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Applied Microbiology and Biotechnology (v.95, #4)

Fungal cytochrome P450 sterol 14α-demethylase (CYP51) and azole resistance in plant and human pathogens by Rayko Becher; Stefan G. R. Wirsel (pp. 825-840).

Azoles have been applied widely to combat pathogenic fungi in medicine and agriculture and, consequently, loss of efficacy has occurred in populations of some species. Often, but not always, resistance was found to result from amino acid substitutions in the molecular target of azoles, 14α-sterol demethylase (CYP51 syn. ERG11). This review summarizes CYP51 function, evolution, and structure. Furthermore, we compare the occurrence and contribution of CYP51 substitutions to azole resistance in clinical and field isolates of important fungal pathogens. Although no crystal structure is available yet for any fungal CYP51, homology modeling using structures from other origins as template allowed deducing models for fungal orthologs. These models served to map amino acid changes known from clinical and field isolates. We conclude with describing the potential consequences of these changes on the topology of the protein to explain CYP51-based azole resistance. Knowledge gained from molecular modeling and resistance research will help to develop novel azole structures.

Keywords: CYP51; ERG11; 14α-Demethylase; Azole; Resistance; Homology model; Fungicide

Downstream processing of biotechnological produced succinic acid by Ke-Ke Cheng; Xue-Bing Zhao; Jing Zeng; Ru-Chun Wu; Yun-Zhen Xu; De-Hua Liu; Jian-An Zhang (pp. 841-850).

Succinic acid is a promising chemical which has a wide range of applications and can be biologically produced. The separation of succinic acid from fermentation broth makes more than 50 % of the total costs in their microbial production. This review summarizes the present state of methods studied for the recovery and purification of biologically produced succinate. Previous studies on the separation of succinic acid primarily include direct crystallization, precipitation, membrane separation, extraction, chromatography, and in situ separation. No single method has proved to be simple and efficient, and improvements are especially needed with regard to yield, purity, and energy consumption. It is argued that separation technologies coupled with upstream technology, in situ product removal, and biorefining strategy deserve more attentions in the future.

Keywords: Bioconversion; Broth; Fermentation; Recovery; Succinic acid

Stress-tolerant P-solubilizing microorganisms by N. Vassilev; B. Eichler-Löbermann; M. Vassileva (pp. 851-859).

Drought, high/low temperature, and salinity are abiotic stress factors accepted as the main reason for crop yield losses in a world with growing population and food price increases. Additional problems create nutrient limitations and particularly low P soil status. The problem of phosphate fertilizers, P plant nutrition, and existing phosphate bearing resources can also be related to the scarcity of rock phosphate. The modern agricultural systems are highly dependent on the existing fertilizer industry based exclusively of this natural, finite, non-renewable resource. Biotechnology offers a number of sustainable solutions that can mitigate these problems by using plant beneficial, including P-solubilizing, microorganisms. This short review paper summarizes the current and future trends in isolation, development, and application of P-solubilizing microorganisms in stress environmental conditions bearing also in mind the imbalanced cycling and unsustainable management of P. Special attention is devoted to the efforts on development of biotechnological strategies for formulation of P-solubilizing microorganisms in order to increase their protection against adverse abiotic factors.

Keywords: Abiotic stress factors; Microbial P-solubilization; Stress tolerance; Inoculant formulation

Interspecific interactions in mixed microbial cultures in a biodegradation perspective by H. Mikesková; Č. Novotný; K. Svobodová (pp. 861-870).

In recent works, microbial consortia consisting of various bacteria and fungi exhibited a biodegradation performance superior to single microbial strains. A highly efficient biodegradation of synthetic dyes, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and other organic pollutants can be achieved by mixed microbial cultures that combine degradative enzyme activities inherent to individual consortium members. This review summarizes biodegradation results obtained with defined microbial cocultures and real microbial consortia. The necessity of using a proper strategy for the microbial consortium development and optimization was clearly demonstrated. Molecular genetic and proteomic techniques have revolutionized the study of microbial communities, and techniques such as the denaturing gradient gel electrophoresis, rRNA sequencing, and metaproteomics have been used to identify consortium members and to study microbial population dynamics. These analyses could help to further enhance and optimize the natural activities of mixed microbial cultures.

Keywords: Microbial consortia; Consortium development; Biodegradation; Interspecific interactions; PCR-based techniques

Microbial transformation of azaarenes and potential uses in pharmaceutical synthesis by Igor A. Parshikov; Alexander I. Netrusov; John B. Sutherland (pp. 871-889).

Pyridine, quinoline, acridine, indole, carbazole, and other heterocyclic nitrogen-containing compounds (azaarenes) can be transformed by cultures of bacteria and fungi to produce a variety of new derivatives, many of which have biological activity. In many cases, the microbial biotransformation processes are regio- and stereoselective so that the transformation products may be useful for the synthesis of new candidate drugs.

