Microarrays consist of an orderly arrangement of probes (oligonucleotides, DNA fragments, protein... more Microarrays consist of an orderly arrangement of probes (oligonucleotides, DNA fragments, proteins, sugars or lectins) attached to a solid surface. The main advantages of microarray technology are high throughput, parallelism, miniaturization, speed and automation. Despite the fact that microarray analysis is a relatively novel technology, with the publication of the first microarray studies in 1995 , it is now broadly applied and the milestone of nearly 5000 published microarray papers was recorded in 2004 . The scientific and technological background discussed here will be limited to DNA microarrays, excluding the new evolving fields of protein microarrays and glycomics .
Methanotroph microorganisms oxidize methane in four steps, producing methanol, formaldehyde, form... more Methanotroph microorganisms oxidize methane in four steps, producing methanol, formaldehyde, formate intermediers and eventually degrade methane to carbon dioxide and water. It is possible to separate the pathway into four steps in the cell free extract or after partial purification of the various enzymes. The key enzyme is a metalloenzyme, methane monooxygenase (MMO) which catalyses the oxidation of methane to methanol. MMO is also capable of biodegrading exceptionally harmful and stable chlorinated hydrocarbons. Produced by various industrial activities, most chlorinated hydrocarbons are toxic, potential and/or proven carcinogens and their decomposition challenges water treatment technologies.
a combination of biochemical and serological typing. However, primary strain isolation and tradit... more a combination of biochemical and serological typing. However, primary strain isolation and traditional serotyping is time-consuming and faster methods would be desirable. A microarray, based on two housekeeping and two virulence marker genes (atpD, gyrB, fliC and fljB), has been developed for the detection and identification of the two species of Salmonella (S. enterica and S. bongori), the five subspecies of S. enterica (II, IIIa, IIIb, IV, VI) and 43 S. enterica ssp. enterica serovars (covering the most prevalent ones in Austria and the UK). A comprehensive set of probes (n = 240), forming 119 probe units, was developed based on the corresponding sequences of 148 Salmonella strains, successfully validated with 57 Salmonella strains and subsequently evaluated with 35 blind samples including isolated serotypes and mixtures of different serotypes. Results demonstrated a strong discriminatory ability of the microarray among Salmonella serovars. Threshold for detection was 1 colony forming unit per 25 g of food sample following overnight (14 h) enrichment.
A microbial diagnostic microarray for the detection of the most relevant bacterial food-and water... more A microbial diagnostic microarray for the detection of the most relevant bacterial food-and water-borne pathogens and indicator organisms was developed and thoroughly validated. The microarray platform based on sequence-specific end labelling of oligonucleotides and the pyhylogenetically robust gyrB marker gene allowed a highly specific (resolution on genus/species level) and sensitive (0.1% relative and 10 4 cfu absolute detection sensitivity) detection of the target pathogens. Validation was performed using a set of reference strains and a set of spiked environmental samples. Reliability of the obtained data was additionally verified by independent analysis of the samples via fluorescence in situ hybridization (FISH) and conventional microbiological reference methods. The applicability of this diagnostic system for food analysis was demonstrated through extensive validation using artificially and naturally contaminated spiked food samples. The microarray-based pathogen detection was compared with the corresponding microbiological reference methods (performed according to the ISO norm). Microarray results revealed high consistency with the reference microbiological data.
Salmonella enterica subsp. enterica is one of the main causative agents of food-borne disease in ... more Salmonella enterica subsp. enterica is one of the main causative agents of food-borne disease in man, and can also be the cause of serious systemic illness. Organisms belonging to this genus have traditionally been classified on the basis of the antigenic properties of the cell-surface lipopolysaccharide and of the phase 1 and phase 2 flagellar proteins. Primary isolation, biochemical identification, and serotyping are laborious and time consuming. Molecular identification based on suitable marker genes could be an attractive alternative to conventional bacteriological and serological methods. We have assessed the applicability of two housekeeping genes, gyrB, atpD, in combination with the flagellin genes fliC and fljB in multilocus sequence typing of Salmonella. Sequencing and comparative analysis of sequence data was performed on multiple strains from Austria, the United Kingdom, and Switzerland, representing all subspecies and 22 of the more prevalent non-typhoid S. enterica subsp. enterica serovars. A combination of these four marker genes allowed for a clear differentiation of all the strains analysed, indicating their applicability in molecular typing. The term MLST-v, for multilocus sequence typing based on virulence genes, is proposed to distinguish this approach from MLST based solely on housekeeping genes. An assortative recombination of the fliC gene was found in seven of the analysed serovars indicating multiple phylogenetic origin of these serovars.
