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Applied Microbiology and Biotechnology (v.81, #2)

Modifications and applications of the Acinetobacter venetianus RAG-1 exopolysaccharide, the emulsan complex and its components by Hanna Dams-Kozlowska; Michael P. Mercaldi; Bruce J. Panilaitis; David L. Kaplan (pp. 201-210).

Since its discovery in the late 1970s, emulsan has been the subject of significant interest for fundamental biosynthesis and structure–function relationships as well as for its potential industrial applications. These studies initially examined the emulsification properties of the compound, while more recent efforts have focused on potential biomedical applications. As a result of this change of focus, it became necessary to more completely characterize the structure of the emulsan molecule and to develop a more reproducible purification process. We review previous studies with emulsan and explain how prior notions were recently shown to be incorrect through the development of a new purification process. More recent genetic modification of the relevant operon is also reviewed. Finally, the potential applications for the new purified polymer will be discussed.

Keywords: Exopolysaccharide; Emulsan; Amphiphile; Acinetobacter; Emulsification

Proline as a stress protectant in yeast: physiological functions, metabolic regulations, and biotechnological applications by Hiroshi Takagi (pp. 211-223).

Proline is an important amino acid in terms of its biological functions and biotechnological applications. In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. However, it has been shown that proline levels are not increased under various stress conditions in the yeast Saccharomyces cerevisiae cells. Proline is believed to serve multiple functions in vitro such as protein and membrane stabilization, lowering the T _m of DNA, and scavenging of reactive oxygen species, but the mechanisms of these functions in vivo are poorly understood. Yeast cells biosynthesize proline from glutamate in the cytoplasm via the same pathway found in bacteria and plants and also convert excess proline to glutamate in the mitochondria. Based on the fact that proline has stress-protective activity, S. cerevisiae cells that accumulate proline were constructed by disrupting the PUT1 gene involved in the degradation pathway and by expressing the mutant PRO1 gene encoding the feedback inhibition-less sensitive γ-glutamate kinase to enhance the biosynthetic activity. The engineered yeast strains successfully showed enhanced tolerance to many stresses, including freezing, desiccation, oxidation, and ethanol. However, the appropriate cellular level and localization of proline play pivotal roles in the stress-protective effect. These results indicate that the increased stress protection is observed in yeast cells under the artificial condition of proline accumulation. Proline is expected to contribute to yeast-based industries by improving the production of frozen dough and alcoholic beverages or breakthroughs in bioethanol production.

Keywords: Proline; Saccharomyces cerevisiae ; Stress tolerance; Metabolic engineering

Microencapsulated bile salt hydrolase producing Lactobacillus reuteri for oral targeted delivery in the gastrointestinal tract by Christopher Martoni; Jasmine Bhathena; Aleksandra Malgorzata Urbanska; Satya Prakash (pp. 225-233).

This is the first study of its kind to screen probiotic lactic acid bacteria for the purpose of microencapsulating a highly bile salt hydrolase (BSH)-active strain. A Lactobacillus reuteri strain and a Bifidobacterium longum strain were isolated as the highest BSH producers among the candidates. Microcapsules were prepared with a diameter of 619 ± 31 μm and a cell load of 5 × 10⁹ cfu/ml. Post de Man, Rogosa, and Sharpe broth-acid challenge, L. reuteri microcapsules metabolized glyco- and tauro-conjugated bile salts at rates of 10.16 ± 0.46 and 1.85 ± 0.33 μmol/g microcapsule per hour, respectively, over the first 2 h. Microencapsulated B. longum had minimal BSH activity and were significantly (P < 0.05) more susceptible to acid challenge. Further testing of L. reuteri microcapsules in a simulated human gastrointestinal (GI) model showed an improved rate, with 49.4 ± 6.21% of glyco-conjugates depleted after 60 min and complete deconjugation after 4 h. Microcapsules protected the encased cells in the simulated stomach maintaining L. reuteri viability above 10⁹, 10⁸, and 10⁶ cfu/ml after 2 h at pH 3.0, 2.5, and 2.0, respectively. Results show excellent potential for this highly BSH-active microencapsulation system in vitro, highlighted by improved viability and substrate utilization in simulated GI transit.

