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Applied Microbiology and Biotechnology (v.77, #3)
Aspergillus flavus hydrolases: their roles in pathogenesis and substrate utilization by Jay E. Mellon; Peter J. Cotty; Michael K. Dowd (pp. 497-504).
Aspergillus flavus is a fungus that principally obtains resources for growth in a saprophytic mode. Yet, it also possesses the characteristics of an opportunistic pathogen with a wide, non-specific host range (plants, animals, and insects). It has attained a high level of agricultural significance due to production of the carcinogen aflatoxin, which significantly reduces the value of contaminated crops. To access a large variety of nutrient substrates and penetrate host tissues, A. flavus possesses the capacity to produce numerous extracellular hydrolases. Most work on A. flavus hydrolases has focused on the serine and metalloproteinases, pectinase P2c, and amylase. Many hydrolases are presumed to function in polymer degradation and nutrient capture, but the regulation of hydrolase secretion is complex and substrate dependent. Proteinases are employed not only to help access protein substrates, such as elastin that is found in mammals and insects, but may also play roles in fungal defense and virulence. Secretion of the endopolygalacturonase P2c is strongly correlated with isolate virulence (against plants) and maceration of cotton boll tissues. In some hosts, secretion of α-amylase is critical for starch digestion and may play a critical role in induction of aflatoxin biosynthesis. Despite a significant body of work, much remains to be learned about hydrolase production and utilization by A. flavus. This information may be critical for the formulation of successful strategies to control aflatoxin contamination in affected commodities.
Keywords: Aspergillus flavus ; Hydrolases; Pathogenesis; Nutrient capture; Saprophytic
An update on microbial carotenoid production: application of recent metabolic engineering tools by Amitabha Das; Sang-Hwal Yoon; Sook-Hee Lee; Jae-Yean Kim; Deok-Kun Oh; Seon-Won Kim (pp. 505-512).
Carotenoids are ubiquitous pigments synthesized by plants, fungi, algae, and bacteria. Industrially, carotenoids are used in pharmaceuticals, neutraceuticals, and animal feed additives, as well as colorants in cosmetics and foods. Scientific interest in dietary carotenoids has increased in recent years because of their beneficial effects on human health, such as lowering the risk of cancer and enhancement of immune system function, which are attributed to their antioxidant potential. The availability of carotenoid genes from carotenogenic microbes has made possible the synthesis of carotenoids in non-carotenogenic microbes. The increasing interest in microbial sources of carotenoid is related to consumer preferences for natural additives and the potential cost effectiveness of creating carotenoids via microbial biotechnology. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms with particular focus on the potential of Escherichia coli for improved carotenoid productivity.
Keywords: Carotenoids; Metabolic engineering; MEP; Mevalonate; Isopentenyl diphosphate; Prenyl diphosphate
Yeast expression platforms by Erik Böer; Gerhard Steinborn; Gotthard Kunze; Gerd Gellissen (pp. 513-523).
Yeasts provide attractive expression platforms. They combine ease of genetic manipulations and the option for a simple fermentation design of a microbial organism with the capabilities of an eukaryotic organism to secrete and to modify a protein according to a general eukaryotic scheme. For platform applications, a range of yeast species has been developed during the last decades. We present in the following review a selection of established and newly defined expression systems. The review is concluded by the description of a wide-range vector system that allows the assessment of the selected organisms in parallel for criteria like secretion or appropriate processing and modification in a given case.
Keywords: Heterologous gene expression; Transformation; Wide-range vector system; Yeast expression platforms
Production of recombinant bacteriocin divercin V41 by high cell density Escherichia coli batch and fed-batch cultures by Selcuk Yildirim; Daniel Konrad; Ségolène Calvez; Djamel Drider; Hervé Prévost; Christophe Lacroix (pp. 525-531).
To increase the yield of heterologous production of the class II bacteriocin DvnRV41 with Escherichia coli Origami (DE3) (pLysS/pCR03), induction of bacteriocin gene expression was optimized by varying the inducer isopropyl β-d-thiogalactopyranoside (IPTG) concentration (0–2 mM), and controlled batch and fed-batch cultures were tested on a 2-L scale. A concentration of 0.5 mM IPTG was found to be optimal for cell growth and bacteriocin production. Shake flask cultivation of E. coli Origami (DE3) (pLysS/pCR03) gave biomass and bacteriocin yields of 1.54 ± 0.06 g cdw/l and 18 ± 1 mg DvnRV41/l, respectively. Biomass (2.70 ± 0.06 and 6.8 ± 0.6 g cdw/l, respectively) and bacteriocin yields (30 and 74 mg DvnRV41 per liter, respectively) were both increased with batch and fed-batch compared to shake flask cultures. Bacteriocin yields reported in this study are among the highest published for other heterologous expression systems in shake flasks.
