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Applied Microbiology and Biotechnology (v.82, #3)

Antibiotic resistance genes in water environment by Xu-Xiang Zhang; Tong Zhang; Herbert H. P. Fang (pp. 397-414).

The use of antibiotics may accelerate the development of antibiotic resistance genes (ARGs) and bacteria which shade health risks to humans and animals. The emerging of ARGs in the water environment is becoming an increasing worldwide concern. Hundreds of various ARGs encoding resistance to a broad range of antibiotics have been found in microorganisms distributed not only in hospital wastewaters and animal production wastewaters, but also in sewage, wastewater treatment plants, surface water, groundwater, and even in drinking water. This review summarizes recently published information on the types, distributions, and horizontal transfer of ARGs in various aquatic environments, as well as the molecular methods used to detect environmental ARGs, including specific and multiplex PCR (polymerase chain reaction), real-time PCR, DNA sequencing, and hybridization based techniques.

Keywords: Antibiotic resistance gene; Environmental pollution; Gene transfer; Molecular detection method; Water environment

Biological nitrate removal in industrial wastewater treatment: which electron donor we can choose by Jae Yeon Park; Young Je Yoo (pp. 415-429).

Biological denitrification was reviewed regarding its potential application to treating nitrate in industrial wastewater. Although heterotrophic denitrification is an efficient and well-developed process, some carbon content in wastewater is essential to maintain bacterial activity. Because of the high operating cost of heterotrophic denitrification caused by the required addition of a carbon source and potential “carbon breakthrough”, the study of autotrophic denitrification has attracted the interest of numerous researchers. Many advances in autotrophic processes have been made in the application of novel concepts and reaction schemes. While the main advantage of autotrophic bacteria rests on the reduction of operating costs by the replacement of an external carbon source with a cheaper electron donor, further decrease in cost requires additional refinement of these processes, including further improvement of reactor structure and optimization of reaction conditions. In the long term, new concepts are required for a compact wastewater treatment process. This review addresses the state of the art of each electron donor candidate for its potential application to the treatment of industrial wastewater containing nitrate.

Keywords: Nitrate; Biological treatment; Wastewater; Electron donor

Localization of human (pro)renin receptor lacking the transmembrane domain on budded baculovirus of Autographa californica multiple nucleopolyhedrovirus by Tatsuya Kato; Dongning Du; Fumiaki Suzuki; Enoch Y. Park (pp. 431-437).

Human (pro)renin receptor (hPRR), a construct with native transmembrane and cytoplasmic domains (hPRR-wTM), and hPRR lacking both (hPRR-w/oTM) were expressed using insect cells. The hPRR-wTM was expressed in the peripheral domains of the nucleus in infected Sf-9 cells, and its localization was observed in endoplasmic reticulum (ER). However, it could not be extracted from recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by Triton X-100 treatment at 4°C. In contrast, hPRR-w/oTM was observed in punctate domains in the cytoplasm of infected Sf-9 cells, but intracellular hPRR-w/oTM did not co-localize in the Golgi apparatus and lysosomes. This indicates that hPRR-wTM and hPRR-w/oTM is localized in the ER and cytoplasmic organelles of Sf-9 cell, respectively. Moreover, the localization of hPRR-w/oTM in budded baculovirus of recombinant AcMNPV was confirmed by Western blotting. This is the first finding of the association of a foreign protein lacking a transmembrane domain with a baculovirus. If this finding is available for double displaying system, being capable of expression on the envelope and the capsid of baculovirus, it will lead to new methodology of baculovirus display system for tissue- and cell-specific targeting and intracellular targeting.

Keywords: Human (pro)renin receptor; AcMNPV; Transmembrane domain; Budded baculovirus; Localization

Large-scale production of biologically active human keratinocyte growth factor-2 by Xiaoping Wu; Haishan Tian; Yadong Huang; Sixian Wu; Xiaoju Liu; Cong Wang; Xiaojie Wang; Zhifeng Huang; Jian Xiao; Wenke Feng; Xiaokun Li (pp. 439-444).

