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Applied Microbiology and Biotechnology (v.76, #5)
Bacillus megaterium—from simple soil bacterium to industrial protein production host by Patricia S. Vary; Rebekka Biedendieck; Tobias Fuerch; Friedhelm Meinhardt; Manfred Rohde; Wolf-Dieter Deckwer; Dieter Jahn (pp. 957-967).
Bacillus megaterium has been industrially employed for more than 50 years, as it possesses some very useful and unusual enzymes and a high capacity for the production of exoenzymes. It is also a desirable cloning host for the production of intact proteins, as it does not possess external alkaline proteases and can stably maintain a variety of plasmid vectors. Genetic tools for this species include transducing phages and several hundred mutants covering the processes of biosynthesis, catabolism, division, sporulation, germination, antibiotic resistance, and recombination. The seven plasmids of B. megaterium strain QM B1551 contain several unusual metabolic genes that may be useful in bioremediation. Recently, several recombinant shuttle vectors carrying different strong inducible promoters and various combinations of affinity tags for simple protein purification have been constructed. Leader sequences-mediated export of affinity-tagged proteins into the growth medium was made possible. These plasmids are commercially available. For a broader application of B. megaterium in industry, sporulation and protease-deficient as well as UV-sensitive mutants were constructed. The genome sequence of two different strains, plasmidless DSM319 and QM B1551 carrying seven natural plasmids, is now available. These sequences allow for a systems biotechnology optimization of the production host B. megaterium. Altogether, a “toolbox” of hundreds of genetically characterized strains, genetic methods, vectors, hosts, and genomic sequences make B. megaterium an ideal organism for industrial, environmental, and experimental applications.
Keywords: Bacillus megaterium ; High level production; Biosafety; Metabolic flux analysis; Genome sequencing
Ultrahigh bioproductivity from algae by Jeffrey M. Gordon; Juergen E. W. Polle (pp. 969-975).
The potential for dramatic increases in bioproductivity in algal photobioreactors relative to current biomass approaches, e.g., for converting sunlight into biofuels, by an unorthodox integration of photonics and biotechnologies is described. The key to greater biomass yields—projected as high as 100 g dry weight m−2 h−1—is a pronounced heightening of algal flux tolerance, achieved by tailoring the photonic temporal, spectral and intensity characteristics with pulsed light-emitting diodes. Such tailored photonic input is applied in concert with thin-channel ultradense culture photobioreactors with flow patterns that produce rapid light/dark algae exposure cycles. The artificial-light scheme is globally feasible only with electricity generated from renewables. Recent advances in ultra-efficient concentrator photovoltaics, as well as high-performance light-emitting diodes, create a practical reality for converting sunlight into pulsed red light and delivering it to indoor photobioreactors, with characteristic pulse times and intensities optimally suited to the rate-limiting dark reactions of photosynthesis. Cellular engineering built upon recent progress in modifying algal chlorophyll antenna size, in combination with metabolic engineering, could further enhance bioproductivity. The proposed strategy requires no major advances for implementation and adopts existing technologies.
Keywords: Photosynthesis; Algae; Photonics; Solar energy; Light-emitting diodes; Biofuels
Understanding nonaflatoxigenicity of Aspergillus sojae: a windfall of aflatoxin biosynthesis research by Perng-Kuang Chang; Kenichiro Matsushima; Tadashi Takahashi; Jiujiang Yu; Keietsu Abe; Deepak Bhatnagar; Gwo-Fang Yuan; Yasuji Koyama; Thomas E. Cleveland (pp. 977-984).
Aspergillus section Flavi includes aflatoxin-producing and nonproducing fungi. Aspergillus sojae is unable to produce aflatoxins and is generally recognized as safe for food fermentation. However, because of its taxonomical relatedness to aflatoxin-producing Aspergillus parasiticus and A. flavus, it is necessary to decipher the underlying mechanisms for its inability to produce aflatoxins. This review addresses the relationship between A. sojae and A. parasiticus and the advances that have been made in aflatoxin biosynthesis research, especially with regard to gene structure, genome organization, and gene regulation in A. parasiticus and A. flavus and how this has been used to assure the safety of A. sojae as an organism for food fermentation. The lack of aflatoxin-producing ability of A. sojae results primarily from an early termination point mutation in the pathway-specific aflR regulatory gene, which causes the truncation of the transcriptional activation domain of AflR and the abolishment of interaction between AflR and the AflJ co-activator. Both are required for gene expression. In addition, a defect in the polyketide synthase gene also contributes to its nonaflatoxigenicity.
Lipid accumulation in Schizochytrium G13/2S produced in continuous culture by E. Ganuza; M. S. Izquierdo (pp. 985-990).
