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Protein microarrays: a chance to study microorganisms?

Within the last 5 years, protein microarrays have been developed and applied to multiple approaches: identification of protein-protein interactions or protein-small molecule interactions, cancer profiling, detection of microorganisms and toxins, and identification of antibodies due to allergens, autoantigens, and pathogens. Protein microarrays are small size (typically in the microscopy slide format) planar analytical devices with probes arranged in high density to provide the ability to screen several hundred to thousand known substrates (e.g., proteins, peptides, antibodies) simultaneously. Due to their small size, only minute amounts of spotted probes and analytes (e.g., serum) are needed; this is a particularly important feature, for these are limited or expensive. In this review, different types of protein microarrays are reviewed: protein microarrays (PMAs), with spotted proteins or peptides; antibody microarrays (AMAs), with spotted antibodies or antibody fragments (e.g., scFv); reverse phase protein microarrays (RPMAs), a special form of PMA where crude protein mixtures (e.g., cell lysates, fractions) are spotted; and nonprotein microarrays (NPMAs) where macromolecules other than proteins and nucleic acids (e.g., carbohydrates, monosaccharides, lipopolysaccharides) are spotted. In this study, exemplary experiments for all types of protein arrays are discussed wherever applicable with regard to investigations of microorganisms.

Animals↗

Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples.

Due to the advanced development of fluorogenic chemistry, quantitative real-time polymerase chain reaction (qRT-PCR) has become an emerging technique for the detection and quantification of microorganisms in the environment. Compared with the conventional hybridization- and PCR-based techniques, qRT-PCR not only has better sensitivity and reproducibility, but it is also quicker to perform and has a minimum risk of amplicon carryover contamination. This article reviews the principle of this emerging technique, its detection reagents, target DNAs, quantification procedures, and affecting factors. The applications of qRT-PCR for the quantification of microorganisms in the environment are also summarized.

Archaea↗

Remaining acetamide in acetonitrile degradation using nitrile hydratase- and amidase-producing microorganisms.

The tandem conversion process involving nitrile hydratase- and amidase-producing microorganisms has potential for use in the treatment of acetonitrile-containing wastes. In that process, the acetamide hydrolysis step catalyzed by amidase is very slow compared with the acetonitrile hydration step catalyzed by nitrile hydratase, and a small amount of acetamide remains in the resulting solution. This study aimed to improve the efficiency of the acetamide hydrolysis step. An amidase-producing microorganism, Rhodococcus sp. S13-4, was newly obtained, whose use enabled rapid acetamide degradation. Though residual acetamide was still detected, it was successfully reduced by the addition of cation/anion mixed ion exchange resin or calcium hydroxide after the acetamide hydrolysis reaction using Rhodococcus sp. S13-4 cells. This result implies that acetamide hydrolysis and acetamide formation are in equilibrium. The incubation of Rhodococcus sp. S13-4 cells with high concentrations of ammonium acetate produced acetamide. The purified amidase from Rhodococcus sp. S13-4 revealed the acetamide formation activity (specific activity of 30.6 U/mg protein). This suggests that the amidase-catalyzed amide formation may cause the remaining of acetamide in the acetonitrile conversion process.

Acetamides↗

Plate screening methods for the detection of polysaccharase-producing microorganisms.

Polysaccharide-degrading enzymes (polysaccharases) are widely applied in industry. One of the sources of these enzymes are polysaccharide-degrading microorganisms. To obtain such microorganisms from enrichment cultures, strain collections or gene libraries, efficient plate screening methods are required that discriminate between intact and degraded polysaccharide. This can be achieved by making use of specific physicochemical properties of the polysaccharide, such as complex formation with dyes and gelling capacity, or by the application of dye-labelled polysaccharides. This review presents a survey of plate methods based on these principles. Both theoretical and practical aspects of the methods are discussed.

Bacteria↗

Killing of microorganisms by pulsed electric fields.

Lethal effects of pulsed electric fields (PEF) on suspensions of various bacteria, yeast, and spores in buffer solutions and liquid foodstuffs were examined. Living-cell counts of vegetative cell types were reduced by PEF treatment by up to more than four orders of magnitude (> 99.99%). On the other hand, endo- and ascospores were not inactivated or killed to any great extent. The killing of vegetative cell types depends on the electrical field strength of the pulses and on the treatment time (the product of the pulse number and the decay time constant of the pulses). For each cell type, a specific critical electric field strength (Ec) and a specific critical treatment time (tc) were determined. Above these critical values, the fractions of surviving cells were reduced drastically. The "limits" Ec and tc depend on the cell characteristics as well as on the type of medium in which the cells are suspended. Especially in acid media living-cell counts were sufficiently decreased at very low energy inputs. In addition to the inactivation of microorganisms, the effect of PEF on food components such as whey proteins, enzymes and vitamins, and on the taste of foodstuffs was studied. The degree of destruction of these food components by PEF was very low or negligible. Moreover, no significant deterioration of the taste of foodstuffs was detected after PEF treatment. Disintegration of cells by PEF treatment in order to harvest intracellular products was also studied. Yeast cells, suspended in buffer solution, were not disintegrated by electric pulses. Hence, PEF treatment is an excellent process for inactivation of microorganisms in acid and in thermosensive media, but not for complete disintegration of microbial cells.