A rapid and efficient expression and purification system has been developed for large-scale production of biologically active recombinant human keratinocyte growth factor-2 (rhKGF-2). The gene encoding human KGF-2 was cloned into the expression vector pET3c and transformed into Escherichia coli BL21(DE3)/pLys S. Under optimal conditions in a 30-l fermentor, the average bacterial yield and the average expression level of rhKGF-2 of three batches were up to 732 g and 32%, respectively. The recombinant protein was purified by cation exchange and heparin-affinity chromatography. One hundred and sixty five milligrams of pure rhKGF-2 was achieved per liter culture. A preliminary biochemical characterization of purified rhKGF-2 was performed by Western blotting and mitogenic activity analysis, and the results demonstrated that purified rhKGF-2 could react with anti-human KGF-2 antibody and stimulate the proliferation of HaCat cells.

Keywords: Human keratinocyte growth factor-2; Large-scale production; Expression; Purification; Mitogenic activity

Enhanced levan production using chitin-binding domain fused levansucrase immobilized on chitin beads by Chung-Jen Chiang; Jen-You Wang; Po-Ting Chen; Yun-Peng Chao (pp. 445-451).

Levan is a homopolymer of fructose which can be produced by the transfructosylation reaction of levansucrase (EC 2.4.1.10) from sucrose. In particular, levan synthesized by Zymomonas mobilis has found a wide and potential application in the food and pharmaceutical industry. In this study, the immobilization of Z. mobilis levansucrae (encoded by levU) was attempted for repeated production of levan. By fusion levU with the chitin-binding domain (ChBD), the hybrid protein was overproduced in a soluble form in Escherichia coli. After direct absorption of the protein mixture from E. coli onto chitin beads, levansucrase tagged with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indicated that the linkage between the enzyme and chitin beads was substantially stable. Furthermore, with 20% sucrose, the production of levan was enhanced by 60% to reach 83 g/l using the immobilized levansucrase as compared to that by the free counterpart. This production yield accounts for 41.5% conversion yield (g/g) on the basis of sucrose. After all, a total production of levan with 480 g/l was obtained by recycling of the immobilized enzyme for seven times. It is apparent that this approach offers a promising way for levan production by Z. mobilis levansucrase immobilized on chitin beads.

Keywords: Levan; Bioconversion; Enzyme immobilization; Z. mobilis levansucrase

A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris by Huiying Luo; Yaru Wang; Hui Wang; Jun Yang; Yuhui Yang; Huoqing Huang; Peilong Yang; Yingguo Bai; Pengjun Shi; Yunliu Fan; Bin Yao (pp. 453-461).

Using degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR, a 1,347-bp full-length complementary DNA fragment encompassing the gene man5A, which encodes a 429-amino acid β-mannanase with a calculated mass of 46.8 kDa, was cloned from acidophilic Bispora sp. MEY-1. The deduced amino acid sequence (catalytic domain) displayed highest identity (54.1%) with the Emericella nidulans endo-β-1,4-d-mannanase, a member of the glycoside hydrolase family 5. Recombinant MAN5A was overexpressed in Pichia pastoris, and its activity in the culture medium reached 500 U ml⁻¹. The enzyme was acidophilic, with highest activity at pH 1.0–1.5, lower than any known mannanases, and optimal temperature for activity was 65°C. MAN5A had good pH adaptability, excellent thermal and pH stability, and high resistance to both pepsin and trypsin. The specific activity, K _m, and V _max for locust bean gum substrate was 3,373 U mg⁻¹, 1.56 mg ml⁻¹, and 6,587.6 μmol min⁻¹ mg⁻¹, respectively. The enzymatic activity was not significantly affected by ions such as Ca²⁺, Cr³⁺, Co²⁺, Zn²⁺, Na⁺, K⁺, and Mg²⁺ and enhanced by Ni²⁺, Fe³⁺, Mn²⁺ and Ag⁺. These favorable properties make MAN5A a potential candidate for use in various industrial applications.

Keywords: Acidophilic fungus; Bispora sp. MEY-1; β-mannanase; Pichia pastoris

Improving aquaporin Z expression in Escherichia coli by fusion partners and subsequent condition optimization by Jiazhang Lian; Shinghua Ding; Jin Cai; Danping Zhang; Zhinan Xu; Xiaoning Wang (pp. 463-470).

Aquaporin Z (AqpZ), a typical orthodox aquaporin with six transmembrane domains, was expressed as a fusion protein with TrxA in E. coli in our previous work. In the present study, three fusion partners (DsbA, GST and MBP) were employed to improve the expression level of this channel protein in E. coli. The result showed that, compared with the expression level of TrxA-AqpZ, five- to 40-fold increase in the productivity of AqpZ with fusion proteins was achieved by employing these different fusion partners, and MBP was the most efficient fusion partner to increase the expression level. By using E. coli C43 (DE3)/pMAL-AqpZ, the effects of different expression conditions were investigated systematically to improve the expression level of MBP-AqpZ in E. coli. The high productivity of MBP-AqpZ (200 mg/l) was achieved under optimized conditions. The present work provides a novel approach to improve the expression level of membrane proteins in E. coli.