Lipid and docosahexaenoic acid (DHA) accumulation into Schizochytrium G13/2S was studied under batch and continuous culture. Different glucose and glutamate concentrations were supplemented in a defined medium. During batch cultivation, lipid accumulation, 35% total fatty acids (TFA) occurred at the arithmetic growth phase but ceased when cell growth stopped. When continuous culture was performed under different glutamate concentrations, nitrogen-growth-limiting conditions induced the accumulation of 30–28% TFA in Schizochytrium. As the dilution rate decreased from 0.08 to 0.02 h−1, both cell dry weight and TFA content of the cell increased. Under a constant dilution rate of 0.04 h−1, carbon-limiting conditions decreased the TFA to 22%. Fatty acid profile was not affected by the different nutrient concentrations provided during continuous culture. Consequently, lipid accumulation can be induced through the carbon and nitrogen source concentration in the medium to maximise the TFA and subsequently DHA productivity by this microorganism.
Keywords: Continuous culture; Docosahexaenoic acid; Lipid accumulation; Schizochytrium
Ubiquinone and carotene production in the Mucorales Blakeslea and Phycomyces by Vera Kuzina; Enrique Cerdá-Olmedo (pp. 991-999).
The filamentous fungi Phycomyces blakesleeanus and Blakeslea trispora (Zygomycota, Mucorales) are actual or potential industrial sources of β-carotene and lycopene. These chemicals and the large terpenoid moiety of ubiquinone derive from geranylgeranyl pyrophosphate. We measured the ubiquinone and carotene contents of wild-type and genetically modified strains under various conditions. Light slightly increased the ubiquinone content of Blakeslea and had no effect on that of Phycomyces. Oxidative stress modified ubiquinone production in Phycomyces and carotene production in both fungi. Sexual interaction and mutations in both organisms made the carotene content vary from traces to 23 mg/g dry mass, while the ubiquinone content remained unchanged at 0.3 mg/g dry mass. We concluded that the biosyntheses of ubiquinone and carotene are not coregulated. The specific regulation for carotene biosynthesis does not affect even indirectly the production of ubiquinone, as would be expected if terpenoids were synthesized through a branched pathway that could divert precursor flows from one branch to another.
Biocatalytic racemization of sec-alcohols and α-hydroxyketones using lyophilized microbial cells by Bettina M. Nestl; Constance V. Voss; Anne Bodlenner; Ursula Ellmer-Schaumberger; Wolfgang Kroutil; Kurt Faber (pp. 1001-1008).
Biocatalytic racemization of aliphatic and aryl-aliphatic sec-alcohols and α-hydroxyketones (acyloins) was accomplished using whole resting cells of bacteria, fungi, and one yeast. The mild (physiological) reaction conditions ensured the suppression of undesired side reactions, such as elimination or condensation. Cofactor and inhibitor studies suggest that the racemization proceeds through an equilibrium-controlled enzymatic oxidation–reduction sequence via the corresponding ketones or α-diketones, respectively, which were detected in various amounts. Ketone formation could be completely suppressed by exclusion of molecular oxygen. Figure Biocatalytic racemization whole microbial cells
Keywords: sec-alcohols; Acyloins; Isomerization; Biotransformation; Racemization
Substantially monodispersed poly(ɛ-l-lysine)s frequently occurred in newly isolated strains of Streptomyces sp. by Hideo Hirohara; Masayuki Saimura; Munenori Takehara; Masahiro Miyamoto; Atsushi Ikezaki (pp. 1009-1016).
The presence of poly(ɛ-l-lysine) (ɛ-PL) was found quite frequently by screening various strains of Streptomyces sp. Most of the ten newly obtained ɛ-PLs, when they were produced from glucose, showed a polydispersity index of M w/M n = 1.01 using ion-pair chromatography analysis. The polymers were classified into five groups according to their chain lengths. The average numbers of residues in the five groups were 32, 28, 25, 19, and 16, respectively. The use of glycerol instead of glucose resulted in decreases of 10 to 20% in the M n and slight increases in the M w/M n. These observations indicated the chain length and polydispersity of ɛ-PL were primarily determined by each producer strain. Proton and 13C NMR analysis revealed the signals of glycerol-derived ester at the C terminus of the polymer from several producers including the first discovered S. albulus strain, although the percentages of the ester were low under our culture conditions. These results, coupled with the previous observation that $$ { ext{SO}}^{{{ ext{2}} - }}_{{ ext{4}}} $$ was essential for the polymer production, led to discussion on the mechanistic aspects of monomer activation, elongation, and termination in the biosynthesis of ɛ-PL.
Keywords: Biocompatible; Biosynthesis; Molecular weight distribution; Poly(ɛ-l-lysine); Screening; Streptomyces sp.
Partial purification and chemical characterization of a glycoprotein (putative hydrocolloid) emulsifier produced by a marine bacterium Antarctobacter by Tony Gutiérrez; Barbara Mulloy; Charlie Bavington; Kenny Black; David H. Green (pp. 1017-1026).
