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Applied Microbiology and Biotechnology (v.86, #1)

The transcriptome of Mycobacterium tuberculosis by Rachel Haller; Meghann Kennedy; Nick Arnold; Robert Rutherford (pp. 1-9).

In this review, we summarize the present understanding of the Mycobacterium tuberculosis transcriptome and catalog both experimentally verified findings and computationally derived predictions. We also provide a new analysis for a range of discoveries by comparing the results of previously independent research papers. Bringing these data together and improving their accessibility should help catalyze further discoveries. This minireview also provides some general insights that may be valuable to those working to characterize the transcriptome of less-studied prokaryotes.

Keywords: Transcript structure; Operon; Transcription unit; Transcription factor; Predicted; Regulon

Biotransformation of nicotine by microorganism: the case of Pseudomonas spp. by Hongjuan Li; Xuemei Li; Yanqing Duan; Ke-Qin Zhang; Jinkui Yang (pp. 11-17).

Several bacterial species are capable of using nicotine, the main alkaloid in tobacco plants, as a substrate for growth. The dominant species include members of two genera, Pseudomonas and Arthrobacter. The degradation pathway and genetic structure of nicotine catabolism in Arthrobacter nicotinovorans were recently reviewed (Brandsch Appl Microbiol Biotechnol 69:493–498, 2006). Here, we present up-to-date information on biodegradation of nicotine by Pseudomonas spp. Species in this genus capable of degrading nicotine are summarized and analyzed phylogenetically. Their metabolic intermediates and nicotine degradation-related genes were summarized, and the nicotine-biotransformation pathways were compared and discussed.

Keywords: Nicotine degradation; Pseudomonas spp.; Metabolic products; Nicotine degradation-related genes; Biotransformation pathway

Improvement of secondary metabolite production in Streptomyces by manipulating pathway regulation by Yihua Chen; Michael J. Smanski; Ben Shen (pp. 19-25).

Titer improvement is a constant requirement in the fermentation industry. The traditional method of “random mutation and screening” has been very effective despite the considerable amount of time and resources it demands. Rational metabolic engineering, with the use of recombinant DNA technology, provides a novel, alternative strategy for titer improvement that complements the empirical method used in industry. Manipulation of the specific regulatory systems that govern secondary metabolite production is an important aspect of metabolic engineering that can efficiently improve fermentation titers. In this review, we use examples from Streptomyces secondary metabolism, the most prolific source of clinically used drugs, to demonstrate the power and utility of exploiting natural regulatory networks, in particular pathway-specific regulators, for titer improvement. Efforts to improve the titers of fredericamycin, C-1027, platensimycin, and platencin in our lab are highlighted.

Keywords: Titer improvement; Streptomyces ; Secondary metabolite; Regulator; Biosynthesis

Biotechnological production of lutein and its applications by José M. Fernández-Sevilla; F. G. Acién Fernández; E. Molina Grima (pp. 27-40).

Lutein is an antioxidant that has gathered increasing attention due to its potential role in preventing or ameliorating age-related macular degeneration. Currently, it is produced from marigold oleoresin, but continuous reports of lutein-producing microalgae pose the question if those microorganisms can become an alternative source. Several microalgae have higher lutein contents than most marigold cultivars and have been shown to yield productivities hundreds of times higher than marigold crops on a per square meter basis. Microalgae and marigold are opposite alternatives in the use of resources such as land and labor and the prevalence of one or the other could change in the future as the lutein demand rises and if labor or land becomes more restricted or expensive in the producing countries. The potential of microalgae as a lutein source is analyzed and compared to marigold. It is suggested that, in the current state of the art, microalgae could compete with marigold even without counting on any of the improvements in microalgal technology that can be expected in the near future.

Keywords: Lutein; Microalgae; Extraction; Photobioreactor; Mass production

Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology by Regine Eibl; Stephan Kaiser; Renate Lombriser; Dieter Eibl (pp. 41-49).

Disposable bioreactors have increasingly been incorporated into preclinical, clinical, and production-scale biotechnological facilities over the last few years. Driven by market needs, and, in particular, by the developers and manufacturers of drugs, vaccines, and further biologicals, there has been a trend toward the use of disposable seed bioreactors as well as production bioreactors. Numerous studies documenting their advantages in use have contributed to further new developments and have resulted in the availability of a multitude of disposable bioreactor types which differ in power input, design, instrumentation, and scale of the cultivation container. In this review, the term “disposable bioreactor” is defined, the benefits and constraints of disposable bioreactors are discussed, and critical phases and milestones in the development of disposable bioreactors are summarized. An overview of the disposable bioreactors that are currently commercially available is provided, and the domination of wave-mixed, orbitally shaken, and, in particular, stirred disposable bioreactors in animal cell-derived productions at cubic meter scale is reported. The growth of this type of reactor system is attributed to the recent availability of stirred disposable benchtop systems such as the Mobius CellReady 3 L Bioreactor. Analysis of the data from computational fluid dynamic simulation studies and first cultivation runs confirms that this novel bioreactor system is a viable alternative to traditional cell culture bioreactors at benchtop scale.

Keywords: Biomanufacturing; Bioreactor types; Cell cultures; CFD; Disposable bioreactor; Microorganisms; Mobius CellReady 3 L bioreactor

New tools for the genetic manipulation of filamentous fungi by Ulrich Kück; Birgit Hoff (pp. 51-62).

Filamentous fungi have a long-standing tradition as industrial producers of primary and secondary metabolites. Initially, industrial scientists selected production strains from natural isolates that fulfilled both microbiological and technical requirements for economical production processes. Subsequently, genetically modified strains with novel properties were obtained through traditional strain improvement programs relying mostly on random mutagenesis. In recent years, however, recombinant technologies have contributed significantly to improve the capacities of production and have also allowed the design of genetically manipulated strains. These major advances were only made possible by basic research bringing deeper and novel insights into cellular and molecular fungal processes, thus allowing the design of genetically manipulated strains. This better understanding of fundamental genetic processes in model organisms has resulted in the design and generation of new experimental transformation strategies to manipulate specifically gene expression and function in diverse filamentous fungi, including those having a biotechnical significance. In this review, we summarize recent developments in the application of homologous DNA recombination and RNA interference to manipulate fungal recipients for further improvement of physiology and development in regards to biotechnical and pharmaceutical applications.

Keywords: Homologous recombination; RNA interference; Filamentous fungi; Molecular genetic tools

Microbially induced diseases of Agaricus bisporus: biochemical mechanisms and impact on commercial mushroom production by Michèle L. Largeteau; Jean-Michel Savoie (pp. 63-73).

The button mushroom, Agaricus bisporus (Lange) Imbach, the most common cultivated mushroom, is susceptible to a wide range of virus, bacterial, and fungal diseases. However, only some diseases were studied for the mechanisms involved in the host–microorganism interaction. This review deals with biochemical mechanisms related to cavity disease (Burkholderia gladioli) and to the interaction between A. bisporus and the causal agents responsible for the most severe diseases, namely the bacteria Pseudomonas tolaasii and Pseudomonas reactans and the fungi Trichoderma aggressivum and Lecanicillium fungicola.

