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Applied Microbiology and Biotechnology (v.92, #1)

Peptidoglycan turnover and recycling in Gram-positive bacteria by Jan Reith; Christoph Mayer (pp. 1-11).

Bacterial cells are protected by an exoskeleton, the stabilizing and shape-maintaining cell wall, consisting of the complex macromolecule peptidoglycan. In view of its function, it could be assumed that the cell wall is a static structure. In truth, however, it is steadily broken down by peptidoglycan-cleaving enzymes during cell growth. In this process, named cell wall turnover, in one generation up to half of the preexisting peptidoglycan of a bacterial cell is released from the wall. This would result in a massive loss of cell material, if turnover products were not be taken up and recovered. Indeed, in the Gram-negative model organism Escherichia coli, peptidoglycan recovery has been recognized as a complex pathway, named cell wall recycling. It involves about a dozen dedicated recycling enzymes that convey cell wall turnover products to peptidoglycan synthesis or energy pathways. Whether Gram-positive bacteria also recover their cell wall is currently questioned. Given the much larger portion of peptidoglycan in the cell wall of Gram-positive bacteria, however, recovery of the wall material would provide an even greater benefit in these organisms compared to Gram-negatives. Consistently, in many Gram-positives, orthologs of recycling enzymes were identified, indicating that the cell wall may also be recycled in these organisms. This mini-review provides a compilation of information about cell wall turnover and recycling in Gram-positive bacteria during cell growth and division, including recent findings relating to muropeptide recovery in Bacillus subtilis and Clostridium acetobutylicum from our group. Furthermore, the impact of cell wall turnover and recycling on biotechnological processes is discussed.

Keywords: Peptidoglycan turnover; Murein hydrolase; Muramidase; Cell wall recycling; Autolysins; Muropeptide recovery

Thermophilic, lignocellulolytic bacteria for ethanol production: current state and perspectives by Tinghong Chang; Shuo Yao (pp. 13-27).

Lignocellulosic biomass contains a variety of carbohydrates, and their conversion into ethanol by fermentation requires an efficient microbial platform to achieve high yield, productivity, and final titer of ethanol. In recent years, growing attention has been devoted to the development of cellulolytic and saccharolytic thermophilic bacteria for lignocellulosic ethanol production because of their unique properties. First of all, thermophilic bacteria possess unique cellulolytic and hemicellulolytic systems and are considered as potential sources of highly active and thermostable enzymes for efficient biomass hydrolysis. Secondly, thermophilic bacteria ferment a broad range of carbohydrates into ethanol, and some of them display potential for ethanologenic fermentation at high yield. Thirdly, the establishment of the genetic tools for thermophilic bacteria has allowed metabolic engineering, in particular with emphasis on improving ethanol yield, and this facilitates their employment for ethanol production. Finally, different processes for second-generation ethanol production based on thermophilic bacteria have been proposed with the aim to achieve cost-competitive processes. However, thermophilic bacteria exhibit an inherent low tolerance to ethanol and inhibitors in the pretreated biomass, and this is at present the greatest barrier to their industrial application. Further improvement of the properties of thermophilic bacteria, together with the optimization production processes, is equally important for achieving a realistic industrial ethanol production.

Keywords: Thermophilic bacteria; Cellulolytic and hemicellulolytic enzymes; Ethanol production

Pullulan: biosynthesis, production, and applications by Kuan-Chen Cheng; Ali Demirci; Jeffrey M. Catchmark (pp. 29-44).

Pullulan is a linear glucosic polysaccharide produced by the polymorphic fungus Aureobasidium pullulans, which has long been applied for various applications from food additives to environmental remediation agents. This review article presents an overview of pullulan’s chemistry, biosynthesis, applications, state-of-the-art advances in the enhancement of pullulan production through the investigations of enzyme regulations, molecular properties, cultivation parameters, and bioreactor design. The enzyme regulations are intended to illustrate the influences of metabolic pathway on pullulan production and its structural composition. Molecular properties, such as molecular weight distribution and pure pullulan content, of pullulan are crucial for pullulan applications and vary with different fermentation parameters. Studies on the effects of environmental parameters and new bioreactor design for enhancing pullulan production are getting attention. Finally, the potential applications of pullulan through chemical modification as a novel biologically active derivative are also discussed.