During screening for novel emulsifiers and surfactants, a marine alphaproteobacterium, Antarctobacter sp. TG22, was isolated and selected for its production of an extracellular emulsifying agent, AE22. This emulsifier was produced optimally in a low-nutrient seawater medium supplemented with glucose and was extractable by cold ethanol precipitation of the high-molecular-weight fraction (>100 kDa). Production of AE22 commenced towards the late exponential phase of growth, with maximum emulsifying activity detected after approximately 4 days of the cells entering the death phase. Chemical, chromatographic and nuclear magnetic resonance spectroscopic analysis confirmed AE22 to be a high-molecular-weight (>2,000 kDa) glycoprotein with high uronic acids content, thus denoting an apparent polyanionic structure. Functional characterization showed this polymer to compare well to xanthan gum and gum arabic as an emulsion-stabilizing agent for a range of different food oils. However, AE22 exhibited better stabilizing than emulsifying properties, which could be conferred by its viscosifying effect in solution or from certain chemical groups found on the polysaccharide or protein moieties of the polymer. This new high-molecular-weight glycoprotein exhibits interesting functional qualities that are comparable to other biopolymers of this type and shows particular promise as an emulsion-stabilizing agent in biotechnological applications.
Enhancement of substrate recognition ability by combinatorial mutation of β-glucosidase displayed on the yeast cell surface by Takeshi Fukuda; Michiko Kato-Murai; Tetsuya Kadonosono; Hiroshi Sahara; Yoji Hata; Shin-ichiro Suye; Mitsuyoshi Ueda (pp. 1027-1033).
Recently, in family 3 β-glucosidase (BGL), the catalytically important Asp nucleophile has been identified in the SDW segment of the SDWG sequence by site-directed mutagenesis. However, the details about the roles of each amino acid residue of the SDWG sequence have not been investigated. W293 of the SDW segment, which is the residue next to the nucleophile (D292) in family 3 BGL, is very important for hydrolytic reaction as a binder to a substrate. G294 of the SDWG sequence might play an important role in catalysis. In this study, to obtain a functional BGL1 mutant by the substitution of G294 using a genetic engineering method, the library of mutant BGL1 from Aspergillus oryzae was rapidly constructed by yeast cell surface engineering, and the hydrolytic activities of mutants were comprehensively detected. Consequently, G294F, G294W, and G294Y, in which G was substituted with aromatic amino acids, showed higher activities for substrate recognition than the parent strain (1.5-, 1.5-, and 1.6-fold, respectively). These results suggest the presence of some interaction between the sugar rings and aromatic ring of W293 at the entrance of the catalytic pocket, which enhances the substrate recognition of β-glucosidase.
Keywords: Yeast cell surface engineering; SDW segment; Family 3 β-glucosidase; Substrate recognition; Comprehensive library of mutants
Escherichia coli hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities by Toshinari Maeda; Viviana Sanchez-Torres; Thomas K. Wood (pp. 1035-1042).
In the past, it has been difficult to discriminate between hydrogen synthesis and uptake for the three active hydrogenases in Escherichia coli (hydrogenase 1, 2, and 3); however, by combining isogenic deletion mutations from the Keio collection, we were able to see the role of hydrogenase 3. In a cell that lacks hydrogen uptake via hydrogenase 1 (hyaB) and via hydrogenase 2 (hybC), inactivation of hydrogenase 3 (hycE) decreased hydrogen uptake. Similarly, inactivation of the formate hydrogen lyase complex, which produces hydrogen from formate (fhlA) in the hyaB hybC background, also decreased hydrogen uptake; hence, hydrogenase 3 has significant hydrogen uptake activity. Moreover, hydrogen uptake could be restored in the hyaB hybC hycE and hyaB hybC fhlA mutants by expressing hycE and fhlA, respectively, from a plasmid. The hydrogen uptake results were corroborated using two independent methods (both filter plate assays and a gas-chromatography-based hydrogen uptake assay). A 30-fold increase in the forward reaction, hydrogen formation by hydrogenase 3, was also detected for the strain containing active hydrogenase 3 activity but no hydrogenase 1 or 2 activity relative to the strain lacking all three hydrogenases. These results indicate clearly that hydrogenase 3 is a reversible hydrogenase.
Keywords: E. coli hydrogenase 3; Reversible hydrogenase; Hydrogen production; Hydrogen uptake
Evaluation of colors in green mutants isolated from purple bacteria as a host for colorimetric whole-cell biosensors by Kazuyuki Yoshida; Daiki Yoshioka; Koichi Inoue; Shinichi Takaichi; Isamu Maeda (pp. 1043-1050).