Keywords: Bacterial blotch; Cavity disease; Dry bubble; Green mould

The mechanisms of citrate on regulating the distribution of carbon flux in the biosynthesis of uridine 5′-monophosphate by Saccharomyces cerevisiae by Yong Chen; Shuya Li; Jian Xiong; Zhenjiang Li; Jianxin Bai; Lei Zhang; Qi Ye; Pingkai Ouyang; Hanjie Ying (pp. 75-81).

A whole cell biocatalytic process for uridine 5′-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. The concentration of UMP was increased by 23% when 1 g l⁻¹ sodium citrate was fed into the broth. Effects of citrate addition on UMP production were investigated. Glucose-6-phosphate pool was elevated by onefold, while FBP and pyruvate were decreased by 42% and 40%, respectively. Organic acid pools such as acetate and succinate were averagely decreased by 30% and 49%. The results demonstrated that manipulation of citrate levels could be used as a novel tool to regulate the metabolic fluxes distribution among glycolysis, pentose phosphate pathway, and TCA cycle.

Keywords: Citrate; Carbon flux; Saccharomyces cerevisiae ; Uridine 5′-monophosphate

Bioproduction of chiral mandelate by enantioselective deacylation of α-acetoxyphenylacetic acid using whole cells of newly isolated Pseudomonas sp. ECU1011 by Xin Ju; Hui-Lei Yu; Jiang Pan; Dong-Zhi Wei; Jian-He Xu (pp. 83-91).

Substrate-directed screening was carried out to find bacteria that could deacylate O-acetylated mandelic acid from environmental samples. From more than 200 soil isolates, we identified for the first time that Pseudomonas sp. ECU1011 biocatalytically deacylated (S)-α-acetoxyphenylacetic acid with high enantioselectivity (E > 200), yielding (S)-mandelic acid with 98.1% enantiomeric excess (ee) at a 45.5% conversion rate. The catalytic deacylation of (S)-α-acetoxyphenylacetic acid by the resting cell was optimized using a single-factor method to yield temperature and pH optima of 30°C and 6.5, respectively. These optima help to reduce the nonselective spontaneous hydrolysis of the racemic substrate. It was found that substrate concentrations up to 60 mM could be used. 2-Propanol was used as a moderate cosolvent to help the substrate disperse in the aqueous phase. Under optimized reaction conditions, the ee of the residual (R)-α-acetoxyphenylacetic acid could be improved further, to greater than 99%, at a 60% conversion rate. Furthermore, using this newly isolated strain of Pseudomonas sp. ECU1011, three kinds of optically pure analogs of (S)-mandelic acid and (R)-α-acetoxyphenylacetic acid were successfully prepared at high enantiomeric purity.

Keywords: Microbial esterase; Pseudomonas sp.; Enantioselective deacylation; α-Acetoxyphenylacetic acid; Enantiopure mandelic acid

Increased product formation induced by a directed secondary substrate limitation in a batch Hansenula polymorpha culture by Kirsten Kottmeier; Carsten Müller; Robert Huber; Jochen Büchs (pp. 93-101).

By the use of directed limitations of secondary substrates, the metabolic flux should be deflected from biomass production to product formation. In order to study the impact of directed limitations caused by various secondary substrates on the growth and product formation of the methylotrophic yeast Hansenula polymorpha, the cultivation systems respiration activity monitoring system (RAMOS) and BioLector were used in parallel. While the RAMOS device allows the online monitoring of the oxygen transfer rate in shake flasks, the BioLector enables in microtiter plates the monitoring of scattered light and the fluorescence intensity of the green fluorescent protein (GFP). Secondary substrate limitations of phosphate, potassium, and magnesium were analyzed in batch fermentations. The sole carbon source was either 10 g/L glucose or 10 g/L glycerol. The expression of the GFP gene is controlled by the FMD promoter (formate dehydrogenase). In batch cultures with glucose as carbon source, a directed limitation of phosphate increased the GFP production 1.87-fold, compared to phosphate unlimited conditions. Under potassium-limited conditions with glycerol as sole carbon source, the GFP production was 1.41-fold higher compared to unlimited conditions. A limitation of the substrate magnesium resulted in a 1.22-fold increase GFP formation in the case of glycerol as carbon source.

Keywords: Respiration activity monitoring system; BioLector; Hansenula polymorpha ; Secondary substrate limitation; Process development

Application of oscillation for efficiency improvement of continuous ethanol fermentation with Saccharomyces cerevisiae under very-high-gravity conditions by Yu Shen; X. M. Ge; Feng Wu Bai (pp. 103-108).

Compared with steady state, oscillation in continuous very-high-gravity ethanol fermentation with Saccharomyces cerevisiae improved process productivity, which was thus introduced for the fermentation system composed of a tank fermentor followed by four-stage packed tubular bioreactors. When the very-high-gravity medium containing 280 g l⁻¹ glucose was fed at the dilution rate of 0.04 h⁻¹, the average ethanol of 15.8% (v/v) and residual glucose of 1.5 g l⁻¹ were achieved under the oscillatory state, with an average ethanol productivity of 2.14 g h⁻¹ l⁻¹. By contrast, only 14.8% (v/v) ethanol was achieved under the steady state at the same dilution rate, and the residual glucose was as high as 17.1 g l⁻¹, with an ethanol productivity of 2.00 g h⁻¹ l⁻¹, indicating a 7% improvement under the oscillatory state. When the fermentation system was operated under the steady state at the dilution rate of 0.027 h⁻¹ to extend the average fermentation time to 88 h from 59 h, the ethanol concentration increased slightly to 15.4% (v/v) and residual glucose decreased to 7.3 g l⁻¹, correspondingly, but the ethanol productivity was decreased drastically to 1.43 g h⁻¹ l⁻¹, indicating a 48% improvement under the oscillatory state at the dilution rate of 0.04 h⁻¹.

Keywords: Continuous ethanol fermentation; Very-high-gravity; Saccharomyces cerevisiae ; Oscillation; Steady state

Isolation of an unusual metabolite 2-allyloxyphenol from a marine actinobacterium, its biological activities and applications by Meyyappan Arumugam; Anindita Mitra; Parasuraman Jaisankar; Shreya Dasgupta; Tuhinadri Sen; Ratan Gachhui; Ujjal Kumar Mukhopadhyay; Joydeep Mukherjee (pp. 109-117).

