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Applied Microbiology and Biotechnology (v.80, #3)

Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities by Ursel M. E. Schütte; Zaid Abdo; Stephen J. Bent; Conrad Shyu; Christopher J. Williams; Jacob D. Pierson; Larry J. Forney (pp. 365-380).

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.

Keywords: T-RFLP; Microbial communities; 16S rRNA genes; Multivariate statistics

Fungal secretomes—nature’s toolbox for white biotechnology by Henning Bouws; Andreas Wattenberg; Holger Zorn (pp. 381-388).

Adapting their metabolism to varying carbon and nitrogen sources, saprophytic fungi produce an arsenal of extracellular enzymes, the secretome, which allows for an efficient degradation of lignocelluloses and further biopolymers. Based on fundamental advances in electrophoretic, chromatographic, and mass spectrometric techniques on the one hand and the availability of annotated fungal genomes and sophisticated bioinformatic software tools on the other hand, a detailed analysis of fungal secretomes has become feasible. While a number of reports on ascomycetous secretomes of, e.g., Aspergillus, Trichoderma, and Fusarium species are already available, studies on basidiomycetes have been mainly focused on the two model organisms Phanerochaete chrysosporium and Coprinopsis cinerea so far. Though an impressive number and diversity of fungal biocatalysts has been revealed by secretome analyses, the identity and function of many extracellular proteins still remains to be elucidated. A comprehensive understanding of the qualitative and quantitative composition of fungal secretomes, together with their synergistic actions and kinetic expression profiles, will allow for the development of optimized enzyme cocktails for white biotechnology.

Keywords: Ascomycetes; Basidiomycetes; 2-D electrophoresis; Tandem-mass spectrometry

Laccase-induced C–N coupling of substituted p-hydroquinones with p-aminobenzoic acid in comparison with known chemical routes by Annett Mikolasch; Anastasia Matthies; Michael Lalk; Frieder Schauer (pp. 389-397).

Fungal laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) from Pycnoporus cinnabarinus and Myceliophthora thermophila were used as biocatalysts for enzymatic reaction of halogen-, alkyl-, alkoxy-, and carbonyl-substituted p-hydroquinones (laccase substrates) with p-aminobenzoic acid (no laccase substrate). During this reaction, the laccase substrate was oxidized to the corresponding quinones, which react with p-aminobenzoic acid by amination of the laccase substrate. The different substitutions at the hydroquinone substrates were used to prove whether the substituents influence the position of amination and product yields. The cross-coupling of methoxy-p-hydroquinone (alkoxylated) and 2,5-dihydroxybenzaldehyd (carbonyl-substituted) with p-aminobenzoic acid resulted in the formation of one monoaminated product (yield alkoxylated 52%). If monohalogen- or monoalkyl-substituted p-hydroquinones were used as laccase substrates, two monoaminated products (constitution isomers) were formed. The simultaneous formation of two different monoaminated products from the same hydroquinone substrate is the first report for laccase-mediated synthesis of aminated constitution isomers. Depending from the type of substituent of the hydroquinone, the positions of the two monoaminations are different. While the amination at the monoalkylated hydroquinone occurs at the 5- and 6-positions (yield 38%), the amination at monohalogenated hydroquinones was detectable at the 3- and 5-positions (yield 53%). The same product pattern could be achieved if instead of the biocatalyst laccase the chemical catalyst sodium iodate was used as the oxidant. However, the yields were partially much lower (0–45% of the yields with laccase).

Keywords: Laccase; Coupling; Hydroquinones; p-aminobenzoic acid

Characterization of a modular enzyme of exo-1,5-α-l-arabinofuranosidase and arabinan binding module from Streptomyces avermitilis NBRC14893 by Hitomi Ichinose; Makoto Yoshida; Zui Fujimoto; Satoshi Kaneko (pp. 399-408).

