The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenbu... more The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. Samples stored in PBS-ethanol were diluted and treated by mild sonication. A 10-ml aliquot of a 1:40 dilution was filtered onto a 0.2-mm-pore-size type GTTP polycarbonate filter (Millipore, Eschborn, Germany). Hybridization and microscopic counting of hybridized and 49,69-diamidino-2-phenylindole (DAPI)-stained cells were performed as described previously from Snaidr et al. (1997, http://aem.asm.org/content/63/7/2884.full.pdf). Details of probes and formamide concentrations which were used are listed in futher details.. Means were calculated by using 10 to 20 randomly chosen fields for each filter section, which corresponded to 800 to 1,000 DAPI-stained cells. Counting results were always corrected by subtracting signals obs...
Fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes were used... more Fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard). Hybridization and microscopy counts of hybridized and 4',6'-diamidino-2-phenylindole (DAPI)-stained cells were performed as described previously from Snaidr et al. (1997, http://aem.asm.org/content/63/7/2884.full.pdf). Means were calculated from 10 to 20 randomly chosen fields on each filter section, corresponding to 800 to 1,000 DAPI-stained cells. Counting results were always corrected by subtracting signals observed with the probe NON338. Formamide concentrations are given in further details. FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment. Up to 65.4% ± 7.5% of total DAPI cell counts hybridized to the bacterial probe EUB338, and up to 4.9% ± 1.5% hybridized to the archaeal probe ARCH915. Besides delta-proteobacterial sulfate-reducing bacte...
A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. ... more A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with Myxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the gamma subclass of the class Proteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity wi...
Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psyc... more Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psychrophilic aerobic bacteria on cellulose, xylan, chitin, and starch. A variety of species belonging to Alpha- and Gammaproteobacteria and to Flavobacteria were isolated from sediment depths between 12 and 42 mbsf. Metagenomic DNA purified from the pooled enrichments was sequenced and analyzed for phylogenetic composition and presence of genes encoding carbohydrate-active enzymes. More than 200 open reading frames coding for glycoside hydrolases were identified, and more than 60 of them relevant for enzymatic degradation of lignocellulose. Four genes encoding β-glucosidases with less than 52% identities to characterized enzymes were chosen for recombinant expression in Escherichia coli. In addition one endomannanase, two endoxylanases, and three β-xylosidases were produced recombinantly. All genes could be actively expressed. Functional analysis revealed discrepancies and additional variability for the recombinant enzymes as compared to the sequence-based predictions.
ABSTRACT Die weiße Biotechnologie ist ein hochaktuelles, schnell wachsendes Feld in der angewandt... more ABSTRACT Die weiße Biotechnologie ist ein hochaktuelles, schnell wachsendes Feld in der angewandten Forschung. Die Bedeutung von Biokatalysatoren für Prozesse in der Chemikalienherstellung, der Lebensmittel- und der Textilindustrie sowie im Bereich der Bioraffinerie nimmt rapide zu. Während in der Vergangenheit zumeist Enzyme aus mesophilen Mikroorganismen als Alternative in traditionell chemischen Prozessen eingesetzt wurden, verschiebt sich der Fokus des Interesses zurzeit immer mehr zugunsten von Biokatalysatoren aus extremophilen Mikroben, welche als Extremozyme bezeichnet werden. Mikroorganismen, die beispielsweise aus besonders heißen, sauren oder salzigen Habitaten isoliert wurden, beherbergen einen großen Schatz an industriell nutzbaren Extremozymen. Diese zeigen zumeist optimale Aktivität unter extremen Umweltbedingungen und sind daher für biotechnologische Anwendungen besonders interessant.Extremozymes for biotechnological applicationsIndustrial biotechnology is a fast growing and proliferating field of research. Biocatalysis gradually replaces chemical processes and is widely used in textile or food industry or in the sustainable production of fine chemicals. Although currently most of the enzymes in industry are of mesophilic origin, the focus is changing towards more robust biocatalysts from extremophilic organisms. Research on extremophiles will progressively supply novel extremozymes for biotechnological applications. In particular (hyper-)thermophiles, acidophiles or salt-tolerant microorganisms are a rich source of industrial applicable and robust extremozymes with optimal activity under harsh conditions.