Keywords: Microcapsule; Probiotic bacteria; L. reuteri ; Bile salt hydrolase; Oral delivery; Gastrointestinal tract

Increasing the cell viability and heterologous protein expression of Pichia pastoris mutant deficient in PMR1 gene by culture condition optimization by Hong Liang Zhao; Chong Xue; Yang Wang; Xue Qin Yao; Zhi Min Liu (pp. 235-241).

In this study, we assessed the potential of PMR1-disrupted Pichia pastoris (Pppmr1) expressing human serum albumin and interferon alpha2b fusion protein (HSA-IFN-alpha2b) in large-scale fermentation. The high osmotic pressure of standard basal salts medium (BSM) was detrimental to the growth and viability of Pppmr1. HSA-IFN-alpha2b was secreted into a supernatant with a concentration of up to 112 mg/L after 20 h of induction and then began to decline. In vitro stability tests indicated that the disappearance of HSA-IFN-alpha2b was ascribed to proteolytic degradation. Decreasing the salt concentration of BSM medium to one quarter of the original formula improved the growth and viability of Pppmr1. As a result of reduced cell lysis and protease release, HSA-IFN-alpha2b was stable in the supernatant, which enabled a longer production phase (30 h) and a higher expression level (215 mg/L). Lowering the culture temperature to 20°C increased the cell viability during carbon source transition and alleviated the oxygen and methanol limitation, which extended the production phase to 40 h and increased the expression level to 680 mg/L. The addition of 2% Soytone prolonged the production phase to 60 h and increased the expression level to 1,260 mg/L, which was more than tenfold higher than that of Pppmr1 cultured under the conditions recommended by Invitrogen.

Keywords: Pichia pastoris ; PMR1 disruption; Large-scale fermentation

Expression of protein engineered NADP⁺-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae by Akinori Matsushika; Seiya Watanabe; Tsutomu Kodaki; Keisuke Makino; Hiroyuki Inoue; Katsuji Murakami; Osamu Takimura; Shigeki Sawayama (pp. 243-255).

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD⁺-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP⁺. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP⁺-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP⁺-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.

Keywords: Xylitol dehydrogenase; Coenzyme specificity; Saccharomyces cerevisiae ; Xylose fermentation; Ethanol production

Nonpungent Capsicum fermentation by Bacillus subtilis and the addition of Rapidase by Seon-Mi Lee; Seong-Yeong Kim; Junsoo Lee; Kwang-Won Yu; Inseop Chang; Hyung Joo Suh (pp. 257-262).

To reduce the pungency of Capsicum without the loss of its biological activity, a Capsicum sp. was fermented by Bacillus subtilis with the addition of Rapidase enzyme. At 1 day of fermentation, the capsaicin content of the Capsicum ferment with Rapidase had sharply decreased from an initial content of 11.8 to 2.8 μg/ml. The Rapidase-fermented Capsicum had higher total flavonoid and phenolic contents than the Capsicum ferment without Rapidase. In addition, ABTS radical scavenging activity was enhanced in the Rapidase-fermented Capsicum as compared to that without Rapidase. Overall, fermentation using B. subtilis and Rapidase was an efficient method to produce a non-pungent Capsicum with antioxidant properties.

Keywords: ABTS radical scavenging activity; Bacillus subtilis ; Capsaicin; Capsicum fermentation; Rapidase

TADH, the thermostable alcohol dehydrogenase from Thermus sp. ATN1: a versatile new biocatalyst for organic synthesis by Volker Höllrigl; Frank Hollmann; Andreas C. Kleeb; Katja Buehler; Andreas Schmid (pp. 263-273).

The alcohol dehydrogenase from Thermus sp. ATN1 (TADH) was characterized biochemically with respect to its potential as a biocatalyst for organic synthesis. TADH is a NAD(H)-dependent enzyme and shows a very broad substrate spectrum producing exclusively the (S)-enantiomer in high enantiomeric excess (>99%) during asymmetric reduction of ketones. TADH is active in the presence of 10% (v/v) water-miscible solvents like 2-propanol or acetone, which permits the use of these solvents as sacrificial substrates in substrate-coupled cofactor regeneration approaches. Furthermore, the presence of a second phase of a water-insoluble solvent like hexane or octane had only minor effects on the enzyme, which retained 80% of its activity, allowing the use of these solvents in aqueous/organic mixtures to increase the availability of low-water soluble substrates. A further activity of TADH, the production of carboxylic acids by dismutation of aldehydes, was investigated. This reaction usually proceeds without net change of the NAD⁺/NADH concentration, leading to equimolar amounts of alcohol and carboxylic acid. When applying cofactor regeneration at high pH, however, the ratio of acid to alcohol could be changed, and full conversion to the carboxylic acid was achieved.