Keywords: Divercin V41; Bacteriocin; Heterologous expression system; Recombinant protein production
Direct production of L-lysine from raw corn starch by Corynebacterium glutamicum secreting Streptococcus bovis α-amylase using cspB promoter and signal sequence by Toshihiro Tateno; Hideki Fukuda; Akihiko Kondo (pp. 533-541).
Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes α-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40°C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37°C, respectively. The α-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence.
Keywords: Corynebacterium glutamicum ; Raw starch; cspB promoter; α-Amylase
Enhanced secondary metabolite (tanshinone) production of Salvia miltiorrhiza hairy roots in a novel root–bacteria coculture process by Jian-Yong Wu; Janet Ng; Ming Shi; Shu-Jing Wu (pp. 543-550).
Salvia miltiorrhiza Bunge (Lamiaceae) hairy root cultures were inoculated (at 0.02 and 0.2% v/v) and co-cultured with Bacillus cereus bacteria. The root biomass growth was inhibited significantly by the bacteria inoculated to the root culture on the first day (day 0) but not by the bacteria inoculated on days 14 or 21 (in a 28-day overall period). On the other hand, the growth of the bacteria in the hairy root culture was also strongly inhibited by the hairy roots, partially because of the antibacterial activity of the secondary compounds produced by the roots. Most interestingly, the tanshinone production was promoted by the inoculation of bacteria at any of these days but more significantly by an earlier bacteria inoculation. With 0.2% bacteria inoculated on day 0, for example, the total tanshinone content of roots was increased by more than 12-fold (from 0.20 to 2.67 mg g−1 dry weight), and the volumetric tanshinone yield increased by more than sixfold (from 1.40 to 10.4 mg l−1). The tanshinone production was also stimulated by bacterial water extract and bacterial culture supernatant but less significantly than by the inoculation of live bacteria. The results suggest that the stimulation of tanshinone production by live bacteria in the root cultures may be attributed to the elicitor compounds originating from the bacteria, and the hairy root–bacteria coculture may be an effective strategy for improving secondary metabolite production in plant tissue cultures.
Keywords: Salvia miltiorrhiza ; Hairy roots; Bacillus cereus ; Coculture; Diterpenoid tanshinones; Plant–microbe interaction
Composition and distribution of internal resistance in three types of microbial fuel cells by Peng Liang; Xia Huang; Ming-Zhi Fan; Xiao-Xin Cao; Cheng Wang (pp. 551-558).
High internal resistance is a key problem limiting the power output of the microbial fuel cell (MFC). Therefore, more knowledge about the internal resistance is essential to enhance the performance of the MFC. However, different methods are used to determine the internal resistance, which makes the comparison difficult. In this study, three different types of MFCs were constructed to study the composition and distribution of internal resistance. The internal resistance (R i) is partitioned into anodic resistance (R a), cathodic resistance (R c), and ohmic resistance ( $$R_{Omega } $$ ) according to their origin and the design of the MFCs. These three resistances were then evaluated by the “current interrupt” method and the “steady discharging” method based on the proposed equivalent circuits for MFCs. In MFC-A, MFC-B, and MFC-C, the R i values were 3.17, 0.35, and 0.076 Ω m2, the $$R_{Omega } $$ values were 2.65, 0.085, and 0.008 Ω m2, the R a values were 0.055, 0.115, and 0.034 Ω m2, and the R c values were 0.466, 0.15, and 0.033 Ω m2, respectively. For MFC-B and MFC-C, the remarkable decrease in R i compared with the two-chamber MFC was mainly ascribed to the decline in $$R_{Omega } $$ and R c. In MFC-C, the membrane electrodes’ assembly lowered the ohmic resistance and facilitated the mass transport through the anode and cathode electrodes, resulting in the lowest R i among the three types.