The change in carotenoid-based bacterial color from yellow to red can be applied to whole-cell biosensors. We generated several green mutants to emphasize the color change in such biosensors. The blue-green crtI-deleted mutant, Rhodopseudomonas palustris no.711, accumulated the colorless carotenoid precursor, phytoene. Green Rhodovulum sulfidophilum M31 accumulated neurosporene, a downstream product of phytoene. Another green mutant, Rhodobacter sphaeroides Ga, accumulated neurosporene and chloroxanthin, which are both downstream products of phytoene. All green mutants accumulated bacteriochlorophyll a. Photosynthetic membrane obtained from the green mutants all exhibited decreased absorption of wavelength range at 510–570 nm. Therefore, these indicate that the greenish bacterial colors were mainly caused by the existence of bacteriochlorophyll a and the changes in carotenoid composition in photosynthetic membrane. The colors of the green mutants and their wild-type strains were plotted in the CIE-L*a*b* color space, and the color difference (ΔE*ab) values between a green mutant and its wild type were calculated. ΔE*ab values were higher in the green mutants than in Rdv. sulfidophilum CDM2, the yellowish host strain of reported biosensors. These data indicate that change in bacterial color from green to red is more distinguishable than that from yellow to red as a reporter signal of carotenoid-based whole-cell biosensors.
Development of a selection system for the detection of L-ribose isomerase expressing mutants of Escherichia coli by Cassandra De Muynck; Jef Van der Borght; Marjan De Mey; Sofie L. De Maeseneire; Inge N. A. Van Bogaert; Joeri Beauprez; Wim Soetaert; Erick Vandamme (pp. 1051-1057).
L-Arabinose isomerase (E.C. 5.3.1.14) catalyzes the reversible isomerization between L-arabinose and L-ribulose and is highly selective towards L-arabinose. By using a directed evolution approach, enzyme variants with altered substrate specificity were created and screened in this research. More specifically, the screening was directed towards the identification of isomerase mutants with L-ribose isomerizing activity. Random mutagenesis was performed on the Escherichia coli L-arabinose isomerase gene (araA) by error-prone polymerase chain reaction to construct a mutant library. To enable screening of this library, a selection host was first constructed in which the mutant genes were transformed. In this selection host, the genes encoding for L-ribulokinase and L-ribulose-5-phosphate-4-epimerase were brought to constitutive expression and the gene encoding for the native L-arabinose isomerase was knocked out. L-Ribulokinase and L-ribulose-5-phosphate-4-epimerase are necessary to ensure the channeling of the formed product, L-ribulose, to the pentose phosphate pathway. Hence, the mutant clones could be screened on a minimal medium with L-ribose as the sole carbon source. Through the screening, two first-generation mutants were isolated, which expressed a small amount of L-ribose isomerase activity.
Keywords: L-arabinose isomerase; L-ribose isomerase; Directed evolution; Screening; Gene disruption
Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae by Feng Jie Jin; Taisuke Watanabe; Praveen Rao Juvvadi; Jun-ichi Maruyama; Manabu Arioka; Katsuhiko Kitamoto (pp. 1059-1068).
In this study, we investigated the effects of proteinase gene disruption on heterologous protein production by Aspergillus oryzae. The human lysozyme (HLY) was selected for recombinant production as a model for the heterologous protein. A tandem HLY construct fused with α-amylase (AmyB) was expressed by A. oryzae in which the Kex2 cleavage site was inserted at the upstream of HLY. HLY was successfully processed from AmyB and produced in the medium. We performed a systematic disruption analysis of five proteinase genes (pepA, pepE, alpA, tppA, and palB) in the HLY-producing strain with the adeA selectable marker. Comparative analysis indicated that disruption of the tppA gene encoding a tripeptidyl peptidase resulted in the highest increase (36%) in the HLY production. We further deleted the tppA gene in the pepE or palB disruptant with another selectable marker, argB. Consequently, a double disruption of the tppA and pepE genes led to a 63% increase in the HLY production compared to the control strain. This is the first study to report that the double disruption of the tppA and pepE genes improved the production level of a heterologous protein by filamentous fungi.
Keywords: Aspergillus oryzae ; Proteinase; Heterologous protein production
Transcriptional regulation of two cellobiohydrolase encoding genes (cel1 and cel2) from the wood-degrading basidiomycete Polyporus arcularius by Yuka Ohnishi; Mitsutoshi Nagase; Tsuyoshi Ichiyanagi; Yutaka Kitamoto; Tadanori Aimi (pp. 1069-1078).
In the current studies, we sequenced and characterized the genomic and complementary deoxyribonucleic acid clones encoding the cellobiohydrolase encoding genes cel1 and cel2 of Polyporus arcularius. The predicted amino acid sequences of Cel1 and Cel2 are similar to glycosyl hydrolase family 7 and 6 proteins, respectively. The expression of cel1 and cel2 was induced by microcrystalline cellulose (Avicel) and cellopentaose but repressed by glucose, cellobiose, cellotriose, and cellotetraose. There was a very low level of cel1 and cel2 transcription regardless of the carbon source. These results suggest that P. arcularius cells constitutively express a very low level of cellulase that can degrade insoluble crystalline cellulose and that the transcription of cel1 and cel2 in the cells is induced by products produced by these endoglucanases such as cellooligosaccharides.