Bacillus cereus↗

Safe biotechnology. 7. Classification of microorganisms on the basis of hazard. Working Party "Safety in Biotechnology" of the European Federation Biotechnology.

The current systems for classifying human pathogens on the basis of hazard are well developed and their basic criteria are in general agreement one with another. Of more importance, the safety practices based on these classifications have generally been successful. They have enabled extensive research activities, medical practice and industrial production to be conducted on an ever-increasing scale, involving dangerous microorganisms (e.g. in vaccine production and treatment of infected patients) with a very low incidence of adverse effects on the workers involved and the general public. Although the EU has adopted a harmonised list of agents in groups 1-4 there is as yet no complete agreement among member states and individual microbiologists. The purpose of this paper is to present a historical survey and to discuss the current processes for identifying and classifying the hazards posed by the use of microorganisms in research and technology. This is essential in the design of appropriate methods of counteracting potential risks.

Bacteria↗

Utilization of sorbed compounds by microorganisms specifically isolated for that purpose.

A bacterium obtained by enrichment on nonsorbed phenanthrene was unable to degrade phenanthrene sorbed to polyacrylic beads and had little activity on phenanthrene sorbed to lake-bottom sediment. A bacterium obtained by enrichment on phenanthrene sorbed to polyacrylic beads readily mineralized the compound sorbed to the beads or the sediment. Degradation by the second bacterium of phenanthrene sorbed to beads 38-63 microns or 63-150 microns in diameter was more rapid than the rate of desorption of the hydrocarbon in the absence of the bacterium. Little degradation of sorbed, nonleachable phenanthrene in soil was effected by another isolate obtained by enrichment with the nonsorbed hydrocarbon, but a mixed culture and the bacterium obtained by enrichment on the sorbed compound extensively degraded phenanthrene. Because microorganisms specifically obtained for their capacity to degrade sorbed phenanthrene are more active than species not specialized for use of the bound compound, we suggest that microorganisms enriched on nonsorbed compounds may not be appropriate for evaluation of biodegradation and bioremediation of sorbed compounds.

Bacteria↗

Processes of liquefaction/solubilization of Spanish coals by microorganisms

Several fundamental aspects of microbial coal liquefaction/solubilization were studied. The liquefied/solubilized products from coal by microorganisms were analysed. The liquid products analysed by IR titration and UV/visible spectrometry showed some alterations with regard to the original coal. Humic acids extracted from the liquefied lignite showed a reduction in the average molecular weight and a increase in the condensation index, probably due to depolymerization caused by microorganisms. The mechanisms implicated in coal biosolubilization by two fungal strains, M2 (Trichoderma sp.) and M4 (Penicillium sp.) were also studied. Extracellular peroxidase, esterase and phenoloxidase enzymes appear to be involved in coal solubilization.

Journal Article↗

Regulatory Oversight of Genetically Engineered Microorganisms: Has Regulation Inhibited Innovation?

/ Using detailed interviews with company representatives and researchers in the field, this paper examines the factors that might account for the slow pace of development of genetically engineered microorganisms (GEMs) intended for environmental release. We specifically analyzed the role of the regulatory system in shaping innovation. We identified at least two cases where industry decided to discontinue the development of a genetically engineered microbial product because of concerns over regulatory oversight. However, most often industry decisions to continue or halt development of GEMs were based on an evaluation of the particular product's efficacy and potential for profitability. Thus the inability of GEMs to perform up to expectations in the field, rather than the regulatory constraints, appears to be the factor responsible for the slow pace of development. KEY WORDS: Genetically engineered microorganisms; Biotechnology; Regulation of biotechnology; Innovation; Environmental release

Journal Article↗

Effects of DL-malate on in vitro forage fiber digestion by mixed ruminal microorganisms.