Keywords: Azaarenes; Biotransformation; Drugs; Heterocyclic compounds; Pharmacology

Genetic linkage mapping in fungi: current state, applications, and future trends by Marie Foulongne-Oriol (pp. 891-904).

Genetic mapping is a basic tool for eukaryotic genomic research. Linkage maps provide insights into genome organization and can be used for genetic studies of traits of interest. A genetic linkage map is a suitable support for the anchoring of whole genome sequences. It allows the localization of genes of interest or quantitative trait loci (QTL) and map-based cloning. While genetic mapping has been extensively used in plant or animal models, this discipline is more recent in fungi. The present article reviews the current status of genetic linkage map research in fungal species. The process of linkage mapping is detailed, from the development of mapping populations to the construction of the final linkage map, and illustrated based on practical examples. The range of specific applications in fungi is browsed, such as the mapping of virulence genes in pathogenic species or the mapping of agronomically relevant QTL in cultivated edible mushrooms. Future prospects are finally discussed in the context of the most recent advances in molecular techniques and the release of numerous fungal genome sequences.

Keywords: Genetic mapping; Map-based cloning; Molecular markers; QTL

Variation of the by-product spectrum during α-ketoglutaric acid production from raw glycerol by overexpression of fumarase and pyruvate carboxylase genes in Yarrowia lipolytica by Christina Otto; Venelina Yovkova; Andreas Aurich; Stephan Mauersberger; Gerold Barth (pp. 905-917).

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric, isocitric, pyruvic (PA), and α-ketoglutaric (KGA) acids, triggered by growth limitation and excess of carbon source. This is leading to an increased interest in this non-conventional yeast for biotechnological applications. To improve the KGA production by Y. lipolytica for an industrial application, it is necessary to reduce the amounts of by-products, e.g., fumarate (FU) and PA, because production of by-products is a main disadvantage of the KGA production by this yeast. We have examined whether the concentration of secreted organic acids (main product KGA and PA as major by-product and FU, malate (MA), and succinate (SU) as minor by-products) can be influenced by a gene–dose-dependent overexpression of fumarase (FUM) or pyruvate carboxylase (PYC) genes under KGA production conditions. Recombinant Y. lipolytica strains were constructed, which harbor multiple copies of the respective FUM1, PYC1 or FUM1, and PYC1 genes. Overexpression of the genes FUM1 and PYC1 resulted in strongly increased specific enzyme activities during cultivation of these strains on raw glycerol as carbon source in bioreactors. The recombinant Y. lipolytica strains showed different product selectivity of the secreted organic acids KGA, PA, FU, MA, and SU. Concentrations of the by-products FU, MA, SU, and PA decreased significantly at overproduction of FUM and increased at overproduction of PYC and also of FUM and PYC simultaneously. In contrast, the production of KGA with the multicopy strains H355A(FUM1) and H355A(FUM1-PYC1) was comparable with the wild-type strain H355 or slightly lower in case of H355(PYC1). KGA productivity was not changed significantly compared with strain H355 whereas product selectivity of the main product KGA was increased in H355A(FUM1).

Keywords: Fumarase; Pyruvate carboxylase; α-Ketoglutaric acid; Product selectivity; Raw glycerol; Yarrowia lipolytica

A marine-derived Streptomyces sp. MS449 produces high yield of actinomycin X₂ and actinomycin D with potent anti-tuberculosis activity by Caixia Chen; Fuhang Song; Qian Wang; Wael M. Abdel-Mageed; Hui Guo; Chengzhang Fu; Weiyuan Hou; Huanqin Dai; Xueting Liu; Na Yang; Feng Xie; Ke Yu; Ruxian Chen; Lixin Zhang (pp. 919-927).

In the course of our screening program for anti-Mycobacterium bovis bacillus Calmette-Guérin (BCG) and anti-Mycobacterium tuberculosis H37Rv (MTB H37Rv) agents from our marine natural product library, a newly isolated actinomycete strain, designated as MS449, was picked out for further investigation. The strain MS449, isolated from a sediment sample collected from South China Sea, produced actinomycin X₂ and actinomycin D in substantial quantities, which showed strong inhibition of BCG and MTB H37Rv. The structures of actinomycins were elucidated by nuclear magnetic resonance and mass spectrometric analysis. The strain MS449 was taxonomically characterized on the basis of morphological and phenotypic characteristics, genotypic data, and phylogenetic analysis. The 16S rRNA gene sequence of the strain was determined and a database search indicated that the strain was closely associated with the type strain of Streptomyces avermitilis (99.7 % 16S rRNA gene similarity). S. avermitilis has not been previously reported to produce actinomycins. The marine-derived strain of Streptomyces sp. MS449 produced notably higher quantities of actinomycin X₂ (1.92 mg/ml) and actinomycin D (1.77 mg/ml) than previously reported actinomycins producing strains. Thus, MS449 was considered of great potential as a new industrial producing strain of actinomycin X₂ and actinomycin D.