A gene encoding an α-l-arabinofuranosidase, designated SaAraf43A, was cloned from Streptomyces avermitilis. The deduced amino acid sequence implies a modular structure consisting of an N-terminal glycoside hydrolase family 43 module and a C-terminal family 42 carbohydrate-binding module (CBM42). The recombinant enzyme showed optimal activity at pH 6.0 and 45°C and was stable over the pH range of 5.0–6.5 at 30°C. The enzyme hydrolyzed p-nitrophenol (PNP)-α-l-arabinofuranoside but did not hydrolyze PNP-α-l-arabinopyranoside, PNP-β-d-xylopyranoside, or PNP-β-d-galactopyranoside. Debranched 1,5-arabinan was hydrolyzed by the enzyme but arabinoxylan, arabinogalactan, gum arabic, and arabinan were not. Among the synthetic regioisomers of arabinofuranobiosides, only methyl 5-O-α-l-arabinofuranosyl-α-l-arabinofuranoside was hydrolyzed by the enzyme, while methyl 2-O-α-l-arabinofuranosyl-α-l-arabinofuranoside and methyl 3-O-α-l-arabinofuranosyl-α-l-arabinofuranoside were not. These data suggested that the enzyme only cleaves α-1,5-linked arabinofuranosyl linkages. The analysis of the hydrolysis product of arabinofuranopentaose suggested that the enzyme releases arabinose in exo-acting manner. These results indicate that the enzyme is definitely an exo-1,5-α-l-arabinofuranosidase. The C-terminal CBM42 did not show any affinity for arabinogalactan and debranched arabinan, although it bound arabinan and arabinoxylan, suggesting that the CBM42 bound to branched arabinofuranosyl residues. Removal of the module decreased the activity of the enzyme with regard to debranched arabinan. The CBM42 plays a role in enhancing the debranched arabinan hydrolytic action of the catalytic module in spite of its preference for binding arabinofuranosyl side chains.

Keywords: α-l-Arabinofuranosidase; Glycoside hydrolase family 43; Carbohydrate binding module family 42; Arabinan binding module; Streptomyces avermitilis

Enhancing survival of Escherichia coli by expression of azoreductase AZR possessing quinone reductase activity by Guangfei Liu; Jiti Zhou; Ruofei Jin; Mi Zhou; Jing Wang; Hong Lu; Yuanyuan Qu (pp. 409-416).

Quinone reductase activity of azoreductase AZR from Rhodobacter sphaeroides was reported. High homologies were found in the cofactor/substrate-binding regions of quinone reductases from different domains. 3D structure comparison revealed that AZR shared a common overall topology with mammal NAD(P)H/quinone oxidoreductase NQO1. With menadione as substrate, the optimal pH value and temperature were pH 8–9 and 50°C, respectively. Following the ping-pong kinetics, AZR transferred two electrons from NADPH to quinone substrate. It could reduce naphthoquinones and anthraquinones, such as menadione, lawsone, anthraquinone-2-sulfonate, and anthraquinone-2,6-disulfonate. However, no activity was detected with 1,4-benzoquinone. Dicoumarol competitively inhibited AZR’s quinone reductase activity with respect to NADPH, with an obtained K _i value of 87.6 μM. Significantly higher survival rates were obtained in Escherichia coli YB overexpressing AZR than in the control strain when treated by heat shock and oxidative stressors such as H₂O₂ and menadione.

Keywords: Quinone reductase; Azoreductase; Oxidative stress; Heat shock; Survival

A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions by Huoqing Huang; Huiying Luo; Yaru Wang; Dawei Fu; Na Shao; Guozeng Wang; Peilong Yang; Bin Yao (pp. 417-426).

Two novel phytase genes belonging to the histidine acid phosphatase family were cloned from Yersinia rohdei and Y. pestis and expressed in Pichia pastoris. Both the recombinant phytases had high activity at pH 1.5–6.0 (optimum pH 4.5) with an optimum temperature of 55°C. Compared with the major commercial phytases from Aspergillus niger, Escherichia coli, and a potential commercial phytase from Y. intermedia, the Y. rohdei phytase was more resistant to pepsin, retained more activity under gastric conditions, and released more inorganic phosphorus (two to ten times) from soybean meal under simulated gastric conditions. These superior properties suggest that the Y. rohdei phytase is an attractive additive to animal feed. Our study indicated that, in order to better hydrolyze the phytate and release more inorganic phosphorus in the gastric passage, phytase should have high activity and stability, simultaneously, at low pH and high protease concentration.