The objective of this study was to evaluate the effects of 0, 4, 8, and 12 mM DL-malate on the in vitro mixed ruminal microorganism fermentation of alfalfa hay and Coastal bermudagrass hay. When alfalfa hay was the substrate, 4 and 8 mM DL-malate numerically increased propionate concentration, and 12 mM DL-malate increased (P < 0.10) propionate. All three concentrations of DL-malate decreased (P < 0.05) the acetate:propionate ratio. In Coastal bermudagrass hay fermentations, all three DL-malate concentrations increased (P < 0.05) propionate and decreased (P < 0.05) the acetate:propionate ratio, while 4 and 12 mM DL-malate numerically increased in vitro dry matter disappearance. When mixed ruminal microorganisms were incubated with 6.25 mM DL-lactic acid and alfalfa hay, 8 and 12 mM DL-malate increased (P < 0.05) final pH, and 12 mM DL-malate increased (P < 0.10) propionate and decreased (P < 0.10) the acetate:propionate ratio. DL-Malate treatment had little effect on in vitro dry matter disappearance. Addition of 8 and 12 mM DL-malate to Coastal bermudagrass hay plus DL-lactic acid fermentations increased (P < 0.05) final pH, and 8 mM DL-malate increased (P < 0.10) in vitro dry matter disappearance. Even though DL-malate treatment consistently increased final pH values in fermentations that included DL-lactic acid, there was not a corresponding increase in in vitro dry matter disappearance of either alfalfa hay or Coastal bermudagrass hay in the 48-h batch culture incubations.

Animals↗

Screening of compactin-resistant microorganisms capable of converting compactin to pravastatin.

A simple method of using compactin for effective screening of microbial strains with high hydroxylation activity at the 6beta position of compactin was developed. Agar plates containing different carbon sources and 500 microg compactin mL(-1) were used to screen the microorganisms that can convert compactin to pravastatin. About 100 compactin-resistant strains were isolated from the Basal agar containing 7% (w/v) mannitol as a carbon source, in which two bacteria, Pseudomocardia autotrophica BCRC 12444 and Streptomyces griseolus BCRC 13677, capable of converting compactin to pravastatin with the yield of 20 and 32% (w/w), respectively, were found. High-performance liquid chromatography using C-18 column and two sequential mobile phases, 30% and 50% (v/v) acetonitrile, was also established to simultaneously determine the concentration of compactin and pravastatin in the culture broth. As such, about 2% of target microorganisms could be obtained from the screening program.

Bacteria↗

Antimicrobial activity of propolis on oral microorganisms.

Formation of dental caries is caused by the colonization and accumulation of oral microorganisms and extracellular polysaccharides that are synthesized from sucrose by glucosyltransferase of Streptococcus mutans. The production of glucosyltransferase from oral microorganisms was attempted, and it was found that Streptococcus mutans produced highest activity of the enzyme. Ethanolic extracts of propolis (EEP) were examined whether EEP inhibit the enzyme activity and growth of the bacteria or not. All EEP from various regions in Brazil inhibited both glucosyltransferase activity and growth of S. mutans, but one of the propolis from Rio Grande do Sul (RS2) demonstrated the highest inhibition of the enzyme activity and growth of the bacteria. It was also found that propolis (RS2) contained the highest concentrations of pinocembrin and galangin.

Actinomyces↗

Effects of chlorhexidine diacetate on ruminal microorganisms.

The objectives of this study were to examine the effects of chlorhexidine diacetate on growth and L-lactate production by Streptococcus bovis JB1 as well as the effects of this antimicrobial compound on the mixed ruminal microorganism fermentation. Addition of 1.8 microM chlorhexidine diacetate to glucose medium resulted in a lag in growth by S. bovis JB1, and growth was completely inhibited in the presence of 3.6, 9.0, and 18 microM chlorhexidine. When 6.2 microM chlorhexidine diacetate was added to glucose medium after 2 h of incubation, glucose utilization and L-lactate production by S. bovis JB1 were reduced. Phosphoenolpyruvate-dependent phosphorylation of 14C-glucose by toluene-treated cells of S. bovis JB1 was inhibited by increasing concentrations (1.8 to 18 microM) of chlorhexidine, whereas only the 18 microM concentration reduced the membrane potential (delta psi). Chlorhexidine diacetate was a potent inhibitor of L-lactate and methane production from glucose fermentation by mixed ruminal microorganisms. However, because chlorhexidine also decreased acetate and propionate concentrations and increased ammonia concentrations in mixed-culture incubations, this antimicrobial compound may have limited application as a ruminant feed additive.

Animals↗

Potential of halotolerant and halophilic microorganisms for biotechnology.

Halotolerant or halophilic microorganisms, able to live in saline environments, offer a multitude of actual or potential applications in various fields of biotechnology. The technical applications of bacteriorhodopsin comprise holography, spatial light modulators, optical computing, and optical memories. Compatible solutes are useful as stabilizers of biomolecules and whole cells, salt antagonists, or stress-protective agents. Biopolymers, such as biosurfactants and exopolysaccharides, are of interest for microbially enhanced oil recovery. Other useful biosubstances are enzymes, such as new isomerases and hydrolases, that are active and stable at high salt contents. Halotolerant microorganisms play an essential role in food biotechnology for the production of fermented food and food supplements. The degradation or transformation of a range of organic pollutants and the production of alternative energy are other fields of applications of these groups of extremophiles.