A marine actinobacterium isolated from the Bay of Bengal, India and previously found to be producing an antimicrobial and cytotoxic terpenoid was further investigated for antimicrobial metabolites. The bacterium was preliminarily identified as a new species of the genus Streptomyces (strain MS1/7). The cell-free culture broth was extracted with n-butanol and purified using silica gel column chromatography and high-performance liquid chromatography. Molecular characterization was done using ESI mass, IR and ¹H and ¹³C NMR spectrometry. 2-Allyloxyphenol (MW 150; C₉H₁₀O₂), a synthetic drug and chemical intermediate, was obtained as a natural product for the first time. Serendipitous natural occurrence provided new insights into the synthetic molecule. 2-Allyloxyphenol was found to be inhibitory to 21 bacteria and three fungi in the minimum range 0.2–1.75 mg mL⁻¹ determined by agar dilution method. 2-Allyoxyphenol possesses strong antioxidant property (IC₅₀ 22 μg mL⁻¹, measured by 1, 1-diphenyl-2-picryl hydrazyl scavenging activity). Hydroxyl and allyloxy groups in 2-allyloxyphenol were responsible for antimicrobial and antioxidant activities. 2-Allyloxyphenol has marked resemblance to smoky aroma and is two to three times more active as an antimicrobial than some commercial smoke-flavour compounds. Absence of hemolytic toxicity, potential carcinogenicity, cytotoxicity and reports of toxic reactions in literature suggest possible application of 2-allyloxyphenol as a food preservative and an oral disinfectant.

Keywords: Sundarbans ; 2-Allyloxyphenol; Streptomyces ; Antimicrobial; Antioxidant; Smoke flavour

Chitinase of Bacillus licheniformis from oyster shell as a probe to detect chitin in marine shells by Shah Md. Asraful Islam; Kye Man Cho; Sun Joo Hong; Renukaradhya K. Math; Jong Min Kim; Myoung Geun Yun; Ji Joong Cho; Jae Young Heo; Young Han Lee; Hoon Kim; Han Dae Yun (pp. 119-129).

Bacillus licheniformis CBFOS-03 is a chitinase producing bacteria isolated from oyster (Crassostrea gigas) shell waste. We have cloned and expressed the chi18B gene of B. licheniformis CBFOS-03, which encodes a glycohydrolase family 18 chitinase (GH18). Chi18B is a predicted 598 amino acid protein that consists of a catalytic domain (GH18), a fibronectin type III domain (Fn3), and a chitin binding domain (CBD). Purified Chi18B showed optimum chitinase activity at pH 9 and 55 °C, and activity was stimulated with 25 mM Mn²⁺. In kinetic analysis, Chi18B showed Km values of 9.07 ± 0.65 μM and 129.27 ± 0.38 μM with the substrates 4-methylumbelliferyl-N-N′-diacetylchitobiose and α-chitin, respectively. Studies of C-terminal deletion constructs revealed that the GH18 domain with one amino acid in C-terminal region was sufficient for chitinase activity; however, fusions of full length and CBD-deleted constructs to green florescent protein (GFP) and yellow florescent protein (YFP) suggest that the C-terminus is supposedly important in binding to shell powder. Full length Chi18B with GFP showed green fluorescence with oyster shell powder, but GH18+Fn3 with GFP did not. Similarly, full length Chi18B with YFP showed yellow fluorescence with clam (Chamelea gallina) shell and disk abalone (Haliotis discus) shell powder, but GH18+Fn3 with YFP construct did not. So, the CBD domain of Chi18B appears to play an important role in binding of oyster and other marine shells. It is likely to be used as a probe to identify the presence of chitin in marine shells like oyster shell, clam shell, and disk abalone shell using fusions of Chi18B with fluorescent proteins.

Keywords: Oyster shell; Bacillus licheniformis ; Chitinase; Chitin binding domain; Fibronectin; Enzyme binding

A novel α-amylase from the cyanobacterium Nostoc sp. PCC 7119 by Francisco M. Reyes-Sosa; Fernando P. Molina-Heredia; Miguel A. De la Rosa (pp. 131-141).

Little information is yet available on the α-amylases of cyanobacteria. Here, the presence of an α-amylase in the cyanobacterium Nostoc sp. PCC 7119 is first demonstrated. A gene (amy1) encoding a cytoplasmic α-amylase (Amy1) protein has been identified, cloned, and overexpressed in Escherichia coli cells. The recombinant protein is a 56.7-kDa monomer, which has been purified to electrophoretic homogeneity by affinity chromatography. The substrate specificity and end product analyses confirm that it is a calcium-dependent α-amylase enzyme, which exhibits its maximum activity at 31°C and at pH between 6.5 and 7.5. The Amy1 protein breaks down mainly starch, is also able to cleave glycogen and dextrin, and exhibits no activity against xylan or pullulan. So the enzyme cannot efficiently attack the maltodextrins with degrees of polymerization below that of maltooctaose. Maltotriose, maltose, and maltotetraose are the major products of the enzymatic reaction with starch as substrate. The enzyme shows a very high turnover number against soluble potato starch (3,420 ± 270 s⁻¹), as compared with other α-amylases reported in the literature. The high catalytic efficiency and relatively low optimum temperature of the Nostoc Amy1 protein make this previously unexplored group of cyanobacterial enzymes of great interest for further physiological studies and industrial applications.

Keywords: Affinity chromatography; α-amylase; Cyanobacteria; Endoglycosyl hydrolase; Heterologous expression; Nostoc ; Starch

Characterization and kinetic analysis of a thermostable GH3 β-glucosidase from Penicillium brasilianum by Kristian B. R. M. Krogh; Paul V. Harris; Carsten L. Olsen; Katja S. Johansen; Jesper Hojer-Pedersen; Johan Borjesson; Lisbeth Olsson (pp. 143-154).

A GH3 β-glucosidase (BGL) from Penicillium brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated temperatures with no loss in activity after 24 h of incubation at 60°C at pH 4–6, and the BGL was shown to have significantly higher stability at these conditions in comparison to Novozym 188 and to other fungal GH3 BGLs reported in the literature. The BGL had significant lower affinity for cellobiose compared with the artificial substrate para-nitrophenyl-β-d-glucopyranoside (pNP-Glc) and further, pronounced substrate inhibition using pNP-Glc. Kinetic studies demonstrated the high importance of using cellobiose as substrate and glucose as inhibitor to describe the inhibition kinetics of BGL taking place during cellulose hydrolysis. A novel assay was developed to characterize this glucose inhibition on cellobiose hydrolysis. The assay uses labelled glucose-¹³C₆ as inhibitor and subsequent mass spectrometry analysis to quantify the hydrolysis rates.

Keywords: Cellulose hydrolysis; Glucose inhibition; Purification; Fungal expression; Kinetics

Characterization of a chimeric enzyme comprising feruloyl esterase and family 42 carbohydrate-binding module by Takuya Koseki; Keiji Mochizuki; Hiroe Kisara; Akimasa Miyanaga; Shinya Fushinobu; Tetsuya Murayama; Yoshihito Shiono (pp. 155-161).