Keywords: Microbial fuel cell; Internal resistance; Flat-type
Enzymatic synthesis and characterization of arbutin glucosides using glucansucrase from Leuconostoc mesenteroides B-1299CB by Young Hwan Moon; Seung Hee Nam; Jin Kang; Young-Min Kim; Jin-Ha Lee; Hee-Kyung Kang; Vincent Breton; Woo-Jin Jun; Ki-Deok Park; Atsuo Kimura; Doman Kim (pp. 559-567).
Two arbutin glucosides were synthesized via the acceptor reaction of a glucansucrase from Leuconostoc mesenteroides B-1299CB with arbutin and sucrose. The glucosides were purified by Bio-gel P-2 column chromatography and high-performance liquid chromatography, and the structures were elucidated as 4-hydroxyphenyl β-isomaltoside (arbutin-G1), 4-hydroxyphenyl β-isomaltotrioside (arbutin-G2), according to the results of 1H, 13C, heteronuclear single-quantum coherence, 1H-1H COSY, and heteronuclear multiple-bond correlation analyses. Arbutin glucoside (4-hydroxyphenyl β-isomaltoside) exhibited slower effects on 1,1-diphenyl-2-picrylhydrazyl radical scavenging and similar effects on tyrosinase inhibition, and increased inhibitory effect on matrix metalloproteinase-1 production induced by UVB than arbutin.
Keywords: Leuconostoc mesenteroides ; Dextransucrase; Arbutin; Glucoside; Acceptor reaction
Directed evolution of a beta-galactosidase from Pyrococcus woesei resulting in increased thermostable beta-glucuronidase activity by Ai-Sheng Xiong; Ri-He Peng; Jing Zhuang; Xian Li; Yong Xue; Jin-Ge Liu; Feng Gao; Bin Cai; Jian-Min Chen; Quan-Hong Yao (pp. 569-578).
We performed directed evolution on a chemically synthesized 1,533-bp recombinant beta-galactosidase gene from Pyrococcus woesei. More than 200,000 variant colonies in each round of directed evolution were screened using the pYPX251 vector and host strain Rosetta-Blue (DE3). One shifted beta-galactosidase to beta-glucuronidase mutant, named YG6762, was obtained after four rounds of directed evolution and screening. This mutant had eight mutated amino acid residues. T29A, V213I, L217M, N277H, I387V, R491C, and N496D were key mutations for high beta-glucuronidase activity, while E414D was not essential because the mutation did not lead to a change in beta-glucuronidase activity. The amino acid site 277 was the most essential because mutating H back to N resulted in a 50% decrease in beta-glucuronidase activity at 37°C. We also demonstrated that amino acid 277 was the most essential site, as the mutation from N to H resulted in a 1.5-fold increase in beta-glucuronidase activity at 37°C. Although most single amino acid changes lead to less than a 20% increase in beta-glucuronidase activity, the YG6762 variant, which was mutated at all eight amino acid sites, had a beta-glucuronidase activity that was about five and seven times greater than the wild-type enzyme at 37 and 25°C, respectively.
Keywords: Beta-galactosidase; Beta-glucuronidase; Pyrococcus woesei ; Directed evolution; Enzyme properties; Structure–function analysis
Enzymatic synthesis of cephalosporins. The immobilized acylase from Arthrobacter viscosus: A new useful biocatalyst by Marco Terreni; Daniela Ubiali; Teodora Bavaro; Massimo Pregnolato; Roberto Fernández-Lafuente; José M. Guisán (pp. 579-587).
The acylase from Arthrobacter viscosus was immobilized, studied in the enzymatic synthesis of some cephalosporins by kinetically controlled N-acylation (kcNa) of different cephem nuclei, and compared with the penicillin G acylase (PGA) from Escherichia coli. The reaction outcomes were dependent on the acylase microbial source and on the type of immobilization support. Generally, both enzymes, when immobilized onto hydrophilic resins such as glyoxyl-agarose (activated with aldehyde groups), displayed higher synthetic performances in comparison with hydrophobic acrylic epoxy-supports like Eupergit C. The kcNa of 7-amino cephalosporanic acid catalyzed by A. viscosus immobilized on glyoxyl-agarose afforded a quantitative conversion in 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ3-cephem-4-carboxylic acid, a useful intermediate for the synthesis of Cefamandole and Cefonicid. Similar results were obtained in the synthesis of these cephalosporins by direct acylation of the corresponding 3′-functionalized nucleus. In these reactions, A. viscosus displayed higher synthetic performances than the PGA from E. coli.