Keywords: Gastric conditions; Inorganic phosphorus; Phytase; Yersinia rohdei ; Y. pestis

Genome mining of cyanide-degrading nitrilases from filamentous fungi by Lacy J. Basile; Richard C. Willson; B. Trevor Sewell; Michael J. Benedik (pp. 427-435).

A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. In this paper, we report on two previously unknown and uncharacterized cyanide hydratases from Neurospora crassa and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N. crassa, A. nidulans, Gibberella zeae, and Gloeocercospora sorghi were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in Escherichia coli, and purified using immobilized metal affinity chromatography. These enzymes were compared according to their relative specific activity, pH activity profiles, thermal stability, and ability to remediate cyanide contaminated waste water from silver and copper electroplating baths. Although all four were similar, the N. crassa cyanide hydratase (CHT) has the greatest thermal stability and widest pH range of >50% activity. N. crassa also demonstrated the highest rate of cyanide degradation in the presence of both heavy metals. The CHT of A. nidulans has the highest reaction rate of the four fungal nitrilases evaluated in this work. These data will help determine optimization procedures for the possible use of these enzymes in the bioremediation of cyanide-containing waste. Similar to known plant pathogenic fungi, both N. crassa and A. nidulans were induced to express CHT by growth in the presence of KCN.

Keywords: Cyanide; Cyanide hydratase; Bioremediation

Efficient secretion of Bacillus subtilis lipase A in Saccharomyces cerevisiae by translational fusion to the Pir4 cell wall protein by María Mormeneo; Isabel Andrés; Cristina Bofill; Pilar Díaz; Jesús Zueco (pp. 437-445).

Both the secretion and the cell surface display of Bacillus subtilis lipase A (Lip A) in Saccharomyces cerevisiae was investigated using different domains of the cell wall protein Pir4 as translational fusion partners. LipA gene minus its leader peptide was fused inframe in two places of PIR4 to achieve cell wall targeting, or substituting most of the PIR4 sequence, after the signal peptide and the Kex2 processed subunit I of Pir4 to achieve secretion to the growth medium. Expression of the recombinant fusion proteins was investigated in a standard and a glycosylation-deficient strain of S. cerevisiae, grown in selective or rich medium. Fusion proteins intended to be retained at the cell wall were secreted to the growth medium, most likely as result of the degradation of the Pir4 moiety containing the cell wall retention domain, giving low levels of lipase activity. However, the fusion intended for secretion was efficiently secreted in a percentage of close to 90% and remained stable even in rich medium at high cell density cultures, yielding values of over 400 IU of lipase activity per milliliter of cell supernatant. This is, to our knowledge, the first report of the efficient production, as a secreted protein, of lipase A of B. subtilis in baker’s yeast.

Keywords: Lipase A; Bacillus subtilis ; Saccharomyces cerevisiae ; Secretion of recombinant proteins; Pir cell wall proteins

Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display by Jonas Kügler; Simone Nieswandt; Gerald F. Gerlach; Jochen Meens; Thomas Schirrmann; Michael Hust (pp. 447-458).

The identification of immunogenic polypeptides of pathogens is helpful for the development of diagnostic assays and therapeutic applications like vaccines. Routinely, these proteins are identified by two-dimensional polyacrylamide gel electrophoresis and Western blot using convalescent serum, followed by mass spectrometry. This technology, however, is limited, because low or differentially expressed proteins, e.g. dependent on pathogen–host interaction, cannot be identified. In this work, we developed and improved a M13 genomic phage display-based method for the selection of immunogenic polypeptides of Mycoplasma hyopneumoniae, a pathogen causing porcine enzootic pneumonia. The fragmented genome of M. hyopneumoniae was cloned into a phage display vector, and the genomic library was packaged using the helperphage Hyperphage to enrich open reading frames (ORFs). Afterwards, the phage display library was screened by panning using convalescent serum. The analysis of individual phage clones resulted in the identification of five genes encoding immunogenic proteins, only two of which had been previously identified and described as immunogenic. This M13 genomic phage display, directly combining ORF enrichment and the presentation of the corresponding polypeptide on the phage surface, complements proteome-based methods for the identification of immunogenic polypeptides and is particularly well suited for the use in mycoplasma species.