We engineered a chimeric enzyme (AwFaeA-CBM42) comprising of type-A feruloyl esterase from Aspergillus awamori (AwFaeA) and family 42 carbohydrate-binding module (AkCBM42) from glycoside hydrolase family 54 α-l-arabinofuranosidase of Aspergillus kawachii. The chimeric enzyme was successfully produced in Pichia pastoris and accumulated in the culture broth. The purified chimeric enzyme had an apparent relative molecular mass (M _r) of 53,000. The chimeric enzyme binds to arabinoxylan; this indicates that the AkCBM42 in AwFaeA-CBM42 binds to arabinofuranose side chain moiety of arabinoxylan. The thermostability of the chimeric enzyme was greater than that of AwFaeA. No significant difference of the specific activity toward methyl ferulate was observed between the AwFaeA and chimeric enzyme, but the release of ferulic acid from insoluble arabinoxylan by the chimeric enzyme was approximately 4-fold higher than that achieved by AwFaeA alone. In addition, the chimeric enzyme and xylanase acted synergistically for the degradation of arabinoxylan. In conclusion, the findings of our study demonstrated that the components of the AwFaeA-CBM42 chimeric enzyme act synergistically to bring about the degradation of complex substrates and that the family 42 carbohydrate-binding module has potential for application in the degradation of polysaccharides.

Keywords: Chimeric enzyme; Feruloyl esterase; α-l-arabinofuranosidase; CBM42; Arabinoxylan

A cytochrome P450 class I electron transfer system from Novosphingobium aromaticivorans by Stephen G. Bell; Alison Dale; Nicholas H. Rees; Luet-Lok Wong (pp. 163-175).

Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from Novosphingobium aromaticivorans are heme monooxygenases that catalyze the hydroxylation of a range of terpenoid compounds. CYP101D1 and CYP101D2 oxidized camphor to 5-exo-hydroxycamphor. CYP101B1 and CYP101C1 oxidized β-ionone to predominantly 3-R-hydroxy-β-ionone and 4-hydroxy-β-ionone, respectively. CYP111A2 oxidized linalool to 8-hydroxylinalool. Physiologically, these CYP enzymes could receive electrons from Arx, a [2Fe-2S] ferredoxin equivalent to putidaredoxin from the CYP101A1 system from Pseudomonas putida. A putative ferredoxin reductase (ArR) in the N. aromaticivorans genome, with high amino acid sequence homology to putidaredoxin reductase, has been over-produced in Escherichia coli and found to support substrate oxidation by these CYP enzymes via Arx with both high activity and coupling of product formation to NADH consumption. The ArR/Arx electron-transport chain has been co-expressed with the CYP enzymes in an E. coli host to provide in vivo whole-cell substrate oxidation systems that could produce up to 6.0 g L⁻¹ of 5-exo-hydroxycamphor at rates of up to 64 μM (gram of cell dry weight)⁻¹ min⁻¹. These efficient biocatalytic systems have potential uses in preparative scale whole-cell biotransformations.

Keywords: Cytochrome P450; Novosphingobium aromaticivorans ; Electron transfer; Ferredoxin reductase; Whole-cell biotransformations

Characterization of two proline dipeptidases (prolidases) from the hyperthermophilic archaeon Pyrococcus horikoshii by Casey M. Theriot; Sherry R. Tove; Amy M. Grunden (pp. 177-188).

Prolidases hydrolyze the unique bond between X-Pro dipeptides and can also cleave the P–F and P–O bonds found in organophosphorus compounds, including the nerve agents, soman and sarin. The advantages of using hyperthermophilic enzymes in biodetoxification strategies are based on their enzyme stability and efficiency. Therefore, it is advantageous to examine new thermostable prolidases for potential use in biotechnological applications. Two thermostable prolidase homologs, PH1149 and PH0974, were identified in the genome of Pyrococcus horikoshii based on their sequences having conserved metal binding and catalytic amino acid residues that are present in other known prolidases, such as the previously characterized Pyrococcus furiosus prolidase. These P. horikoshii prolidases were expressed recombinantly in the Escherichia coli strain BL21 (λDE3), and both were shown to function as proline dipeptidases. Biochemical characterization of these prolidases shows they have higher catalytic activities over a broader pH range, higher affinity for metal and are more stable compared to P. furiosus prolidase. This study has important implications for the potential use of these enzymes in biotechnological applications and provides further information on the functional traits of hyperthermophilic proteins, specifically metalloenzymes.

Keywords: Prolidase; Pyrococcus horikoshii ; Hyperthermophile; Metalloenzyme; Cobalt enzyme

Modular organisation and functional analysis of dissected modular β-mannanase CsMan26 from Caldicellulosiruptor Rt8B.4 by Anwar Sunna (pp. 189-200).

CsMan26 from Caldicellulosiruptor strain Rt8.B4 is a modular β-mannanase consisting of two N-terminal family 27 carbohydrate-binding modules (CBMs), followed by a family 35 CBM and a family 26 glycoside hydrolase catalytic module (mannanase). A functional dissection of the full-length CsMan26 and a comprehensive characterisation of the truncated derivatives were undertaken to evaluate the role of the CBMs. Limited proteolysis was used to define biochemically the boundaries of the different structural modules in CsMan26. The full-length CsMan26 and three truncated derivatives were produced in Escherichia coli, purified and characterised. The systematic removal of the CBMs resulted in a decrease in the optimal temperature for activity and in the overall thermostability of the derivatives. Kinetic experiments indicated that the presence of the mannan-specific family 27 CBMs increased the affinity of the enzyme towards the soluble galactomannan substrate but this was accompanied by lower catalytic efficiency. The full-length CsMan26 and its truncated derivatives were unable to hydrolyse mannooligosaccharides with degree of polymerisation (DP) of three or less. The major difference in the hydrolysis pattern of larger mannooligosaccharides (DP >3) by the derivatives was determined by their abilities to further hydrolyse the intermediate sugar mannotetraose.

Keywords: Modular enzyme; β-Mannanase; Carbohydrate-binding module; Catalytic module; Limited proteolysis

Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1 by Laura Saa; Arrate Jaureguibeitia; Eneko Largo; María J. Llama; Juan L. Serra (pp. 201-211).

Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His₆PheA1 and His₆PheA2 were purified and its catalytic activity characterized. His₆PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His₆PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His₆PheA1 and His₆PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.

Keywords: Phenol hydroxylase; Cloning; Purification; Characterization; Rhodococcus erythropolis UPV-1

Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production and characterization of an Aspergillus oryzae catechol oxidase by Chiara Gasparetti; Greta Faccio; Mikko Arvas; Johanna Buchert; Markku Saloheimo; Kristiina Kruus (pp. 213-226).

A homology search against public fungal genome sequences was performed to discover novel secreted tyrosinases. The analyzed proteins could be divided in two groups with different lengths (350–400 and 400–600 residues), suggesting the presence of a new class of secreted enzymes lacking the C-terminal domain. Among them, a sequence from Aspergillus oryzae (408 aa, AoCO4) was selected for production and characterization. AoCO4 was expressed in Trichoderma reesei under the strong cbh1 promoter. Expression of AoCO4 in T. reesei resulted in high yields of extracellular enzyme, corresponding to 1.5 g L⁻¹ production of the enzyme. AoCO4 was purified with a two-step purification procedure, consisting of cation and anion exchange chromatography. The N-terminal analysis of the protein revealed N-terminal processing taking place in the Kex2/furin-type protease cleavage site and removing the first 51 amino acids from the putative N-terminus. AoCO4 activity was tested on various substrates, and the highest activity was found on 4-tert-butylcatechol. Because no activity was detected on L-tyrosine and on l-dopa, AoCO4 was classified as a catechol oxidase. AoCO4 showed the highest activity within an acidic and neutral pH range, having an optimum at pH 5.6. AoCO4 showed good pH stability within a neutral and alkaline pH range and good thermostability up to 60°C. The UV–visible and circular dichroism spectroscopic analysis suggested that the folding of the protein was correct.

