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Applied Microbiology and Biotechnology (v.75, #2)
Quantification of uncultured microorganisms by fluorescence microscopy and digital image analysis by Holger Daims; Michael Wagner (pp. 237-248).
Traditional cultivation-based methods to quantify microbial abundance are not suitable for analyses of microbial communities in environmental or medical samples, which consist mainly of uncultured microorganisms. Recently, different cultivation-independent quantification approaches have been developed to overcome this problem. Some of these techniques use specific fluorescence markers, for example ribosomal ribonucleic acid targeted oligonucleotide probes, to label the respective target organisms. Subsequently, the detected cells are visualized by fluorescence microscopy and are quantified by direct visual cell counting or by digital image analysis. This article provides an overview of these methods and some of their applications with emphasis on (semi-)automated image analysis solutions.
Keywords: Uncultured microorganisms; Quantification; Fluorescence in situ hybridization; Digital image analysis; Microbial ecology
Novel enzyme reactions related to the tricarboxylic acid cycle: phylogenetic/functional implications and biotechnological applications by Miho Aoshima (pp. 249-255).
The tricarboxylic acid (TCA) cycle is an energy-producing pathway for aerobic organisms. However, it is widely accepted that the phylogenetic origin of the TCA cycle is the reductive TCA cycle, which is a non-Calvin-type carbon-dioxide-fixing pathway. Most of the enzymes responsible for the oxidative and reductive TCA cycles are common to the two pathways, the difference being the direction in which the reactions operate. Because the reductive TCA cycle operates in an energetically unfavorable direction, some specific mechanisms are required for the reductive TCA-cycle-utilizing organisms. Recently, the molecular mechanism for the “citrate cleavage reaction” and the “reductive carboxylating reaction from 2-oxoglutarate to isocitrate” in Hydrogenobacter thermophilus have been demonstrated. Both of these reactions comprise two distinct consecutive reactions, each catalyzed by two novel enzymes. Sequence analyses of the newly discovered enzymes revealed phylogenetic and functional relationships between other TCA-cycle-related enzymes. The occurrence of novel enzymes involved in the citrate-cleaving reaction seems to be limited to the family Aquificaceae. In contrast, the key enzyme in the reductive carboxylation of 2-oxoglutarate appears to be more widely distributed in extant organisms. The four newly discovered enzymes have a number of potential biotechnological applications.
Keywords: Citryl-CoA synthetase; Citryl-CoA lyase; 2-Oxoglutarte carboxylase; Oxalosuccinate reductase; Hydrogenobacter thermophilus; Reductive TCA cycle
Microbial enzymes involved in lactone compound metabolism and their biotechnological applications by Michihiko Kataoka; Kohsuke Honda; Keiji Sakamoto; Sakayu Shimizu (pp. 257-266).
Lactone compounds are widely distributed in nature and play important roles in organisms. These compounds are synthesized and metabolized enzymatically in vivo; however, detailed investigation of these enzymes lags behind that of other common enzymes. In this paper, recent work on the enzymes involved in the metabolism of lactone compounds will be reviewed. In particular, fundamental and application studies on lactonases and Baeyer-Villiger monooxgenases of microbial origin are described.
Keywords: Lactone; Lactonase; Hydrolase; Asymmetric synthesis; Perhydrolase
Strategies to unravel the function of orphan biosynthesis pathways: recent examples and future prospects by Harald Gross (pp. 267-277).
The recent increase and availability of whole genome sequences have revised our view of the metabolic capabilities of microorganisms. From these data, a large number of orphan biosynthesis pathways have been identified by bio-informatics. Orphan biosynthetic pathways are gene clusters for which the encoded natural product is unknown. It is worthy to note that the number of orphan pathways coding for putative natural products outnumbers by far the number of currently known metabolites for a given organism. Whilst Streptomyces coelicolor was known to produce only 4 secondary metabolites, the genome analysis revealed 18 additional orphan biosynthetic pathways. It is intriguing to note that this is not a particular case because analysis of other microbial genomes originating from myxobacteria, cyanobacteria and filamentous fungi showed the presence of a comparable or even larger number of orphan pathways. The discovery of these numerous pathways represents a treasure trove, which is likely to grow exponentially in the future, uncovering many novel and possibly bio-active compounds. The few natural products that have been correlated with their orphan pathway are merely the tip of the iceberg, whilst plenty of metabolites await discovery. The recent strategies and methods to access these promising hidden natural products are discussed in this review.