Keywords: Streptomyces avermitilis ; Anti-BCG; Anti-tuberculosis; Actinomycins

Production of 10-hydroxystearic acid from oleic acid and olive oil hydrolyzate by an oleate hydratase from Lysinibacillus fusiformis by Bi-Na Kim; Young-Chul Joo; Yeong-Su Kim; Kyoung-Rok Kim; Deok-Kun Oh (pp. 929-937).

A recombinant enzyme from Lysinibacillus fusiformis was expressed, purified, and identified as an oleate hydratase because the hydration activity of the enzyme was the highest for oleic acid (with a k _cat of 850 min⁻¹ and a K _m of 540 μM), followed by palmitoleic acid, γ-linolenic acid, linoleic acid, myristoleic acid, and α-linolenic acid. The optimal reaction conditions for the enzymatic production of 10-hydroxystearic acid were pH 6.5, 35 °C, 4% (v/v) ethanol, 2,500 U ml⁻¹ (8.3 mg ml⁻¹) of enzyme, and 40 g l⁻¹ oleic acid. Under these conditions, 40 g l⁻¹ (142 mM) oleic acid was converted into 40 g l⁻¹ (133 mM) 10-hydroxystearic acid for 150 min, with a molar yield of 94% and a productivity of 16 g l⁻¹ h⁻¹, and olive oil hydrolyzate containing 40 g l⁻¹ oleic acid was converted into 40 g l⁻¹ 10-hydroxystearic acid for 300 min, with a productivity of 8 g l⁻¹ h⁻¹.

Keywords: Lysinibacillus fusiformis ; Oleic acid; 10-Hydroxystearic acid production; Oleate hydratase; Olive oil hydrolyzate

Bactericidal activity of Musca domestica cecropin (Mdc) on multidrug-resistant clinical isolate of Escherichia coli by X. Lu; J. Shen; X. Jin; Y. Ma; Y. Huang; H. Mei; F. Chu; J. Zhu (pp. 939-945).

The housefly (Musca domestica) larvae have been used clinically to cure osteomyelitis, decubital necrosis, lip boil, ecthyma and malnutritional stagnation ever since the Ming/Qing Dynasty (1368 Anno Domini) till now, in China. In prior research, we have cloned and characterized a new gene of antimicrobial peptide cecropin from M. domestica larvae. This peptide was potently active against Gram-positive and Gram-negative bacteria standard strain. In the present study, we evaluated the possibility of Mdc to be a potential bactericidal agent against clinical isolates of multidrug-resistant (MDR) Escherichia coli and to elucidate the related antimicrobial mechanisms. Antimicrobial activity assays indicated a minimal inhibitory concentration (MIC) of 1.56 μM. Bactericidal kinetics at MIC showed that Mdc rapid killing of MDR E. coli. Lipopolysaccharide (LPS) dose-dependently suppressed Mdc antibacterial potency indicates that LPS is the initial binding site of Mdc in E. coli. Propidium iodide-based flow cytometry revealed that Mdc causes E. coli membrane permeabilization. Transmission electron micrographs further indicated that a remarkable damage in the bacteria’s outer and inner membrane, even the leakage of cytoplasmic contents induced by Mdc. DNA binding experimental result implies that DNA is one of the possible intracellular targets of Mdc. Of note, Mdc did not show a perceptible cytotoxic effect on human red blood cells. Altogether, these results suggest that Mdc could be an excellent candidate for the development of more efficacious bactericidal agents.

Keywords: Musca domestica cecropin; Multidrug resistant; Flow cytometry; Transmission electron; Lipopolysaccharide

A novel thermoacidophilic and thermostable endo-β-1,4-glucanase from Phialophora sp. G5: its thermostability influenced by a distinct β-sheet and the carbohydrate-binding module by Junqi Zhao; Pengjun Shi; Huoqing Huang; Zhongyuan Li; Tiezheng Yuan; Peilong Yang; Huiying Luo; Yingguo Bai; Bin Yao (pp. 947-955).