Keywords: Fungal; Catechol oxidase; Tyrosinase; Secreted; Aspergillus ; Trichoderma

Overexpression and molecular characterization of Aga50D from Saccharophagus degradans 2-40: an exo-type β-agarase producing neoagarobiose by Hee Taek Kim; Saeyoung Lee; Dongho Lee; Hyun-Soo Kim; Won-Gi Bang; Kyoung Heon Kim; In-Geol Choi (pp. 227-234).

β-Agarases are mostly categorized into three glycoside hydrolase (GH) families 16, 50, and 86. Recent genomic analysis of Saccharophagus degradans 2–40 revealed the presence of five agarase genes belonging to these GH families. Among the five agarases, Aga50D (a member of GH50) had neither been functionally characterized nor overexpressed. In this report, we present soluble overexpression and molecular characterization of Aga50D. Aga50D was expressed in an active form resulting in a single major product from agarose without intermediates. While known GH50 agarases have both endo-lytic and exo-lytic activities, which produce neoagarobiose as a final product through the intermediate, neoagaro-oligosaccharides, identification and analysis of the reaction product by mass spectrometry and ¹³C NMR showed that Aga50D had unique exo-lytic activity and was able to produce neoagarobiose directly from agarose. The optimum pH and temperature for the activity were 7.0 and 30°C, respectively. The K _m and V _max for agarose were 41.9 mg/ml (4.2 mM) and 17.9 U/mg, respectively.

Keywords: β-Agarase; GH50; Exo-lytic activity; Neoagarobiose; Saccharification

Proline reduces the binding of transcriptional regulator ArgR to upstream of argB in Corynebacterium glutamicum by Soo Youn Lee; Hwa Sung Shin; Jin-Soo Park; Yang-Hoon Kim; Jiho Min (pp. 235-242).

In this study, the ArgR-binding sites on the arg operon Corynbebacterium glutamicum were characterized by in vivo chromatin immunoprecipitation (ChIP). In addition, the ArgR-binding affinity in the presence of glutamate, proline, or arginine was examined to get further information on expression control. The ChIP assay showed that the ArgR protein binds specifically to the upstream regions of argC, argB, argF, and argG. Upon proline supplementation, ArgR-binding affinity was significantly reduced upstream of argB, resulting in increased ornithine production. In contrast, there was no change in the binding affinity of ArgR to the upstream regions of argC, argF, argG, or argB following the addition of glutamate and arginine. These results suggest that the upstream region of argB on the arg operon plays an important role in interacting with ArgR under proline-supplemented conditions and that proline causes an increase in the endogenous level of ornithine by reducing the binding affinity of ArgR to the upstream region of argB.

Keywords: Proline; Upstream of argB; Corynebacterium glutamicum ; ChIP assay; Ornithine

Improved secretion of the cancer-testis antigen SSX2 in Pichia pastoris by deletion of its nuclear localization signal by Chung-Jr Huang; Kyle A. Anderson; Leonardo M. Damasceno; Gerd Ritter; Lloyd J. Old; Carl A. Batt (pp. 243-253).

The cancer-testis (CT) antigen synovial sarcoma X break point 2 (SSX2) was expressed in Pichia pastoris as a means to produce a delayed-type hypersensitivity skin test reagent for monitoring SSX2-specific anti-cancer immune responses. SSX2 was detected intracellularly in P. pastoris despite the addition of the Saccharomyces cerevisiae alpha-mating factor secretion signal. Increasing the SSX2 gene copy number did not improve its secretion but did enhance intracellular SSX2 levels. SSX2 with its C-terminal nuclear localization signal (NLS) deleted (SSX2NORD), however, was secreted. Indirect immunofluorescence indicated that SSX2 containing the NLS did not translocate to the nucleus but accumulated in the endoplasmic reticulum (ER). Experimental results further suggested that SSX2 containing the NLS was misfolded in the ER, while deletion of the NLS facilitated correct folding of SSX2 inside the ER and improved its secretion. Production of SSX2NORD was scaled-up to a 2-L fermentor using a fed-batch protocol to maintain methanol at a concentration of 1 g L⁻¹. Decreasing the cultivation temperature from 25 °C to 16 °C improved protein stability in the culture supernatant. In this process, after 120 h cultivation, the wet cell weight of P. pastoris reached 280 mg mL⁻¹, and the yield of SSX2NORD was 21.6 mg L⁻¹.

Keywords: Cancer-testis antigen; SSX2; Recombinant protein secretion; Pichia pastoris ; Nuclear localization signal; Delayed-type hypersensitivity

Alteration of hydrogen metabolism of ldh-deleted Enterobacter aerogenes by overexpression of NAD(+)-dependent formate dehydrogenase by Yuan Lu; Hongxin Zhao; Chong Zhang; Qiheng Lai; Xi Wu; Xin-Hui Xing (pp. 255-262).

The NAD⁺-dependent formate dehydrogenase FDH1 gene (fdh1), cloned from Candida boidinii, was expressed in the ldh-deleted mutant of Enterobacter aerogenes IAM1183 strain. The plasmid of pCom10 driven by the PalkB promoter was used to construct the fdh1 expression system and thus introduce a new dihydronicotinamide adenine dinucleotide (NADH) regeneration pathway from formate in the ldh-deleted mutant. The knockout of NADH-consuming lactate pathway affected the whole cellular metabolism, and the hydrogen yield increased by 11.4% compared with the wild strain. Expression of fdh1 in the ldh-deleted mutant caused lower final cell concentration and final pH after 16 h cultivation, and finally resulted in 86.8% of increase in hydrogen yield per mole consumed glucose. The analysis of cellular metabolites and estimated redox state balance in the fdhl-expressed strain showed that more excess of reducing power was formed by the rewired NADH regeneration pathway, changing the metabolic distribution and promoting the hydrogen production.

Keywords: Anaerobic fermentation; Enterobacter aerogenes ; Formate dehydrogenase; Hydrogen production; Lactate dehydrogenase; NADH regeneration

N- and O-linked oligosaccharides completely lack galactose residues in the gms1och1 mutant of Schizosaccharomyces pombe by Takao Ohashi; Kaoru Takegawa (pp. 263-272).