Keywords: Aureobasidium pullulans ; Pullulan; Applications; Production; Biosynthesis

Impact of the Penicillium chrysogenum genome on industrial production of metabolites by Marco Alexander van den Berg (pp. 45-53).

The genome sequence of Penicillium chrysogenum has initiated a range of fundamental studies, deciphering the genetic secrets of the industrial penicillin producer. More than 60 years of classical strain improvement has resulted in major but delicate rebalancing of the intracellular metabolism leading to the impressive penicillin titres of the current production strains. Several leads for further improvement are being followed up, including the use of P. chrysogenum as a cell factory for other products than β-lactam antibiotics.

Keywords: Penicillium chrysogenum ; Genomics; Antibiotics; Metabolites; Industrial production

Potentials and limitations of miniaturized calorimeters for bioprocess monitoring by Thomas Maskow; Torsten Schubert; Antje Wolf; Friederike Buchholz; Lars Regestein; Jochen Buechs; Florian Mertens; Hauke Harms; Johannes Lerchner (pp. 55-66).

In theory, heat production rates are very well suited for analysing and controlling bioprocesses on different scales from a few nanolitres up to many cubic metres. Any bioconversion is accompanied by a production (exothermic) or consumption (endothermic) of heat. The heat is tightly connected with the stoichiometry of the bioprocess via the law of Hess, and its rate is connected to the kinetics of the process. Heat signals provide real-time information of bioprocesses. The combination of heat measurements with respirometry is theoretically suited for the quantification of the coupling between catabolic and anabolic reactions. Heat measurements have also practical advantages. Unlike most other biochemical sensors, thermal transducers can be mounted in a protected way that prevents fouling, thereby minimizing response drifts. Finally, calorimetry works in optically opaque solutions and does not require labelling or reactants. It is surprising to see that despite all these advantages, calorimetry has rarely been applied to monitor and control bioprocesses with intact cells in the laboratory, industrial bioreactors or ecosystems. This review article analyses the reasons for this omission, discusses the additional information calorimetry can provide in comparison with respirometry and presents miniaturization as a potential way to overcome some inherent weaknesses of conventional calorimetry. It will be discussed for which sample types and scientific question miniaturized calorimeter can be advantageously applied. A few examples from different fields of microbiological and biotechnological research will illustrate the potentials and limitations of chip calorimetry. Finally, the future of chip calorimetry is addressed in an outlook.

Keywords: Calorimetry; Biothermodynamics; Bioprocess control; Biofilms; Chip calorimetry; FIA; At-line

Homo-d-lactic acid production from mixed sugars using xylose-assimilating operon-integrated Lactobacillus plantarum by Shogo Yoshida; Kenji Okano; Tsutomu Tanaka; Chiaki Ogino; Akihiko Kondo (pp. 67-76).

In order to achieve efficient d-lactic acid fermentation from a mixture of xylose and glucose, the xylose-assimilating xylAB operon from Lactobacillus pentosus (PXylAB) was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (ΔldhL1-xpk1::tkt-Δxpk2) strain in which the phosphoketolase 1 gene (xpk1) was replaced with the transketolase gene (tkt) from Lactococcus lactis, and the phosphoketolase 2 (xpk2) gene was deleted. Two copies of xylAB introduced into the genome significantly improved the xylose fermentation ability, raising it to the same level as that of ΔldhL1-xpk1::tkt-Δxpk2 harboring a xylAB operon-expressing plasmid. Using the two-copy xylAB integrated strain, successful homo-d-lactic acid production was achieved from a mixture of 25 g/l xylose and 75 g/l glucose without carbon catabolite repression. After 36-h cultivation, 74.2 g/l of lactic acid was produced with a high yield (0.78 g per gram of consumed sugar) and an optical purity of d-lactic acid of 99.5%. Finally, we successfully demonstrated homo-d-lactic acid fermentation from a mixture of three kinds of sugar: glucose, xylose, and arabinose. This is the first report that describes homo-d-lactic acid fermentation from mixed sugars without carbon catabolite repression using the xylose-assimilating pathway integrated into lactic acid bacteria.