Keywords: Aquaporin Z; MBP; GST; DsbA; Membrane protein

A novel α-galactosidase from Βifidobacterium bifidum with transgalactosylating properties: gene molecular cloning and heterologous expression by Theodoros Goulas; Athanasios Goulas; George Tzortzis; Glenn R. Gibson (pp. 471-477).

A genomic library of Bifidobacterium bifidum (NCIMB 41171) DNA was constructed in Escherichia coli RA11r (melA⁻B⁺) and one α-galactosidase encoding gene was isolated. Conceptual translation combined with insertional mutagenesis analysis indicated an open reading frame (ORF) of 759 amino acid (aa) residues encoding an α-galactosidase (named as MelA) of 82.8 kDa. Partial purification and characterisation showed that the enzyme had an apparent native molecular mass of ≈243 kDa and a subunit size of ≈85 kDa. The enzyme belongs to glycosyl hydrolases 36 family with high aa sequence similarities (≈73%) to other known α-galactosidases of bifidobacterial origin. Under optimum pH conditions for activity (pH 6.0) and high melibiose concentration (40% w/v), the enzyme was able to form oligosaccharides with degree of polymerisation (DP) ≥3 at higher concentration than DP = 2, with a total yield of 20.5% (w/w).

Keywords: Bifidobacteria; Galactosidase; Prebiotic

Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase by Noriyuki Doukyu; Kanpei Shibata; Hiroyasu Ogino; Martin Sagermann (pp. 479-490).

Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53–62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. K _m and V _max values of the cholesterol oxidase were estimated from Lineweaver–Burk plots. The V _max/K _m ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures.

Keywords: Cholesterol oxidase; Cloning; Thermal stability; Organic solvent; Detergent; Chromobacterium

Identification of new secreted proteins and secretion of heterologous amylase by C. glutamicum by Nobuaki Suzuki; Keiro Watanabe; Naoko Okibe; Yoshiki Tsuchida; Masayuki Inui; Hideaki Yukawa (pp. 491-500).

In this study, secreted Corynebacterium glutamicum proteins were investigated by two-dimensional gel electrophoresis. Around 100 spots observed in the pH range 4.5–5.5 had molecular masses that varied from 10 to 50 kDa. Upon N-terminal amino acid sequence analysis by Edman degradation, two of them were hits to two hypothetical proteins encoded by cgR_1176 and cgR_2070 on C. glutamicum R genome, respectively. Active-form α-amylase derived from Geobacillus stearothermophilus was successfully secreted by using the predicted cgR_1176 and cgR_2070 signal sequences, indicating that these hypothetical proteins were secreted proteins. Analysis using a disruption mutant of the twin-arginine translocation (Tat) export pathway machinery of C. glutamicum suggested that one is Tat pathway dependent secretion while the other is independent of the pathway. Our results demonstrate that C. glutamicum can secrete exoproteins by using its own signal sequences, indicating its potential as a host for protein productions.

Keywords: Secretion; C. glutamicum ; Signal sequence; Sec; Tat

NdgR, an IclR-like regulator involved in amino-acid-dependent growth, quorum sensing, and antibiotic production in Streptomyces coelicolor by Yung-Hun Yang; Eunjung Song; Eun-Jung Kim; Kwangwon Lee; Woo-Seong Kim; Sung-Soo Park; Ji-Sook Hahn; Byung-Gee Kim (pp. 501-511).