Unlike their counterparts in budding yeast Saccharomyces cerevisiae, the glycoproteins of Schizosaccharomyces pombe contain, in addition to α-d-mannose (Man), a large number of α-d-galactose (Gal) residues. In both yeasts, large outer chains are attached to the oligosaccharide cores of glycoproteins during export via Golgi. Formation of the yeast-specific large outer chain is initiated by α-1,6-mannosylatransferase encoded by the och1 ⁺ gene, the disruption of which blocked outer chain elongation. We previously reported that N-linked oligosaccharide structures of S. pombe och1Δ mutant consisted of Gal_2–6Man₉GlcNAc₂ with α-linked Gal residues attached to the core oligosaccharide moiety. The disruption of gms1 ⁺, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, abolished cell surface galactosylation in S. pombe. In this study, we constructed a gms1Δoch1Δ double mutant and determined the N- and O-linked oligosaccharide structures present on the cell surface. Oligosaccharides were liberated from glycoproteins by hydrazinolysis and labeled with the fluorophore, 2-aminopyridine. The pyridylaminated N-linked oligosaccharides were analyzed by high-performance liquid chromatography in combination with α1,2-mannosidase digestion and partial acetolysis. These analyses revealed that the N-linked oligosaccharides of gms1Δoch1Δ cells consisted of α1,2-linked Man-extended core oligosaccharides (Man_8–12GlcNAc₂) from which the fission yeast-specific α-linked Gal residues were completely absent.

Keywords: Schizosaccharomyces pombe ; Gms1; Och1; N-Linked oligosaccharide; Galactosylation

Hyaluronic acid production is enhanced by the additional co-expression of UDP-glucose pyrophosphorylase in Lactococcus lactis by Shashi Bala Prasad; Guhan Jayaraman; K. B. Ramachandran (pp. 273-283).

Hyaluronic acid (HA) production was metabolically engineered in Lactococcus lactis by introducing the HA synthetic machinery from the has operon of the pathogenic bacterium Streptococcus zooepidemicus. This study shows that the insertion of uridine diphosphate (UDP)-glucose pyrophosphorylase (hasC) gene in addition to the HA synthase (hasA) and UDP-glucose dehydrogenase (hasB) genes has a significant impact on increasing HA production. The recombinant L. lactis NZ9000 strain transformed with the plasmid pSJR2 (co-expressing hasA and hasB genes only) produced a maximum of 107 mg/l HA in static flask experiments with varying initial glucose concentrations, while the corresponding experiments with the transformant SJR3 (co-expressing hasA, hasB, and hasC genes) gave a maximum yield of 234 mg/l HA. The plasmid cloned with the insertion of the full has operon comprising of five different genes (hasA, hasB, hasC, hasD, and hasE) exhibited structural instability. The HA yield was further enhanced in batch bioreactor experiments with controlled pH and aeration, and a maximum of 1.8 g/l HA was produced by the SJR3 culture.

Keywords: Hyaluronic acid; Metabolic engineering; Lactococcus lactis ; UDP-glucose pyrophosphorylase; hasC gene

Distinguishable codon usage and amino acid composition patterns among substrates of leaderless secretory pathways from proteobacteria by Ināra Kampenusa; Pēteris Zikmanis (pp. 285-293).

The combined set of codon usage frequencies (61 sense codons) from the 111 annotated sequences of leaderless secreted type I, type III, type IV, and type VI proteins from proteobacteria were subjected to the forward and backward selection to obtain a combination of most effective predictor variables for classification/prediction purposes. The group of 24 codon frequencies displayed a strong discriminatory power with an accuracy of 100% for originally grouped and 97.3 ± 1.6% for cross-validated (LOOCV) cases and an acceptable error rate (0.062 ± 0.012) in k-fold (k = 6) cross-validation (KCV). The summary frequencies of synonymous codons for ten amino acids as the alternative predictor variables revealed a comparable discriminatory power (92.8 ± 2.5% for LOOCV), however at somewhat lower levels of prediction accuracy (0.106 ± 0.015 of KCV). A number of significant (p < 0.001) differences were found among indices of codon usage and amino acid composition depending on a definite secretion type. About 60% of secretion substrates were characterized as apparently originated from horizontal gene transfer events or putative alien genes and found to be unequally allocated in respect of groups. The proposed prediction approaches could be used to specify secretome proteins from genomic sequences as well as to assess the compatibility between bacterial secretion pathways and secretion substrates.

Keywords: Protein secretion; Codon usage bias; Amino acid composition; Discriminant analysis; Proteobacteria

Biosynthetic pathway of sugar nucleotides essential for welan gum production in Alcaligenes sp. CGMCC2428 by Hui Li; Hong Xu; Hao Xu; Sha Li; Ping-Kai Ouyang (pp. 295-303).

Welan gum is a microbial polysaccharide produced by Alcaligenes sp. CGMCC2428 that has d-glucose, d-glucuronic acid, d-glucose, and l-rhamnose as the main structural unit. The biosynthetic pathway of sugar nucleotides essential for producing welan gum in this strain was established in the following ways: (1) the detection of the presence of several intermediates and key enzymes; (2) the analysis of the response upon addition of precursors to the culture medium; (3) the correlation of the activities between several key enzymes with the yields of welan gum. With addition of 200-μM glucose-6-phosphate and fructose-6-phosphate, the production of welan gum was improved by 18%. The activities of phosphoglucomutase, phosphomannose isomerase, UDP-glucose pyrophosphorylase, and dTDP-glucose pyrophosphorylase, correlated well with the yields of welan gum. According to these findings, the biosynthetic pathway was proposed to involve the metabolism of glucose via two discrete systems. The first involves conversion of glucose to glucose-6-phosphate, with further reactions producing glucose-1-phosphate and fructose-6-phosphate, which are metabolized to the nucleotide sugar precursors of welan gum. The second system involves metabolism of glucose to synthesize the basic structural skeleton of the cell via central metabolic pathways, including the Entner–Doudoroff pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle.

Keywords: Alcaligenes sp. CGMCC2428; Welan gum; Sugar nucleotides; Biosynthetic pathway

Potential therapeutic efficacy of a bactericidal–immunomodulatory fusion peptide against methicillin-resistant Staphylococcus aureus skin infection by Qingtian Li; Yuhua Zhou; Ke Dong; Xiaokui Guo (pp. 305-309).

To enhance the potential therapeutic efficacy of an antimicrobial peptide human β-defensin 3, two fusion peptides, a bactericidal–immunomodulatory fusion peptide human β-defensin 3-mannose-binding lectin and a bactericidal–bactericidal fusion peptide human β-defensin 3-lysozyme were synthesized and the bactericidal activities in vitro and in vivo against methicillin-resistant Staphylococcus aureus N315 were demonstrated in this study. Peptide human β-defensin 3-lysozyme showed the best bactericidal activity in vitro, but human β-defensin 3-mannose-binding lectin showed a significant improvement in angiogenesis and tissue reconstruction. Our results illustrated that outstanding bactericidal activity in vitro is not essential in the development of antimicrobial peptides. Fusion strategy and immunomodulatory factors should be utilized in novel antimicrobial peptide development.