Keywords: Alcohol dehydrogenase; Cofactor regeneration; Asymmetric reduction; Aldehyde dismutation; Substrate specificity; Organic solvent tolerance

Recovery of choline oxidase activity by in vitro recombination of individual segments by Birgit Heinze; Nina Hoven; Timothy O’Connell; Karl-Heinz Maurer; Sebastian Bartsch; Uwe T. Bornscheuer (pp. 275-282).

Initial attempts to express a choline oxidase from Arthrobacter pascens (APChO-syn) in Escherichia coli starting from a synthetic gene only led to inactive protein. However, activity was regained by the systematic exchange of individual segments of the gene with segments from a choline oxidase-encoding gene from Arthrobacter globiformis yielding a functional chimeric enzyme. Next, a sequence alignment of the exchanged segment with other choline oxidases revealed a mutation in the APChO-syn, showing that residue 200 was a threonine instead of an asparagine, which is, thus, crucial for confering enzyme activity and, hence, provides an explanation for the initial lack of activity. The active recombinant APChO-syn-T200N variant was biochemically characterized showing an optimum at pH 8.0 and at 37°C. Furthermore, the substrate specificity was examined using N,N-dimethylethanolamine, N-methylethanolamine and 3,3-dimethyl-1-butanol.

Keywords: Choline oxidase; In vitro recombination; Chimeragenesis

Cloning and characterization of a novel l-arabinose isomerase from Bacillus licheniformis by Ponnandy Prabhu; Manish Kumar Tiwari; Marimuthu Jeya; Paramasamy Gunasekaran; In-Won Kim; Jung-Kul Lee (pp. 283-290).

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.

Keywords: l-Arabinose isomerase; Bacillus licheniformis ; Characterization; Rare sugar; Substrate specificity

Expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase of Corynebacterium glutamicum is regulated by the global regulator SugR by Koichi Toyoda; Haruhiko Teramoto; Masayuki Inui; Hideaki Yukawa (pp. 291-301).

Regulation of expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase essential for glycolysis in Corynebacterium glutamicum was studied. We applied DNA affinity beads to isolate proteins binding to the promoter region of the gapA gene and obtained SugR, which has been shown to be a repressor of pts genes involved in sugar transport system. The results of electrophoretic mobility shift assays revealed that SugR specifically bound to the gapA promoter and the consensus sequence TGTTTG in the promoter region was required for its binding. We examined expression of the gapA gene in a sugR deletion mutant. Effect of mutation in the SugR binding site on gapA-lacZ fusion expression was also examined. These assays revealed that SugR acts as a negative transcriptional regulator of the gapA gene in the absence of sugar, and repression by SugR is alleviated in the presence of sugar, i.e., fructose and sucrose. Fructose-1-phosphate and fructose-1,6-bisphosphate revealed negative effects on binding of SugR to the gapA promoter, indicating that the sugar metabolites are involved in the derepression of gapA expression.

Keywords: Glyceraldehyde-3-phosphate dehydrogenase; Corynebacterium glutamicum ; Gene expression; Glycolysis

New available SCAR markers: potentially useful in distinguishing a commercial strain of the superior type from other strains of Lentinula edodes in China by Hai-Bo Li; Xue-Qian Wu; Hua-Zheng Peng; Li-Zhong Fu; Hai-Long Wei; Qing-Qi Wu; Qun-Ying Jin; Nan Li (pp. 303-309).

At present, more than 100 strains of Lentinula edodes are cultivated on a commercial scale in China. A simple, reliable, and effective method to distinguish some commercial strains of the superior type from other commercial strains is very important for the Lentinula industry. In this study, 23 commercial strains of L. edodes cultivated widely in China at present were collected and analyzed with randomly amplified polymorphic DNA (RAPD) technique. Three informative dominant sequence characterized amplified region (SCAR) markers were developed by designing three pairs of specific SCAR primers from three sequenced differential RAPD bands, respectively. Based on the three SCAR markers, three different multiplex polymerase chain reaction (PCR) phenotypes were detected among the 23 studied commercial strains and in which a multilocus phenotype characterizing a commercial strain Cr02 of the superior type could potentially be used to distinguish this strain from the other 22 studied commercial strains. To our knowledge, this study is the first to describe the development of a multiplex PCR technique based on SCAR markers for detecting the molecular phenotypes among commercial strains of L. edodes in China.