Slot-blot hybridization of rRNA with domain-specific oligonucleotide probes targeting the 16S rRN... more Slot-blot hybridization of rRNA with domain-specific oligonucleotide probes targeting the 16S rRNA of Archaea and Bacteria was utilized to assess the relative abundance of these domains along a thermal gradient at a shallow submarine hydrothermal vent near Milos Island (Greece). The highest prokaryotic rRNA concentrations (defined as the sum of bacterial and archaeal rRNA) were found in the uppermost sediment surface (0-20 mm), decreasing strongly with depth. This indicates that the microbial activity was mainly occurring in the surface layer of this hydrothermal vent. Furthermore, rRNA concentrations were higher in regions closer to the vent, suggesting that the hydrothermal activity stimulated microbial activity. Archaea seemed to be a minor component of the microbial community at this vent site, even in the zones with higher temperatures. Bacteria made up at least 78% (mean 95%) of the prokaryotic rRNA. However, along the steepest temperature gradient, the proportion of archaeal ...
Extremozymes are enzymes derived from extremophilic microorganisms that are able to withstand har... more Extremozymes are enzymes derived from extremophilic microorganisms that are able to withstand harsh conditions in industrial processes that were long thought to be destructive to proteins. Heat-stable and solvent-tolerant biocatalysts are valuable tools for processes in which for example hardly decomposable polymers need to be liquefied and degraded, while cold-active enzymes are of relevance for food and detergent industries. Extremophilic microorganisms are a rich source of naturally tailored enzymes, which are more superior over their mesophilic counterparts for applications at extreme conditions. Especially lignocellulolytic, amylolytic, and other biomass processing extremozymes with unique properties are widely distributed in thermophilic prokaryotes and are of high potential for versatile industrial processes.
A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. ... more A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with Myxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the gamma subclass of the class Proteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity wi...
Thirteen psychrophilic sulfate-reducing isolates from two permanently cold fjords of the Arctic i... more Thirteen psychrophilic sulfate-reducing isolates from two permanently cold fjords of the Arctic island Spitsbergen (Hornsund and Storfjord) were phylogenetically analyzed. They all belonged to the delta subclass of Proteobacteria and were widely distributed within this group, indicating that psychrophily is a polyphyletic property. A new 16S rRNA-directed oligonucleotide probe was designed against the largest coherent cluster of these isolates. The new probe, as well as a set of available probes, was applied in rRNA slot blot hybridization to investigate the composition of the sulfate-reducing bacterial community in the sediments. rRNA related to the new cluster of incompletely oxidizing, psychrophilic isolates made up 1.4 to 20.9% of eubacterial rRNA at Storfjord and 0.6 to 3. 5% of eubacterial rRNA at Hornsund. This group was the second-most-abundant group of sulfate reducers at these sites. Denaturing gradient gel electrophoresis and hybridization analysis showed bands identical ...
Recently, four Thiomicrospira strains were isolated from a coastal mud flat of the German Wadden ... more Recently, four Thiomicrospira strains were isolated from a coastal mud flat of the German Wadden Sea (T. Brinkhoff and G. Muyzer, Appl. Environ. Microbiol. 63:3789-3796, 1997). Here we describe the use of a polyphasic approach to investigate the functional role of these closely related bacteria. Microsensor measurements showed that there was oxygen penetration into the sediment to a depth of about 2.0 mm. The pH decreased from 8.15 in the overlaying water to a minimum value of 7.3 at a depth of 1.2 mm. Further down in the sediment the pH increased to about 7.8 and remained constant. Most-probable-number (MPN) counts of chemolithoautotrophic sulfur-oxidizing bacteria revealed nearly constant numbers along the vertical profile; the cell concentration ranged from 0.93 x 10(5) to 9.3 x 10(5) cells per g of sediment. A specific PCR was used to detect the presence of Thiomicrospira cells in the MPN count preparations and to determine their 16S rRNA sequences. The concentration of Thiomicr...