Keywords: Fusion peptide; MRSA; Antimicrobial peptide; Human ß-defensin 3

An intracellular pH gradient in the anammox bacterium Kuenenia stuttgartiensis as evaluated by ³¹P NMR by Wouter R. L. van der Star; Cor Dijkema; Pieter de Waard; Cristian Picioreanu; Marc Strous; Mark C. M. van Loosdrecht (pp. 311-317).

The cytoplasm of anaerobic ammonium oxidizing (anammox) bacteria consists of three compartments separated by membranes. It has been suggested that a proton motive force may be generated over the membrane of the innermost compartment, the “anammoxosome”. ³¹P nuclear magnetic resonance (NMR) spectroscopy was employed to investigate intracellular pH differences in the anammox bacterium Kuenenia stuttgartiensis. With in vivo NMR, spectra were recorded of active, highly concentrated suspensions of K. stuttgartiensis in a wide-bore NMR tube. At different external pH values, two stable and distinct phosphate peaks were apparent in the recorded spectra. These peaks were equivalent with pH values of 7.3 and 6.3 and suggested the presence of a proton motive force over an intracytoplasmic membrane in K. stuttgartiensis. This study provides for the second time—after discovery of acidocalcisome-like compartments in Agrobacterium tumefaciens—evidence for an intracytoplasmic pH gradient in a chemotrophic prokaryotic cell.

Keywords: Anammox; Kuenenia stuttgartiensis ; Internal pH; In vivo NMR; Nuclear magnetic resonance spectroscopy; ³¹P NMR

Lactic acid bacteria display on the cell surface cytosolic proteins that recognize yeast mannan by Yoshio Katakura; Ryosuke Sano; Takashi Hashimoto; Kazuaki Ninomiya; Suteaki Shioya (pp. 319-326).

Fluorescent-labeled invertase, a hyperglycosylated mannoprotein from Saccharomyces cerevisiae, was found to bind to Lactococcus lactis IL1403 at acidic pH. Proteins on the cell wall of the bacterium affinity-purified using invertase as a ligand were identified to be heat shock proteins such as DnaK and GroEL and glycolytic enzymes such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase. DnaK bound to both the bacterium and yeast at pH 4 and aggregated them at above 0.1 mg/ml, whereas no significant difference between the circular dichroism spectra of DnaK at pH 4 and 7 was observed. These results indicate that the cytosolic proteins, including DnaK displayed on the cell wall, cause the lactic acid bacterium to adhere to the yeast.

Keywords: Lactic acid bacteria; Yeast; Mannan; Cell wall; Symbiosis; DnaK; Adhesion

Oxygen-induced expression of ∆⁶-, ∆⁹- and ∆¹²-desaturase genes modulates fatty acid composition in Mucor rouxii by Rawisara Ruenwai; Supapon Cheevadhanarak; Sansanalak Rachdawong; Morakot Tanticharoen; Kobkul Laoteng (pp. 327-334).

The effect of oxygen availability on the molecular mechanisms of fatty acid biosynthesis was investigated in Mucor rouxii, a Mucorale fungus capable of producing γ-linolenic acid through perturbation of the gaseous environment. Shifting of the M. rouxii culture from anaerobic to aerobic conditions resulted in an increase of the biomass and total fatty acid content of the M. rouxii culture. In addition, the levels of unsaturated fatty acids were enhanced accompanied by a decrease in the levels of medium- and long-chain saturated fatty acids. These results correspond to the levels of expressions of the ∆⁹-, ∆¹²- and ∆⁶-desaturases genes, all of which were coordinately up-regulated after the shift. The transcriptional response observed was rapid and transient, with the maximal mRNA levels detected between 0.5 h and 1.0 h after the shift. Together, our findings indicate that the anaerobic M. rouxii culture acclimatised to oxygen exposure by modulating fatty acid composition that was transcriptionally co-regulated by the ∆⁹-, ∆¹²- and ∆⁶-desaturase genes.

Keywords: Mucor rouxii ; Desaturase; Oxygen; Gene expression; Gamma-linolenic acid

Description of the novel perchlorate-reducing bacteria Dechlorobacter hydrogenophilus gen. nov., sp. nov. and Propionivibrio militaris, sp. nov. by J. Cameron Thrash; Jarrod Pollock; Tamas Torok; John D. Coates (pp. 335-343).

Novel dissimilatory perchlorate-reducing bacteria (DPRB) were isolated from enrichments conducted under conditions different from those of all previously described DPRB. Strain LT-1^T was enriched using medium buffered at pH 6.6 with 2-(N-morpholino)ethanesulfonic acid (MES) and had only 95% 16S rRNA gene identity with its closest relative, Azonexus caeni. Strain MP^T was enriched in the cathodic chamber of a perchlorate-reducing bioelectrical reactor (BER) and together with an additional strain, CR (99% 16S rRNA gene identity), had 97% 16S rRNA gene identity with Propionivibrio limicola. The use of perchlorate and other electron acceptors distinguished strains MP^T and CR from P. limicola physiologically. Strain LT-1^T had differences in electron donor utilization and optimum growth temperatures from A. caeni. Strains LT-1^T and MP^T are the first DPRB to be described in the Betaproteobacteria outside of the Dechloromonas and Azospira genera. On the basis of phylogenetic and physiological features, strain LT-1^T represents a novel genus in the Rhodocyclaceae; strain MP^T represents a novel species within the genus Propionivibrio. The names Dechlorobacter hydrogenophilus gen. nov., sp. nov and Propionivibrio militaris sp. nov. are proposed.

Biotransformation of fluorobiphenyl by Cunninghamella elegans by Jessica Amadio; Cormac D. Murphy (pp. 345-351).

The fungus Cunninghamella elegans is a useful model of human catabolism of xenobiotics. In this paper, the biotransformation of fluorinated biphenyls by C. elegans was investigated by analysis of the culture supernatants with a variety of analytical techniques. 4-Fluorobiphenyl was principally transformed to 4-fluoro-4′-hydroxybiphenyl, but other mono- and dihydroxylated compounds were detected in organic extracts by gas chromatography–mass spectrometry. Additionally, fluorinated water-soluble products were detected by ¹⁹F NMR and were identified as sulphate and β-glucuronide conjugates. Other fluorobiphenyls (2-fluoro-, 4,4′-difluoro- and 2,3,4,5,6-pentafluoro-biphenyl) were catabolised by C. elegans, yielding mono- and dihydroxylated products, but phase II metabolites were detected from 4,4′-difluorobiphenyl only.

Keywords: Fluorine; Biphenyl; Metabolism; F-19 NMR

Functional consortium for denitrifying sulfide removal process by Chuan Chen; Nanqi Ren; Aijie Wang; Lihong Liu; Duu-Jong Lee (pp. 353-358).