Keywords: Lentinula edodes ; Commercial strain; Superior type; SCAR marker; Multiplex PCR; Molecular phenotype

High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in Escherichia coli by Tingmei Ye; Zhihua Lin; Huanzong Lei (pp. 311-317).

Antibodies currently constitute the most rapidly growing class of human therapeutics; however, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. High expression of active single-chain antibody fragment (scFv) in Escherichia coli has not been successful, as the protein contains three intramolecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the scFv gene against VEGF165 with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO–scFv fusion gene that was highly expressed in the BL21(DE3) strain. The optimal expression level of the soluble fusion protein, SUMO–scFv, was up to 28.5% of the total cellular protein. The fusion protein was purified by Ni nitrilotriacetic acid (NTA) affinity chromatography and cleaved by a SUMO-specific protease to obtain the native scFv, which was further purified by Ni-NTA affinity chromatography. The result of the high-performance liquid chromatography showed that the purity of the recombinant cleaved scFv was greater than 98%. The primary structure of the purified scFv was confirmed by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analysis. In vitro activity assay demonstrated that the recombinant scFv could dose-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cell proliferation. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant scFv with native sequences.

Keywords: Vascular endothelial growth factor; scFv; Small ubiquitin-related modifier; Purification

Factors that influence the extracellular expression of streptavidin in Escherichia coli using a bacteriocin release protein by Gerhard Miksch; Stella Ryu; Joe Max Risse; Erwin Flaschel (pp. 319-326).

Aiming to increase production of recombinant streptavidin in Escherichia coli, the effect of different leader sequences, different promoter strengths of the bacteriocin release protein (kil), host strain and medium composition on the expression and secretion into the medium was investigated. Expression vectors containing an expression or secretion unit were constructed with different combinations of leader sequence for the streptavidin gene and promoters for the kil gene and streptavidin gene. Results showed that a high-level extracellular production of streptavidin could be accomplished with E. coli BL21(DE3) by using the leader sequence of the phoA gene, a strong stationary-phase promoter for the kil gene and supplementation of the medium by glycine. Using a stationary-phase promoter for the expression of streptavidin had a negative effect.

Keywords: Escherichia coli ; Streptavidin; Secretion; Promoter; Leader sequence; Glycine; Bacteriocin release protein

Comparative proteome analysis of robust Saccharomyces cerevisiae insights into industrial continuous and batch fermentation by Jing-Sheng Cheng; Bin Qiao; Ying-Jin Yuan (pp. 327-338).

A robust Saccharomyces cerevisiae strain has been widely applied in continuous and batch/fed-batch industrial fermentation. However, little is known about the molecular basis of fermentative behavior of this strain in the two realistic fermentation processes. In this paper, we presented comparative proteomic profiling of the industrial yeast in the industrial fermentation processes. The expression levels of most identified protein were closely interrelated with the different stages of fermentation processes. Our results indicate that, among the 47 identified protein spots, 17 of them belonging to 12 enzymes were involved in pentose phosphate, glycolysis, and gluconeogenesis pathways and glycerol biosynthetic process, indicating that a number of pathways will need to be inactivated to improve ethanol production. The differential expressions of eight oxidative response and heat-shock proteins were also identified, suggesting that it is necessary to keep the correct cellular redox or osmotic state in the two industrial fermentation processes. Moreover, there are significant differences in changes of protein levels between the two industrial fermentation processes, especially these proteins associated with the glycolysis and gluconeogenesis pathways. These findings provide a molecular understanding of physiological adaptation of industrial strain for optimizing the performance of industrial bioethanol fermentation.

Keywords: Continuous fermentation; Batch/fed-batch fermentation; Proteomic analysis; Bioethanol; Saccharomyces cerevisiae

Fluorescence anisotropy analysis of the mechanism of action of mesenterocin 52A: speculations on antimicrobial mechanism by Jordane Jasniewski; Catherine Cailliez-Grimal; Mohamed Younsi; Jean-Bernard Millière; Anne-Marie Revol-Junelles (pp. 339-347).