Keywords: Cellobiohydrolase; Polyporus arcularius ; Microcrystalline cellulose; Real time PCR; Cellopentaose; Transcription
Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624 by Kenji Yamagishi; Toshiyuki Kimura; Sigeru Oita; Tatsuki Sugiura; Hirofumi Hirai (pp. 1079-1091).
Phanerochaete sordida YK-624 is a hyper lignin-degrading basidiomycete possessing greater ligninolytic selectivity than either P. chrysosporium or Trametes versicolor. To construct a gene transformation system for P. sordida YK-624, uracil auxotrophic mutants were generated using a combination of ultraviolet (UV) radiation and 5-fluoroorotate resistance as a selection scheme. An uracil auxotrophic strain (UV-64) was transformed into a uracil prototroph using the marker plasmid pPsURA5 containing the orotate phosphoribosyltransferase gene from P. sordida YK-624. This system generated approximately 50 stable transformants using 2 × 107 protoplasts. Southern blot analysis demonstrated that the transformed pPsURA5 was ectopically integrated into the chromosomal DNA of all transformants. The enhanced green fluorescent protein (EGFP) gene was also introduced into UV-64. The transformed EGFP was expressed in the co-transformants driven by P. sordida glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator regions.
Keywords: Phanerochaete sordida ; EGFP; Genetic transformation; PsURA5 ; Uracil auxotroph
Comparative global transcription analysis of sodium hypochlorite, peracetic acid, and hydrogen peroxide on Pseudomonas aeruginosa by David A. Small; Wook Chang; Freshteh Toghrol; William E. Bentley (pp. 1093-1105).
Disinfectants are routinely used in hospitals and health care facilities for surface sterilization. However, the mechanisms by which these disinfectants kill and the extent to which bacteria, including Pseudomonas aeruginosa, are resistant remains unclear. Consequently, P. aeruginosa nosocomial infections result in considerable casualties and economic hardship. Previously, DNA microarrays were utilized to analyze the genome-wide transcription changes in P. aeruginosa after oxidative antimicrobial (sodium hypochlorite, peracetic acid, and hydrogen peroxide) exposure. Simultaneous analysis of these transcriptome datasets provided a comprehensive understanding of the differential responses to these disinfectants. An analysis of variance, functional classification analysis, metabolic pathway analysis, Venn diagram analysis, and principal component analysis revealed that sodium hypochlorite exposure resulted in more genome-wide changes than either peracetic acid or hydrogen peroxide exposures.
Keywords: Pseudomonas aeruginosa ; Sodium hypochlorite; Peracetic acid; Hydrogen peroxide; Microarrays
Elucidation of veA-dependent genes associated with aflatoxin and sclerotial production in Aspergillus flavus by functional genomics by J. W. Cary; G. R. OBrian; D. M. Nielsen; W. Nierman; P. Harris-Coward; J. Yu; D. Bhatnagar; T. E. Cleveland; G. A. Payne; A. M. Calvo (pp. 1107-1118).
The aflatoxin-producing fungi, Aspergillus flavus and A. parasiticus, form structures called sclerotia that allow for survival under adverse conditions. Deletion of the veA gene in A. flavus and A. parasiticus blocks production of aflatoxin as well as sclerotial formation. We used microarray technology to identify genes differentially expressed in wild-type veA and veA mutant strains that could be involved in aflatoxin production and sclerotial development in A. flavus. The DNA microarray analysis revealed 684 genes whose expression changed significantly over time; 136 of these were differentially expressed between the two strains including 27 genes that demonstrated a significant difference in expression both between strains and over time. A group of 115 genes showed greater expression in the wild-type than in the veA mutant strain. We identified a subgroup of veA-dependent genes that exhibited time-dependent expression profiles similar to those of known aflatoxin biosynthetic genes or that were candidates for involvement in sclerotial production in the wild type.
Keywords: Aspergillus flavus ; veA ; Aflatoxin; Sclerotia; Microarray
pH shock induces overexpression of regulatory and biosynthetic genes for actinorhodin productionin Streptomyces coelicolor A3(2) by Yoon Jung Kim; Jae Yang Song; Myung Hee Moon; Colin P. Smith; Soon-Kwang Hong; Yong Keun Chang (pp. 1119-1130).
Actinorhodin production is markedly enhanced when an acidic pH shock is applied to a surface-grown culture of Streptomyces coelicolor A3(2). For an in-depth study of this phenomenon, transcriptional analyses using DNA microarrays and reverse transcription polymerase chain reaction and proteomic analysis were performed. Investigated were expression levels of the regulators and enzymes responsible for signal transduction and actinorhodin biosynthesis and enzymes involved in some major metabolic pathways. Regulators PkaG, AfsR, AfsS and/or another unidentified regulator and ActII-ORF4, in sequence, were observed to be activated by pH shock. In addition, a number of genes associated with actinorhodin production and secretion and the major central metabolic pathways investigated were observed to be upregulated with pH shock. Fatty acid degradation was particularly promoted by pH shock, while fatty acid biosynthesis was suppressed; it is envisaged that this enriches the precursor pool (acetyl-CoA) and building blocks for actinorhodin biosynthesis. Furthermore, glucose 6-phosphate dehydrogenases, initiating the pentose phosphate pathway, were highly activated by pH shock, enriching the reduced nicotinamide adenine dinucleotide phosphate (NADPH) pool for biosynthesis in general. It is deduced that these metabolic changes caused by pH shock have positively contributed to the stimulation of actinorhodin biosynthesis in a concerted manner.