Denitrifying sulfide removal (DSR) process simultaneously converts sulfide, nitrate, and chemical oxygen demand from industrial wastewaters to elemental sulfur, nitrogen gas, and carbon dioxide, respectively. This investigation utilizes a dilution-to-extinction approach at 10⁻² to 10⁻⁶ dilutions to elucidate the correlation between the composition of the microbial community and the DSR performance. In the original suspension and in 10⁻² dilution, the strains Stenotrophomonas sp., Thauera sp., and Azoarcus sp. are the heterotrophic denitrifiers and the strains Paracoccus sp. and Pseudomonas sp. are the sulfide-oxidizing denitrifers. The 10⁻⁴ dilution is identified as the functional consortium for the present DSR system, which comprises two functional strains, Stenotrophomonas sp. strain Paracoccus sp. At 10⁻⁶ dilution, all DSR performance was lost. The functions of the constituent cells in the DSR granules were discussed based on data obtained using the dilution-to-extinction approach.

Keywords: Denitrifying sulfide removal; Functional consortium; Denitrifier

Effects of nitrite inhibition on anaerobic ammonium oxidation by Yuya Kimura; Kazuichi Isaka; Futaba Kazama; Tatsuo Sumino (pp. 359-365).

In order to assess the stability of nitrogen removal systems utilizing anaerobic ammonium oxidation (anammox), it is necessary to study the toxic effects of nitrite on these biochemical reactions. In this study, the effects of nitrite on anammox bacteria entrapped in gel carriers were investigated using batch and continuous feeding tests. The results showed that the nitrite concentration in a reactor must be less than 274-mg N/L in order to prevent a decrease in the anammox activity, which occurred when the gel carriers were soaked in nitrite solutions with concentrations greater than 274-mg N/L in a batch test. In a continuous feeding test, nitrite inhibition was not observed at low concentrations of nitrite. However, the anammox activity decreased to 10% when the nitrite concentration increased to 750-mg N/L over a 7-day period in the reactor. In addition, it was shown that the effects of nitrogen on the anammox reaction were reversible because the anammox activity completely recovered within 3 days when the influent nitrite concentration was decreased to less than 274-mg N/L.

Keywords: Anammox; Nitrite; Ammonium; Denitrification; Immobilization

Microbial community structure changes during oyster mushroom substrate preparation by Balázs Vajna; Adrienn Nagy; Enikő Sajben; László Manczinger; Nóra Szijártó; Zsófia Kádár; Diána Bordás; Károly Márialigeti (pp. 367-375).

Although oyster mushroom (Pleurotus spp.) is a valuable food, cultivated worldwide on an industrial scale, still very little is known about the microbial dynamics during oyster mushroom substrate preparation. Therefore, the characterization of the microbial dynamics by chemical and biological tools was the objective of this study. During substrate preparation, enzymatic digestibility of the substrate improved by 77%, whereas the cellulose and hemicellulose to lignin ratios decreased by 9% and 19%, respectively. Fluorescein diacetate hydrolysis reached its minimum value at the temperature maximum of the process during the composting phase and exceeded the initial level at the end of the process. Fungal species played part in the initial mesophilic phase of the substrate preparation process, but they disappeared after pasteurization in tunnels at constant elevated temperatures. Changes in the microbiota showed a marked bacterial community succession during substrate preparation investigated by 16S ribosomal deoxyribonucleic acid-based terminal restriction fragment length polymorphism (T-RFLP). Mature samples represented the least variance, which indicated the effect of the standardized preparation protocol. The relation between mushroom yield and the bacterial community T-RFLP fingerprints was investigated, but the uniformity of mushroom yields did not support any significant correlation.

Keywords: Oyster mushroom; Substrate production; Microbial succession

Method to detect only viable cells in microbial ecology by Jian-Fei Luo; Wei-Tie Lin; Yong Guo (pp. 377-384).

Propidium monoazide can limit the analysis of microbial communities derived from genetic fingerprints to viable cells with intact cell membranes. However, PMA treatment cannot completely suppress polymerase chain reaction (PCR) amplification when the targeted gene is too short. PMA treatment in combination with two-step nested PCR was designed to overcome this problem. Four experiments were performed to determine the limitation of PMA treatment and to evaluate the suitability of the method by applying the following samples: (1) pure cultures of Escherichia coli O157:H7, Enterobacter aerogenes, and Alcaligenes faecalis; (2) pond water samples spiked with heat-killed E. coli O157:H7 and E. aerogenes; (3) anaerobic sludge samples exposed to increasing heat stress; and (4) selected natural samples of estuarine sediment and lake mud. Results from the first two experiments show that PMA treatment cannot efficiently suppress dead cells from PCR amplification when the targeted gene is as short as 190 bp, however, the two-step nested PCR can overcome this problem. The last two experiments indicate the method that PMA treatment in combination with two-step nested PCR is useful for viable cells detection in microbial ecology.

Keywords: Propidium monoazide (PMA); Denaturing gradient gel electrophoresis (DGGE); Viable cells; Microbial community; Environmental samples

A standard quantitative method to measure acid tolerance of probiotic cells by Eng-Seng Chan; Peh-Phong Lee; Pogaku Ravindra; Kamatan Krishnaiah; Wan-Ping Voo (pp. 385-391).

The aim of this work was to develop a standard quantitative method to measure the acid tolerance of probiotic cells when exposed to a simulated gastric fluid. Three model strains of different cell concentrations were exposed to a standard simulated gastric fluid of fixed volume. The fluid pH ranged from pH 1.5 to 2.5. In general, the death kinetics followed an exponential trend. The overall death constant, k _d, for all strains was found to be in a power relationship with the pH value and the initial cell concentration, and it can be expressed as $$ {k_{ ext{d}}}{ ext{ = }}{k_{ ext{AII}}}left( {{ ext{p}}{{ ext{H}}^{ - { ext{9}}.0}}{N_0}^{ - 0.{ ext{19}}}} ight) $$ where k _AII is defined as the acid intolerance indicator and N ₀ is the initial cell concentration (CFU/ml). This equation was validated with the experimental data with an average R ² of 0.98. The acid intolerance of cells can be quantitatively expressed by the k _AII values, where higher value indicates higher intolerance. In conclusion, a standard quantitative method has been developed to measure the acid tolerance of probiotic cells. This could facilitate the selection of probiotic strains and processing technologies.

Keywords: Acid tolerance; Probiotic; Simulated gastric fluid; pH; Initial cell concentration; Survival kinetics

Erratum to: Characterization of two proline dipeptidases (prolidases) from the hyperthermophilic archaeon Pyrococcus horikoshii by Casey M. Theriot; Sherry R. Tove; Amy M. Grunden (pp. 393-393).

Erratum to: Characterization of an adenylate cyclase gene (cyaB) deletion mutant of Corynebacterium glutamicum ATCC 13032 by Pu Hyeon Cha; Sun-Yang Park; Min-Woo Moon; Bindu Subhadra; Tae-Kwang Oh; Eungbin Kim; Jihyun F. Kim; Jung-Kee Lee (pp. 395-395).

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Applied Microbiology and Biotechnology (v.86, #1)

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Applied Microbiology and Biotechnology (v.86, #1)