Differential expression of cellulases and xylanases by Cellulomonas flavigena grown on different carbon sources by Leticia M. Sánchez-Herrera; Ana C. Ramos-Valdivia; Mayra de la Torre; Luis M. Salgado; Teresa Ponce-Noyola (pp. 589-595).
The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.
Keywords: Cellulases; Xylanases; Two-dimensional gel electrophoresis; Protein pattern
Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface by Norihiko Okochi; Michiko Kato-Murai; Tetsuya Kadonosono; Mitsuyoshi Ueda (pp. 597-603).
Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.
Keywords: Catalytic triad; Proteolytic antibody light chain; Cell surface display; Site-directed mutagenesis
Enzyme activities in activated sludge flocs by Guang-Hui Yu; Pin-Jing He; Li-Ming Shao; Duu-Jong Lee (pp. 605-612).
This study quantified the activities of enzymes in extracellular polymeric substances (EPS) and in pellets. Seven commonly adopted extraction schemes were utilized to extract from aerobic flocs the contained EPS, which were further categorized into loosely bound (LB) and tightly bound (TB) fractions. Ultrasonication effectively extracted the EPS from sludge flocs. Enzyme assay tests showed that the protease activity was localized mainly on the pellets, α-amylase and α-glucosidase activities were largely bound with LB-EPS, and few protease, α-amylase, or α-glucosidase activities were associated with the TB-EPS fraction. There exists no correlation between the biochemical compositions of EPS and the distribution of enzyme activities in the sludge matrix. The 44–65% of α-amylase and 59–100% of α-glucosidase activities noted with the LB-EPS indicate heterogeneous hydrolysis patterns in the sludge flocs with proteins and carbohydrates.
Keywords: Enzymatic distribution; Enzymes; Extracellular polymeric substances; Extraction process; Infrared spectra; Sludge
Purification and biochemical characterization of a laccase from the aquatic fungus Myrioconium sp. UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene by C. Martin; M. Pecyna; H. Kellner; N. Jehmlich; C. Junghanns; D. Benndorf; M. von Bergen; D. Schlosser (pp. 613-624).
Myrioconium sp. strain UHH 1-13-18-4 is an ascomycete anamorph isolated from the river Saale, Central Germany. An extracellular, monomeric, and glycosylated laccase with a molecular mass of 72.7 kDa as determined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and an isoelectric point below 2.8 was purified from CuSO4 and vanillic acid amended liquid fungal cultures grown in malt extract medium. The catalytic efficiencies (k cat/K m) for the oxidation of syringaldazine, 2,6-dimethoxyphenol, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) were 67.3, 46.9, and 28.2 s−1 mM−1, respectively, with K m values of 4.2, 67.8, and 104.9 μM. After pre-incubation at different pH values and temperatures for 1 h, more than 80% of the initial laccase activity was retained between pH 4 to 6 and 15°C. The laccase-encoding gene was identified and sequenced at both the genomic and complementary DNA (cDNA) level, and corresponding structural characteristics and putative regulatory elements of the promoter region are reported. The identification of two tryptic peptides of the purified enzyme by mass spectrometry confirmed the identity of the functional laccase protein with the translated genomic sequence of the Myrioconium sp. laccase. Myrioconium sp. laccase shows the highest degree of identity with laccases from ascomycetes belonging to the family Sclerotiniaceae, order Helotiales.
Keywords: Ascomycete; Aquatic fungi; Laccase; Characterization; Gene; Expression
Characterization of a novel two-component regulatory system involved in the regulation of both actinorhodin and a type I polyketide in Streptomyces coelicolor by Yinhua Lu; Weihua Wang; Dan Shu; Weiwen Zhang; Lei Chen; Zhongjun Qin; Sheng Yang; Weihong Jiang (pp. 625-635).