An endo-β-1,4-glucanase gene, egG5, was cloned from the fungus Phialophora sp. G5. The 1,290-bp open reading frame encodes a bimodular cellulase composed of an N-terminal family 1 carbohydrate-binding module (CBM) and a C-terminal family 5 glycoside hydrolase catalytic module. Recombinant EgG5 produced in Pichia pastoris exhibited maximal activity at pH 4.0–5.0 and 70 °C, retained 40% of the maximal activity at pH 2.0, and was stable at pH 2.0–10.0. When compared with its closest homolog in Trichoderma sp. C-4 (70.6% identity), EgG5 had better thermostability (51.6% activity at 65 °C for 12 h vs 10% activity at 60 °C for 20 min). Sequence–structure analysis indicated that the distinct β-sheet in EgG5 in place of a linking loop in Trichoderma sp. C-4 endoglucanase might be the reason. To verify its function, two mutants, EgG5-Mut (disrupting the β-sheet with four amino acid substitutions) and EgG5-CBM (removing the CBM), were constructed, expressed in P. pastoris, and characterized. Both mutants had similar pH optima (pH 4.0) and temperature optima (70 °C) but varied in pH stabilities (pH 2.0–10.0 and pH 2.0–7.0, respectively) and thermostabilities. The thermostability of EgG5-Mut (13.4% activity vs 52.5% of EgG5 at 65 °C for 12 h) confirmed the effect of β-sheet on enzyme thermostability. EgG5-CBM was more thermostable (94.9% activity at 65 °C for 12 h and 15.5% activity at 80 °C for 30 min) and had higher specific activity (711.6 vs 60.3 U mg⁻¹ of EgG5). This study presents an excellent endoglucanase with potential use in the bioconversion of lignocellulosic materials and provides good ideas for the improvement of enzyme thermostability.

Keywords: Carbohydrate-binding module (CBM); Endo-β-1,4-glucanase; Phialophora sp. G5; Thermostability

Codon-optimized glucoamylase sGAI of Aspergillus awamori improves starch utilization in an industrial yeast by Lorenzo Favaro; Tania Jooste; Marina Basaglia; Shaunita H. Rose; Maryna Saayman; Johann F. Görgens; Sergio Casella; Willem H. van Zyl (pp. 957-968).

The development of a yeast that converts raw starch to ethanol in one step (called consolidated bioprocessing) could yield large cost reductions in the bioethanol industry. The aim of this study was to develop an efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production. A native and codon-optimized variant of the Aspergillus awamori glucoamylase gene were expressed in the S. cerevisiae Y294 laboratory strain. Codon optimization resulted to be effective and the synthetic sequence sGAI was then δ-integrated into a S. cerevisiae strain with promising industrial fermentative traits. The mitotically stable recombinant strains showed high enzymatic capabilities both on soluble and raw starch (2425 and 1140 nkat/g dry cell weight, respectively). On raw corn starch, the engineered yeasts exhibited improved fermentative performance with an ethanol yield of 0.42 (g/g), corresponding to 75 % of the theoretical maximum yield.

Keywords: Industrial yeast; Codon optimization; δ-integration; Raw starch; Ethanol production

Enhancement of photoheterotrophic biohydrogen production at elevated temperatures by the expression of a thermophilic clostridial hydrogenase by Shou-Chen Lo; Shau-Hua Shih; Jui-Jen Chang; Chun-Ying Wang; Chieh-Chen Huang (pp. 969-977).

The working temperature of a photobioreactor under sunlight can be elevated above the optimal growth temperature of a microorganism. To improve the biohydrogen productivity of photosynthetic bacteria at higher temperatures, a [FeFe]-hydrogenase gene from the thermophile Clostridium thermocellum was expressed in the mesophile Rhodopseudomonas palustris CGA009 (strain CGA-CThydA) using a log-phase expression promoter P pckA to drive the expression of heterogeneous hydrogenase gene. In contrast, a mesophilic Clostridium acetobutylicum [FeFe]-hydrogenase gene was also constructed and expressed in R. palustris (strain CGA-CAhydA). Both transgenic strains were tested for cell growth, in vivo hydrogen production rate, and in vitro hydrogenase activity at elevated temperatures. Although both CGA-CThydA and CGA-CAhydA strains demonstrated enhanced growth over the vector control at temperatures above 38 °C, CGA-CThydA produced more hydrogen than the other strains. The in vitro hydrogenase activity assay, measured at 40 °C, confirmed that the activity of the CGA-CThydA hydrogenase was higher than the CGA-CAhydA hydrogenase. These results showed that the expression of a thermophilic [FeFe]-hydrogenase in R. palustris increased the growth rate and biohydrogen production at elevated temperatures. This transgenic strategy can be applied to a broad range of purple photosynthetic bacteria used to produce biohydrogen under sunlight.

Keywords: Rhodopseudomonas palustris ; Clostridium thermocellum ; Hydrogen production; [FeFe]-hydrogenase; Thermostable; Thermophiles

Substrate-specific transcription of the enigmatic GH61 family of the pathogenic white-rot fungus Heterobasidion irregulare during growth on lignocellulose by Igor Yakovlev; Gustav Vaaje-Kolstad; Ari M. Hietala; Emil Stefańczyk; Halvor Solheim; Carl Gunnar Fossdal (pp. 979-990).