Keywords: Genome mining; Data mining; Genomics; Orphan biosynthetic gene cluster; Natural products
Increased Hepatitis B surface antigen production by recombinant Aspergillus niger through the optimization of agitation and dissolved oxygen concentration by Emmanuel R. James; Willem H. van Zyl; Johann F. Görgens (pp. 279-288).
The capacity of the filamentous fungi Aspergillus niger to produce and assemble complex immunogenic viral proteins into virus-like particles (VLPs) in batch culture was enhanced by optimizing the bioprocessing parameters, agitation intensity and dissolved oxygen (dO2) concentration. Response surface methodology (RSM) and a two-factor-two-level central composite rotatable design (CCRD) were employed to evaluate the interactive response pattern between parameters and their optimum combination. The recombinant hepatitis B surface antigen (HBsAg) was used as a model VLP system to determine the effect of these parameters on biomass yield, fungal morphology, HBsAg production and bioreactor kinetics. The response surface model predicted optimum cultivation conditions at an agitation of rate of 100 rpm and a dO2 concentration of 25%, obtaining highest intracellular membrane-associated HBsAg levels of $$ { ext{3}}{ ext{.4}};{ ext{mg}};{ ext{l}}^{{{ ext{ - 1}}}}_{{{ ext{culture}}}} $$ . HBsAg production levels were increased tenfold compared to yields obtained in shake flask cultivation. Although hepatitis B VLPs mostly accumulated intracellularly, optimal bioreactor conditions resulted in significant HBsAg release in culture supernatant. These results compare favourably with other recombinant VLP systems in batch culture, and therefore, indicate a substantial potential for further engineering of the A. niger production system for the high level of intracellular and extracellular VLP production.
Keywords: VLP production; HBsAg; A. niger ; Dissolved oxygen concentration; Agitation intensity
Anaerobic glycerol production by Saccharomyces cerevisiae strains under hyperosmotic stress by Tobias Modig; Katarina Granath; Lennart Adler; Gunnar Lidén (pp. 289-296).
Glycerol formation is vital for reoxidation of nicotinamide adenine dinucleotide (reduced form; NADH) under anaerobic conditions and for the hyperosmotic stress response in the yeast Saccharomyces cerevisiae. However, relatively few studies have been made on hyperosmotic stress under anaerobic conditions. To study the combined effect of salt stress and anaerobic conditions, industrial and laboratory strains of S. cerevisiae were grown anaerobically on glucose in batch-cultures containing 40 g/l NaCl. The time needed for complete glucose conversion increased considerably, and the specific growth rates decreased by 80–90% when the cells were subjected to the hyperosmotic conditions. This was accompanied by an increased yield of glycerol and other by-products and reduced biomass yield in all strains. The slowest fermenting strain doubled its glycerol yield (from 0.072 to 0.148 g/g glucose) and a nearly fivefold increase in acetate formation was seen. In more tolerant strains, a lower increase was seen in the glycerol and in the acetate, succinate and pyruvate yields. Additionally, the NADH-producing pathway from acetaldehyde to acetate was analysed by overexpressing the stress-induced gene ALD3. However, this had no or very marginal effect on the acetate and glycerol yields. In the control experiments, the production of NADH from known sources well matched the glycerol formation. This was not the case for the salt stress experiments in which the production of NADH from known sources was insufficient to explain the formed glycerol.
Keywords: ALD3 ; Glycerol; Osmotic stress; Saccharomyces cerevisiae
Synthesis of chiral α-hydroxy amides by two sequential enzymatic catalyzed reactions by Yeritzia Salinas; Rosa María Oliart; Mario Ramírez-Lepe; Arturo Navarro-Ocaña; Gerardo Valerio-Alfaro (pp. 297-302).
Enantiomerically pure α-hydroxy amides have been prepared from the corresponding α-oxo esters by the use of a double sequence reaction involving in a first step the highly enantioselective Saccharomyces cerevisiae bioreduction and then in a second step, the resulting α-hydroxy esters followed a non-enantiospecific lipase catalyzed aminolysis with n-butylamine reaction. In the first non-organic solvent process, the moistened baker’s yeast reduced seven α-oxo esters with high conversions degree (93% for one substrate and >99% for the others) and high enantioselectivities [>99% for all the substrates except for ketopantoyl lactone, which gave 88% of enantiomeric excess (ee)]. At the same way, the isolated resulting chiral α-hydroxy esters were subjected to the second Candida antarctica lipase fraction B (CAL-B) catalyzed aminolysis in dioxane conducting to the corresponding chiral α-hydroxy amides with high conversions degree, between 88 and 99%. Both processes were carried out at 28–30°C.