To seek more information on function of two-component regulatory systems (TCSs) in Streptomyces coelicolor, a dozen TCS-knockout mutants were generated, and phenotype changes were determined. One TCS (SCO5403/5404)-deleted mutant with phenotype change was obtained. Here, we report the characterization of this novel TCS, designated as RapA1/A2 (regulation of both actinorhodin and a type I polyketide), using genetic and proteomic approaches. Although growth and morphological analyses showed no difference between the knockout mutant and wild-type strain M145, a visible decrease of the production of actinorhodin (Act) was observed in rapA1/A2 mutant. The decrease can be restored by introducing rapA1/A2 genes on an integrative vector. A 2D-gel based proteomic analysis showed that knockout of rapA1/A2 resulted in reduced expression of a putative 3-oxoacyl-[acyl-carrier protein] reductase that is part of a biosynthetic cluster for a cryptic type I polyketide. Further reverse-transcriptase-polymerase chain reaction (RT-PCR) analyses confirmed that expression levels of several biosynthetic genes and the respective pathway-specific regulatory genes actII-ORF4 and kasO for these two clusters were all down-regulated in the rapA1/A2 mutant, compared to M145. Taken together, the results demonstrated that RapA1/A2 may serve as a positive regulator for biosynthesis of both Act and the uncharacterized polyketide in S. coelicolor, and the effects exerted by RapA1/A2 were dependent on the pathway-specific regulatory genes.
Keywords: Two-component regulatory system; Proteomics; Secondary metabolism; Streptomyces coelicolor
Engineering the halophilic bacterium Halomonas elongata to produce β-carotene by Marta Rodríguez-Sáiz; Cristina Sánchez-Porro; Juan Luis De La Fuente; Encarnación Mellado; José Luis Barredo (pp. 637-643).
Engineering halophilic bacteria to produce carotenoids is a subject of great scientific and commercial interest, as carotenoids are desirable products used as additives and colorants in the food industry, with β-carotene the most prominent. With this target, we expressed the β-carotene biosynthetic genes crtE, crtY, crtI, and crtB from Pantoea agglomerans and the cDNA encoding isopentenyl pyrophosphate isomerase from Haematococcus pluvialis in the halophilic bacterium Halomonas elongata obtaining a strain able to produce practically pure β-carotene. Reverse transcription-polymerase chain reaction analysis showed crtY, crtI, and crtB heterologous expression in a selected exconjugant of H. elongata. Biosynthesis of β-carotene was dependent on NaCl concentration in the culture medium, with the highest production (560 μg per g of dry weight) in 2% NaCl. On the contrary, no β-carotene was detected in 15% NaCl. Successful construction of the β-carotene biosynthetic pathway in H. elongata opens the possibility of engineering halophilic bacteria for carotenoid production.
Keywords: Carotene; Halophilic; Halomonas ; Engineering; Carotenoid
Improving PCR and qPCR detection of hydrogenase A (hydA) associated with Clostridia in pure cultures and environmental sludges using bovine serum albumin by Mei-Yun Wang; Betty H. Olson; Jo-Shu Chang (pp. 645-656).
Detection of hydA genes of Clostridia spp. using degenerative and species specific primers for C. butyricum were optimized by the addition of bovine serum albumin (BSA) to polymerase chain reaction (PCR) and quantitative PCR (qPCR) reactions. BSA concentrations ranging from 100 to 400 ng/μl were examined using pure cultures and a variety of environmental samples as test targets. A BSA concentration of 100 ng/μl, which is lower than previously reported in the literature, was found to be most effective in improving the detection limit. The brightness of amplicons with 100 ng/μl BSA increased in ethidium bromide-treated gels, the minimum detection limit with BSA was at least one log greater, and cycle threshold (C T) values were lower than without BSA in qPCR indicating improved detection of target deoxyribonucleic acid for most samples tested. Although amplicon visualization was improved at BSA concentrations greater than or equal to 100 ng/μl, gene copy numbers detected by qPCR were less, CT values were increased, and T m values were altered. SYBR Green dissociation curves of qPCR products of DNA from pure culture or sludge samples showed that BSA at 100 ng/μl reduced the variability of peak areas and T m values.
Keywords: Bovine serum albumin; Clostridia; Hydrogenase; PCR; qPCR
Transcriptional profile induced by furazolidone treatment of Shigella flexneri by Hua Fu; Wenchuan Leng; Jing Wang; Wenliang Zhang; Junping Peng; Lingling Wang; Qi Jin (pp. 657-667).
Shigella flexneri is a facultative intracellular pathogen responsible for endemic shigellosis especially in developing countries. Furazolidone, a nitrofuran derivative, is very effective against the infection with S. flexneri. To examine potential effects of furazolidone on this germ, a whole-genome DNA microarray was constructed and transcriptional profiles of the responses to furazolidone were determined. The expressing data revealed adaptive responses of S. flexneri to oxidative stress induced by furazolidone treatment. Iron metabolism was found to be disturbed by furazolidone through derepression of the iron uptake regulon. In addition, energy metabolism, amino acid metabolism, cofactors metabolism, and DNA repair system were also affected by the drug. These data establish a potential for furazolidone to enhance free radical reactions through reductive activation by oxygen-sensitive nitroreductase. Moreover, we provide evidence that furazolidone is able to cause metabolic dysfunction, which cannot always be attributed to oxidative stress, and interactions between reductive metabolites of furazolidone and S. flexneri should be considered.