Keywords: Lactic acid; Co-fermentation; Xylose; Fermentation

Xylitol does not inhibit xylose fermentation by engineered Saccharomyces cerevisiae expressing xylA as severely as it inhibits xylose isomerase reaction in vitro by Suk-Jin Ha; Soo Rin Kim; Jin-Ho Choi; Myeong Soo Park; Yong-Su Jin (pp. 77-84).

Efficient fermentation of xylose, which is abundant in hydrolysates of lignocellulosic biomass, is essential for producing cellulosic biofuels economically. While heterologous expression of xylose isomerase in Saccharomyces cerevisiae has been proposed as a strategy to engineer this yeast for xylose fermentation, only a few xylose isomerase genes from fungi and bacteria have been functionally expressed in S. cerevisiae. We cloned two bacterial xylose isomerase genes from anaerobic bacteria (Bacteroides stercoris HJ-15 and Bifidobacterium longum MG1) and introduced them into S. cerevisiae. While the transformant with xylA from B. longum could not assimilate xylose, the transformant with xylA from B. stercoris was able to grow on xylose. This result suggests that the xylose isomerase (BsXI) from B. stercoris is functionally expressed in S. cerevisiae. The engineered S. cerevisiae strain with BsXI consumed xylose and produced ethanol with a good yield (0.31 g/g) under anaerobic conditions. Interestingly, significant amounts of xylitol (0.23 g xylitol/g xylose) were still accumulated during xylose fermentation even though the introduced BsXI might not cause redox imbalance. We investigated the potential inhibitory effects of the accumulated xylitol on xylose fermentation. Although xylitol inhibited in vitro BsXI activity significantly (K _I = 5.1 ± 1.15 mM), only small decreases (less than 10%) in xylose consumption and ethanol production rates were observed when xylitol was added into the fermentation medium. These results suggest that xylitol accumulation does not inhibit xylose fermentation by engineered S. cerevisiae expressing xylA as severely as it inhibits the xylose isomerase reaction in vitro.

Keywords: Cellulosic biomass; Ethanol; Xylose isomerase; Xylitol

Application of immobilized thrombin for production of S-thanatin expressed in Escherichia coli by Guoqiu Wu; Xuepeng Deng; Xiaofang Li; Xiyong Wang; Shenglan Wang; Hanmei Xu (pp. 85-93).

S-thanatin, a small antimicrobial peptide with 21 amino acid residues, was expressed as a fusion protein containing thrombin cleavage site in Escherichia coli BL21 (DE3). To reduce the production cost, immobilization of thrombin in polyacrylamide gel for cleavage was studied in this work. The immobilized thrombin exhibited excellent activity within wider ranges of pH value and temperature for reaction than free enzyme, and the residual activity could remain above 75% after ten times of usage. Tricine–SDS–PAGE result showed that the immobilized thrombin could cleave the S-thanatin fusion protein effectively. After cleavage, recombinant S-thanatin was purified by preparative reversed-phase high-performance liquid chromatography and mass spectrum showed that the molecular weight (2,448.86) was close to the theoretical value (2,448.98). After purification, about 7 mg of S-thanatin was obtained from 1 l of culture and the recombinant exhibited excellent bioactivity to E. coli ATCC 25922, with the minimum inhibitory concentration of 12 μg/ml. The purification method could be applied to prepare other peptides with similar properties at low cost.

Keywords: Antimicrobial peptide; Thanatin; Immobilized thrombin; Fusion expression; Escherichia coli

Enzymatic treatment for preventing biofilm formation in the paper industry by Claudia Esperanza Torres; Giles Lenon; Delphine Craperi; Reinhard Wilting; Ángeles Blanco (pp. 95-103).