NdgR (regulator for nitrogen source-dependent growth and antibiotic production), an IclR-like regulator, has been initially identified as a binding protein to the promoters of doxorubicin biosynthetic genes in Streptomcyes peucetius by DNA affinity capture assay method. NdgR is well conserved throughout the Streptomcyes species and many other bacteria such as Mycobacteria and Corynebacteria. In Streptomcyes coelicolor, ndgR deletion mutant showed slow cell growth and defects in differentiation and enhances the production of actinorhodin (ACT) in minimal media containing certain amino acids where wild-type strain could not produce ACT. Although deletion mutant of ndgR showed different antibiotic production in minimal media containing Leu or Gln, it only showed reduced mRNA expression levels of the genes involved in leucine metabolism. Neither NdgR-dependent expression of glnA nor direct binding of NdgR protein to glnA, glnII, and glnR promoters was observed. However, ScbR, which is governed by NdgR shown by gel mobility shift assay, binds to promoter of glnR, suggesting indirect regulation of glutamine metabolism by NdgR. NdgR protein binds to intergenic region of ndgR–leuC, and scbR–scbA involved in γ-butyrolactone. Two-dimensional gel analysis has shown a global effect of ndgR deletion in protein expression, including up-regulated proteins involved in ACT synthesis and down-regulation of chaperones such as GroEL, GroES, and DnaK. These results suggest a global regulatory role for NdgR in amino acid metabolisms, quorum sensing, morphological changes, antibiotic production, and expression of chaperonines in S. coelicolor.

Keywords: Streptomyces coelicolor ; Streptomyces peucetius ; IclR-like regulator; DNA affinity capture assay; scbR

α- and β-tubulin from Phytophthora capsici KACC 40483: molecular cloning, biochemical characterization, and antimicrotubule screening by Bon-Sung Koo; Haechul Park; Satish Kalme; Hye-Yeon Park; Jin Wook Han; Yun-Soo Yeo; Sang-Hong Yoon; Soo-Jin Kim; Chang-Muk Lee; Moon-Young Yoon (pp. 513-524).

Internal fragments of α- and β-tubulin genes were generated using reverse transcription polymerase chain reaction (RT-PCR), and the termini were isolated using 5′- and 3′-rapid amplification of cDNA ends. Phytophthora capsici α- and β-tubulin specific primers were then used to generate full-length cDNA by RT-PCR. The recombinant α- and β-tubulin genes were expressed in Escherichia coli BL21 (DE3), purified under denaturing conditions, and average yields were 3.38–4.5 mg of α-tubulin and 2.89–4.0 mg of β-tubulin, each from 1-l culture. Optimum conditions were obtained for formation of microtubule-like structures. A value of 0.12 mg/ml was obtained as the critical concentration of polymerization of P. capsici tubulin. Benomyl inhibited polymerization with half-maximal inhibition (IC₅₀) = 468 ± 20 μM. Approximately 18.66 ± 0.13 cysteine residues per tubulin dimer were accessible to 5,5′-dithiobis-(2-nitrobenzoic acid), a quantification reagent of sulfhydryl and 12.43 ± 0.12 residues were accessible in the presence of 200 μM benomyl. The order of preference for accessibility to cysteines was benomyl > colchicine > GTP > taxol, and cysteine accessibility changes conformed that binding sites of these ligands in tubulin were folding correctly. Fluorescence resonance energy transfer technique was used for high throughput screening of chemical library in search of antimitotic agent. There was significant difference in relative fluorescence by 210-O-2 and 210-O-14 as compared to colchicine.

Keywords: Phytophthora capsici ; Recombinant tubulin; Benomyl; DTNB; FRET

5-Fluorouracil reduces biofilm formation in Escherichia coli K-12 through global regulator AriR as an antivirulence compound by Can Attila; Akihiro Ueda; Thomas K. Wood (pp. 525-533).

The uracil analog, 5-fluorouracil (5-FU), reduces virulence and biofilm formation for Pseudomonas aeruginosa PA14 without affecting its growth. As 5-FU is an approved anticancer drug, its antivirulence attributes in P. aeruginosa prompted us to examine the effect of this compound on three different Escherichia coli K-12 strains and its effect on virulence genes in E. coli O157:H7 (EHEC); the mechanism by which it functions was also examined. 5-FU decreased biofilm formation in a dose-dependent manner in E. coli K-12 and repressed the expression of virulence genes in EHEC. Five other uracil analogs were also tested for their effects on biofilm formation, and none of these compounds affected the biofilm formation in E. coli K-12. Whole-transcriptome analysis revealed that 5-FU induced the expression of 157 genes and repressed the expression of 19 genes. Biofilm formation with the addition of 5-FU was checked in 21 isogenic knockout mutants whose gene expression was induced in the microarray data; we found that 5-FU does not decrease biofilm formation of the cells that lack AriR, a global DNA regulator that controls acid resistance in E. coli. Hence, 5-FU represses biofilm formation of E. coli K-12 through AriR and is a novel antivirulence compound for this strain.

