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Applied Microbiology and Biotechnology (v.85, #2)

Biodegradation of aromatic compounds: current status and opportunities for biomolecular approaches by Bin Cao; Karthiga Nagarajan; Kai-Chee Loh (pp. 207-228).

Biodegradation can achieve complete and cost-effective elimination of aromatic pollutants through harnessing diverse microbial metabolic processes. Aromatics biodegradation plays an important role in environmental cleanup and has been extensively studied since the inception of biodegradation. These studies, however, are diverse and scattered; there is an imperative need to consolidate, summarize, and review the current status of aromatics biodegradation. The first part of this review briefly discusses the catabolic mechanisms and describes the current status of aromatics biodegradation. Emphasis is placed on monocyclic, polycyclic, and chlorinated aromatic hydrocarbons because they are the most prevalent aromatic contaminants in the environment. Among monocyclic aromatic hydrocarbons, benzene, toluene, ethylbenzene, and xylene; phenylacetic acid; and structurally related aromatic compounds are highlighted. In addition, biofilms and their applications in biodegradation of aromatic compounds are briefly discussed. In recent years, various biomolecular approaches have been applied to design and understand microorganisms for enhanced biodegradation. In the second part of this review, biomolecular approaches, their applications in aromatics biodegradation, and associated biosafety issues are discussed. Particular attention is given to the applications of metabolic engineering, protein engineering, and “omics” technologies in aromatics biodegradation.

Keywords: Biodegradation; Aromatics; Molecular approaches; Metabolic engineering; Protein engineering; Omics technologies

Aptamers and riboswitches: perspectives in biotechnology by Julia E. Weigand; Beatrix Suess (pp. 229-236).

Aptamers are short, single stranded nucleic acids which bind a wide range of different ligands with extraordinary high binding affinity and specificity. The steadily increasing number of aptamers is accompanied by an expanding range of applications in biotechnology. We will describe new developments in the field including the use of aptamers for conditional gene regulation and as biosensors. In addition, we will discuss the potential of aptamers as tags to visualize RNA and protein distribution in living cells and as therapeutics. Furthermore, we will consider biotechnological applications of riboswitches for gene regulation and as drug target.

Keywords: RNA; Aptamer; Riboswitch; Conditional gene expression; Biosensor

DNA profiling of complex bacterial populations: toxic cyanobacterial blooms by Martin Saker; Cristiana Moreira; Joana Martins; Brett Neilan; Vitor Manuel Vasconcelos (pp. 237-252).

Cyanobacteria are prokaryotic photosynthetic living organisms that inhabit our planet for over three billion years. With a worldwide distribution, they can be found in all types of environments: fresh, brackish and saltwater as well as terrestrial. Though beneficial in the development of life on earth, they also constitute a serious risk to our ecosystems since they can biologically produce harmful secondary metabolites named cyanotoxins. When studying cyanobacteria and their cyanotoxins, several methodologies have been applied with an increasing relevance to molecular methods. Therefore, the aim of this review is to describe alternative molecular methods that can be used as alternative methods for the identification of cyanobacteria. More traditional chemotaxonomic methods are discussed briefly as are the standard and somewhat dated techniques for assessing genome content for taxonomic classification schemes. The use of DNA amplification technology has been applied to the systematics and phylogeny of many bacterial groups, and the optimisation of methods for rapid identification and classification of cyanobacteria are presented. Together with novel methods developed for these photosynthetic microorganisms, the generated DNA profiles have been utilised to study cyanobacterial bloom population diversity and prediction of strain toxigenicity. Finally, the genotypes found were applied to a variety of phylogenetic analyses; trees were reconstructed and compared to the current morphological system of classification. The ecology and diversity of the cyanobacteria is discussed with respect to the derived molecular phylogenies and systematics.

Keywords: Cyanobacteria; Toxins; DNA profiling

Tolerance and stress response to ethanol in the yeast Saccharomyces cerevisiae by Junmei Ding; Xiaowei Huang; Lemin Zhang; Na Zhao; Dongmei Yang; Keqin Zhang (pp. 253-263).

Eukaryotic cells have developed diverse strategies to combat the harmful effects of a variety of stress conditions. In the model yeast Saccharomyces cerevisiae, the increased concentration of ethanol, as the primary fermentation product, will influence the membrane fluidity and be toxic to membrane proteins, leading to cell growth inhibition and even death. Though little is known about the complex signal network responsible for alcohol stress responses in yeast cells, several mechanisms have been reported to be associated with this process, including changes in gene expression, in membrane composition, and increases in chaperone proteins that help stabilize other denatured proteins. Here, we review the recent progresses in our understanding of ethanol resistance and stress responses in yeast.