The GH61 represents the most enigmatic Glycoside Hydrolase family (GH) regarding enzymatic activity and importance in cellulose degradation. Heterobasidion irregulare is a necrotizing pathogen and white-rot fungus that causes enormous damages in conifer forests. The genome of H. irregulare allowed identification of ten HiGH61 genes. qRT-PCR analysis separate the HiGH61 members into two groups; one that show up regulation on lignocellulosic substrates (HiGH61A, HiGH61B, HiGH61D, HiGH61G, HiGH61H, and HiGH61I) and a second showing either down-regulation or constitutive expression (HiGH61C, HiGH61E, HiGH61F, and HiGH61J). HiGH61H showed up to 17,000-fold increase on spruce heartwood suggesting a pivotal role in cellulose decomposition during saprotrophic growth. Sequence analysis of these genes reveals that all GH61s except HiGH61G possess the conserved metal-binding motif essential for activity. The sequences also divide into groups having either an insert near the N terminus or an insert near the second catalytic histidine, which may represent extensions of the substrate-binding surface. Three of the HiGH61s encode cellulose-binding modules (CBM1). Interestingly, HiGH61H and HiGH61I having CBM1s are up-regulated on pure cellulose. There was a common substrate-specific induction patterns of the HiGH61s with several reference cellulolytic and hemicellulolytic GHs, this taken together with their low transcript levels on media lacking lignocellulose, reflect the concerted nature of cell wall polymer degradation.

Keywords: Heterobasidion spp.; Glycoside hydrolases; GH61; Wood degradation; Gene expression

Lactobacillus reuteri CRL 1101 highly produces mannitol from sugarcane molasses as carbon source by Maria Eugenia Ortiz; María José Fornaguera; Raúl R. Raya; Fernanda Mozzi (pp. 991-999).

Mannitol is a natural polyol extensively used in the food industry as low-calorie sugar being applicable for diabetic food products. We aimed to evaluate mannitol production by Lactobacillus reuteri CRL 1101 using sugarcane molasses as low-cost energy source. Mannitol formation was studied in free-pH batch cultures using 3–10% (w/v) molasses concentrations at 37 °C and 30 °C under static and agitated conditions during 48 h. L. reuteri CRL 1101 grew well in all assayed media and heterofermentatively converted glucose into lactic and acetic acids and ethanol. Fructose was used as an alternative electron acceptor and reduced it to mannitol in all media assayed. Maximum mannitol concentrations of 177.7 ± 26.6 and 184.5 ± 22.5 mM were found using 7.5% and 10% molasses, respectively, at 37 °C after 24-h incubation. Increasing the molasses concentration from 7.5% up to 10% (w/v) and the fermentation period up to 48 h did not significantly improve mannitol production. In agitated cultures, high mannitol values (144.8 ± 39.7 mM) were attained at 8 h of fermentation as compared to static ones (5.6 ± 2.9 mM), the highest mannitol concentration value (211.3 ± 15.5 mM) being found after 24 h. Mannitol 2-dehydrogenase (MDH) activity was measured during growth in all fermentations assayed; the highest MDH values were obtained during the log growth phase, and no correlation between MDH activities and mannitol production was observed in the fermentations performed. L. reuteri CRL 1101 successfully produced mannitol from sugarcane molasses being a promising candidate for microbial mannitol synthesis using low-cost substrate.

Keywords: Mannitol; Lactobacillus ; Lactic acid bacteria; Sugarcane molasses

Lactobacillus reuteri CRL 1101 highly produces mannitol from sugarcane molasses as carbon source by Maria Eugenia Ortiz; María José Fornaguera; Raúl R. Raya; Fernanda Mozzi (pp. 991-999).

Keywords: Mannitol; Lactobacillus ; Lactic acid bacteria; Sugarcane molasses

Physiological characterization of recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase pathway: validation of activity through ¹³C-based metabolic flux analysis by Marta Papini; Intawat Nookaew; Verena Siewers; Jens Nielsen (pp. 1001-1010).

Several bacterial species and filamentous fungi utilize the phosphoketolase pathway (PHK) for glucose dissimilation as an alternative to the Embden–Meyerhof–Parnas pathway. In Aspergillus nidulans, the utilization of this metabolic pathway leads to increased carbon flow towards acetate and acetyl CoA. In the first step of the PHK, the pentose phosphate pathway intermediate xylulose-5-phosphate is converted into acetylphosphate and glyceraldehyde-3-phosphate through the action of xylulose-5-phosphate phosphoketolase, and successively acetylphosphate is converted into acetate by the action of acetate kinase. In the present work, we describe a metabolic engineering strategy used to express the fungal genes of the phosphoketolase pathway in Saccharomyces cerevisiae and the effects of the expression of this recombinant route in yeast. The phenotype of the engineered yeast strain MP003 was studied during batch and chemostat cultivations, showing a reduced biomass yield and an increased acetate yield during batch cultures. To establish whether the observed effects in the recombinant strain MP003 were due directly or indirectly to the expression of the phosphoketolase pathway, we resolved the intracellular flux distribution based on ¹³C labeling during chemostat cultivations. From flux analysis it is possible to conclude that yeast is able to use the recombinant pathway. Our work indicates that the utilization of the phosphoketolase pathway does not interfere with glucose assimilation through the Embden–Meyerhof–Parnas pathway and that the expression of this route can contribute to increase the acetyl CoA supply, therefore holding potential for future metabolic engineering strategies having acetyl CoA as precursor for the biosynthesis of industrially relevant compounds.