Keywords: Chiral; Bioreduction; α-oxo esters; α-Hydroxy esters; α-Hydroxy amides; CAL-B; Saccharomyces cerevisiae
l-Arabinose metabolism in Candida arabinofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012: influence of sugar and oxygen on product formation by César Fonseca; Isabel Spencer-Martins; Bärbel Hahn-Hägerdal (pp. 303-310).
l-Arabinose utilization by the yeasts Candida arabinofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012 was investigated in aerobic batch cultures and compared, under similar conditions, to d-glucose and d-xylose metabolism. At high aeration levels, only biomass was formed from all the three sugars. When oxygen became limited, ethanol was produced from d-glucose, demonstrating a fermentative pathway in these yeasts. However, pentoses were essentially respired and, under oxygen limitation, the respective polyols accumulated—arabitol from l-arabinose and xylitol from d-xylose. Different l-arabinose concentrations and oxygen conditions were tested to better understand l-arabinose metabolism. P. guilliermondii PYCC 3012 excreted considerably more arabitol from l-arabinose (and also xylitol from d-xylose) than C. arabinofermentans PYCC 5603T. In contrast to the latter, P. guilliermondii PYCC 3012 did not produce any traces of ethanol in complex l-arabinose (80 g/l) medium under oxygen-limited conditions. Neither sustained growth nor active metabolism was observed under anaerobiosis. This study demonstrates, for the first time, the oxygen dependence of metabolite and product formation in l-arabinose-assimilating yeasts.
Keywords: l-Arabinose; Pentose; Fermentation conditions; Yeast; Hemicellulose; Candida arabinofermentans ; Pichia guilliermondii
Studies on the Pycnoporus sanguineus CCT-4518 laccase purified by hydrophobic interaction chromatography by Telma Alves Garcia; Mariângela Fontes Santiago; Cirano José Ulhoa (pp. 311-318).
A laccase from Pycnoporus sanguineus was purified by two steps using phenyl-Sepharose columm. A typical procedure provided 54.1-fold purification, with a yield of 8.37%, using syringaldazine as substrate. The molecular weight of the purified laccase was 69 and 68 kDa as estimated by 12% (w/v) SDS-PAGE gel and by gel filtration, respectively. The K m values for the substrates ABTS, syringaldazine, and guaiacol were 58, 8.3, and 370 μM, respectively. The enzyme’s pH optimum for syringaldazine was 4.2 and optimal activity was 50°C. The enzyme showed to be thermostable because when kept at 50°C for 24 and 48 h it retained 93 and 76% activity. This laccase was inhibited by l-cysteine, β-mercaptoethanol, NaN3, NaF, and HgCl2.
Keywords: Pycnoporus sanguineus ; Thermostable laccase; Purification; Characterization; Hydrophobic interaction
Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases by Yi Feng; Cheng-Jie Duan; Hao Pang; Xin-Chun Mo; Chun-Feng Wu; Yuan Yu; Ya-Lin Hu; Jie Wei; Ji-Liang Tang; Jia-Xun Feng (pp. 319-328).
A metagenomic cosmid library was prepared in Escherichia coli from DNA extracted from the contents of rabbit cecum and screened for cellulase activities. Eleven independent clones expressing cellulase activities (four endo-β-1,4-glucanases and seven β-glucosidases) were isolated. Subcloning and sequencing analysis of these clones identified 11 cellulase genes; the encoded products of which shared less than 50% identities and 70% similarities to cellulases in the databases. All four endo-β-1,4-glucanases and all seven β-glucosidases, respectively, belonged to glycosyl hydrolase family 5 (GHF 5) and family 3 (GHF 3) and formed two separate branches in the phylogenetic tree. Ten of the 11 cloned cellulases exhibited highest activities at pH 5.5 ∼ 7.0 and 40 ∼ 55°C, a condition similar to that in the rabbit cecum. All the four endo-β-1,4-glucanases could hydrolyze a wide range of β-1,4-, β-1,4/β-1,3- or β-1,3/β-1,6-linked polysaccharides. One endo-β-1, 4-glucanase gene, umcel5G, was overexpressed in E. coli, and the purified recombinant enzyme was characterized in detail. The enzymes cloned in this work represented at least some of the cellulases operating efficiently in the rabbit cecum. This work provides the first snapshot on the cellulases produced by bacteria in rabbit cecum.