Keywords: Biofilm; Escherichia coli ; Antivirulence; 5-Fluorouracil

Characterization of extracellular polymeric substances from denitrifying organism Comamonas denitrificans by Sofia Andersson; Gunnel Dalhammar; Carl Johan Land; Gunaratna Kuttuva Rajarao (pp. 535-543).

Extracellular polymeric substances (EPS) play an important role in the formation and activity of biofilms in wastewater treatment (WWT). The EPS of the denitrifying biomarker Comamonas denitrificans strain 110, produced in different culture media and growth modes, were characterized. The EPS mainly contained protein (3–37%), nucleic acids (9–50%), and carbohydrates (3–21%). The extracellular DNA was found to be important for initial biofilm formation since biofilm, but not planktonic growth, was inhibited in the presence of DNase. The polysaccharide fraction appeared to consist of at least two distinct polymers, one branched fraction (A) made up of glucose and mannose with a molecular weight around 100 kDa. The other fraction (B) was larger and consisted of ribose, mannose, glucose, rhamnose, arabinose, galactose, and N-acetylglucosamine. Fraction B polysaccharides were mainly found in capsular EPS which was the dominant type in biofilms and agar-grown colonies. Fraction A was abundant in the released EPS, the dominant type in planktonic cultures. Biofilm and agar-grown EPS displayed similar overall properties while planktonic EPS showed clear compositional disparity. This study presents results on the physiology of a key WWT organism, which may be useful in the future development of improved biofilm techniques for WWT purposes.

Keywords: Biofilm; Comamonas denitrificans ; GC–MS; Extracellular polymeric substances; HPAEC; Polysaccharides

Physico-chemical properties of polyhydroxyalkanoate produced by mixed-culture nitrogen-fixing bacteria by Meeta Patel; Daniel J. Gapes; Roger H. Newman; Peter H. Dare (pp. 545-555).

Ultra-high molecular weight polyhydroxyalkanoates (PHAs) with low polydispersity index (PDI = 1.3) were produced in a novel, pilot scale application of mixed cultures of nitrogen-fixing bacteria. The number average molecular weight (M _n) of the poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was determined to be 2.4 × 10⁶ and 2.5 × 10⁶ g mol⁻¹, respectively. Using two types of carbon sources, biomass contents of the P(3HB) and P(3HB-co-3HV) were 18% and 30% (PHA in dry biomass), respectively. The extracted polymers were analysed for their physical properties using analytical techniques such as nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC) and gel permeation chromatography (GPC). NMR confirmed the formation of homopolymer and copolymer. DSC showed a single melting endotherm peak for both polymers, with enthalpies that indicated crystallinity indices of 44% and 37% for P(3HB) and P(3HB-co-3HV), respectively. GPC showed a sharp unimodal trace for both polymers, reflecting the homogeneity of the polymer chains. The work described here emphasises the potential of mixed colony nitrogen-fixing bacteria cultures for producing biodegradable polymers which have properties that are very similar to those from their pure-culture counterparts and therefore making a more economically viable route for obtaining biopolyesters.

Keywords: Biopolymer; Differential scanning calorimetry; Nitrogen-fixing bacteria; Nuclear magnetic resonance; Polyhydroxyalkanoates

Effects of NO₂ ⁻ and NO₃ ⁻ on the Fe(III)EDTA reduction in a chemical absorption–biological reduction integrated NO x removal system by Shi-Han Zhang; Ling-Lin Cai; Yun Liu; Yao Shi; Wei Li (pp. 557-563).

The biological reduction of Fe(III) ethylenediaminetetraacetic acid (EDTA) is a key step for NO removal in a chemical absorption–biological reduction integrated process. Since typical flue gas contain oxygen, NO₂ ⁻ and NO₃ ⁻ would be present in the absorption solution after NO absorption. In this paper, the interaction of NO₂ ⁻, NO₃ ⁻, and Fe(III)EDTA reduction was investigated. The experimental results indicate that the Fe(III)EDTA reduction rate decrease with the increase of NO₂ ⁻ or NO₃ ⁻ addition. In the presence of 10 mM NO₂ ⁻ or NO₃ ⁻, the average reduction rate of Fe(III)EDTA during the first 6-h reaction was 0.076 and 0.17 mM h⁻¹, respectively, compared with 1.07 mM h⁻¹ in the absence of NO₂ ⁻ and NO₃ ⁻. Fe(III)EDTA and either NO₂ ⁻ or NO₃ ⁻ reduction occurred simultaneously. Interestingly, the reduction rate of NO₂ ⁻ or NO₃ ⁻ was enhanced in presence of Fe(III)EDTA. The inhibition patterns observed during the effect of NO₂ ⁻ and NO₃ ⁻ on the Fe(III)EDTA reduction experiments suggest that Escherichia coli can utilize NO₂ ⁻, NO₃ ⁻, and Fe(III)EDTA as terminal electron acceptors.