Mesenterocin 52A (Mes 52A) is a class IIa bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, active against Listeria sp. The interaction of Mes 52A with bacterial membranes of two sensitive Listeria strains has been investigated. The Microbial Adhesion to Solvents test used to study the physico-chemical properties of the surface of the two strains indicated that both surfaces were rather hydrophilic and bipolar. The degree of insertion of Mes 52A in phospholipid bilayer was studied by fluorescence anisotropy measurements using two probes, 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and DPH, located at different positions in the membrane. TMA-DPH reflects the fluidity at the membrane surface and DPH of the heart. With Listeria ivanovii CIP 12510, Mes 52A induced an increase only in the TMA-DPH fluorescence anisotropy, indicating that this bacteriocin affects the membrane surface without penetration into the hydrophobic core of the membrane. No significant K⁺ efflux was measured, whereas the ΔΨ component of the membrane potential was greatly affected. With Listeria innocua CIP 12511, Mes 52A caused an increase in the fluorescence of TMA-DPH and DPH, indicating that this peptide inserts deeply in the cytoplasmic membrane of this sensitive strain. This insertion led to K⁺ efflux, without perturbation of ΔpH and a weak modification of ΔΨ, and is consistent with pore formation. These data indicate that Mes 52A interacts at different positions of the membrane, with or without pore formation, suggesting two different mechanisms of action for Mes 52A depending on the target strain.

Keywords: Bacteriocin; Anisotropy; Antimicrobial peptide; MIC

Investigating the effect of patulin, penicillic acid and EDTA on biofilm formation of isolates from dental unit water lines by Iram Liaqat; Robert Th. Bachmann; Anjum Nasim Sabri; Robert G. J. Edyvean; Catherine A. Biggs (pp. 349-358).

This study investigated the effect of patulin and penicillic acid, two known quorum-sensing inhibitors, and the common biocide ethylenediaminetetraacetic acid (EDTA) on the biofilm formation and auto-inducer (AI)-2 production of three isolates from dental unit water lines, Klebsiella sp., Bacillus subtilis and Bacillus cereus. Penicillic acid on its own had no effect on the biofilm formation of all isolates, whereas in combination with EDTA, it enhanced biofilm formation significantly in Klebsiella sp. and B. cereus. EDTA at concentrations greater than 10 μM promoted biofilm formation in B. cereus and B. subtilis. Patulin was found to promote biofilm formation in B. cereus up to 25 μM. A significant increase in biofilm formation was observed in B. cereus and B. subtilis at concentrations greater than 10 μM of patulin when combined with EDTA. The Vibrio harveyi BB170 AI-2 bioassay showed a positive response for Klebsiella sp. AI-2 production with a maximum fold induction at the late exponential growth phase. Addition of glucose prolonged the AI-2 production phase considerably. No significant effect of patulin, penicillic acid alone as well as in combination with EDTA was observed on AI-2 production by Klebsiella sp. The findings have important implications for the design of biofilm prevention and eradication strategies.

Keywords: DUWL isolates; Patulin; Penicillic acid; Biocide; Biofilm assay; AI-2 assay

Mutations in the N-terminal coding region of the harpin protein Hpa1 from Xanthomonas oryzae cause loss of hypersensitive reaction induction in tobacco by Xiao-yu Wang; Cong-feng Song; Wei-guo Miao; Zhao-lin Ji; Xiben Wang; Yan Zhang; Jia-huan Zhang; John S. Hu; Wayne Borth; Jin-sheng Wang (pp. 359-369).

Harpins encoded by many gram-negative phytopathogenic bacterial hrp genes induce hypersensitive response (HR) and associated defense responses on nonhost plants. Hpa1_Xoo and Hpa1_Xoc, two harpin proteins from Xanthomonas oryzae pathovars, induce HR when infiltrated into tobacco leaves. N- and C-terminal mutations of Hpa1_Xoo and Hpa1_Xoc, respectively, were tested for their ability to elicit HR on tobacco. Deletion of codons for 12 highly hydrophilic amino acids (H₂N-QGISEKQLDQLL-COOH) that partially overlap the N-terminal α-helical regions of respective proteins was found to be critical for the elicitation of HR in tobacco. Furthermore, two single missense mutants Hpa1_Xoo (L51P) and Hpa1_Xoc (L53P) that are predicted to destroy the coiled-coil integrity and inhibit the dimer formation eliminated HR elicitation activity in tobacco. However, both wild-type proteins and derivative mutants retained the ability to induce systemic acquired resistance in tobacco against tobacco mosaic virus. Accumulations of npr1 (nonexpressor of pathogenesis-related protein 1), hsr515 (hypersensitivity-related protein 515), and pr2 (pathogenesis-related protein 2) transcripts were found in tobacco plants infiltrated with wild-type or mutated proteins.