Keywords: Cellulase; Cloning; Metagenome; Rabbit cecum; Expression; Enzyme; Properties
Molecular basis for thermal properties of Streptomyces thermovulgaris fumarase C hinge at hydrophilic amino acids R163, E170 and S347 by Wenjie Lin; Maurice Chan; Liuh-Ling Goh; Tiow-Suan Sim (pp. 329-335).
Industrially, the use of high temperatures (40–60°C) in the l-malate production process could result in rapid inactivation of the mesophilic fumarases, warranting constant replenishment of the biocatalyst. Thus, a thermostable fumarase C that is active and stable at high temperatures would be ideal. Biochemical studies using recombinant fumarase C from thermophilic Streptomyces thermovulgaris (stFUMC) indicated that it was optimally active at 50°C and highly stable even after 24 h of incubation at 40°C. The same gene from mesophilic Streptomyces coelicolor (scfumC) was also cloned and expressed as soluble proteins for comparison in thermal properties of both enzymes. In contrast to stFUMC, scFUMC exhibited a lower temperature optima of 30°C and was rapidly denatured at 50°C. The specific activity of stFUMC was also higher than that of scFUMC by 20-fold. After primary sequence comparison, three hydrophilic amino acid residues, R163, E170 and S347, were forged into the thermolabile scFUMC either singly or in combination for the investigation of their contributions in the thermal properties of the mutant enzymes. Of the mutants studied, the A347S scFUMC mutant resulted in the highest increase in optimum temperature of 10°C and a fourfold enhancement in specific activity. G163R/G170E and G163R/G170E/A347S scFUMC mutants are more thermostable than wild-type scFUMC. These findings support stFUMC as a highly efficient, thermostable fumarase C with industrial potential and suggest that R163, E170 and S347 are involved in the enhancement of thermal properties in fumarase C.
Keywords: Streptomyces; Site-directed mutagenesis; Thermostability; Hydrophilic residues; Fumarase C
Biochemical and molecular characterization of a cellobiohydrolase from Trametes versicolor by Karim Lahjouji; Reginald Storms; Zhizhuang Xiao; Kwang-Bo Joung; Yun Zheng; Justin Powlowski; Adrian Tsang; Luc Varin (pp. 337-346).
A cellobiohydrolase-encoding cDNA, Tvcel7a, from Trametes versicolor has been cloned and expressed in Aspergillus niger. The deduced amino acid sequence shows that Tvcel7a encodes a 456-amino acid polypeptide belonging to glycosyl hydrolase family 7. TvCel7a possesses a 19-amino acid secretion signal but does not possess a linker region nor a carbohydrate-binding domain. Two peaks of activity were obtained after TvCel7a was purified to apparent homogeneity by gel-filtration followed by anion-exchange chromatography. Mass spectrometry performed on the purified proteins confirmed that both peaks corresponded to the predicted sequence of the T. versicolor cellulase. The biochemical properties of the purified TvCel7a obtained from both peaks were studied in detail. The pH and temperature optima were 5.0 and 40°C, respectively. The enzyme was stable over a pH range extending from pH 3.0 to 9.0 and at temperatures lower than 50°C. The kinetic parameters with the substrate p-nitrophenyl β-d-cellobioside (pNPC) were 0.58 mM and 1.0 μmol/min/mg protein for the purified TvCel7a found in both peaks 1 and 2. TvCel7a catalyzes the hydrolysis of pNPC, filter paper, β-glucan, and avicel to varying extents, but no detectable hydrolysis was observed when using the substrates carboxymethylcellulose, laminarin and pNPG.
Keywords: Cellulase; Cellobiohydrolase; Trametes versicolor ; Expression; Substrate specificity; Enzymatic properties
Enhanced stability of heterologous proteins by supramolecular self-assembly by Jin-Seung Park; Ji-Young Ahn; Sung-Hyun Lee; Hyewon Lee; Kyung-Yeon Han; Hyuk-Seong Seo; Keum-Young Ahn; Bon Hong Min; Sang Jun Sim; Insung S. Choi; Yang Hoon Kim; Jeewon Lee (pp. 347-355).
