the genes involved in glutathione synthesis in the platform host strain increased the extracellular glutathione production by up to 17.1-fold compared to the parental strain. Overall, the metabolic engineering of the glutathione synthesis, degradation, and transport increased the total (extracellular + intracellular) glutathione production. The extracellular glutathione fermentation method developed in this study has the potential to overcome the limitations of the present intracellular glutathione fermentation process in yeast.

Keywords: Extracellular glutathione production; Glutathione transport; Yeast; ABC protein; Cell factory

Study on carvacrol and cinnamaldehyde polymeric films: mechanical properties, release kinetics and antibacterial and antibiofilm activities by A. Nostro; R. Scaffaro; M. D’Arrigo; L. Botta; A. Filocamo; A. Marino; G. Bisignano (pp. 1029-1038).

Polyethylene-co-vinylacetate (EVA) films with different concentrations (3.5 wt% and 7 wt%) of essential oil constituents, carvacrol or cinnamaldehyde, were prepared and characterized by mechanical, antibacterial and antibiofilm properties. The incorporation of the compounds into copolymer films affected their elastic modulus, tensile stress and elongation at break. Carvacrol and cinnamaldehyde act as plasticizers which reduce the intermolecular forces of polymer chains, thus improving the flexibility and extensibility of the film. The analysis of the surface characteristics demonstrated that essential oil constituents lowered the contact angle values without causing any remarkable variation of the surface roughness. The films allowed progressive diffusion of the bioactive molecules and the kinetic of release was correlated with the damaging effect on bacterial growth. The kill curves proved that the film with essential oil constituents (7 wt%) had a significant bactericidal effect (reduction of 4 and 2 log CFU) against Staphylococcus aureus and Escherichia coli and a bacteriostatic effect against Staphylococcus epidermidis and Listeria monocytogenes (reduction of about 1 log CFU). With regard to biofilm formation the biomass formed on polymeric films surface was significantly reduced if compared with the pure copolymer control. The results were confirmed by fluorescence microscopy images by Live/dead staining. The reduction in the surface tension coupled to an inherent bactericidal property of carvacrol and cinnamaldehyde could in turn affect the initial attachment phase of bacteria and compromise the normal biofilm development.

Keywords: Carvacrol; Cinnamaldehyde; Polymeric film; Mechanical properties; Release kinetic; Biofilm

Validation of the use of multiple internal control genes, and the application of real-time quantitative PCR, to study esterase gene expression in Oenococcus oeni by Krista M. Sumby; Paul R. Grbin; Vladimir Jiranek (pp. 1039-1047).

The study of gene expression and accurate quantitation of target genes in any organism depends on correct normalisation. Due to the increase in studies on Oenococcus oeni gene expression, there is a clear need for alternative reference genes in order to reliably measure expression levels. In this manuscript, we propose the approach of using multiple reference genes to provide a more robust basis for establishing a reference gene set. The identification and evaluation of a panel of nine reference genes, including the commonly used ldhD, for real-time PCR normalisation was performed in O. oeni. Expression levels of these reference genes were then measured by real-time qPCR in an independent set of O. oeni samples (n = 30). The nine genes were ranked according to their stability of gene expression measure (M) using geNorm to identify the most consistently expressed reference genes. This approach resulted in the identification of multiple reference genes that may be used for a screening and more robust normalisation of target gene expression measured by real-time RT-qPCR. Expression of esterase genes was then measured in these O. oeni samples in the presence of known esterase substrates. The results give an indication of how these genes may be involved in ester synthesis and hydrolysis in O. oeni.

Keywords: qPCR; Reference genes; Oenococcus oeni ; Esterase

Discriminating experimental Listeria monocytogenes infections in mice using serum profiling by James R. Hocker; Douglas A. Drevets; Marilyn J. Dillon; Jay S. Hanas (pp. 1049-1058).