Keywords: Phosphoketolase; Metabolic engineering; ¹³C-based metabolic flux analysis; Acetyl CoA supply; Glycolysis

Volatile sulphur compounds and pathways of l-methionine catabolism in Williopsis yeasts by Amelia W. J. Tan; Pin-Rou Lee; Yi-Xin Seow; Peter K. C. Ong; Shao-Quan Liu (pp. 1011-1020).

Volatile sulphur compounds (VSCs) are important to the food industry due to their high potency and presence in many foods. This study assessed for the first time VSC production and pathways of l-methionine catabolism in yeasts from the genus Williopsis with a view to understanding VSC formation and their potential flavour impact. Five strains of Williopsis saturnus (var. saturnus, var. subsufficiens, var. suavolens, var. sargentensis and var. mrakii) were screened for VSC production in a synthetic medium supplemented with l-methionine. A diverse range of VSCs were produced including dimethyl disulphide, dimethyl trisulphide, 3-(methylthio)-1-propanal (methional), 3-(methylthio)-1-propanol (methionol), 3-(methylthio)-1-propene, 3-(methylthio)-1-propyl acetate, 3-(methylthio)-1-propanoic acid (methionic acid) and ethyl 3-(methylthio)-1-propanoate, though the production of these VSCs varied between yeast strains. W. saturnus var. saturnus NCYC22 was selected for further studies due to its relatively high VSC production. VSC production was characterised step-wise with yeast strain NCYC22 in coconut cream at different l-methionine concentrations (0.00–0.20%) and under various inorganic sulphate (0.00–0.20%) and nitrogen (ammonia) supplementation (0.00–0.20%), respectively. Optimal VSC production was obtained with 0.1% of l-methionine, while supplementation of sulphate had no significant effect. Nitrogen supplementation showed a dramatic inhibitory effect on VSC production. Based on the production of VSCs, the study suggests that the Ehrlich pathway of l-methionine catabolism is operative in W. saturnus yeasts and can be manipulated by adjusting certain nutrient parameters to control VSC production.

Keywords: Sulphur; Flavour; Williopsis saturnus ; l-Methionine; Fermentation; Yeast

Comparison of the transient responses of Escherichia coli to a glucose pulse of various intensities by Sirichai Sunya; Frank Delvigne; Jean-Louis Uribelarrea; Carole Molina-Jouve; Nathalie Gorret (pp. 1021-1034).

Dynamic stimulus-responses of Escherichia coli DPD2085, yciG::LuxCDABE reporter strain, to glucose pulses of different intensities (0.08, 0.4 and 1 g L⁻¹) were compared using glucose-limited chemostat cultures at dilution rate close to 0.15 h⁻¹. After at least five residence times, the steady-state cultures were disturbed by a pulse of glucose, engendering conditions of glucose excess with concomitant oxygen limitation. In all conditions, glucose consumption, acetate and formate accumulations followed a linear relationship with time. The resulting specific uptake and production rates as well as respiratory rates were rapidly increased within the first seconds, which revealed a high ability of E. coli strain to modulate its metabolism to a new environment. For transition from glucose-excess to glucose-limited conditions, the cells rapidly re-established its pseudo-steady state. The dynamics of transient responses at the macroscopic viewpoint were shown to be independent on the glucose pulse intensity in the tested range. On the contrary, the E. coli biosensor yciG::luxCDABE revealed a transcriptional induction of yciG gene promoter depending on the quantities of the glucose added, through in situ and online monitoring of the bioluminescence emitted by the cells. Despite many studies describing the dynamics of the transient response of E. coli to glucose perturbations, it is the first time that a direct comparison is reported, using the same experimental design (strain, medium and experimental set up), to study the impact of the glucose pulse intensity on the dynamics of microbial behaviour regarding growth, respiration and metabolite productions.

Keywords: Dynamic responses; Escherichia coli biosensor yciG::luxCDABE ; Effect of the glucose intensity; Glucose pulse; In situ and online bioluminescence monitoring

Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitata by Manuela D. Machado; Eduardo V. Soares (pp. 1035-1042).