Recently, we reported on the dual function of human ferritin heavy chain (hFTN-H) used for the fusion expression and solubility enhancement of various heterologous proteins: (1) high-affinity interaction with HSP70 chaperone DnaK and (2) formation of self-assembled supramolecules with limited and constant sizes. Especially the latter, the self-assembly function of hFTN-H is highly useful in avoiding the undesirable formation of insoluble macroaggregates of heterologous proteins in bacterial cytoplasm. In this study, using enhanced green fluorescent protein (eGFP) and several deletion mutants of Mycoplasma arginine deiminase (ADI132–410) as reporter proteins, we confirmed through TEM image analysis that the recombinant fusion proteins (hFTN-H::eGFP and hFTN-H::ADI132–410) formed intracellular spherical particles with nanoscale diameter (≈10 nm), i.e., noncovalently cross-linked supramolecules. Surprisingly, the supramolecular eGFP and ADI showed much enhanced stability in bioactivity. That is, the activity level was much more stably maintained for the prolonged period of time even at high temperature, at high concentration of Gdn–HCl, and in wide range of pH. The stability enhancement by supramolecular self-assembly may make it possible to utilize the protein supramolecules as novel means for drug delivery, enzymatic material conversion (biotransformation), protein chip/sensor, etc. where the maintenance of protein/enzyme stability is strictly required.
Keywords: Human ferritin heavy chain; Self-assembly; Supramolecules; Enhanced stability
Purification and characterization of Vibrio parahaemolyticus extracellular chitinase and chitin oligosaccharide deacetylase involved in the production of heterodisaccharide from chitin by K. Kadokura; A. Rokutani; M. Yamamoto; T. Ikegami; H. Sugita; S. Itoi; W. Hakamata; T. Oku; T. Nishio (pp. 357-365).
A chitin-degrading bacterial strain, KN1699, isolated from Yatsu dry beach (Narashino, Chiba Prefecture, Japan), was identified as Vibrio parahaemolyticus. Treatment of powdered chitin with crude enzyme solution prepared from the supernatant of KN1699 cultures yielded a disaccharide, β-d-N-acetylglucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN), as the primary chitin degradation product. The extracellular enzymes involved in the production of this heterodisaccharide, chitinase (Pa-Chi; molecular mass, 92 kDa) and chitin oligosaccharide deacetylase (Pa-COD; molecular mass, 46 kDa), were isolated from the crude enzyme solution, and their hydrolysis specificities were elucidated. These studies confirmed that (1) Pa-Chi hydrolyzes chitin to produce (GlcNAc)2 and (2) Pa-COD hydrolyzes the acetamide group of reducing end GlcNAc residue of (GlcNAc)2. These findings indicate that GlcNAc-GlcN is produced from chitin by the cooperative hydrolytic reactions of both Pa-Chi and Pa-COD.
Characterization of a hyperthermostable Fe-superoxide dismutase from hot spring by Yong-Zhi He; Ke-Qiang Fan; Cui-Juan Jia; Zhi-Jun Wang; Wu-Bin Pan; Li Huang; Ke-Qian Yang; Zhi-Yang Dong (pp. 367-376).
A new gene encoding a thermostable Fe-superoxide dismutase (tcSOD) was identified from a metagenomic library prepared from a hot spring sample. The open reading frame of tcSOD encoded a 211 amino acid protein. The recombinant protein was overexpressed in Escherichia coli and confirmed to be a Fe–SOD with a specific activity of 1,890 U/mg using the pyrogallol method. The enzyme was highly stable at 80°C and retained 50% activity after heat treatment at 95°C for 2 h. It showed striking stability across a wide pH span from 4 to 11. The native form of the enzyme was determined as a homotetramer by analytical ultracentrifugation and gradient native polyacrylamide gel electrophoresis. Fe2+ was found to be important to SOD activity and to the stability of tcSOD dimer. Comparative modeling analyses of tcSOD tetramer indicate that its high thermostability is mainly due to the presence of a large number of intersubunit ion pairs and hydrogen bonds and to a decrease in solvent accessible hydrophobic surfaces.
An azoreductase, aerobic NADH-dependent flavoprotein discovered from Bacillus sp.: functional expression and enzymatic characterization by Toshihiko Ooi; Takeshi Shibata; Reiko Sato; Hiroaki Ohno; Shinichi Kinoshita; Tran Linh Thuoc; Seiichi Taguchi (pp. 377-386).