The first gene cluster encoding for a membrane bound [NiFe] hydrogenase from a methanotroph, Meth... more The first gene cluster encoding for a membrane bound [NiFe] hydrogenase from a methanotroph, Methylococcus capsulatus (Bath), was cloned and sequenced. The cluster consisted of the structural genes hupS and hupL and accessory genes hupE, hupC and hupD. A vhupSL deletion mutant of Mc. capsulatus was constructed by marker exchange mutagenesis. Membrane associated hydrogenase activity disappeared. The membrane associated hydrogenase appeared to have a hydrogen uptake function in vivo. ß
Oligonucleotide microarrays offer a fast, high-throughput alternative for the parallel detection ... more Oligonucleotide microarrays offer a fast, high-throughput alternative for the parallel detection of microbes from virtually any sample. The application potential spreads across most sectors of life sciences, including environmental microbiology and microbial ecology; human, veterinary, food and plant diagnostics; water quality control; industrial microbiology, and so on. The past two years have witnessed a rapid increase of research in this field. Many alternative techniques were developed and validated as seen in 'proof-of-concept' articles. Publications reporting on the application of oligonucleotide microarray technology for microbial diagnostics in microbiology driven projects have just started to appear. Current and future technical and bioinformatics developments will inevitably improve the potential of this technology further.
Oligonucleotide microarrays are highly parallel hybridization platforms, allowing rapid and simul... more Oligonucleotide microarrays are highly parallel hybridization platforms, allowing rapid and simultaneous identification of many different microorganisms and viruses in a single assay. In the past few years, researchers have been confronted with a dramatic increase in the number of studies reporting development and/or improvement of oligonucleotide microarrays for microbial diagnostics, but use of the technology in routine diagnostics is still constrained by a variety of factors. Careful development of microarray essentials (such as oligonucleotide probes, protocols for target preparation and hybridization, etc.) combined with extensive performance testing are thus mandatory requirements for the maturation of diagnostic microarrays from fancy technological gimmicks to robust and routinely applicable tools. D
Landfills are large sources of CH 4 , but a considerable amount of CH 4 can be removed in situ by... more Landfills are large sources of CH 4 , but a considerable amount of CH 4 can be removed in situ by methanotrophs if their activity can be stimulated through the addition of nitrogen. Nitrogen can, however, lead to increased N 2 O production. To examine the effects of nitrogen and a selective inhibitor on CH 4 oxidation and N 2 O production in situ, 0.5 M of NH 4 Cl and 0.25 M of KNO 3 , with and without 0.01% (w/v) phenylacetylene, were applied to test plots at a landfill in Kalamazoo, MI from 2007 November to 2009 July. Nitrogen amendments stimulated N 2 O production but had no effect on CH 4 oxidation. The addition of phenylacetylene stimulated CH 4 oxidation while reducing N 2 O production. Methanotrophs possessing particulate methane monooxygenase and archaeal ammonia-oxidizers (AOAs) were abundant. The addition of nitrogen reduced methanotrophic diversity, particularly for type I methanotrophs. The simultaneous addition of phenylacetylene increased methanotrophic diversity and the presence of type I methanotrophs. Clone libraries of the archaeal amoA gene showed that the addition of nitrogen increased AOAs affiliated with Crenarchaeal group 1.1b, while they decreased with the simultaneous addition of phenylacetylene. These results suggest that the addition of phenylacetylene with nitrogen reduces N 2 O production by selectively inhibiting AOAs and/or type II methanotrophs.
With the advent of molecular biological techniques, especially next-generation sequencing and met... more With the advent of molecular biological techniques, especially next-generation sequencing and metagenomics, the number of microbial biogeography studies is rapidly increasing. However, these studies involve the synthesis of data generated by different laboratories using different protocols, chemicals, etc., all with inherent biases. The aim of this study was to assess inter-as well as intralaboratory variations in microbial community composition when standardized protocols are applied to a single soil sample. Aliquots from a homogenized soil sample from a rice field in Italy were sent to five participating laboratories. DNA was extracted by two investigators per laboratory using an identical protocol. All DNA samples were sent to one laboratory to perform DNA quantification, quantitative PCR (QPCR), and microarray and denaturing gradient gel electrophoresis (DGGE) analyses of methanotrophic communities. Yields, as well as purity of DNA, were significantly different between laboratories but in some cases also between investigators within the same laboratory. The differences in yield and quality of the extracted DNA were reflected in QPCR, microarray, and DGGE analysis results. Diversity indices (Shannon-Wiener, evenness, and richness) differed significantly between laboratories. The observed differences have implications for every project in which microbial communities are compared in different habitats, even if assessed within the same laboratory. To be able to make sensible comparisons leading to valid conclusions, intralaboratory variation should be assessed. Standardization of DNA extraction protocols and possible use of internal standards in interlaboratory comparisons may help in rendering a "quantifiable" bias.