Microbiological control programmes at industrial level should aim at reducing both the detrimental effects of microorganisms on the process and the environmental impact associated to the use of biocides as microbiological control products. To achieve this target, new efficient and environmentally friendly products are required. In this paper, 17 non-specific, commercial enzymatic mixtures were tested to assess their efficacy for biofilm prevention and control at laboratory and pilot plant scale. Pectin methylesterase, an enzyme found in the formulation of two of the mixtures tested, was identified as an active compound able to reduce biofilm formation by 71% compared to control tests.

Keywords: Enzymes; Biofilm; Microbiological control; Pectin methylesterase; Paper industry

Large-scale production of tannase using the yeast Arxula adeninivorans by Erik Böer; Friederike Sophie Breuer; Michael Weniger; Sylvia Denter; Michael Piontek; Gotthard Kunze (pp. 105-114).

Tannase (tannin acyl hydrolase, EC 3.1.1.20) hydrolyses the ester and depside bonds of gallotannins and gallic acid esters and is an important industrial enzyme. In the present study, transgenic Arxula adeninivorans strains were optimised for tannase production. Various plasmids carrying one or two expression modules for constitutive expression of tannase were constructed. Transformant strains that overexpress the ATAN1 gene from the strong A. adeninivorans TEF1 promoter produce levels of up to 1,642 U L⁻¹ when grown in glucose medium in shake flasks. The effect of fed-batch fermentation on tannase productivity was then investigated in detail. Under these conditions, a transgenic strain containing one ATAN1 expression module produced 51,900 U of tannase activity per litre after 142 h of fermentation at a dry cell weight of 162 g L⁻¹. The highest yield obtained from a transgenic strain with two ATAN1 expression modules was 31,300 U after 232 h at a dry cell weight of 104 g L⁻¹. Interestingly, the maximum achieved yield coefficients [Y(P/X)] for the two strains were essentially identical.

Keywords: Arxula adeninivorans ; ATAN1 ; Fed-batch fermentation; Tannase; TEF1 promoter; Yeast

siRNA targeting mCD14 inhibits TNF-α, MIP-2, and IL-6 secretion and NO production from LPS-induced RAW264.7 cells by Ming Lei; Hanwei Jiao; Tao Liu; Li Du; Ying Cheng; Donglin Zhang; Yongchang Hao; Churiga Man; Fengyang Wang (pp. 115-124).

Innate immunity plays a key role in protecting a host against invading microorganism, including Gram-negative bacteria. Cluster of differentiation antigen 14 (CD14) is an important innate immunity molecule, existing as a soluble (sCD14) and membrane-associated (mCD14) protein. Endotoxin [lipopolysaccharide (LPS)] is recognized as a key molecule in the pathogenesis of sepsis and septic shock caused by Gram negative bacteria. Emerging evidences indicate that upstream inhibition of bacterial LPS/Toll-like receptor 4(TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. RNA interference (RNAi) provides a promising approach to down-regulate gene expression specifically. To explore the possibility of using RNAi against mCD14 as a strategy for inhibiting the secretion of cytokines and the nitric oxide (NO) production from LPS-activated RAW264.7 cells, four different short interfering RNA (siRNA) molecules corresponding to the sequence of mCD14 gene were designed and synthesized. We then tested the inhibition effects of these siRNA molecules on mCD14 expression by real-time quantitative RT-PCR and Western blot. After effective siRNA molecule (mCD14–siRNA-224), which is capable of reducing messenger RNA (mRNA) accumulation and protein expression of mCD14 specifically, was identified, RAW264.7 cells pretreated with mCD14–siRNA-224 were stimulated with LPS, and the secretion of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2) and interleukin-6 (IL-6) and the NO production were evaluated. The results indicated that mCD14–siRNA-224 effectively inhibited TNF-α, MIP-2, and IL-6 release and NO production from LPS-stimulated RAW 264.7 cells by down-regulating mRNA accumulation and protein expression of mCD14 specifically. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for endotoxin-related diseases.