Adaptation, Physiological↗

Screening and characterization of fructosyl-valine-utilizing marine microorganisms.

We describe the isolation of microorganisms utilizing fructosyl-amine (Amadori compound) from the marine environment and of fructosyl-amine oxidase from a marine yeast. Using fructosyl-valine (Fru-Val), a model Amadori compound for glycated hemoglobin, we isolated 12 microbial strains that grow aerobically in a minimal medium supplemented with Fru-Val as the sole nitrogen source. Among these strains, a yeast strain identified as Pichia sp. N1-1, produced a Fru-Val-oxidizing enzyme. The enzyme was purified in its active form, a single-polypeptide water-soluble protein of 54 kDa by gel electrophoresis, producing H(2)O(2) with the oxidation of Fru-Val. By its substrate specificity, the enzyme was categorized as a novel fructosyl-amine oxidase. This is the first study on the isolation of microorganisms utilizing fructosyl-amine in the marine environment and of fructosyl-amine oxidase from budding yeast.

Journal Article↗

Cometabolic reduction of bromate by a mixed culture of microorganisms using hydrogen gas in a gas-lift reactor.

The discharge of bromate, a suspected carcinogen, will be restricted in the near future. To assess the possibility of biotechnological treatment of bromate-containing wastewaters, the removal of bromate by chlorate-reducing microorganisms was studied. The removal of bromate and chlorate was studied in laboratory gas-lift bioreactors supplied with hydrogen gas as electron donor in the absence of molecular oxygen. In these reactors, bromate was reduced cometabolically by chlorate-respiring microorganisms. To allow the cometabolic reduction of bromate, a chlorate:bromate molar ratio of at least 3:1 was required. The cometabolic conversion permitted almost complete reduction of bromate into bromide at hydraulic retention times of at least 6 h. Optimal bromate reduction activity was observed at approximately 35 degrees C. The pH optimum was between 7 and 8. Bromate reduction in excess of 80% and a maximum bromate reduction rate of 2.3 g l(-1) day(-1) in a pilot-scale gas-lift bioreactor demonstrates that the process is sustainable.

Air Pollutants↗

Genomic insights into the iron uptake mechanisms of the biomining microorganism Acidithiobacillus ferrooxidans.

Commercial bioleaching of copper and the biooxidation of gold is a cost-effective and environmentally friendly process for metal recovery. A partial genome sequence of the acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans is available from two public sources. This information has been used to build preliminary models that describe how this microorganism confronts unusually high iron loads in the extremely acidic conditions (pH 2) found in natural environments and in bioleaching operations. A. ferrooxidans contains candidate genes for iron uptake, sensing, storage, and regulation of iron homeostasis. Predicted proteins exhibit significant amino acid similarity with known proteins from neutrophilic organisms, including conservation of functional motifs, permitting their identification by bioinformatics tools and allowing the recognition of common themes in iron transport across distantly related species. However, significant differences in amino acid sequence were detected in pertinent domains that suggest ways in which the periplasmic and outer membrane proteins of A. ferrooxidans maintain structural integrity and relevant protein-protein contacts at low pH. Unexpectedly, the microorganism also contains candidate genes, organized in operon-like structures that potentially encode at least 11 siderophore systems for the uptake of Fe(III), although it does not exhibit genes that could encode the biosynthesis of the siderophores themselves. The presence of multiple Fe(III) uptake systems suggests that A. ferrooxidans can inhabit aerobic environments where iron is scarce and where siderophore producers are present. It may also help to explain why it cannot tolerate high Fe(III) concentrations in bioleaching operations where it is out-competed by Leptospirillum species.

Acidithiobacillus↗

Isolation and characterization of a novel thraustochytrid-like microorganism that efficiently produces docosahexaenoic acid.

A thraustochytrid-like microorganism (strain 12B) was isolated from the mangrove area of Okinawa, Japan. On the basis of its ectoplasmic net structure and biflagellate zoospores we determined strain 12B to be a novel member of the phylum Labyrinthulomycota in the kingdom Protoctista. When grown on glucose/seawater at 28 degrees C, it had a lipid content of 58% with docosahexaenoic acid (DHA; 22:6 n-3) at 43% of the total fatty acids. It had a growth rate of 0.38 h(-1). The DHA production rate of 2.8 +/- 0.7 g l(-1) day(-1) is the highest value reported for any microorganism.

Docosahexaenoic Acids↗