Keywords: Shigella flexneri ; Furazolidone; microarray
Endophytic fungi as models for the stereoselective biotransformation of thioridazine by Keyller Bastos Borges; Warley De Souza Borges; Mônica Tallarico Pupo; Pierina Sueli Bonato (pp. 669-674).
The stereoselective kinetic biotransformation of thioridazine, a phenothiazine neuroleptic drug, by endophytic fungi was investigated. In general, the sulfur of lateral chain (position 2) or the sulfur of phenothiazinic ring (position 5) were oxidated yielding the major human metabolites thioridazine-2-sulfoxide and thioridazine-5-sulfoxide. The quantity of metabolites biosynthesized varied among the 12 endophytic fungi evaluated. However, mono-2-sulfoxidation occurred in higher ratio and frequency. Among the 12 fungi evaluated, 4 of them deserve prominence for presenting an evidenced stereoselective biotransformation: Phomopsis sp. (TD2), Glomerella cingulata (VA1), Diaporthe phaseolorum (VR4), and Aspergillus fumigatus (VR12). Both enantiomers of thioridazine were consumed by the fungi; however, the 2-sulfoxidation yielded preferentially the R configuration at the sulfur atom.
Keywords: Stereoselective biotransformation; Thioridazine; Endophytic fungi
Influence of wine fermentation temperature on the synthesis of yeast-derived volatile aroma compounds by Ana M. Molina; Jan H. Swiegers; Cristian Varela; Isak S. Pretorius; Eduardo Agosin (pp. 675-687).
The yeast Saccharomyces cerevisiae synthesises a variety of volatile aroma compounds during wine fermentation. In this study, the influence of fermentation temperature on (1) the production of yeast-derived aroma compounds and (2) the expression of genes involved in aroma compounds’ metabolism (ADH1, PDC1, BAT1, BAT2, LEU2, ILV2, ATF1, ATF2, EHT1 and IAH1) was assessed, during the fermentation of a defined must at 15 and 28°C. Higher concentrations of compounds related to fresh and fruity aromas were found at 15°C, while higher concentrations of flowery related aroma compounds were found at 28°C. The formation rates of volatile aroma compounds varied according to growth stage. In addition, linear correlations between the increases in concentration of higher alcohol and their corresponding acetates were obtained. Genes presented different expression profiles at both temperatures, except ILV2, and those involved in common pathways were co-expressed (ADH1, PDC1 and BAT2; and ATF1, EHT1 and IAH1). These results demonstrate that the fermentation temperature plays an important role in the wine final aroma profile, and is therefore an important control parameter to fine-tune wine quality during winemaking.
Keywords: Fermentation temperature; Wine aroma; Wine fermentation; Wine yeast
Kinetics of consumption of fermentation products by anode-respiring bacteria by César I. Torres; Andrew Kato Marcus; Bruce E. Rittmann (pp. 689-697).
We determined the kinetic response of a community of anode-respiring bacteria oxidizing a mixture of the most common fermentation products: acetate, butyrate, propionate, ethanol, and hydrogen. We acclimated the community by performing three consecutive batch experiments in a microbial electrolytic cell (MEC) containing a mixture of the fermentation products. During the consecutive-batch experiments, the coulombic efficiency and start-up period improved with each step. We used the acclimated biofilm to start continuous experiments in an MEC, in which we controlled the anode potential using a potentiostat. During the continuous experiments, we tested each individual substrate at a range of anode potentials and substrate concentrations. Our results show low current densities for butyrate and hydrogen, but high current densities for propionate, acetate, and ethanol (maximum values are 1.6, 9.0, and 8.2 A/m2, respectively). Acetate showed a high coulombic efficiency (86%) compared to ethanol and propionate (49 and 41%, respectively). High methane concentrations inside the MEC during ethanol experiments suggest that methanogenesis is one reason why the coulombic efficiency was lower than that of acetate. Our results provide kinetic parameters, such as the anode overpotential, the maximum current density, and the Monod half-saturation constant, that are needed for model development when using a mixture of fermentation products. When we provided no electron donor, we measured current due to endogenous decay of biomass (~0.07 A/m2) and an open-cell potential (−0.54 V vs Ag/AgCl) associated with biomass components active in endogenous respiration.