Keywords: Xanthomonas oryzae ; Hpa1; Hypersensitive reaction; Induced resistance; N-terminal α-helix

Utilization of arylaliphatic nitriles by haloalkaliphilic Halomonas nitrilicus sp. nov. isolated from soda soils by A. Chmura; A. A. Shapovalova; S. van Pelt; F. van Rantwijk; T. P. Tourova; G. Muyzer; D. Yu. Sorokin (pp. 371-378).

An enrichment culture from saline soda soils, using acetate as carbon and energy source and 2-phenylpropionitrile as nitrogen source (PPN) at pH 10, resulted in the isolation of strain ANL-αCH3. The strain was identified as a representative of the genus Halomonas in the Gammaproteobacteria. The bacterium was capable of PPN utilization as a nitrogen source only, while phenylacetonitrile (PAN) served both as carbon, energy and nitrogen source. This capacity was not described previously for any other haloalkaliphilic bacteria. Apart from the nitriles mentioned above, resting cells of ANL-αCH3 also hydrolyzed mandelonitrile, benzonitrile, acrylonitrile, and phenylglycinonitrile, presumably using nitrilase pathway. Neither nitrile hydratase nor amidase activity was detected. The isolate showed a capacity to grow with benzoate and salicylate as carbon and energy source and demonstrated the ability to completely mineralize PAN. These clearly indicated a potential to catabolize aromatic compounds. On the basis of unique phenotype and distinct phylogeny, strain ANL-αCH3 is proposed as a novel species of the genus Halomonas—Halomonas nitrilicus sp. nov.

Keywords: Nitriles; Phenylpropionitrile (PPN); Phenylacetonitrile (PAN); Haloalkaliphilic; Halomonas ; Nitrilase

Anaerobic ammonium oxidation (anammox) irreversibly inhibited by methanol by Kazuichi Isaka; Yuichi Suwa; Yuya Kimura; Takao Yamagishi; Tatsuo Sumino; Satoshi Tsuneda (pp. 379-385).

Methanol inhibition of anaerobic ammonium oxidation (anammox) activity was characterized. An enrichment culture entrapped in a polyethylene glycol gel carrier was designed for practical uses of wastewater treatment. Batch experiments demonstrated that anammox activity decreased with increases in methanol concentration, and relative activity reached to 29% of the maximum when 5 mM methanol was added. Also, batch experiments were conducted using anammox sludge without immobilization. Anammox activity was evaluated by quantifying ¹⁴N¹⁵N (²⁹N) emission by combined gas chromatography-quadrupole mass spectrometry, and the anammox activity was found to be almost as sensitive to methanol as in the earlier trials in which gel carriers were used. These results indicated that methanol inhibition was less severe than previous studies. When methanol was added in the influent of continuous feeding system, relative activity was decreased to 46% after 80 h. Although the addition was halted, afterwards the anammox activity was not resumed in another 19 days of cultivation, suggesting that methanol inhibition to anammox activity was irreversible. It is notable that methanol inhibition was not observed if anammox activity was quiescent when substrate for anammox was not supplied. These results suggest that methanol itself is not inhibitory and may not directly inhibit the anammox activity.

Keywords: Anammox; Ammonium; Denitrification; Methanol; Ethanol; Inhibition

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates by Joaquin Sanchis; Layla Fernández; J. Daniel Carballeira; Jullien Drone; Yosephine Gumulya; Horst Höbenreich; Daniel Kahakeaw; Sabrina Kille; Renate Lohmer; Jérôme J.-P. Peyralans; John Podtetenieff; Shreenath Prasad; Pankaj Soni; Andreas Taglieber; Sheng Wu; Felipe E. Zilly; Manfred T. Reetz (pp. 387-397).

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

Keywords: Directed evolution; Saturation mutagenesis; PCR; Megaprimer; Antiprimer; Difficult-to-amplify templates

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Applied Microbiology and Biotechnology (v.81, #2)

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Applied Microbiology and Biotechnology (v.81, #2)