Methane oxidation can occur in both aerobic and anaerobic environments; however, these are comple... more Methane oxidation can occur in both aerobic and anaerobic environments; however, these are completely different processes involving different groups of prokaryotes. Aerobic methane oxidation is carried out by aerobic methanotrophs, and anaerobic methane oxidizers, discovered recently, thrive under anaerobic conditions and use sulfate or nitrate as electron donors for methane oxidation . This review will focus on the aerobic oxidation of methane.
A method was developed for the mRNA-based application of microbial diagnostic microarrays to dete... more A method was developed for the mRNA-based application of microbial diagnostic microarrays to detect active microbial populations. DNA-and mRNA-based analyses of environmental samples were compared and confirmed via quantitative PCR. Results indicated that mRNA-based microarray analyses may provide additional information on the composition and functioning of microbial communities.
ARChip Epoxy and ARChip UV are presented as novel chip platforms for oligonucleotide immobilizati... more ARChip Epoxy and ARChip UV are presented as novel chip platforms for oligonucleotide immobilization. ARChip Epoxy is made of reactive epoxy resin available commercially. ARChip UV consists of photoactivatable poly(styrene-co-4-vinylbenzylthiocyanate). Both ARChip surfaces are tested in a model assay based on oligonucleotide probes from a real-life genotyping project and are evaluated in comparison with Wve commercial chip surfaces based on nitrocellulose, epoxy, and aldehyde polymer, and two diVerent aminosilanes. Optimum print buVer, spotter compatibility, and data normalization are discussed. 2004 Elsevier Inc. All rights reserved.
Microarrays consist of an orderly arrangement of probes (oligonucleotides, DNA fragments, protein... more Microarrays consist of an orderly arrangement of probes (oligonucleotides, DNA fragments, proteins, sugars or lectins) attached to a solid surface. The main advantages of microarray technology are high throughput, parallelism, miniaturization, speed and automation. Despite the fact that microarray analysis is a relatively novel technology, with the publication of the first microarray studies in 1995 , it is now broadly applied and the milestone of nearly 5000 published microarray papers was recorded in 2004 . The scientific and technological background discussed here will be limited to DNA microarrays, excluding the new evolving fields of protein microarrays and glycomics .
Methanotroph microorganisms oxidize methane in four steps, producing methanol, formaldehyde, form... more Methanotroph microorganisms oxidize methane in four steps, producing methanol, formaldehyde, formate intermediers and eventually degrade methane to carbon dioxide and water. It is possible to separate the pathway into four steps in the cell free extract or after partial purification of the various enzymes. The key enzyme is a metalloenzyme, methane monooxygenase (MMO) which catalyses the oxidation of methane to methanol. MMO is also capable of biodegrading exceptionally harmful and stable chlorinated hydrocarbons. Produced by various industrial activities, most chlorinated hydrocarbons are toxic, potential and/or proven carcinogens and their decomposition challenges water treatment technologies.
a combination of biochemical and serological typing. However, primary strain isolation and tradit... more a combination of biochemical and serological typing. However, primary strain isolation and traditional serotyping is time-consuming and faster methods would be desirable. A microarray, based on two housekeeping and two virulence marker genes (atpD, gyrB, fliC and fljB), has been developed for the detection and identification of the two species of Salmonella (S. enterica and S. bongori), the five subspecies of S. enterica (II, IIIa, IIIb, IV, VI) and 43 S. enterica ssp. enterica serovars (covering the most prevalent ones in Austria and the UK). A comprehensive set of probes (n = 240), forming 119 probe units, was developed based on the corresponding sequences of 148 Salmonella strains, successfully validated with 57 Salmonella strains and subsequently evaluated with 35 blind samples including isolated serotypes and mixtures of different serotypes. Results demonstrated a strong discriminatory ability of the microarray among Salmonella serovars. Threshold for detection was 1 colony forming unit per 25 g of food sample following overnight (14 h) enrichment.