Keywords: Phage display; Mycoplasma; Screening methods; Open reading frame enrichment

Evaluation of ergosterol and its esters in the pileus, gill, and stipe tissues of agaric fungi and their relative changes in the comminuted fungal tissues by Jian-Ping Yuan; Hui-Cong Kuang; Jiang-Hai Wang; Xin Liu (pp. 459-465).

A gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the rapid determination of free ergosterol, ergosteryl esters, and ergocalciferol. The HPLC method was used to evaluate the distribution of ergosterol and ergosteryl esters in the different parts (stipe, pileus, and gills) of the agaric fungi, Agrocybe aegerita, Termitomyces albuminosus, and Lentinus edodes, and the relative changes of free and esterified ergosterols during the degradation of ergosterol in the comminuted fungal tissues. The results showed that total ergosterol levels and the relative abundances of free to esterified ergosterols were different among the various species and in the different parts of these agaric fungi. The results also indicated that ergosteryl esters were more stable than free ergosterol. While the content of free ergosterol markedly decreased, substantial amounts of ergosteryl esters remained for a long period, and even an increase in the contents of ergosteryl esters was also found in some comminuted fungal tissues. Therefore, it is possible that free ergosterol in the cell membrane of the dead fungal hyphae undergoes degradation or esterification, by which excess free ergosterol may be removed, and stored in cytosolic lipid particles. It is suggested that free ergosterol (not total ergosterol) should be used as a biomarker for fungal biomass.

Keywords: Agaric fungi; Ergosterol and its esters; Gills; Stipe; Pileus; Biomarker

Partially saturated canthaxanthin purified from Aspergillus carbonarius induces apoptosis in prostrate cancer cell line by Nallasamy Kumaresan; Konasur R. Sanjay; Kundumani S. Venkatesh; Ravi-Kumar Kadeppagari; Govindaswamy Vijayalakshmi; Sukumaran Umesh-Kumar (pp. 467-473).

A mutant Aspergillus carbonarius selected for temperature tolerance after UV treatment, when grown in shake flasks, produced mycelia bearing yellow pigment. Since the mutant was affected in sterol biosynthetic pathway, the pigment was apparently produced to maintain membrane fluidity and rigidity for growth sustenance in low-pH culture broth. Nuclear magnetic resonance analyses characterizing the pigment as a partially saturated canthaxanthin, containing β-ionone end rings, suggested its application as a retinoid. When tested for this property in retinoic acid receptor expressing prostate cancer cell line, LNCaP, the fungal partially saturated canthaxanthin induced apoptosis. Low apoptosis percentage in DU145 prostrate cancer cells that does not express functional retinoic acid receptor-β (RAR-β) suggested binding specificity of the partially saturated canthaxanthin for RAR-β.

Keywords: Aspergillus carbonarius ; Partially saturated canthaxanthin; Apoptosis; LNCaP; DU145; HeLa; Retinoic acid receptors

Regulation of ferulic catabolic genes in Pseudomonas fluorescens BF13: involvement of a MarR family regulator by C. Calisti; A. G. Ficca; P. Barghini; M. Ruzzi (pp. 475-483).