Membrane integrity has been used as a criterion for the definition of cell viability. In the present work, staining conditions (time and dye concentration) for the evaluation of membrane integrity in a fluorescence microplate reader, using the membrane-impermeant nucleic-acid dye SYTOX Green, were optimized. Incubating Pseudokirchneriella subcapitata algal cells with 0.5 μmol/l SYTOX Green for 40 min allowed a clear discrimination between live (intact plasma membrane) and dead cells (with compromised plasma membrane). Algal cell suspensions, labelled with SYTOX Green, exhibited a green fluorescence proportional to the fraction of the cells with a permeabilized plasma membrane. The optimized staining conditions were used to assess the toxicity of 1-pentanol on P. subcapitata in a short-term exposure (6 h) assay. The loss of membrane integrity in the cell population increased with the concentration of 1-pentanol. The 6-h EC₁₀ and EC₅₀ values were 7,617 mg/l 1-pentanol (95 % confidence limits 4,670–9,327) and 12,818 mg/l 1-pentanol (95 % confidence limits 10,929–15,183), respectively. The developed microplate-based short-term assay can be useful in the high-throughput screening of toxics or environmental samples using the alga P. subcapitata.

Keywords: Membrane integrity; Microplate assay; Selenastrum capricornutum ; 1-Pentanol; SYTOX Green; Acute toxicity

Assessment of the anti-biofouling potentials of a copper iodide-doped nylon mesh by Tetsuya Sato; Yoshie Fujimori; Tsuruo Nakayama; Yasuo Gotoh; Yoshihiko Sunaga; Michiko Nemoto; Tadashi Matsunaga; Tsuyoshi Tanaka (pp. 1043-1050).

We propose a copper iodide (CuI)-doped nylon mesh prepared using polyiodide ions as a precursor toward anti-biofouling polymer textile. The CuI-doped nylon mesh was subjected to the prevention of biofouling in marine environments. The attachment of the marine organisms was markedly inhibited on the CuI-doped nylon mesh surface until 249 days. Scanning electron microscopy–energy dispersive X-ray analysis indicated that copper compounds were maintained in the nylon mesh after the field experiment, although copper content in the nylon mesh was reduced. Therefore, the copper ions slowly dissolved from nylon mesh will contribute to the long-term prevention of biofouling. Furthermore, electron spin resonance analysis revealed the generation of reactive oxygen species (ROS) from CuI-doped nylon mesh after the field experiment. One of the possibilities for toxic action of copper ions will be the direct effect of Cu⁺-induced ROS on biofilm forming on nylon mesh surface. The proposed polymer textile can be applied to fishing and aquafarming nets, mooring rope for ship, or silt fence to restrict polluted water in marine environments.

Keywords: Copper iodide; Anti-biofouling; Nylon mesh; Reactive oxygen species

Characterization of a methane-oxidizing biofilm using microarray, and confocal microscopy with image and geostatic analyses by Tae Gwan Kim; Taewoo Yi; Eun-Hee Lee; Hee Wook Ryu; Kyung-Suk Cho (pp. 1051-1059).

A mixed methane-oxidizing biofilm was characterized, concurrently using a number of advanced techniques. Community analysis results by microarray exhibited that type II members dominated the methanotrophic community, in which Methylocystis was most abundant, followed by Methylosinus. Observation results by fluorescent in situ hybridization and confocal microscopy showed multiple biofilm colonies that were irregular, bell-shaped, with mean thickness of approximately 20 μm. Image analysis results indicated that the relative abundance of methanotrophs peaked at a depth of about 5 μm. Although the biofilm colonies differed in size, methanotrophs accounted for 4–9%. Gaussian and linear regression results between the biofilm volumes and types I (r ² = 0.86) and II volumes (r ² = 0.92), respectively, revealed that type I members played a role in the growth of the biofilm but only below a threshold volume, whereas type II members supported the overall growth. Geostatistical analyses results revealed concentration of types I and II methanotrophic individuals with decreasing depth, and randomness between the spatial locations and population levels. Collectively, the methane-oxidizing biofilm was a highly organized system with methanotrophs and their cohabitants.

Keywords: Methanotrophic biofilm; Methanotrophs; Fluorescent in situ hybridization; Confocal microscopy; Image analysis; Geostatistics

Long-term field fertilization alters the diversity of autotrophic bacteria based on the ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO) large-subunit genes in paddy soil by Hongzhao Yuan; Tida Ge; Xiaohong Wu; Shoulong Liu; Chengli Tong; Hongling Qin; Minna Wu; Wenxue Wei; Jinshui Wu (pp. 1061-1071).