Keywords: Coulombic efficiency; Fermentation products; Microbial electrolytic cell; Open-cell potential
Bacterial defluorination of 4-fluoroglutamic acid by Clár Donnelly; Cormac D. Murphy (pp. 699-703).
Fluorinated amino acids are used as enzyme inhibitors, mechanistic probes and in the production of pharmacologically active peptides. Because enantiomerically pure 4-fluoroglutamate is difficult to prepare, the selective degradation of the l-isomer is a potentially convenient method of obtaining d-4-fluoroglutamate from the racemate. In this paper, we describe our investigations on the degradation of 4-fluoroglutamate by bacteria. Fluoride ion was detected in resting-cell cultures of a number of bacteria that were incubated with racemic 4-fluoroglutamate. Analysis of the culture supernatants by chiral gas chromatography–mass spectrometry revealed that only the l-isomer was degraded. The degradation of 4-fluoroglutamate was also examined in cell-free extracts of Streptomyces cattleya and Proteus mirabilis, and it was observed that equimolar concentrations of fluoride ion and ammonia were generated. The activity was located in the soluble fraction of cell extracts, thus is not related to the l-2-amino-4-chloro-4-pentenoic acid dehydrochlorinase previously identified in membrane fractions of P. mirabilis.
Keywords: Amino acid; Bacteria; Dehalogenation
Anaerobically grown Thauera aromatica, Desulfococcus multivorans, Geobacter sulfurreducens are more sensitive towards organic solvents than aerobic bacteria by Ilka Duldhardt; Ivonne Nijenhuis; Frieder Schauer; Hermann J. Heipieper (pp. 705-711).
The effect of seven important pollutants and three representative organic solvents on growth of Thauera aromatica K172, as reference strain for nitrate-reducing anaerobic bacteria, was investigated. Toxicity in form of the effective concentrations (EC50) that led to 50% growth inhibition of potential organic pollutants such as BTEX (benzene, toluene, ethylbenzene, and xylene), chlorinated phenols and aliphatic alcohols on cells was tested under various anaerobic conditions. Similar results were obtained for Geobacter sulfurreducens and Desulfococcus multivorans as representative for Fe3+-reducing and sulphate-reducing bacteria, respectively, leading to a conclusion that anaerobic bacteria are far more sensitive to organic pollutants than aerobic ones. Like for previous studies for aerobic bacteria, yeast and animal cell cultures, a correlation between toxicity and hydrophobicity (log P values) of organic compounds for different anaerobic bacteria was ascertained. However, compared to aerobic bacteria, all three tested anaerobic bacteria were shown to be about three times more sensitive to the tested substances.
Keywords: Organic pollutants; BTEX; Phenols; Toxicity; Anaerobic bacteria; Thauera aromatica ; Desulfococcus multivorans ; Geobacter sulfurreducens
Influence of bicarbonate, sulfate, and electron donors on biological reduction of uranium and microbial community composition by Wensui Luo; Wei-Min Wu; Tingfen Yan; Craig S. Criddle; Philip M. Jardine; Jizhong Zhou; Baohua Gu (pp. 713-721).
A microcosm study was performed to investigate the effect of ethanol and acetate on uranium(VI) biological reduction and microbial community changes under various geochemical conditions. Each microcosm contained an uranium-contaminated sediment (up to 2.8 g U/kg) suspended in buffer with bicarbonate at concentrations of either 1 or 40 mM and sulfate at either 1.1 or 3.2 mM. Ethanol or acetate was used as an electron donor. Results indicate that ethanol yielded in significantly higher U(VI) reduction rates than acetate. A low bicarbonate concentration (1 mM) was favored for U(VI) bioreduction to occur in sediments, but high concentrations of bicarbonate (40 mM) and sulfate (3.2 mM) decreased the reduction rates of U(VI). Microbial communities were dominated by species from the Geothrix genus and Proteobacteria phylum in all microcosms. However, species in the Geobacteraceae family capable of reducing U(VI) were significantly enriched by ethanol and acetate in low-bicarbonate buffer. Ethanol increased the population of unclassified Desulfuromonales, while acetate increased the population of Desulfovibrio. Additionally, species in the Geobacteraceae family were not enriched in high-bicarbonate buffer, but the Geothrix and the unclassified Betaproteobacteria species were enriched. This study concludes that ethanol could be a better electron donor than acetate for reducing U(VI) under given experimental conditions, and electron donor and groundwater geochemistry alter microbial communities responsible for U(VI) reduction.