In Pseudomonas fluorescens BF13, the cluster of genes essential for degradation of ferulic to vanillic acid (ech, vdh and fcs) is expressed in ferulic but not in succinic-grown cells. In the upstream region, we identified a gene, ferR, encoding a protein homologous to transcriptional regulators of the MarR family. A ferR knockout mutant (BF13–89) showed a 3.5-fold increase in expression of an ech-reporter gene fusion compared with the parent strain in succinic-grown cells, indicating that the ferR gene product negatively regulates expression of the ferulic catabolic operon in P. fluorescens BF13. Consistent with the increased expression of the catabolic genes in the ferR mutant, BF13-89 showed a shorter (relative to its FerR⁺ parent) lag phase during carbon source shift from succinic to ferulic acid. However, expression of ech-lacZ fusion did not increase in BF13–89 grown in the presence of ferulic acid, indicating that FerR has a second function as transcriptional activator. Expression of ech-lacZ in a feruloyl-CoA synthetase-deficient strain revealed unambiguously that FerR-mediated activation of the ferulic catabolic operon is dependent on the thioester product of the feruloyl-CoA synthetase reaction.

Keywords: Ferulic acid catabolism; Pseudomonas ; MarR family regulator; FerR; CoA thioester; Gene transcription

Behavior of the IncP-7 carbazole-degradative plasmid pCAR1 in artificial environmental samples by Masaki Shintani; Kazuhiro Matsui; Tetsuo Takemura; Hisakazu Yamane; Hideaki Nojiri (pp. 485-497).

In artificial environmental samples, the behavior of the IncP-7 conjugative plasmid pCAR1, which is involved in the catabolism of carbazole, was monitored. Sterile soil and water samples supplemented with carbazole were prepared. After inoculation with Pseudomonas putida harboring pCAR1, seven species of the genus Pseudomonas, and three other bacterial species, were monitored for carbazole degradation, bacterial survival, and conjugative transfer of pCAR1. In artificial soils, more than 90% of the carbazole was degraded in samples with high water content, suggesting that the water content is a key factor in carbazole degradation in artificial soils. In three of the artificial environmental water samples, more than 95% of the carbazole was degraded. Transconjugants were detected in some artificial water samples, but not in the artificial soil samples, suggesting that pCAR1 is preferably transferred in aqueous environments. Composition analysis of the artificial water samples and examination of conjugative transfer indicated that the presence of the divalent cations Ca²⁺ and Mg²⁺ promoted the plasmid transfer. The presence of carbazole also increases in incidence of transconjugants, probably by enhancing their growth. In contrast, humic acids in the liquid layer of artificial soil samples appeared to prevent conjugative transfer.

Keywords: IncP-7 plasmid; pCAR1; Conjugative transfer; Carbazole

Significance of fatty acid supplementation on profiles of cell growth, fatty acid, and gene expression of three desaturases in Mucor rouxii by Sakda Khoomrung; Kobkul Laoteng; Sompit Jitsue; Supapon Cheevadhanarak (pp. 499-506).

Molecular and biochemical studies investigating the biosynthesis of gamma-linolenic acid (GLA), a nutritionally important polyunsaturated fatty acid, were implemented in Mucor rouxii by supplementation with exogenous fatty acids. Compared to the fatty acid-free cultures, addition of C18:2Δ^9,12 to the cultures showed up to threefold decrease in the GLA content from the beginning of the culture, whereas a significantly increased GLA amount (P ≤ 0.05) was found after 9 h of growth in the logarithmic cultures fed with stearic acid or oleic acid. Time-course studies of gene expression showed concomitant changes in the messenger RNA (mRNA) levels of the M. rouxii Δ⁶-, Δ⁹-, and Δ¹²-desaturases upon exposure to fatty acids, and these responses were rapid and transient. However, the expression profiles were different depending on the type of exogenous fatty acids supplementing the cultures. Up-regulation of the three desaturase transcripts was detected after addition of C18:0, whereas supplementation of unsaturated fatty acids led to a decrease in mRNA levels. This study showed that the three desaturase genes involved in fatty acid desaturation in M. rouxii are co-regulated in response to exogenous fatty acids.

Keywords: Mucor rouxii ; Desaturase; Cell growth; Fatty acid supplementation; Gamma-linolenic acid; Gene expression

Factors influencing the electrokinetic dispersion of PAH-degrading bacteria in a laboratory model aquifer by L. Shi; S. Müller; H. Harms; L. Y. Wick (pp. 507-515).