The gene coding for an azoreductase, designated as an azrA, was cloned by polymerase chain reaction amplification from the genomic DNA of Bacillus sp. strain B29 isolated from soil. The azrA encoded a protein of 208 amino acids with calculated molecular mass of 22,766 Da. The enzyme was heterologously expressed in Escherichia coli with a strong band of 23 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Purified recombinant AzrA was a homodimer with a native molecular mass of 48 kDa containing two molecules of flavin mononucleotide (FMN; oxidized). This activity was oxygen insensitive and was nicotinamide adenine dinucleotide (reduced form; NADH) dependent. Recombinant AzrA exhibited a broad pH stability between 6 and 10 with a temperature optimum of 60–80°C. The enzyme cleaved the model azo compound of methyl red [MR, 4′-(dimethylamino)-azobenzene-2-carboxylic acid] into 2-aminobenzoic acid and N, N′-dimethyl-p-phenylenediamine by ping-pong mechanism. The enzyme was not only able to decolorize MR but also able to decolorize sulfonated azo dyes such as Orange I and Acid Red 88.
Keywords: Azoreductase; Aerobic azoreductase; Bacillus sp.
Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica by Sriappareddy Tamalampudi; MD Mahabubur Rahman Talukder; Shinji Hama; Takanori Tanino; Yuya Suzuki; Akihiko Kondo; Hideki Fukuda (pp. 387-395).
To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tglA) from A. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of para-nitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.
Keywords: Aspergillus oryzae ; Whole-cell biocatalyst; Candida antarctica lipase B; Enantioselective transesterification
An online monitoring system based on a synthetic sigma32-dependent tandem promoter for visualization of insoluble proteins in the cytoplasm of Escherichia coli by Mario Kraft; Uwe Knüpfer; Rolf Wenderoth; Patricia Pietschmann; Björn Hock; Uwe Horn (pp. 397-406).
The expression of heterologous proteins in the cytoplasm of Escherichia coli is often accompanied by limitations resulting in uncontrollable fermentation processes, increased rates of cell lysis, and thus limited yields of target protein. To deal with these problems, reporter tools are required to improve the folding properties of recombinant protein. In this work, the well-known σ32-dependent promoters ibpAB and fxsA were linked in a tandem promoter (ibpfxs), fused with the luciferase reporter gene lucA to allow enhanced monitoring of the formation of misfolded proteins and their aggregates in E. coli cells. Overexpression of MalE31, a folding-defective variant of the maltose-binding protein, and other partially insoluble heterologous proteins showed that the lucA reporter gene was activated in the presence of these misfolded proteins. Contrary to this, the absence of damaged proteins or overexpression of mostly soluble proteins led to a reduced level of luciferase induction. Through performing expression of aggregation-prone proteins, we were able to demonstrate that the ibpfxs::lucA reporter unit is 2.5–4.5 times stronger than the single reporter units ibp::lucA and fxs::lucA. Data of misfolding studies showed that this reporter system provides an adequate tool for in vivo folding studies in E. coli from microtiter up to fermentation scales.
A chimeric baculovirus displaying bovine herpesvirus-1 (BHV-1) glycoprotein D on its surface and their immunological properties by A. Peralta; P. Molinari; D. Conte-Grand; G. Calamante; O. Taboga (pp. 407-414).
The ability of a recombinant baculovirus containing the ectodomain of the mature sequence of glycoprotein D (gD) fused to the amino-terminus of baculoviral glycoprotein gp64 to display gD on its surface and to serve as an improved immunogen against bovine herpesvirus-1 was tested. The gD–gp64 fusion protein was correctly expressed on the virus particles as revealed by immunomicroscopy assays. Mice immunized with 5 × 108 plaque forming units developed antibodies that specifically reacted in an enzyme-linked immunosorbent assay with recombinant gD and whole bovine herpesvirus-1. These antibodies were able to neutralize bovine herpesvirus-1 in vitro, whereas those elicited by a version of gD expressed in Escherichia coli did not. Our data demonstrated that the display on the virion surface of recombinant baculovirus can provide a tool for the development of recombinant vaccines against bovine herpesvirus-1.
Keywords: Baculovirus display; BHV-1 vaccines; Viral vectors
Extracting the hidden features in saline osmotic tolerance in Saccharomyces cerevisiae from DNA microarray data using the self-organizing map: biosynthesis of amino acids by Gaurav Pandey; Katsunori Yoshikawa; Takashi Hirasawa; Keisuke Nagahisa; Yoshio Katakura; Chikara Furusawa; Hiroshi Shimizu; Suteaki Shioya (pp. 415-426).