Keywords: pH shock; Actinorhodin production; Signal transduction; S. coelicolor
Heterologous MVA-S prime Ad5-S boost regimen induces high and persistent levels of neutralizing antibody response against SARS coronavirus by Lei Ba; Christopher E. Yi; Linqi Zhang; David D. Ho; Zhiwei Chen (pp. 1131-1136).
Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (CoV), SARS-CoV. In previous studies, we showed that a SARS-CoV spike (S) glycoprotein-based modified vaccinia Ankara (MVA-S) vaccine could induce strong neutralizing antibody (Nab) response which might have played a critical role in protecting Chinese rhesus monkeys from the pathogenic viral challenge. To date, however, it remains unknown what the minimal level of Nab is required to achieve sterile immunity in humans. It is therefore important to explore techniques to maximize the level of Nab response in vivo. Here, we evaluate various vaccination regimens using combinations of DNA-S, MVA-S, and adenovirus type 5 (Ad5-S) vaccines. We show that in vaccinated mice and rabbits, a heterologous MVA-S prime with Ad5-S boost regimen induces the highest and most persistent level of Nab response when compared with other combinations. Interestingly, the initial level of Nab after prime does not necessarily predict the magnitude of the secondary response after the boost. Thus, our data provides a promising optimal regimen for vaccine development in humans against SARS-CoV infection.
Keywords: SARS; SARS-CoV; MVA; Ad5; Vaccine
Inhibition of Vibrio biofilm formation by a marine actinomycete strain A66 by JianLan You; XiaoLi Xue; LiXiang Cao; Xin Lu; Jian Wang; LiXin Zhang; ShiNing Zhou (pp. 1137-1144).
China remains by far the largest aquaculture producer in the world. However, biofilms formed by pathogenic Vibrio strains pose serious problems to marine aquaculture. To provide a strategy for biofilm prevention, control, and eradication, extracts from 88 marine actinomycetes were screened. Thirty-five inhibited the biofilm formation of Vibrio harveyi, Vibrio vulnificus, and Vibrio anguillarum at a concentration of 2.5% (v/v). Thirty-three of the actinomycete extracts dispersed the mature biofilm. Six extracts inhibited the quorum-sensing system of V. harveyi by attenuating the signal molecules N-acylated homoserine lactones’ activity. Strain A66, which was identified as Steptomyces albus, both attenuated the biofilms and inhibited their quorum-sensing system. It is suggested that strain A66 is a promising candidate to be used in future marine aquaculture.
Keywords: Vibrio biofilm; Quorum sensing; Biocontrol
Endophytic bacteria in sunflower (Helianthus annuus L.): isolation, characterization, and production of jasmonates and abscisic acid in culture medium by G. Forchetti; O. Masciarelli; S. Alemano; D. Alvarez; G. Abdala (pp. 1145-1152).
This study was designed to isolate and characterize endophytic bacteria from sunflower (Helianthus annuus) grown under irrigation and water stress (drought) conditions, to analyze growth of isolated bacteria under drought condition, and to evaluate the ability of bacteria isolated from plants cultivated under drought to produce jasmonates (JAs) and abscisic acid (ABA). Bacteria were isolated from soil samples collected when sunflower plants were at the end of the vegetative stage. A total of 29 endophytic strains were isolated from plants grown under irrigation or drought condition. Eight strains (termed SF1 through SF8) were selected based on nitrogen-fixing ability. All eight strains showed positive catalase and oxidase activities; five strains (SF2, SF3, SF4, SF5, SF7) solubilized phosphates; none of the strains produced siderophores. Strains SF2, SF3, SF4, and SF5, the ones with the highest phosphate solubilization ability, strongly inhibited growth of the pathogenic fungi Verticillum orense and Sclerotinia sclerotiorum but had less inhibitory effect on Alternaria sp. Among the eight strains, SF2 showed 99.9% sequence homology with Achromobacter xiloxidans or Alcaligenes sp., while the other seven showed 99.9% homology with Bacillus pumilus. Strains SF2, SF3, and SF4 grown in control medium produced jasmonic acid (JA), 12-oxo-phytodienoic acid (OPDA), and ABA. These three strains did not differ in amount of JA or OPDA produced. ABA content was higher than that of JA, and production of both ABA and JA increased under drought condition. The characteristics of these isolated bacterial strains have technological implications for inoculant formulation and improved growth of sunflower crops.