A gene of Thermoanaerobacterium thermosulfurigenes EM1 encoding a protein with similarity to the ... more A gene of Thermoanaerobacterium thermosulfurigenes EM1 encoding a protein with similarity to the maltose-binding protein of Escherichia coli was cloned and sequenced. It was located in the amy gene region of the chromosome downstream of the pullulanase-encoding amyB gene and upstream of amyDC, encoding membrane components of an ABC transport system, and the alpha-amylase gene amyA. The gene was designated amyE. Analysis of mRNA by Northern (RNA) blotting revealed that expression of the amy gene region is repressed during growth on glucose. Maximum levels of mRNA were detected with maltose as a substrate. An operon which was transcribed in the order amyBEDC was identified. However, an additional transcription start point was found in front of amyE. The amyA gene represented a monocistronic operon. Putative -35 and -10 promoter sites were deduced from the three transcription start sites of the amy gene region, and possible regulatory regions mediating induction by maltose and cataboli...
The complete pullulanase gene (amyB) from Thermoanaerobacterium thermosulfurigenes EM1 was cloned... more The complete pullulanase gene (amyB) from Thermoanaerobacterium thermosulfurigenes EM1 was cloned in Escherichia coli, and the nucleotide sequence was determined. The reading frame of amyB consisted of 5,586 bp encoding an exceptionally large enzyme of 205,991 Da. Sequence analysis revealed a composite structure of the pullulanase consisting of catalytic and noncatalytic domains. The N-terminal half of the protein contained a leader peptide of 35 amino acid residues and the catalytic domain, which included the four consensus regions of amylases. Comparison of the consensus regions of several pullulanases suggested that enzymes like pullulanase type II from T. thermosulfurigenes EM1 which hydrolyze alpha-1,4- and alpha-1,6-glycosidic linkages have specific amino acid sequences in the consensus regions. These are different from those of pullulanases type I which only cleave alpha-1,6 linkages. The C-terminal half, which is not necessary for enzymatic function, consisted of at least tw...
Extremophiles : life under extreme conditions, 2014
Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psyc... more Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psychrophilic aerobic bacteria on cellulose, xylan, chitin, and starch. A variety of species belonging to Alpha- and Gammaproteobacteria and to Flavobacteria were isolated from sediment depths between 12 and 42 mbsf. Metagenomic DNA purified from the pooled enrichments was sequenced and analyzed for phylogenetic composition and presence of genes encoding carbohydrate-active enzymes. More than 200 open reading frames coding for glycoside hydrolases were identified, and more than 60 of them relevant for enzymatic degradation of lignocellulose. Four genes encoding β-glucosidases with less than 52% identities to characterized enzymes were chosen for recombinant expression in Escherichia coli. In addition one endomannanase, two endoxylanases, and three β-xylosidases were produced recombinantly. All genes could be actively expressed. Functional analysis revealed discrepancies and additional variab...
ABSTRACT Die weiße Biotechnologie ist ein hochaktuelles, schnell wachsendes Feld in der angewandt... more ABSTRACT Die weiße Biotechnologie ist ein hochaktuelles, schnell wachsendes Feld in der angewandten Forschung. Die Bedeutung von Biokatalysatoren für Prozesse in der Chemikalienherstellung, der Lebensmittel- und der Textilindustrie sowie im Bereich der Bioraffinerie nimmt rapide zu. Während in der Vergangenheit zumeist Enzyme aus mesophilen Mikroorganismen als Alternative in traditionell chemischen Prozessen eingesetzt wurden, verschiebt sich der Fokus des Interesses zurzeit immer mehr zugunsten von Biokatalysatoren aus extremophilen Mikroben, welche als Extremozyme bezeichnet werden. Mikroorganismen, die beispielsweise aus besonders heißen, sauren oder salzigen Habitaten isoliert wurden, beherbergen einen großen Schatz an industriell nutzbaren Extremozymen. Diese zeigen zumeist optimale Aktivität unter extremen Umweltbedingungen und sind daher für biotechnologische Anwendungen besonders interessant.Extremozymes for biotechnological applicationsIndustrial biotechnology is a fast growing and proliferating field of research. Biocatalysis gradually replaces chemical processes and is widely used in textile or food industry or in the sustainable production of fine chemicals. Although currently most of the enzymes in industry are of mesophilic origin, the focus is changing towards more robust biocatalysts from extremophilic organisms. Research on extremophiles will progressively supply novel extremozymes for biotechnological applications. In particular (hyper-)thermophiles, acidophiles or salt-tolerant microorganisms are a rich source of industrial applicable and robust extremozymes with optimal activity under harsh conditions.