Keywords: Ethanol tolerance; Stress response; Membrane composition; Stabilization of proteins; Regulation of gene expression

Achievements and new knowledge unraveled by metagenomic approaches by Carola Simon; Rolf Daniel (pp. 265-276).

Metagenomics has paved the way for cultivation-independent assessment and exploitation of microbial communities present in complex ecosystems. In recent years, significant progress has been made in this research area. A major breakthrough was the improvement and development of high-throughput next-generation sequencing technologies. The application of these technologies resulted in the generation of large datasets derived from various environments such as soil and ocean water. The analyses of these datasets opened a window into the enormous phylogenetic and metabolic diversity of microbial communities living in a variety of ecosystems. In this way, structure, functions, and interactions of microbial communities were elucidated. Metagenomics has proven to be a powerful tool for the recovery of novel biomolecules. In most cases, functional metagenomics comprising construction and screening of complex metagenomic DNA libraries has been applied to isolate new enzymes and drugs of industrial importance. For this purpose, several novel and improved screening strategies that allow efficient screening of large collections of clones harboring metagenomes have been introduced.

Keywords: Metagenomics; Metagenomic library; Biocatalysts; Function-based screens; Sequence-based screens

Continuous hydrolysis of 4-cyanopyridine by nitrilases from Fusarium solani O1 and Aspergillus niger K10 by Anna Malandra; Maria Cantarella; Ondřej Kaplan; Vojtěch Vejvoda; Bronislava Uhnáková; Barbora Štěpánková; David Kubáč; Ludmila Martínková (pp. 277-284).

The operational stabilities of nitrilases from Aspergillus niger K10 and Fusarium solani O1 were examined with 4-cyanopyridine as the substrate in continuous-stirred membrane reactors (CSMRs). The former enzyme was fairly stable at 30 °C with a deactivation constant (k _d) and enzyme half-life of 0.014 h⁻¹ and 50 h, respectively, but the latter exhibited an even higher stability characterized by k _d = 0.008 h⁻¹ and half-life of 87 h at 40 °C. Another advantage of this enzyme was its high chemoselectivity, i.e., selective transformation of nitriles into carboxylic acids, while the amide formed a high ratio of A. niger K10 nitrilase product. High conversion rates (>90%) were maintained for about 52 h using the nitrilase from F. solani O1 immobilized in cross-linked enzyme aggregates (CLEAs). The purity of isonicotinic acid was increased from 98% to >99.9% by using two CSMRs connected in series, the first one containing the F. solani O1 nitrilase and the second the amidase from Rhodococcus erythropolis A4 (both enzymes as CLEAs), the amidase hydrolyzing the by-product isonicotinamide.

Keywords: Nitrilase; Aspergillus niger ; Fusarium solani ; Cyanopyridines; Continuous-stirred membrane reactors; Cross-linked enzyme aggregates

Stimulation of artemisinin synthesis by combined cerebroside and nitric oxide elicitation in Artemisia annua hairy roots by Jian Wen Wang; Li Ping Zheng; Ben Zhang; Ting Zou (pp. 285-292).

This work examined the accumulation of artemisinin and related secondary metabolism pathways in hairy root cultures of Artemisia annua L. induced by a fungal-derived cerebroside (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine. The presence of the cerebroside induced nitric oxide (NO) burst and artemisinin biosynthesis in the hairy roots. The endogenous NO generation was examined to be involved in the cerebroside-induced biosynthesis of artemisinin by using NO inhibitors, N _ω-nitro-l-arginine methyl ester and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The gene expression and activity of 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate synthase were stimulated by the cerebroside, but more strongly by the potentiation of NO. While the mevalonate pathway inhibitor, mevinolin, only partially inhibited the induced artemisinin accumulation, the plastidic 2-C-methyl-d-erythritol 4-phosphate pathway inhibitor, fosmidomycin, nearly arrested artemisinin accumulation induced by cerebroside and the combination elicitation with an NO donor, sodium nitroprusside (SNP). With the potentiation by SNP at 10 μM, the cerebroside elicitor stimulated artemisinin production in 20-day-old hairy root cultures up to 22.4 mg/l, a 2.3-fold increase over the control. These results suggest that cerebroside plays as a novel elicitor and the involvement of NO in the signaling pathway of the elicitor activity for artemisinin biosynthesis.