Carbon dioxide (CO₂) assimilation by autotrophic bacteria is an important process in the soil carbon cycle with major environmental implications. The long-term impact of fertilizer on CO₂ assimilation in the bacterial community of paddy soils remains poorly understood. To narrow this knowledge gap, the composition and abundance of CO₂-assimilating bacteria were investigated using terminal restriction fragment length polymorphism and quantitative PCR of the cbbL gene [that encodes ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO)] in paddy soils. Soils from three stations in subtropical China were used. Each station is part of a long-term fertilization experiment with three treatments: no fertilizer (CK), chemical fertilizers (NPK), and NPK combined with rice straw (NPKM). At all of the stations, the cbbL-containing bacterial communities were dominated by facultative autotrophic bacteria such as Rhodopseudomonas palustris, Bradyrhizobium japonicum, and Ralstonia eutropha. The community composition in the fertilized soil (NPK and NPKM) was distinct from that in unfertilized soil (CK). The bacterial cbbL abundance (3–8 × 10⁸ copies g soil⁻¹) and RubisCO activity (0.40–1.76 nmol CO₂ g soil⁻¹ min⁻¹) in paddy soils were significantly positively correlated, and both increased with the addition of fertilizer. Among the measured soil parameters, soil organic carbon and pH were the most significant factors influencing the community composition, abundance, and activity of the cbbL-containing bacteria. These results suggest that long-term fertilization has a strong impact on the activity and community of cbbL-containing bacterial populations in paddy soils, especially when straw is combined with chemical fertilizers.

Keywords: RubisCO; cbbL gene; CO₂-assimilating bacteria; Paddy soil

Chemically inhibited ATP synthesis promoted detachment of different-age biofilms from membrane surface by Huijuan Xu; Kaiwen Teo; Huiling Neo; Yu Liu (pp. 1073-1082).

This study investigated the response of different-age biofilms developed on membrane surface to a chemical uncoupler 3, 3′, 4′, 5-tetrachlorosalicylanilide (TCS). Results showed that adenosine triphosphate (ATP) dissipation caused by TCS would promote different-age biofilms detachment, whereas chemically inhibited cellular ATP synthesis subsequently suppressed autoinducer-2 (AI-2) and extracellular polymeric substances (EPS) production. The extent of biofilm detachment was found to be closely related to AI-2-regulated EPS contents of bacteria. It was revealed that energy dissipation induced biofilm detachability was controlled by AI-2 regulated cellular communication via AI-2-mediated EPS secretion. This study would lead to a new cleaning strategy of biologically fouled membrane.

Keywords: Biofilm detachment; Chemical uncoupler; ATP; Autoinducer-2; EPS

Elucidating and reprogramming Escherichia coli metabolisms for obligate anaerobic n-butanol and isobutanol production by Cong T. Trinh (pp. 1083-1094).

Elementary mode (EM) analysis based on the constraint-based metabolic network modeling was applied to elucidate and compare complex fermentative metabolisms of Escherichia coli for obligate anaerobic production of n-butanol and isobutanol. The result shows that the n-butanol fermentative metabolism was NADH-deficient, while the isobutanol fermentative metabolism was NADH redundant. E. coli could grow and produce n-butanol anaerobically as the sole fermentative product but not achieve the maximum theoretical n-butanol yield. In contrast, for the isobutanol fermentative metabolism, E. coli was required to couple with either ethanol- or succinate-producing pathway to recycle NADH. To overcome these “defective” metabolisms, EM analysis was implemented to reprogram the native fermentative metabolism of E. coli for optimized anaerobic production of n-butanol and isobutanol through multiple gene deletion (∼8–9 genes), addition (∼6–7 genes), up- and downexpression (∼6–7 genes), and cofactor engineering (e.g., NADH, NADPH). The designed strains were forced to couple both growth and anaerobic production of n-butanol and isobutanol, which is a useful characteristic to enhance biofuel production and tolerance through metabolic pathway evolution. Even though the n-butanol and isobutanol fermentative metabolisms were quite different, the designed strains could be engineered to have identical metabolic flux distribution in “core” metabolic pathways mainly supporting cell growth and maintenance. Finally, the model prediction in elucidating and reprogramming the native fermentative metabolism of E. coli for obligate anaerobic production of n-butanol and isobutanol was validated with published experimental data.

Keywords: Elementary mode analysis; Metabolic pathway analysis; Metabolic pathway design; Metabolic pathway alignment; Fermentation; Advanced biofuels; Isobutanol; n-Butanol; Ethanol; Rational strain design; Cofactor engineering

Erratum to: Production of 10-hydroxystearic acid from oleic acid and olive oil hydrolyzate by an oleate hydratase from Lysinibacillus fusiformis by Bi-Na Kim; Young-Chul Joo; Yeong-Su Kim; Kyoung-Rok Kim; Deok-Kun Oh (pp. 1095-1096).

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Applied Microbiology and Biotechnology (v.95, #4)

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Applied Microbiology and Biotechnology (v.95, #4)