Serum profiling was used to distinguish mice infected with wild-type or mutant Listeria monocytogenes from noninfected control mice. Identifications of significant electrospray ionization mass spectrometry (ESI-MS) sera peak areas between Listeria-infected- and control mice were performed using t tests. ESI-MS cohort peak distributions differed from mice infected with wild-type or ∆actA Listeria versus control mice with p values of 0.00012 and 0.015, respectively. A “% wild-type Listeria peaks identified” assessment tool yielded values of 64 % for wild-type infection, 51 % for ∆actA infection, and 47 % for no infection. Receiver operator characteristic area discriminatory values were 0.97 (wild-type) and 0.82 (∆actA) versus controls. Predictive value measurements revealed overall test sensitivities of 88 % for wild-type infection and 63 % for ∆actA infection. These studies indicate that ESI-MS serum profiling holds promise for diagnosis of infection with intracellular pathogens such as Listeria and indicate that the technology could be useful in understanding the L. monocytogenes infection process.

Keywords: Serum profiling; Listeria monocytogenes infection; Electrospray; Ionization mass spectrometry; Mouse model system

A novel actinomycete derived from wheat heads degrades deoxynivalenol in the grain of wheat and barley affected by Fusarium head blight by Michihiro Ito; Ikuo Sato; Motoo Koitabashi; Shigenobu Yoshida; Machiko Imai; Seiya Tsushima (pp. 1059-1070).

Deoxynivalenol (DON) is a hazardous and globally prevalent mycotoxin in cereals. It commonly accumulates in the grain of wheat, barley and other small grain cereals affected by Fusarium head blight (caused by several Fusarium species). The concept of reducing DON in naturally contaminated grain of wheat or barley using a DON-degrading bacterium is promising but has not been accomplished. In this study, we isolated a novel DON-utilising actinomycete, Marmoricola sp. strain MIM116, from wheat heads through a novel isolation procedure including an in situ plant enrichment step. Strain MIM116 had background degradation activity, and the activity was enhanced twofold by the consumption of DON. Among Tween 20, Triton X-100 and Tween 80, we selected Tween 80 as a spreading agent of strain MIM116 because it promoted DON degradation and the growth of strain MIM116 in the presence of DON. The inoculation of MIM116 cell suspension plus 0.01% Tween 80 into 1,000 harvested kernels of wheat and barley resulted in a DON decrease from approximately 3 mg kg⁻¹ to less than 1 mg kg⁻¹ of dry kernels, even when cells had only basal levels of DON-degrading activity. To the best of our knowledge, this is the first report that describes (1) the isolation of a DON-degrading bacterium from wheat heads, (2) the effects of surfactants on the biodegradation of DON and (3) the decrease of DON levels in naturally contaminated wheat and barley grain using a DON-degrading bacterium.

Keywords: Deoxynivalenol; Trichothecenes; Mycotoxin degradation; Marmoricola ; Fusarium head blight; Surfactant

Indole-3-acetaldehyde from Rhodococcus sp. BFI 332 inhibits Escherichia coli O157:H7 biofilm formation by Jin-Hyung Lee; Yong-Guy Kim; Chang-Jin Kim; Jae-Chan Lee; Moo Hwan Cho; Jintae Lee (pp. 1071-1078).

Pathogenic biofilms have been associated with persistent infections due to their high resistance to antimicrobial agents. To identify nontoxic biofilm inhibitors for enterohemorrhagic Escherichia coli O157:H7, the spent media of a 4,104 Actinomycetes library was screened. The culture spent medium (1%, v/v) of plant pathogen Rhodococcus sp. BFI 332 markedly inhibited E. coli O157:H7 biofilm formation without affecting the growth of planktonic E. coli O157:H7 cells. Rhodococcus sp. BFI 332 produced significant amounts of indole-3-acetaldehyde and indole-3-acetic acid, and the former of which reduced E. coli O157:H7 biofilm formation. Global transcriptome analyses showed that indole-3-acetaldehyde most repressed two curli operons, csgBAC and csgDEFG, and induced tryptophanase (tnaAB) in E. coli O157:H7 biofilm cells. Electron microscopy showed that spent medium of Rhodococcus sp. BFI 332 and indole-3-acetaldehyde reduced curli production in E. coli O157:H7. The spent medium of Rhodococcus sp. BFI 332 also significantly reduced the biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis. Overall, this study suggests that indole derivatives are present in the Actinomycetes strains and they can be used as biofilm inhibitors against pathogenic bacteria.