Keywords: Artemisinin; Cerebroside elicitor; Nitric oxide; Isoprenoid pathways; Hairy root culture

Secreted production of an elastin-like polypeptide by Pichia pastoris by Roelof Schipperus; Rosalie L. M. Teeuwen; Marc W. T. Werten; Gerrit Eggink; Frits A. de Wolf (pp. 293-301).

Elastin-like polypeptides (ELPs) are biocompatible designer polypeptides with inverse temperature transition behavior in solution. They have a wide variety of possible applications and a potential medical importance. Currently, production of ELPs is done at lab scale in Escherichia coli shake flask cultures. With a view to future large scale production, we demonstrate secreted production of ELPs in methanol-induced fed-batch cultures of Pichia pastoris and purification directly from the culture medium. The production of ELPs by P. pastoris proved to be pH dependent within the experimental pH range of pH 3 to 7, as an increasing yield was found in cultures grown at higher pH. Because ELP produced at pH 7 was partly degraded, a pH optimum for production of ELP was found at pH 6 with a yield of 255 mg of purified intact ELP per liter of cell-free medium.

Keywords: Elastin-like polypeptides; Pichia pastoris ; Secreted expression; Inverse transition cycling; Heterologous protein expression

High-level expression of soluble subunit b of F₁F₀ ATP synthase in Escherichia coli cell-free system by Jiazhang Lian; Yi Ma; Jin Cai; Ming Wu; Jufang Wang; Xiaoning Wang; Zhinan Xu (pp. 303-311).

The overexpression of subunit b of F₁F₀ adenosine triphosphate (ATP) synthase from Escherichia coli is so toxic that it even prevents the transformation of plasmids encoding this protein into E. coli BL21 (DE3). In the present work, E. coli cell-free system was chosen as an alternative to express this highly toxic membrane protein. This protein was either produced as precipitates followed by detergent resolubilization or expressed as a soluble form with detergent addition. Among several types of tested detergents, Brij 58 could effectively solubilize approximately 85% of the target membrane protein within a wide range of concentration (48 to 178 times critical micelle concentration [CMC]) with little effect on the expression level. With the presence of Brij 58 at the final concentration of 96 times CMC in the E. coli cell-free system, 789 μg/mL of soluble subunit b was achieved after 4 h biosynthesis, which is the highest level for the expression of membrane proteins in a batch-mode cell-free expression system. The present work provides a rapid and efficient procedure of expressing one membrane protein with high cytotoxicity in the cell-free system and will be helpful to further exploration of reconstituting F₁F₀ ATP synthase into liposome or polymer vesicle to design a nanoelectromechanical system device.

Keywords: Subunit b of ATP synthase; ATP synthase; Cell-free expression system; Membrane protein; Escherichia coli

Isolation of a thermostable legume chitinase and study on the antifungal activity by Shaoyun Wang; Biao Shao; Hong Fu; Pingfan Rao (pp. 313-321).

Chitinases are listed as one class of pathogenesis-related proteins, and they have become a popular research topic because of their resistance to plant-pathogenic diseases. A chitinase with antifungal activity was isolated from the Canadian cranberry beans (Phaseolus vulgaris). The procedure included extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, CM-Sephadex C-50, and Sephadex G-75. There was an almost 108-fold increase in specific activity of the purified chitinase compared with that of the crude extract. The enzyme exhibited a molecular mass of 30.6 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis both under reducing and non-reducing conditions, indicating that it was a monomeric protein. The pI was determined to be 7.6 by isoelectric-focusing electrophoresis. The optimum pH and the optimum temperature for activity towards N-acetyl-glucosamine was 5.4 and 40–55°C, respectively. It exerted a potent inhibitory action toward fungal species including Botrytis cinerea, Physalospora piricola, Fusarium oxysporum, and Pythium aphanidermatum. The chitinase was thermostable up to 58°C in both enzymatic reaction and antifungal activity. The present findings demonstrated a thermostable chitinase from cranberry beans with potentially exploitable significance.