During saline stress, Saccharomyces cerevisiae changes its metabolic fluxes through the direct accumulation of metabolites such as glycerol and trehalose, which in turn provide tolerance to the cell against stress. Previous research shows that the various controls at both transcriptional and translational levels decide the phenomenon of stress, but details about such transition is still not very clear. This paper attempts to extract some hidden features through the information extraction approach from DNA microarray data during transition to osmotic tolerance, which are expected to be important in directing to the tolerance stage upon encountering osmotic stress in yeast. Time course of DNA microarray data during osmotic tolerance was analyzed by computational approach ‘self-organizing map (SOM) extended with hierarchical clustering’. Since eukaryotic gene expression is governed by short regulatory sequences found upstream in promoter regions, therefore clusters containing the similar profiles obtained by SOM were further analyzed for overrepresentation of known regulatory binding sites in promoter region. It was found that apart from known and expected ‘STRE’ during osmotic stress, the ‘GCN4’ binding site is also found to be significant. Hence, it was suggested that the process of osmotic tolerance proceeds through a stage of amino acid starvation. The intracellular amino acids pool also found to be depleted during transition and restoration is faster in brewing strain than laboratory strain. Experiments involving supplementation of amino acids helps in reducing the lag time for recovery, which was found to be similar to that of brewing strain.
Synergistic effects of Lactobacillus rhamnosus ZDY114 and bovine colostrums on the immunological function of mouse in vivo and in vitro by Hua Wei; Yang Xu; Bocai Cheng; Yonghua Xiong (pp. 427-434).
The synergistic effects of Lactobacillus rhamnosus and bovine colostrums on the immunity of mice in vivo and in vitro were investigated. Eight- to ten-week-old mice were used for two series experiments; one part of mice were immunocompromised by intraperitoneal injections of cyclophosphamide. In series I, immunocompromised mice were continuously fed with diet A (L. rhamnosus ZDY114 5 × 107 CFU/kg), B (bovine colostrums 0.5 g/kg), C (combination of diet A and B), and D (sterile saline) for 4 weeks and killed. Thereof, phagocytosis ratio and index of macrophage to chicken red blood cells in abdominal cavity and lymphocyte transformation rate were determined. In series II, both normal and immunocompromised mice were used to investigate the in vitro stimulation of lymphocyte proliferation by substances from the overnight culture of L. rhamnosus ZDY114 by the MTT colorimetric method. Compared with diet D, in the diet A, B, and C groups, the phagocytosis ratio of macrophages increased by 1.63, 1.54, and 2.3-fold, respectively, and the lymphocyte transformation ratio by 1.78, 2.08, and 2.35-fold, respectively. In vitro test with MTT showed that 3, 10, 50 kD substances from the overnight culture of L. rhamnosus ZDY114 significantly increased the growth of lymphocyte by 1.63, 1.53, and 1.34-fold, respectively. In conclusion, L. rhamnosus and bovine colostrums can enhance the functions of immune system supported by lymphocytes and peritoneal macrophages either in vivo or in vitro.
Keywords: Lactobacillus rhamnosus ; Bovine colostrums; Phagocytosis; Lymphocyte proliferation; Lymphocyte transformation; In vivo; In vitro
Production and identification of a novel compound, 7,10-dihydroxy-8(E)-hexadecenoic acid from palmitoleic acid by Pseudomonas aeruginosa PR3 by Jae-Han Bae; Deuk-Soo Kim; Min-Jung Suh; Sei-Ryang Oh; In-Jung Lee; Sun-Chul Kang; Ching T. Hou; Hak-Ryul Kim (pp. 435-440).
Hydroxy fatty acids are considered as important value-added product for industrial application because of their special properties such as higher viscosity and reactivity. Microbial production of the hydroxy fatty acids from various fatty acid substrates have been actively studied using several microorganisms. The new bacterial isolate Pseudomonas aeruginosa (PR3) had been reported to produce mono-, di-, and tri-hydroxy fatty acids from different unsaturated fatty acids. Of those, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) and 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD) were produced from oleic acid and ricinoleic acid, respectively. Based on the postulated common metabolic pathway involved in DOD and TOD formation by PR3, it was assumed that palmitoleic acid containing a singular 9-cis double bond, common structural property sharing with oleic acid and ricinoleic acid, could be utilized by PR3 to produce hydroxy fatty acid. In this study, we tried to use palmitoleic acid as substrate for production of hydroxy fatty acid by PR3 and firstly confirmed that PR3 could produce 7,10-dihydroxy-8(E)-hexadecenoic acid (DHD) with 23% yield from palmitoleic acid. DHD production was peaked at 72 h after the substrate was added to the 24-h-culture.