Despite growing interest in the electro-bioremediation of contaminated soil it is still largely unknown to which degree weak electric fields influence the fate of contaminant-degrading microorganisms in the sub-surface. Here we evaluate the factors influencing the electrokinetic transport and deposition of fluorene-degrading Sphingomonas sp. LB126 in a laboratory model aquifer exposed to a direct current (DC) electric field (1 V cm⁻¹) typically used in electro-bioremediation measures. The influence of cell size, cell membrane integrity, cell chromosome contents (all assessed by flow cytometry), cell surface charge and cell hydrophobicity on the spatial distribution of the suspended and matrix-bound cells after 15 h of DC-treatment was evaluated. In presence of DC the cells were predominantly mobilised by electroosmosis to the cathode with an apparent velocity of 0.6 cm h⁻¹, whereas a minor fraction only of the cells augmented was mobilised to the anode by electrophoresis. Different electrokinetic behaviour of individual cells could be solely attributed to intra-population heterogeneity of the cell surface charge. In the absence of DC by contrast, a Gaussian-type distribution of bacteria around the point of injection was found. DC had no influence on the deposition efficiency, as the glass beads in presence and absence of an electric field retained quasi-equal fractions of the cells. Propidium iodide staining and flow cytometry analysis of the cells indicated the absence of negative influences of DC on the cell wall integrity of electrokinetically mobilised cells and thus point at unchanged physiological fitness of electrokinetically mobilised bacteria.

Keywords: Electro-bioremediation; Viability; PAH; Sphingomonas; Propidium iodide; Transport

Identification of an uptake hydrogenase for hydrogen-dependent dissimilatory azoreduction by Shewanella decolorationis S12 by Yi-Guo Hong; Jun Guo; Guo-Ping Sun (pp. 517-524).

Shewanella decolorationis S12, a representative dissimilatory azo-reducing bacterium of Shewanella genus, can grow by coupling the oxidation of hydrogen to the reduction of azo compounds as the sole electron acceptor, indicating that an uptake hydrogenase is an important component for electron transfer for azoreduction. For searching to the uptake hydrogenase in the genome of S. decolorationis, two operons, hyd and hya, were cloned and sequenced, which encode periplasmically oriented Fe-only hydrogenase and a Ni–Fe hydrogenase, respectively, according to the homologous comparison with other bacterial hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent azoreduction and growth, hyd- and hya-deficient mutants were generated by gene replacement. Hya was found to be required for hydrogen-dependent reduction of azo compound by resting cell suspensions and to be essential for growth with hydrogen as electron donor and azo compound as electron acceptor. Hyd, in contrast, was not. These findings suggest that Hya is an essential respiratory hydrogenase of dissimilatory azoreduction in S. decolorationis.

Keywords: Hydrogenase; Dissimilatory azoreduction; Shewanella decolorationis S12

Biochemical indication of microbial mass changes using ATP and DNA measurement in biological treatment of thiocyanate by Juntaek Lim; Seungyong Lee; Sang-Don Kim; Seokhwan Hwang (pp. 525-530).

This study was designed to monitor changes in the levels of adenosine 5′-triphosphate (ATP) and deoxyribonucleic acid (DNA) per unit of microbial mass during the autotrophic biodegradation of thiocyanate (SCN⁻). An artificial medium containing trace minerals and 500 mg SCN⁻/L was used as a substrate for bacterial growth. An SCN⁻-degrading bioreactor with a working volume of 6 L, equipped with temperature, pH, and dissolved oxygen controls, was operated in batch mode. During the exponential phase of SCN⁻ biodegradation, the ratios of ATP and DNA to microbial dry weight varied from 0.6 to 1.1 μg ATP/mg of volatile suspended solid (VSS), and from 3.5 to 8.8 μg DNA/mg of VSS, respectively. The ATP and DNA concentrations correlated linearly with microbial mass (r ² > 0.9) within the exponential phase. The linear regression equations were as follows: (1) microbial mass concentration (mg/L) = 0.663 × ATP concentration (μg/L) + 11.1 and (2) microbial concentration (mg/L) = 0.081 × DNA concentration (μg/L) + 10.9. The applicable ranges were 6.8 to 47.4 μg/L for ATP concentration and 41.5 to 395 μg/L for DNA concentration, respectively.