Keywords: Cranberry bean; Chitinase; Thermostable; Chitin-binding; Antifungal

Molecular and biochemical characterization of a novel xylanase from the symbiotic Sphingobacterium sp. TN19 by Junpei Zhou; Huoqing Huang; Kun Meng; Pengjun Shi; Yaru Wang; Huiying Luo; Peilong Yang; Yingguo Bai; Zhigang Zhou; Bin Yao (pp. 323-333).

A xylanase-encoding gene, designated xynA19, was cloned from Sphingobacterium sp. TN19—a symbiotic bacterium isolated from the gut of Batocera horsfieldi larvae—and expressed in Escherichia coli BL21 (DE3). The full-length xynA19 (1,155 bp in length) encodes a 384-residue polypeptide (XynA19) containing a predicted signal peptide of 24 residues and a catalytic domain belonging to glycosyl hydrolase family 10 (GH 10). The deduced amino acid sequence of XynA19 is most similar (53.1% identity) to an endo-1,4-β-xylanase from Prevotella bryantii B₁4. Phylogenetic analysis of GH 10 Bacteroidia xylanases indicated that GH 10 xylanases from Sphingobacteria were separated into two clusters, and XynA19 is more closely related to the xylanases of Bacteroidia from gut or rumen than to those of Flavobacteria and Sphingobacteria from other sources. Recombinant XynA19 (r-XynA19) showed apparent optimal activity at pH 6.5 and 45°C. Compared with thermophilic and mesophilic counterparts, r-XynA19 was more active at low temperatures, retaining >65% of its maximum activity at 20–28°C and ~40% even at 10°C, and modeling indicated that XynA19 has fewer hydrogen bonds and salt bridges. These properties suggest that XynA19 has various potential applications, especially in aquaculture and the food industry.

Keywords: Batocera horsfieldi ; Sphingobacterium sp. TN19; Xylanase; Gut

Heterologous expression and characterization of CpI, OcpA, and novel serine-type carboxypeptidase OcpB from Aspergillus oryzae by Hiroto Morita; Ayako Okamoto; Yohei Yamagata; Ken-ichi Kusumoto; Yoshinao Koide; Hiroki Ishida; Michio Takeuchi (pp. 335-346).

In the genome of Aspergillus oryzae, 12 genes have been predicted to encode serine-type carboxypeptidases. However, the carboxypeptidase activities of the proteins encoded by these genes have not yet been confirmed experimentally. In this study, we have constructed three of these 12 genes overexpressing strains using Aspergillus nidulans and characterized their overproduced recombinant proteins. Of these three genes, one was previously named cpI; the other two have not been reported yet, and hence, we named them ocpA and ocpB. The recombinant proteins released amino acid residues from the C terminus of peptides, and the activity of the enzymes was inhibited by phenylmethylsulfonyl fluoride, indicating the enzymes to be serine-type carboxypeptidases. Recombinant OcpA, OcpB, and CpI were stable at 45°C, 55°C, and 55°C, respectively, at a low pH. The enzymatic properties of recombinant OcpB were different from those of any reported serine-type carboxypeptidase. On the other hand, recombinant OcpA had similar enzymatic properties to A. oryzae carboxypeptidases O1 and O2. The DNA and N-terminal amino acid sequences of carboxypeptidases O1 and O2 from A. oryzae IAM2640 were similar to those of OcpA. Result of transcriptional analysis of ocpA, ocpB, and cpI suggest differences in transcriptional regulation between these genes.

Keywords: Aspergillus oryzae ; Carboxypeptidase; Protease; ocpA ; ocpB ; Characterization

WS-5995 B, an antifungal agent inducing differential gene expression in the conifer pathogen Heterobasidion annosum but not in Heterobasidion abietinum by Nina A. Lehr; Aleksandra Adomas; Frederick O. Asiegbu; Rüdiger Hampp; Mika T. Tarkka (pp. 347-358).