Keywords: siRNA; mCD14; TNF-α; MIP-2; IL-6; NO; LPS; RAW264.7 cell

Engineering Escherichia coli for efficient cellobiose utilization by Parisutham Vinuselvi; Sung Kuk Lee (pp. 125-132).

Escherichia coli normally cannot utilize the β-glucoside sugar cellobiose as a carbon and energy source unless a stringent selection pressure for survival is present. The cellobiose-utilization phenotype can be conferred by mutations in the two cryptic operons, chb and asc. In this study, the cellobiose-utilization phenotype was conferred to E. coli by replacing the cryptic promoters of these endogenous operons with a constitutive promoter. Evolutionary adaptation of the engineered strain CP12CHBASC by repeated subculture in cellobiose-containing minimal medium led to an increase in the rate of cellobiose uptake and cell growth on cellobiose. An efficient cellobiose-metabolizing E. coli strain would be of great importance over glucose-metabolizing E. coli for a simultaneous saccharification and fermentation process, as the cost of the process would be reduced by eliminating one of the three enzymes needed to hydrolyze cellulose into simple sugars.

Keywords: chb operon; Cellobiose metabolism; asc operon; Cryptic genes; Escherichia coli

Molecular fractionation and characterization of a Candida albicans fraction that increases tumor cell adhesion to hepatic endothelium by Andoni Ramirez-Garcia; Natalia Gallot; Ana Abad; Lorea Mendoza; Aitor Rementeria; Fernando Luis Hernando (pp. 133-145).

Systemic candidiasis remains a major complication among patients suffering from hematological malignancies and favors the development of hepatic metastasis. To contribute to the understanding of the underlying mechanisms, the aim of this study was to identify molecules that may increase tumor cell adhesion to hepatic endothelial cells. To this end, a well-established in vitro model was used to determine the enhancement of tumor cell adhesion induced by Candida albicans and its fractions. Different fractions were obtained according to their molecular weight (M _r) (five) or to their isoelectric point (pI) (four), using preparative electrophoresis and preparative isoelectric focusing, respectively, followed by affinity chromatography. The fraction that most enhanced melanoma cell adhesion to endothelium had an M _r range from 45 to 66 kDa. It was characterized using two-dimensional electrophoresis, and 14 proteins were identified by peptide mass fingerprinting: Dor14p, Fba1p, Pdi1p, Pgk1p, Idh2p, Mpg1p, Sfa1p, Ape3p, Ilv5p, Tuf1p, Act1p, Eno1p, Qcr2p, and Adh1p. Of these, several are related to the immunogenic response, and the latter seven belonged to the most reactive fraction according to their pI range, from 5 to 5.6. These findings could represent a step forward in the search for new targets, to suppress the pro-metastatic effect of C. albicans.

Keywords: Cancer; Candida albicans ; Endothelium; Mannoproteins; Melanoma

Long-term preservation of anammox bacteria by Michael J. Rothrock Jr; Matias B. Vanotti; Ariel A. Szögi; Maria Cruz Garcia Gonzalez; Takao Fujii (pp. 147-157).

Deposit of useful microorganisms in culture collections requires long-term preservation and successful reactivation techniques. The goal of this study was to develop a simple preservation protocol for the long-term storage and reactivation of the anammox biomass. To achieve this, anammox biomass was frozen or lyophilized at two different freezing temperatures (−60°C and in liquid nitrogen (−200°C)) in skim milk media (with and without glycerol), and the reactivation of anammox activity was monitored after a 4-month storage period. Of the different preservation treatments tested, only anammox biomass preserved via freezing in liquid nitrogen followed by lyophilization in skim milk media without glycerol achieved stoichiometric ratios for the anammox reaction similar to the biomass in both the parent bioreactor and in the freshly harvested control treatment. A freezing temperature of −60°C alone, or in conjunction with lyophilization, resulted in the partial recovery of the anammox bacteria, with an equal mixture of anammox and nitrifying bacteria in the reactivated biomass. To our knowledge, this is the first report of the successful reactivation of anammox biomass preserved via sub-zero freezing and/or lyophilization. The simple preservation protocol developed from this study could be beneficial to accelerate the integration of anammox-based processes into current treatment systems through a highly efficient starting anammox biomass.