Keywords: Pseudomonas aeruginosa ; Palmitoleic acid; Dihydroxy fatty acid; Bioconversion
Molecular techniques for the early warning of toxic cyanobacteria blooms in freshwater lakes and rivers by Martin L. Saker; Micaela Vale; Dan Kramer; Vitor M. Vasconcelos (pp. 441-449).
The aim of this work was to test the efficacy of molecular techniques for detecting toxigenic cyanobacteria in environmental water samples collected from freshwater lakes, rivers and reservoirs in Portugal. Of 26 environmental samples tested, 21 were found to contain Microcystis using a genus-specific polymerase chain reaction (PCR). Another primer pair was applied to the same DNA template to test for the presence of microcystin synthetase genes. This primer pair resulted in the formation of a PCR product in 15 of the samples containing Microcystis and one sample that did not give a positive result in the Microcystis genus-specific PCR. A restriction assay using the enzyme EcoRV was then applied to show that in most cases, the gene fragment was from toxigenic strains of Microcystis and, in one above-mentioned case, from a microcystin-producing strain of Planktothrix. All environmental samples were examined microscopically to confirm the presence of cyanobacteria species. Samples were also tested for the presence of microcystins using the ELISA plate assay. There was good agreement between the results obtained with molecular techniques and those obtained from microscopy and chemical methods. The PCR techniques applied in this paper were found to be useful, particularly when the concentration of the target organism was very low compared with other organisms. This technique can be used to detect inocula for cyanobacterial populations and therefore provide a useful tool for assessing under which conditions particular species can grow into bloom populations.
Keywords: Cyanobacteria; Environmental monitoring; PCR; Microcystin; Microginin
Effect of dilution rate on the microbial structure of a mesophilic butyrate-degrading methanogenic community during continuous cultivation by Yue-Qin Tang; Toru Shigematsu; Shigeru Morimura; Kenji Kida (pp. 451-465).
We constructed two mesophilic anaerobic chemostats that were continuously fed with synthetic wastewater containing butyrate as the sole source of carbon and energy. Steady-state conditions were achieved at dilution rates between 0.025 and 0.7 day−1. Butyrate, fed into the chemostat, was almost completely mineralized to CH4 and CO2 at dilution rates below 0.5 day−1. The butyrate-degrading methanogenic communities in the chemostats at dilution rates between 0.025 and 0.7 day−1 were monitored based on the 16S rRNA gene, using molecular biological techniques including clone library analysis, denaturing gradient gel electrophoresis, and quantitative real-time polymerase chain reaction. The aceticlastic methanogen Methanosaeta and the hydrogenotrophic methanogen Methanoculleus dominated in methanogens at low dilution rates, whereas the aceticlastic methanogen Methanosaeta, Methanosarcina, the hydrogenotrophic methanogen Methanoculleus, and Methanospirillum dominated at high dilution rates. Bacteria affiliated with the family Syntrophaceae in the phylum Proteobacteria predominated at the low dilution rate of 0.025 day−1, whereas bacteria affiliated with the phylum Firmicutes and Candidate division OP3 predominated at high dilution rates. A significant quantity of bacteria closely related to the genus Syntrophomonas was detected at high dilution rates. Dilution rate showed an apparent effect on archaeal and bacterial communities in the butyrate-fed chemostats.
Keywords: Microbial community; Butyrate; Dilution rate
Staining of extracellular polymeric substances and cells in bioaggregates by Ming-Yuan Chen; Duu-Jong Lee; Joo-Hwa Tay; Kuan-Yeow Show (pp. 467-474).
Multiple fluorochrome experiments with as many fluorochromes as possible are desired for exploring the detailed structure of bioaggregates. Spectral peak interference and other practical limitations, however, restrict the maximum number of stains used simultaneously to three. This current study proposes a sixfold labelled scheme to stain the total cells, dead cells, proteins, lipids, and α- and β-polysaccharides in bioaggregates. Two aerobic granule systems, the phenol-fed and the acetate-fed granules, were utilized as the testing samples for demonstrating the use of the proposed scheme.
Keywords: Staining; EPS; CLSM; Crosstalk