The mycorrhization helper bacterium Streptomyces sp. AcH 505 inhibits Norway spruce root infection and colonisation by the root and butt rot fungus Heterobasidion annosum 005 but not by the congeneric strain Heterobasidion abietinum 331 because of higher sensitivity of H. annosum 005 towards the AcH 505-derived naphthoquinone antibiotic WS-5995 B. Differences in antibiotic sensitivity between two isolates belonging to two species, H. annosum 005 and H. abietinum 331, were investigated by comparative gene expression analysis using macroarrays and quantitative RT-PCR after WS-5995 B, structurally related mollisin and unrelated cycloheximide application. Treatment with 25 µM WS-5995 B for 2 h resulted in a significant up-regulation of expression of inosine-5′-monophosphate dehydrogenase, phosphoglucomutase and GTPase genes, while the expression of genes encoding for thioredoxin and glutathione dependent formaldehyde dehydrogenase was down-regulated in the sensitive fungal strain. No differential expression in the tolerant strain was detected. Application of WS-5995 B at higher concentrations over a time course experiment revealed that H. annosum 005 and H. abietinum 331 responded differently to WS-5995 B. The fungal gene expression levels depended on both the concentration of WS-5995 B and the duration of its application. The WS-5995 B-unrelated cycloheximide caused highly specific changes in patterns of gene expression. Our findings indicate considerable variations in response to bacterial metabolites by the isolates of the conifer pathogen.

Keywords: Heterobasidion ; Plant pathogen; Macroarray; Gene expression; Antibiotic; Streptomyces

Methyl-branched poly(hydroxyalkanoate) biosynthesis from 13-methyltetradecanoic acid and mixed isostearic acid isomer substrates by Richard D. Ashby; Helen L. Ngo; Daniel K. Y. Solaiman; Gary Strahan (pp. 359-370).

Pseudomonas resinovorans, a known medium-chain-length (mcl-) poly(hydroxyalkanoate) (PHA) producer, was grown on 13-methyltetradecanoic acid (13-MTDA) and a mixture of isostearic acid (IA) isomers to produce methyl-branched mcl-PHA polymers. Shake-flask experiments revealed polymer productivities (the percent of the cell mass that is polymer) of 31 ± 1% (n = 3) and 23 ± 3% (n = 3) when grown in 13-MTDA and IA, respectively. Monomer content was determined by a combination of gas chromatography/mass spectrometry (GC/MS) of the acid hydrolyzed, silylated methyl esters, and nuclear magnetic resonance spectroscopy. Results showed that the mcl-PHA polymer derived from 13-MTDA was primarily composed of 3-hydroxy-7-methyloctanoic acid and 3-hydroxy-9-methyldecanoic acid (67 and 16 mol% by GC/MS, respectively). In contrast, the mcl-polymers synthesized from the IA isomeric mixture were more complex, containing both even and odd chain-length monomers as well as varying distributions of methyl-branched derivatives. The PHA distributions among the C8, C10, C12, and C14 carbon chain-length monomers included three isomers of C8, five isomers of C10, seven isomers of C12, and nine isomers of C14 each containing one linear-chain derivative and n−6 methyl-branched derivatives where n equals the total number of carbon atoms in each monomer unit (C8–C14).

Keywords: Biopolymer; Poly(hydroxyalkanoate); Pseudomonas resinovorans ; Isostearic acid; 13-Methyltetradecanoic acid

Isolation and characterization of endophytic plant growth-promoting (PGPB) or stress homeostasis-regulating (PSHB) bacteria associated to the halophyte Prosopis strombulifera by Verónica Sgroy; Fabricio Cassán; Oscar Masciarelli; María Florencia Del Papa; Antonio Lagares; Virginia Luna (pp. 371-381).

This study was designed to isolate and characterize endophytic bacteria from halophyte Prosopis strombulifera grown under extreme salinity and to evaluate in vitro the bacterial mechanisms related to plant growth promotion or stress homeostasis regulation. Isolates obtained from P. strombulifera were compared genotypically by BOX-polymerase chain reaction, grouped according to similarity, and identified by amplification and partial sequences of 16S DNAr. Isolates were grown until exponential growth phase to evaluate the atmospheric nitrogen fixation, phosphate solubilization, siderophores, and phytohormones, such as indole-3-acetic acid, zeatin, gibberellic acid and abscisic acid production, as well as antifungal, protease, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. A total of 29 endophytic strains were grouped into seven according to similarity. All bacteria were able to grow and to produce some phytohormone in chemically defined medium with or without addition of a nitrogen source. Only one was able to produce siderophores, and none of them solubilized phosphate. ACC deaminase activity was positive for six strains. Antifungal and protease activity were confirmed for two of them. In this work, we discuss the possible implications of these bacterial mechanisms on the plant growth promotion or homeostasis regulation in natural conditions.