Keywords: Indole-3-acetaldehyde; Escherichia coli O157:H7; Biofilm inhibition; Actinomycetes

An efficient xylose-fermenting recombinant Saccharomyces cerevisiae strain obtained through adaptive evolution and its global transcription profile by Yu Shen; Xiao Chen; Bingyin Peng; Liyuan Chen; Jin Hou; Xiaoming Bao (pp. 1079-1091).

Factors related to ethanol production from xylose in engineered Saccharomyces cerevisiae that contain an exogenous initial metabolic pathway are still to be elucidated. In the present study, a strain that expresses the xylose isomerase gene of Piromyces sp. Pi-xylA and overexpresses XKS1, RPE1, RKI1, TAL1, and TKL1, with deleted GRE3 and COX4 genes was constructed. The xylose utilization capacity of the respiratory deficiency strain was poor but improved via adaptive evolution in xylose. The μ _max of the evolved strain in 20 g l⁻¹ xylose is 0.11 ± 0.00 h⁻¹, and the evolved strain consumed 17.83 g l⁻¹ xylose within 72 h, with an ethanol yield of 0.43 g g⁻¹ total consumed sugars during glucose–xylose cofermentation. Global transcriptional changes and effect of several specific genes were studied. The result revealed that the increased xylose isomerase acivity, the upregulation of enzymes involved in glycolysis and glutamate synthesis, and the downregulation of trehalose and glycogen synthesis, may have contributed to the improved xylose utilization of the strain. Furthermore, the deletion of PHO13 decreased the xylose growth in the respiration deficiency strain although deleting PHO13 can improve the xylose metabolism in other strains.

Keywords: Xylose isomerase; Respiratory deficiency; Ethanol; PDC6 ; PHO13 ; CWP1

Expression and surface display of Cellulomonas endoglucanase in the ethanologenic bacterium Zymobacter palmae by Motoki Kojima; Tomohiro Akahoshi; Kenji Okamoto; Hideshi Yanase (pp. 1093-1104).

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we developed a tool for cell surface display of cellulolytic enzymes on the ethanologenic bacterium Zymobacter palmae. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, to express and display heterologous cellulolytic enzymes on the Z. palmae cell surface, we utilized the cell-surface display motif of the Pseudomonas ice nucleation protein Ina. The gene encoding Ina from Pseudomonas syringae IFO3310 was cloned, and its product was comprised of three functional domains: an N-terminal domain, a central domain with repeated amino acid residues, and a C-terminal domain. The N-terminal domain of Ina was shown to function as the anchoring motif for a green fluorescence protein fusion protein in Escherichia coli. To express a heterologous cellulolytic enzyme extracellularly in Z. palmae, we fused the N-terminal coding sequence of Ina to the coding sequence of an N-terminal-truncated Cellulomonas endoglucanase. Z. palmae cells carrying the fusion endoglucanase gene were shown to degrade carboxymethyl cellulose. Although a portion of the expressed fusion endoglucanase was released from Z. palmae cells into the culture broth, we confirmed the display of the protein on the cell surface by immunofluorescence microscopy. The results indicate that the N-terminal anchoring motif of Ina from P. syringae enabled the translocation and display of the heterologous cellulase on the cell surface of Z. palmae.

Keywords: Cell surface display; Zymobacter palmae ; Ice nucleation protein; Endoglucanase; Pseudomonas syringae ; Cellulose

Erratum to: Two-component signal transduction in Corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets by Michael Bott; Melanie Brocker (pp. 1105-1110).

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Applied Microbiology and Biotechnology (v.96, #4)

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Applied Microbiology and Biotechnology (v.96, #4)