Keywords: Sunflower; PGPR; Jasmonates; Abscisic acid
Production and characterization of medium-chain-length polyhydroxyalkanoate with high 3-hydroxytetradecanoate monomer content by fadB and fadA knockout mutant of Pseudomonas putida KT2442 by Wenkuan Liu; Guo-Qiang Chen (pp. 1153-1159).
Medium-chain-length polyhydroxyalkanoates (mcl-PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate, 3-hydroxydodecanoate, and high-content 3-hydroxytetradecanoate (HTD) was produced by knockout mutant Pseudomonas putida KT2442 termed P. putida KTOY06. When grown on 6 to14 g/L single-carbon-source tetradecanoic acid, P. putida KTOY06, which β-oxidation pathway was weakened by deleting genes of 3-ketoacyl-coenzyme A (CoA) thiolase (fadA) and 3-hydroxyacyl-CoA dehydrogenase (fadB), for the first time, produced several mcl-PHA including 31 to 49 mol% HTD as a major monomer. HHx contents in these mcl-PHAs remained approximately constant at less than 3 mol%. In addition, large amounts of oligo-HTD were detected in cells, indicating the limited ability of P. putida KTOY06 in polymerizing long-chain-length 3-hydroxyalkanoates. The mcl-PHA containing high HTD monomer contents was found to have both higher crystallinity and improved tensile strength compared with that of typical mcl-PHA.
Keywords: PHB; Polyhydroxyalkanoates; PHA; 3-Hydroxytetradecanoate; Pseudomonas putida
Production of volatile aroma compounds by bacterial strains isolated from different surface-ripened French cheeses by Pawinee Deetae; Pascal Bonnarme; Henry E. Spinnler; Sandra Helinck (pp. 1161-1171).
Twelve bacterial strains belonging to eight taxonomic groups: Brevibacterium linens, Microbacterium foliorum, Arthrobacter arilaitensis, Staphylococcus cohnii, Staphylococcus equorum, Brachybacterium sp., Proteus vulgaris and Psychrobacter sp., isolated from different surface-ripened French cheeses, were investigated for their abilities to generate volatile aroma compounds. Out of 104 volatile compounds, 54 volatile compounds (identified using dynamic headspace technique coupled with gas chromatography-mass spectrometry [GC-MS]) appeared to be produced by the different bacteria on a casamino acid medium. Four out of eight species used in this study: B. linens, M. foliorum, P. vulgaris and Psychrobacter sp. showed a high flavouring potential. Among these four bacterial species, P. vulgaris had the greatest capacity to produce not only the widest varieties but also the highest quantities of volatile compounds having low olfactive thresholds such as sulphur compounds. Branched aldehydes, alcohols and esters were produced in large amounts by P. vulgaris and Psychrobacter sp. showing their capacity to breakdown the branched amino acids. This investigation shows that some common but rarely mentioned bacteria present on the surface of ripened cheeses could play a major role in cheese flavour formation and could be used to produce cheese flavours.
Keywords: Flavour compounds; Ripened cheese; Bacteria; Sulphur compounds; Proteus vulgaris ; Psychrobacter sp.
Quantification of anammox populations enriched in an immobilized microbial consortium with low levels of ammonium nitrogen and at low temperature by Bipin K. Pathak; Futaba Kazama; Yasuhiro Tanaka; Kazuhiro Mori; Tatsuo Sumino (pp. 1173-1179).
Anaerobic ammonium oxidizing (anammox) bacteria present in microbial communities in two laboratory-scale upflow anoxic reactors supplied with small amounts of ammonium (<3 mg/l) at low temperature were detected and quantified. The reactors, operated at 20°C, were seeded with an immobilized microbial consortium (IMC) and anaerobic granules (AG) from an upflow anaerobic sludge blanket (UASB) treating brewery wastewater. Our results showed that complete ammonium and nitrite removal with greater than 92% total nitrogen removal efficiency was achieved in the reactor inoculated with both the IMC and AG, while that of the reactor inoculated with only the IMC was lower than 40%; enrichment was successful after the addition of AG. Quantitative fluorescence in situ hybridization (FISH) analysis confirmed that anammox bacteria were present only in the reactor inoculated with an IMC and AG. The copy number of the 16S-rRNA gene of the anammox bacteria calculated by most probable number-polymerase chain reaction (MPN-PCR) from the total DNA extracted from both reactors (2.5 × 104 copies/μg of DNA) was two orders lower than that of the domain bacteria (2.5 × 106 copies/μg of DNA). The results revealed that immobilized multiple seed sludges were optimal for anammox enrichment at low temperature and ammonium concentrations.
Keywords: Anammox bacteria; FISH; Immobilized microbial consortium; MPN-PCR
Reduction of Fe(II)EDTA-NO by a newly isolated Pseudomonas sp. strain DN-2 in NO x scrubber solution by Shi-Han Zhang; Wei Li; Cheng-Zhi Wu; Han Chen; Yao Shi (pp. 1181-1187).