Keywords: Uranium(VI); Biological reduction; Electron donor; Groundwater; Microbial community; 16S rRNA analysis
Model-based analysis on growth of activated sludge in a sequencing batch reactor by Bing-Jie Ni; Han-Qing Yu (pp. 723-731).
A mathematical model is developed to describe the growth of multiple microbial species such as heterotrophs and autotrophs in activated sludge system. Performance of a lab-scale sequencing batch reactor involving storage process is used to evaluate the model. Results show that the model is appropriate for predicting the fate of major model components, i.e., chemical oxygen demand, storage polymers (X STO), volatile suspended solid (VSS), ammonia, and oxygen uptake rate (OUR). The influence of sludge retention time (SRT) on reactor performance is analyzed by model simulation. The biomass components require different time periods from one to four times of SRT to reach steady state. At an SRT of 20 days, the active bacteria (autotrophs and heterotrophs) constitute about 57% of the VSS; the remaining biomass is not active. The model established demonstrates its capacity of simulating the reactor performance and getting insight in autotrophic and heterotrophic growth in complex activated sludge systems.
Keywords: Activated sludge; Autotrophs; Growth; Heterotrophs; Modeling; Sequencing batch reactor
Horizontal transfer of genetic determinants for degradation of phenol between the bacteria living in plant and its rhizosphere by Yujing Wang; Ming Xiao; Xiaolu Geng; Jiaying Liu; Jun Chen (pp. 733-739).
Phenol and other monocyclic aromatic compounds (MACs) are highly water-soluble and volatile pollutants that plants are unable to completely degrade. Endophytic bacteria with MAC-degrading ability will facilitate phytoremediation, beneficial to plant survival in contaminated soil. Endophytic bacteria, strains FX1–FX3, and rhizosphere bacteria, strains FX0, FX4, and FX5, were isolated from the root tissue of a corn plant (Zea mays) and the corn rhizosphere near a chemical plant, respectively. The strains FX1–FX5 were able to grow on phenol and reduce phenol concentration, but the strain FX0 was unable to. The strains FX1, FX3, and FX4 were classified as Pseudomonas fluorescens and FX0, FX2, and FX5 as Burkholderia cepacia. The plasmids isolated from the strains FX1–FX5 were found to possess similar traits and to be loaded with a gene encoding the catechol 2, 3-dioxygenase (C23O), a key enzyme in the phenol degradation pathway. Alignment and phylogenetic analysis inferred that in situ horizontal transfer of the C23O gene might have occurred. The horizontal transfer of the C23O gene between endophytic and rhizosphere bacteria was proved by using conjugal matings experiment, in which the transconjugants were found to acquire the plasmid with the C23O gene, able to grow on phenol and degrade phenol.
Keywords: Pseudomonas fluorescens ; Bioremediation; Endophytic bacteria; Rhizosphere bacteria
Changes in the potential functional diversity of the bacterial community in biofilters by Jason A. Grove; William A. Anderson; Murray Moo-Young (pp. 741-747).
The bacterial community structure in a biofilter treating ethanol was investigated using community level physiological profiling. Laboratory scale biofilters of two sizes (5 or 11.5 cm internal diameter with 30 or 67 cm packed height, respectively) were packed with compost and a humidified airstream loaded with ethanol passed through them. Good removal efficiencies (82–100%) and elimination capacities (49–205 g ethanol m − 3 h − 1) were observed in all units. Compost packing media samples were extracted and the community level physiological profiles assayed using Biolog Ecoplates. The community structure was found to be similar over a range of a few centimetres. No differences were observed between sample sizes of 0.5–1 and 6 g, and therefore, the smaller sample size (typical of that used in previous studies) is appropriate for use in the future. Two studies of parallel systems showed that the community structure diverged during the acclimation period (10 days) in one pair, but in another pair, no divergence was observed and a similar shift in community profile was observed in both units between 25 and 40 days of operation. Community level physiological profiling with Biolog Ecoplates is a useful method for detecting differences between and changes within the bacterial communities in biofilters.
Keywords: Biofilter; Biofiltration; Biolog; Community-level physiological profiling (CLPP); Microbial community