Keywords: ATP; DNA; Microbial mass; Thiocyanate biodegradation

Enhanced biosorption of mercury(II) and cadmium(II) by cold-induced hydrophobic exobiopolymer secreted from the psychrotroph Pseudomonas fluorescens BM07 by Sheikh Shawkat Zamil; Mun Hwan Choi; Jung Hyun Song; Hyunju Park; Ju Xu; Ki-Whan Chi; Sung Chul Yoon (pp. 531-544).

The cells of psychrotrophic Pseudomonas fluorescens BM07 were found to secrete large amounts of exobiopolymer (EBP) composed of mainly hydrophobic (water insoluble) polypeptide(s) (as contain ∼50 mol% hydrophobic amino acids, lacking cysteine residue) when grown on fructose containing limited M1 medium at the temperatures as low as 0–10 °C but trace amount at high (30 °C, optimum growth) temperature. Two types of nonliving BM07 cells (i.e., cells grown at 30 °C and 10 °C) as well as the freeze-dried EBP were compared for biosorption of mercury (Hg(II)) and cadmium (Cd(II)). The optimum adsorption pH was found 7 for Hg(II) but 6 for Cd(II), irrespective of the type of biomass. Equilibrium adsorption data well fitted the Langmuir adsorption model. The maximum adsorption (Q _max) was 72.3, 97.4, and 286.2 mg Hg(II)/g dry biomass and 18.9, 27.0, and 61.5 mg Cd(II)/g dry biomass for cells grown at 30 °C and 10 °C and EBP, respectively, indicating major contribution of heavy metal adsorption by cold-induced EBP. Mercury(II) binding induced a significant shift of infrared (IR) amide I and II absorption of EBP whereas cadmium(II) binding showed only a very little shift. These IR shifts demonstrate that mercury(II) and cadmium(II) might have different binding sites in EBP, which was supported by X-ray diffraction and differential scanning calorimetric analysis and sorption results of chemically modified biomasses. This study implies that the psychrotrophs like BM07 strain may play an important role in the bioremediation of heavy metals in the temperate regions especially in the inactive cold season.

Keywords: Mercury adsorption; Cadmium adsorption; Cold-induced exobiopolymer; Hydrophobic exopolypeptide; Pseudomonas fluorescens BM07; Psychrotroph

PCR-mediated one-step deletion of targeted chromosomal regions in haploid Saccharomyces cerevisiae by Minetaka Sugiyama; Toshimasa Nakazawa; Kiriko Murakami; Takahiro Sumiya; Atsushi Nakamura; Yoshinobu Kaneko; Masafumi Nishizawa; Satoshi Harashima (pp. 545-553).

Chromosome rearrangements, especially chromosomal deletions, have been exploited as important resources for functional analysis of genomes. To facilitate this analysis, we applied a previously developed method for chromosome splitting for the direct deletion of a designed internal or terminal chromosomal region carrying many nonessential genes in haploid Saccharomyces cerevisiae. The method, polymerase chain reaction (PCR)-mediated chromosomal deletion (PCD), consists of a two-step PCR and one transformation per deletion event. In this paper, we show that the PCD method efficiently deletes internal regions in a single transformation. Of the six chromosomal regions targeted for deletion by this method, five regions (16 to 38 kb in length) containing 10 to 19 nonessential genes were successfully eliminated at high efficiency. The one targeted region on chromosome XIII that was not deleted was subsequently found to contain sequences essential for yeast growth. While 14 individual genes in this region have been reported to be nonessential, synthetic lethal interactions may occur among these nonessential genes. Phenotypic analysis showed that four deletion strains still exhibited normal growth while possible synthetic growth defects were observed in another strain harboring a 19-gene deletion on chromosome XV. These results demonstrate that the PCD method is a useful tool for deleting genes and for analyzing their functions in defined chromosomal regions.

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Applied Microbiology and Biotechnology (v.80, #3)

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Applied Microbiology and Biotechnology (v.80, #3)