Keywords: NO₂ ⁻ ; NO₃ ⁻ ; Fe(III)EDTA; Biological reduction; Inhibition

Enhancement of the microbial community biomass and diversity during air sparging bioremediation of a soil highly contaminated with kerosene and BTEX by Nadja Kabelitz; Jirina Machackova; Gwenaël Imfeld; Maria Brennerova; Dietmar H. Pieper; Hermann J. Heipieper; Howard Junca (pp. 565-577).

In order to obtain insights in complexity shifts taking place in natural microbial communities under strong selective pressure, soils from a former air force base in the Czech Republic, highly contaminated with jet fuel and at different stages of a bioremediation air sparging treatment, were analyzed. By tracking phospholipid fatty acids and 16S rRNA genes, a detailed monitoring of the changes in quantities and composition of the microbial communities developed at different stages of the bioventing treatment progress was performed. Depending on the length of the air sparging treatment that led to a significant reduction in the contamination level, we observed a clear shift in the soil microbial community being dominated by Pseudomonads under the harsh conditions of high aromatic contamination to a status of low aromatic concentrations, increased biomass content, and a complex composition with diverse bacterial taxonomical branches.

Keywords: BTEX; Air sparging; Bioremediation; Biodiversity; Microbiota

Potential natural product discovery from microbes through a diversity-guided computational framework by Eakasit Pacharawongsakda; Sunai Yokwai; Supawadee Ingsriswang (pp. 579-586).

As the occurrence of natural compounds is related to the spatial distribution and evolution of microorganisms for biological and ecological relevance, the data integration of chemistry, geography, and phylogeny within an analytical framework is needed to make better decisions on sourcing the microbes for drug discovery. Such a framework should help researcher to decide on (a) which microorganisms are capable to produce the structurally diverse-bioactive compounds and (b) where those microbes could be found. Here, we present GIST (Geospatial Integrated Species, sites and bioactive compound relationships Tracking tool), a computational framework that could describe and compare how the chemical and genetic diversity varied among microbes in different areas. GIST mainly exploits the measures of bioactive diversity (BD) and phylogenetic diversity (PD), derived from the branch length of bioactive dendrogram and phylogenetic tree, respectively. Based on BD and PD, our framework could provide guidance and tools for measuring, monitoring, and evaluating of patterns and changes in biodiversity of microorganisms to improve the success rate of drug discovery.

Keywords: Bioactive diversity; Phylogenetic diversity; Microbial collection; Natural products; Dendrogram; Drug discovery

Development of a quantitative PCR method to differentiate between viable and nonviable bacteria in environmental water samples by Phillip B. Gedalanga; Betty H. Olson (pp. 587-596).

Ethidium monoazide bromide (EMA) treatment of pure culture and environmental waters at low concentrations (1.0–7.5 µg/ml) indicated effective enumeration of viable and viable but nonculturable Escherichia coli in pure cultures, creek waters, and secondary activated sludge effluent samples by quantitative polymerase chain reaction (qPCR) amplification of the uidA and fliC gene targets at turbidity values <10 NTU. However, EMA treatment was not effective in primary clarifier and secondary trickling filter effluents where turbidities were ≥10 NTU. In viable pure cultures, rapidly dividing and senescent cells were most affected by increasing EMA concentrations. Amplification of heat-killed pure bacterial cultures decreased 4 to 6 logs depending on EMA concentration and culture age. The greatest difference was observed in 5-h cultures using 7.5 μg/ml EMA. Turbidity (≥100 NTU) in environmental samples inhibited EMA effectiveness on viability discrimination. Enumeration of E. coli in certain wastewaters using EMA-qPCR was similar to culture suggesting that EMA treatment could be incorporated into qPCR assays for the quantification of viable bacteria increasing assay time no more than 30 min. Our results indicate that EMA can be used in routine qPCR assays, but optimum conditions for exposure must be identified for each sample type due to sample matrix effects such as turbidity.

Keywords: Cell viability; Escherichia coli ; qPCR; Ethidium monoazide bromide; Environmental and wastewaters

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Applied Microbiology and Biotechnology (v.82, #3)