The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenbu... more The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. Samples stored in PBS-ethanol were diluted and treated by mild sonication. A 10-ml aliquot of a 1:40 dilution was filtered onto a 0.2-mm-pore-size type GTTP polycarbonate filter (Millipore, Eschborn, Germany). Hybridization and microscopic counting of hybridized and 49,69-diamidino-2-phenylindole (DAPI)-stained cells were performed as described previously from Snaidr et al. (1997, http://aem.asm.org/content/63/7/2884.full.pdf). Details of probes and formamide concentrations which were used are listed in futher details.. Means were calculated by using 10 to 20 randomly chosen fields for each filter section, which corresponded to 800 to 1,000 DAPI-stained cells. Counting results were always corrected by subtracting signals obs...
Fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes were used... more Fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard). Hybridization and microscopy counts of hybridized and 4',6'-diamidino-2-phenylindole (DAPI)-stained cells were performed as described previously from Snaidr et al. (1997, http://aem.asm.org/content/63/7/2884.full.pdf). Means were calculated from 10 to 20 randomly chosen fields on each filter section, corresponding to 800 to 1,000 DAPI-stained cells. Counting results were always corrected by subtracting signals observed with the probe NON338. Formamide concentrations are given in further details. FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment. Up to 65.4% ± 7.5% of total DAPI cell counts hybridized to the bacterial probe EUB338, and up to 4.9% ± 1.5% hybridized to the archaeal probe ARCH915. Besides delta-proteobacterial sulfate-reducing bacte...
A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. ... more A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with Myxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the gamma subclass of the class Proteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity wi...
Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psyc... more Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psychrophilic aerobic bacteria on cellulose, xylan, chitin, and starch. A variety of species belonging to Alpha- and Gammaproteobacteria and to Flavobacteria were isolated from sediment depths between 12 and 42 mbsf. Metagenomic DNA purified from the pooled enrichments was sequenced and analyzed for phylogenetic composition and presence of genes encoding carbohydrate-active enzymes. More than 200 open reading frames coding for glycoside hydrolases were identified, and more than 60 of them relevant for enzymatic degradation of lignocellulose. Four genes encoding β-glucosidases with less than 52% identities to characterized enzymes were chosen for recombinant expression in Escherichia coli. In addition one endomannanase, two endoxylanases, and three β-xylosidases were produced recombinantly. All genes could be actively expressed. Functional analysis revealed discrepancies and additional variability for the recombinant enzymes as compared to the sequence-based predictions.
ABSTRACT Die weiße Biotechnologie ist ein hochaktuelles, schnell wachsendes Feld in der angewandt... more ABSTRACT Die weiße Biotechnologie ist ein hochaktuelles, schnell wachsendes Feld in der angewandten Forschung. Die Bedeutung von Biokatalysatoren für Prozesse in der Chemikalienherstellung, der Lebensmittel- und der Textilindustrie sowie im Bereich der Bioraffinerie nimmt rapide zu. Während in der Vergangenheit zumeist Enzyme aus mesophilen Mikroorganismen als Alternative in traditionell chemischen Prozessen eingesetzt wurden, verschiebt sich der Fokus des Interesses zurzeit immer mehr zugunsten von Biokatalysatoren aus extremophilen Mikroben, welche als Extremozyme bezeichnet werden. Mikroorganismen, die beispielsweise aus besonders heißen, sauren oder salzigen Habitaten isoliert wurden, beherbergen einen großen Schatz an industriell nutzbaren Extremozymen. Diese zeigen zumeist optimale Aktivität unter extremen Umweltbedingungen und sind daher für biotechnologische Anwendungen besonders interessant.Extremozymes for biotechnological applicationsIndustrial biotechnology is a fast growing and proliferating field of research. Biocatalysis gradually replaces chemical processes and is widely used in textile or food industry or in the sustainable production of fine chemicals. Although currently most of the enzymes in industry are of mesophilic origin, the focus is changing towards more robust biocatalysts from extremophilic organisms. Research on extremophiles will progressively supply novel extremozymes for biotechnological applications. In particular (hyper-)thermophiles, acidophiles or salt-tolerant microorganisms are a rich source of industrial applicable and robust extremozymes with optimal activity under harsh conditions.