A microbial diagnostic microarray for the detection of the most relevant bacterial food-and water... more A microbial diagnostic microarray for the detection of the most relevant bacterial food-and water-borne pathogens and indicator organisms was developed and thoroughly validated. The microarray platform based on sequence-specific end labelling of oligonucleotides and the pyhylogenetically robust gyrB marker gene allowed a highly specific (resolution on genus/species level) and sensitive (0.1% relative and 10 4 cfu absolute detection sensitivity) detection of the target pathogens. Validation was performed using a set of reference strains and a set of spiked environmental samples. Reliability of the obtained data was additionally verified by independent analysis of the samples via fluorescence in situ hybridization (FISH) and conventional microbiological reference methods. The applicability of this diagnostic system for food analysis was demonstrated through extensive validation using artificially and naturally contaminated spiked food samples. The microarray-based pathogen detection was compared with the corresponding microbiological reference methods (performed according to the ISO norm). Microarray results revealed high consistency with the reference microbiological data.
Salmonella enterica subsp. enterica is one of the main causative agents of food-borne disease in ... more Salmonella enterica subsp. enterica is one of the main causative agents of food-borne disease in man, and can also be the cause of serious systemic illness. Organisms belonging to this genus have traditionally been classified on the basis of the antigenic properties of the cell-surface lipopolysaccharide and of the phase 1 and phase 2 flagellar proteins. Primary isolation, biochemical identification, and serotyping are laborious and time consuming. Molecular identification based on suitable marker genes could be an attractive alternative to conventional bacteriological and serological methods. We have assessed the applicability of two housekeeping genes, gyrB, atpD, in combination with the flagellin genes fliC and fljB in multilocus sequence typing of Salmonella. Sequencing and comparative analysis of sequence data was performed on multiple strains from Austria, the United Kingdom, and Switzerland, representing all subspecies and 22 of the more prevalent non-typhoid S. enterica subsp. enterica serovars. A combination of these four marker genes allowed for a clear differentiation of all the strains analysed, indicating their applicability in molecular typing. The term MLST-v, for multilocus sequence typing based on virulence genes, is proposed to distinguish this approach from MLST based solely on housekeeping genes. An assortative recombination of the fliC gene was found in seven of the analysed serovars indicating multiple phylogenetic origin of these serovars.
The first gene cluster encoding for a membrane bound [NiFe] hydrogenase from a methanotroph, Meth... more The first gene cluster encoding for a membrane bound [NiFe] hydrogenase from a methanotroph, Methylococcus capsulatus (Bath), was cloned and sequenced. The cluster consisted of the structural genes hupS and hupL and accessory genes hupE, hupC and hupD. A vhupSL deletion mutant of Mc. capsulatus was constructed by marker exchange mutagenesis. Membrane associated hydrogenase activity disappeared. The membrane associated hydrogenase appeared to have a hydrogen uptake function in vivo. ß
Oligonucleotide microarrays offer a fast, high-throughput alternative for the parallel detection ... more Oligonucleotide microarrays offer a fast, high-throughput alternative for the parallel detection of microbes from virtually any sample. The application potential spreads across most sectors of life sciences, including environmental microbiology and microbial ecology; human, veterinary, food and plant diagnostics; water quality control; industrial microbiology, and so on. The past two years have witnessed a rapid increase of research in this field. Many alternative techniques were developed and validated as seen in 'proof-of-concept' articles. Publications reporting on the application of oligonucleotide microarray technology for microbial diagnostics in microbiology driven projects have just started to appear. Current and future technical and bioinformatics developments will inevitably improve the potential of this technology further.