Biological reduction of nitric oxide (NO) chelated by ferrous ethylenediaminetetraacetate (Fe(II)EDTA) to N2 is one of the core processes in a chemical absorption–biological reduction integrated technique for nitrogen oxide (NO x ) removal from flue gases. A new isolate, identified as Pseudomonas sp. DN-2 by 16S rRNA sequence analysis, was able to reduce Fe(II)EDTA-NO. The specific reduction capacity as measured by NO was up to 4.17 mmol g DCW−1 h−1. Strain DN-2 can simultaneously use glucose and Fe(II)EDTA as electron donors for Fe(II)EDTA-NO reduction. Fe(III)EDTA, the oxidation of Fe(II)EDTA by oxygen, can also serve as electron acceptor by strain DN-2. The interdependency between various chemical species, e.g., Fe(II)EDTA-NO, Fe(II)EDTA, or Fe (III)EDTA, was investigated. Though each complex, e.g., Fe(II)EDTA-NO or Fe(III)EDTA, can be reduced by its own dedicated bacterial strain, strain DN-2 capable of reducing Fe(III)EDTA can enhance the regeneration of Fe(II)EDTA, hence can enlarge NO elimination capacity. Additionally, the inhibition of Fe(II)EDTA-NO on the Fe(III)EDTA reduction has been explored previously. Strain DN-2 is probably one of the major contributors for the continual removal of NO x due to the high Fe(II)EDTA-NO reduction rate and the ability of Fe(III)EDTA reduction.
Keywords: Pseudomonas sp.; Biological reduction; Fe(II)EDTA-NO; Fe(III)EDTA, NO x
Adsorption of surfactants on a Pseudomonas aeruginosa strain and the effect on cell surface lypohydrophilic property by XingZhong Yuan; FangYi Ren; GuangMing Zeng; Hua Zhong; HaiYan Fu; Jia Liu; XinMiao Xu (pp. 1189-1198).
The adsorption behavior of five surfactants, cetyltrimethylammonium bromide (CTAB), Triton X-100, Tween 80, sodium dodecyl sulfate (SDS), and rhamnolipid, on a Pseudomonas aeruginosa strain and the effect of temperature and ionic strength (IS) on the adsorption were studied. The change of cell surface lypohydrophilic property caused by surfactant adsorption was also investigated. The results showed that the adsorption kinetics of the surfactants on the cell followed the second-order law. CTAB adsorption was the fastest one under the experimental conditions, and it took longest for SDS adsorption to equilibrate because of electric repulsion. The adsorption of Triton X-100 and Tween 80 was characterized by short equilibration time, and rhamnolipid adsorption reached equilibrium in about 90 min. The adsorption isotherms of all the surfactants on the bacterium fitted Freundlich equation well, but the adsorption capacity and mode were variations for the surfactants as indicated by k and n parameters in the equations. The adsorption mode for all the surfactants except SDS is probably hydrophilic interaction because the adsorption totally turned the cell surface to be more hydrophobic. Neither the temperature nor the IS had significant effect on CTAB adsorption, but higher IS significantly enhanced SDS adsorption and modestly strengthened adsorption of Triton X-100, Tween 80, and rhamnolipid. Higher temperature strengthened adsorption of SDS but weakened the adsorption of Triton X-100, Tween 80, and rhamnolipid.
Keywords: Surfactant; Adsorption; Cell surface; Hydrophobicity
Structure and stability of aerobic granules cultivated under different shear force in sequencing batch reactors by Yao Chen; Wenju Jiang; David Tee Liang; Joo Hwa Tay (pp. 1199-1208).
The cultivation of stable aerobic granules as well as granular structure and stability in sequencing batch reactors under different shear force were investigated in this study. Four column sequencing batch reactors (R1–R4) were operated under various shear force, in terms of superficial upflow air velocity of 0.8, 1.6, 2.4, and 3.2 cm s−1, respectively. Aerobic granules were formed in all reactors in the experiment. It was found that the magnitude of shear force has an important impact on the granule stability. At shear force of 2.4 and 3.2 cm s−1, granules can maintain a robust structure and have the potential of long-term operation. Granules developed in low shear force (R1, 0.8 cm s−1 and R2, 1.6 cm s−1) deteriorated to large-sized filamentous granules with irregular shape, loose structure and resulted in poor performance and operation instability. Granules cultivated under high shear force (R3, 2.4 cm s−1 and R4, 3.2 cm s−1) stabilized to clear outer morphology, dense and compact structure, and with good performance in 120 days operation. Fractal dimension (Df) represents the internal structure of granules and can be used as an important indicator to describe the structure and stability of granules. Due to the combined effects of shear force and growth force, the mature granules developed in R3 and R4 also displayed certain differences in granular structure and characteristics.
Keywords: Aerobic granule; Shear force; Stability; Granule structure; Fractal dimension (Df)