Keywords: Anammox; Lyophilization; Stoichiometry; Preservation; Reactivation

Role of bifidobacteria in the activation of the lignan secoisolariciresinol diglucoside by Lucia Roncaglia; Alberto Amaretti; Stefano Raimondi; Alan Leonardi; Maddalena Rossi (pp. 159-168).

Lignans are ubiquitous plant polyphenols, which have relevant health properties being the major phytoestrogens occurring in Western diets. Secoisolariciresinol (SECO) is the major dietary lignan mostly found in plants as secoisolariciresinol diglucoside (SDG). To exert biological activity, SDG requires being deglycosylated to SECO and transformed to enterodiol (ED) and enterolactone (EL) by the intestinal microbes. The involvement of bifidobacteria in the transformation of lignans glucosides has been investigated for the first time in this study. Twenty-eight strains were assayed for SDG and SECO activation. They all failed to transform SECO into reduced metabolites, excluding any role in ED and EL production. Ten Bifidobacterium cultures partially hydrolyzed SDG, giving both SECO and the monoglucoside with yields < 25%. When the cell-free extracts were assayed in SDG transformation, seven additional strains were active in the hydrolysis. Cellobiose induced β-glucosidase activity and caused the enhancement of both the rate of SDG hydrolysis and the final yield of SECO only in the strains capable of SDG bioconversion. The highest SDG conversion to SECO was achieved by Bifidobacterium pseudocatenulatum WC 401, which exhibited 75% yield in cellobiose-based medium after 48 h. These results indicate that SDG hydrolysis is not a common feature in Bifidobacterium genus, but selected probiotic strains can be combined to β-glucoside-based prebiotics to enhance the release of SECO, thus improving its bioavailability for absorption by colonic mucosa and/or the biotransformation to ED and EL by other intestinal microorganisms.

Keywords: Lignan; Bifidobacterium; Secoisolariciresinol; Secoisolariciresinol diglucoside; SECO; SDG

Sorption-assisted surface conjugation: a way to stabilize laccase enzyme by Yannick-Serge Zimmermann; Patrick Shahgaldian; Philippe F. X. Corvini; Gregor Hommes (pp. 169-178).

Enyzme immobilization on solid surfaces is one of the most relevant methods to improve enzyme activity and stability under harsh conditions over extended periods. A typically interesting application is the immobilization of laccases, multicopper enzymes oxidizing aromatic compounds, to solid surfaces in order to develop valuable tools for the elimination of micropollutants in wastewater. Laccase of the white-rot fungus Coriolopsis polyzona has been successfully immobilized on fumed silica nanoparticles using a novel method. It consists in the sorption of the enzyme to amino-modified silica nanoparticles and the subsequent covalent cross-linking using glutaraldehyde as a homobifunctional linker. The so-produced nanoparticulate material has been characterized by means of scanning electron microscopy and Brunauer–Emmett–Teller surface area analysis revealing modifications of the surface structure and area during the coupling procedure. Laccase immobilization on spherical nanoparticles produced according to the method of Stöber has been shown to be much less efficient than on fumed silica nanoparticles. Long-term stability assays revealed that the novel developed method allows a drastic stabilization of the enzyme. In real wastewater, 77% of the laccase activity remained on the nanoparticles over 1 month, whereas the activity of free laccase dropped to 2.5%. The activity loss on the nanoparticles resulted from partial inactivation of the immobilized enzymes and additional release into the surrounding solution with subsequent fast inactivation of the free enzymes, since almost no activity was found in the supernatants.

Keywords: Laccase; Silica nanoparticles; Enzyme immobilization; ABTS

LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila by Xi Lu; Zi-Yao Mo; Hong-Bo Zhao; He Yan; Lei Shi (pp. 179-187).

Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98/107) and 93.33% (56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51/51) and 100% (18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.

Keywords: Loop-mediated isothermal amplification; Legionella pneumophila ; Different diagnosis

Application of real-time PCR in the assessment of the toxic cyanobacterium Cylindrospermopsis raciborskii abundance and toxicological potential by Cristiana Moreira; António Martins; Joana Azevedo; Marisa Freitas; Ana Regueiras; Micaela Vale; Agostinho Antunes; Vitor Vasconcelos (pp. 189-197).

Cyanobacteria are prokaryotic photosynthetic microorganisms that pose a serious threat to aquatic environments because they are able to form blooms under eutrophic conditions and produce toxins. Cylindrospermopsis raciborskii is a planktonic heterocystous filamentous cyanobacterium initially assigned to the tropics but currently being found in more temperate regions such as Portugal, the southernmost record for this species in Europe. Cylindrospermopsin originally isolated from C. raciborskii is a cytotoxic alkaloid that affects the liver, kidney, and other organs. It has a great environmental impact associated with cattle mortality and human morbidity. Aiming in monitoring this cyanobacterium and its related toxin, a shallow pond located in the littoral center of Portugal, Vela Lake, used for agriculture and recreational purposes was monitored for a 2-year period. To accomplish this, we used the real-time PCR methodology in field samples to quantify the variation of specific genetic markers with primers previously described characterizing total cyanobacteria (16S rRNA), C. raciborskii (rpoC1), and cylindrospermopsin synthetase gene (pks). The results report the high abundance of both cyanobacteria and C. raciborskii in Vela Lake, with C. raciborskii representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples. We believe that with the approach developed in this study, it will be possible to monitor C. raciborskii population dynamics and seasonal variation, as well as the potential toxin production in other aquatic environments.

Keywords: C. raciborskii ; Monitoring; Real-time PCR; Cylindrospermopsin genes

End-product induced metabolic shifts in Clostridium thermocellum ATCC 27405 by Thomas Rydzak; David B. Levin; Nazim Cicek; Richard Sparling (pp. 199-209).

When attempting to increase yields of desirable end-products during fermentation, there is the possibility that increased concentrations of one product redirects metabolism towards the synthesis of less desired products. Changes in growth, final end-product concentrations, and activities of enzymes involved in pyruvate catabolism and fermentative end-product formation were studied in Clostridium thermocellum in response to the addition of individual end-products (H₂, acetate, ethanol, formate, and lactate) to the growth medium. These were added to the growth medium at concentrations ten times greater than those found at the end of growth in cultures grown under carbon-limited conditions using cellobiose (1.1 g l⁻¹) as model soluble substrate. Although growth rate and final cell biomass decreased significantly with the addition of all end-products, addition of individual end-products had less pronounced effects on growth. Metabolic shifts, represented by changes in final end-product concentrations, were observed; H₂ and acetate yields increased in the presence of exogenous ethanol and lactate, while ethanol yields increased in the presence of exogenous hydrogen (H₂), acetate, and lactate. Late exponential phase enzyme activity data of enzymes involved in pyruvate catabolism and end-product formation revealed no changes in enzyme levels greater than 2-fold in response to the presence of any given end-product, with the exception of pyruvate:formate lyase (PFL), ferredoxin-dependent hydrogenase (Fd-H₂ase), and pyruvate:ferredoxin oxidoreductase (PFO): PFL and Fd-H₂ase activities increased 2-fold in the presence of ethanol, while PFO activity decreased by 57% in the presence of sodium formate. Changes in enzyme levels did not necessarily correlate with changes in final end-product yields, suggesting that changes in final end-product yields may be governed by thermodynamic considerations rather than levels of enzyme expressed under the conditions tested. We demonstrate that bacterial metabolism may be manipulated in order to selectively improve desired product yields.

Keywords: Clostridium thermocellum ; Exogenous end-product addition; Metabolic shift; Biofuels

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Applied Microbiology and Biotechnology (v.92, #1)

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Applied Microbiology and Biotechnology (v.92, #1)