Keywords: Prosopis strombulifera ; Halophyte; Salinity; Phytohormones; Auxins; Cytokinins; Gibberellins; Abscisic acid; ACC deaminase; Phosphate solubilization; Siderophore production; Nitrogen fixation; Bacillus ; Lysinibacillus ; Pseudomonas ; Achromobacter ; Brevibacterium

Biodiversity in aerobic granule membrane bioreactor at high organic loading rates by Yu-Chuan Juang; Sunil S. Adav; Duu-Jong Lee; Juin-Yih Lai (pp. 383-388).

The aerobic granular sludge process and the membrane bioreactor process are promising, novel environmental bioprocesses for the reclamation of industrial and municipal wastewaters. They are practical and have attracted much research interest. The combination of these two processes in the aerobic granular membrane bioreactor (AGMBR), yields reclaimed water of high quality in a compact reactor. Information on the microbial community in an AGMBR operated at a high organic loading rate is lacking. This study elucidates the microbial dynamics in acetate-fed AGMBR operated in sequencing batch reactor mode. The structure of the microbial community revealed by denaturing gradient gel electrophoresis, demonstrates the dominance of the denitrifying microbial community, which is affiliated with members of the families Comamonadaceae and Alcaligenaceae, of the class Betaproteobacteria. The role of the predominant strains in the AGMBR is discussed.

Keywords: Aerobic granule; Membrane bioreactor; Microbial structure; Organic loading

Effect of nutrient and selective inhibitor amendments on methane oxidation, nitrous oxide production, and key gene presence and expression in landfill cover soils: characterization of the role of methanotrophs, nitrifiers, and denitrifiers by Sung-Woo Lee; Jeongdae Im; Alan A. DiSpirito; Levente Bodrossy; Michael J. Barcelona; Jeremy D. Semrau (pp. 389-403).

Methane and nitrous oxide are both potent greenhouse gasses, with global warming potentials approximately 25 and 298 times that of carbon dioxide. A matrix of soil microcosms was constructed with landfill cover soils collected from the King Highway Landfill in Kalamazoo, Michigan and exposed to geochemical parameters known to affect methane consumption by methanotrophs while also examining their impact on biogenic nitrous oxide production. It was found that relatively dry soils (5% moisture content) along with 15 mg NH ₄ ⁺ (kg soil)⁻¹ and 0.1 mg phenylacetylene∙(kg soil)⁻¹ provided the greatest stimulation of methane oxidation while minimizing nitrous oxide production. Microarray analyses of pmoA showed that the methanotrophic community structure was dominated by Type II organisms, but Type I genera were more evident with the addition of ammonia. When phenylacetylene was added in conjunction with ammonia, the methanotrophic community structure was more similar to that observed in the presence of no amendments. PCR analyses showed the presence of amoA from both ammonia-oxidizing bacteria and archaea, and that the presence of key genes associated with these cells was reduced with the addition of phenylacetylene. Messenger RNA analyses found transcripts of pmoA, but not of mmoX, nirK, norB, or amoA from either ammonia-oxidizing bacteria or archaea. Pure culture analyses showed that methanotrophs could produce significant amounts of nitrous oxide, particularly when expressing the particulate methane monooxygenase (pMMO). Collectively, these data suggest that methanotrophs expressing pMMO played a role in nitrous oxide production in these microcosms.

Keywords: Methanotroph; Landfill; Nitrous oxide; Methane; Ammonia oxidizers

Typing of toxinogenic Microcystis from environmental samples by multiplex PCR by Youness Ouahid; Francisca Fernández del Campo (pp. 405-412).

Microcystin (MC)-producing Microcystis strains from environmental samples were assessed by the simultaneous amplification of up to five DNA sequences, corresponding to specific regions of six mcy genes (mcyA, mcyB, mcyC, mcyD, mcyE and mcyG), codifying for key motifs of the non-ribosomal peptide synthetase and polyketide synthase of the microcystin synthetase complex. Six primer pairs with the same melting temperature, one of them of new design, were used. A crucial point for the good performance of the new multiplex PCR test was the concentration of each primer pair. In the test, cell suspensions from laboratory cultures, field colonies and blooms were directly used as DNA source. The results of the multiplex PCR were consistent with the toxinogenic character of the samples, as checked by high performance liquid chromatography and/or matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. As a whole, the newly developed test could be used for a reliable, rapid and low-cost screening of potential MC-producing Microcystis in field samples, even scattered colonies.

Keywords: mcy genes; NRPS; PKS; Microcystis ; Multiplex PCR; Bloom biomass

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Applied Microbiology and Biotechnology (v.85, #2)