Oligonucleotide microarrays are highly parallel hybridization platforms, allowing rapid and simul... more Oligonucleotide microarrays are highly parallel hybridization platforms, allowing rapid and simultaneous identification of many different microorganisms and viruses in a single assay. In the past few years, researchers have been confronted with a dramatic increase in the number of studies reporting development and/or improvement of oligonucleotide microarrays for microbial diagnostics, but use of the technology in routine diagnostics is still constrained by a variety of factors. Careful development of microarray essentials (such as oligonucleotide probes, protocols for target preparation and hybridization, etc.) combined with extensive performance testing are thus mandatory requirements for the maturation of diagnostic microarrays from fancy technological gimmicks to robust and routinely applicable tools. D
Landfills are large sources of CH 4 , but a considerable amount of CH 4 can be removed in situ by... more Landfills are large sources of CH 4 , but a considerable amount of CH 4 can be removed in situ by methanotrophs if their activity can be stimulated through the addition of nitrogen. Nitrogen can, however, lead to increased N 2 O production. To examine the effects of nitrogen and a selective inhibitor on CH 4 oxidation and N 2 O production in situ, 0.5 M of NH 4 Cl and 0.25 M of KNO 3 , with and without 0.01% (w/v) phenylacetylene, were applied to test plots at a landfill in Kalamazoo, MI from 2007 November to 2009 July. Nitrogen amendments stimulated N 2 O production but had no effect on CH 4 oxidation. The addition of phenylacetylene stimulated CH 4 oxidation while reducing N 2 O production. Methanotrophs possessing particulate methane monooxygenase and archaeal ammonia-oxidizers (AOAs) were abundant. The addition of nitrogen reduced methanotrophic diversity, particularly for type I methanotrophs. The simultaneous addition of phenylacetylene increased methanotrophic diversity and the presence of type I methanotrophs. Clone libraries of the archaeal amoA gene showed that the addition of nitrogen increased AOAs affiliated with Crenarchaeal group 1.1b, while they decreased with the simultaneous addition of phenylacetylene. These results suggest that the addition of phenylacetylene with nitrogen reduces N 2 O production by selectively inhibiting AOAs and/or type II methanotrophs.
With the advent of molecular biological techniques, especially next-generation sequencing and met... more With the advent of molecular biological techniques, especially next-generation sequencing and metagenomics, the number of microbial biogeography studies is rapidly increasing. However, these studies involve the synthesis of data generated by different laboratories using different protocols, chemicals, etc., all with inherent biases. The aim of this study was to assess inter-as well as intralaboratory variations in microbial community composition when standardized protocols are applied to a single soil sample. Aliquots from a homogenized soil sample from a rice field in Italy were sent to five participating laboratories. DNA was extracted by two investigators per laboratory using an identical protocol. All DNA samples were sent to one laboratory to perform DNA quantification, quantitative PCR (QPCR), and microarray and denaturing gradient gel electrophoresis (DGGE) analyses of methanotrophic communities. Yields, as well as purity of DNA, were significantly different between laboratories but in some cases also between investigators within the same laboratory. The differences in yield and quality of the extracted DNA were reflected in QPCR, microarray, and DGGE analysis results. Diversity indices (Shannon-Wiener, evenness, and richness) differed significantly between laboratories. The observed differences have implications for every project in which microbial communities are compared in different habitats, even if assessed within the same laboratory. To be able to make sensible comparisons leading to valid conclusions, intralaboratory variation should be assessed. Standardization of DNA extraction protocols and possible use of internal standards in interlaboratory comparisons may help in rendering a "quantifiable" bias.
Methane oxidation can occur in both aerobic and anaerobic environments; however, these are comple... more Methane oxidation can occur in both aerobic and anaerobic environments; however, these are completely different processes involving different groups of prokaryotes. Aerobic methane oxidation is carried out by aerobic methanotrophs, and anaerobic methane oxidizers, discovered recently, thrive under anaerobic conditions and use sulfate or nitrate as electron donors for methane oxidation . This review will focus on the aerobic oxidation of methane.
A method was developed for the mRNA-based application of microbial diagnostic microarrays to dete... more A method was developed for the mRNA-based application of microbial diagnostic microarrays to detect active microbial populations. DNA-and mRNA-based analyses of environmental samples were compared and confirmed via quantitative PCR. Results indicated that mRNA-based microarray analyses may provide additional information on the composition and functioning of microbial communities.
ARChip Epoxy and ARChip UV are presented as novel chip platforms for oligonucleotide immobilizati... more ARChip Epoxy and ARChip UV are presented as novel chip platforms for oligonucleotide immobilization. ARChip Epoxy is made of reactive epoxy resin available commercially. ARChip UV consists of photoactivatable poly(styrene-co-4-vinylbenzylthiocyanate). Both ARChip surfaces are tested in a model assay based on oligonucleotide probes from a real-life genotyping project and are evaluated in comparison with Wve commercial chip surfaces based on nitrocellulose, epoxy, and aldehyde polymer, and two diVerent aminosilanes. Optimum print buVer, spotter compatibility, and data normalization are discussed. 2004 Elsevier Inc. All rights reserved.
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Papers by L. Bodrossy