Slot-blot hybridization of rRNA with domain-specific oligonucleotide probes targeting the 16S rRN... more Slot-blot hybridization of rRNA with domain-specific oligonucleotide probes targeting the 16S rRNA of Archaea and Bacteria was utilized to assess the relative abundance of these domains along a thermal gradient at a shallow submarine hydrothermal vent near Milos Island (Greece). The highest prokaryotic rRNA concentrations (defined as the sum of bacterial and archaeal rRNA) were found in the uppermost sediment surface (0-20 mm), decreasing strongly with depth. This indicates that the microbial activity was mainly occurring in the surface layer of this hydrothermal vent. Furthermore, rRNA concentrations were higher in regions closer to the vent, suggesting that the hydrothermal activity stimulated microbial activity. Archaea seemed to be a minor component of the microbial community at this vent site, even in the zones with higher temperatures. Bacteria made up at least 78% (mean 95%) of the prokaryotic rRNA. However, along the steepest temperature gradient, the proportion of archaeal ...
Extremozymes are enzymes derived from extremophilic microorganisms that are able to withstand har... more Extremozymes are enzymes derived from extremophilic microorganisms that are able to withstand harsh conditions in industrial processes that were long thought to be destructive to proteins. Heat-stable and solvent-tolerant biocatalysts are valuable tools for processes in which for example hardly decomposable polymers need to be liquefied and degraded, while cold-active enzymes are of relevance for food and detergent industries. Extremophilic microorganisms are a rich source of naturally tailored enzymes, which are more superior over their mesophilic counterparts for applications at extreme conditions. Especially lignocellulolytic, amylolytic, and other biomass processing extremozymes with unique properties are widely distributed in thermophilic prokaryotes and are of high potential for versatile industrial processes.
A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. ... more A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with Myxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the gamma subclass of the class Proteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity wi...
Thirteen psychrophilic sulfate-reducing isolates from two permanently cold fjords of the Arctic i... more Thirteen psychrophilic sulfate-reducing isolates from two permanently cold fjords of the Arctic island Spitsbergen (Hornsund and Storfjord) were phylogenetically analyzed. They all belonged to the delta subclass of Proteobacteria and were widely distributed within this group, indicating that psychrophily is a polyphyletic property. A new 16S rRNA-directed oligonucleotide probe was designed against the largest coherent cluster of these isolates. The new probe, as well as a set of available probes, was applied in rRNA slot blot hybridization to investigate the composition of the sulfate-reducing bacterial community in the sediments. rRNA related to the new cluster of incompletely oxidizing, psychrophilic isolates made up 1.4 to 20.9% of eubacterial rRNA at Storfjord and 0.6 to 3. 5% of eubacterial rRNA at Hornsund. This group was the second-most-abundant group of sulfate reducers at these sites. Denaturing gradient gel electrophoresis and hybridization analysis showed bands identical ...
Recently, four Thiomicrospira strains were isolated from a coastal mud flat of the German Wadden ... more Recently, four Thiomicrospira strains were isolated from a coastal mud flat of the German Wadden Sea (T. Brinkhoff and G. Muyzer, Appl. Environ. Microbiol. 63:3789-3796, 1997). Here we describe the use of a polyphasic approach to investigate the functional role of these closely related bacteria. Microsensor measurements showed that there was oxygen penetration into the sediment to a depth of about 2.0 mm. The pH decreased from 8.15 in the overlaying water to a minimum value of 7.3 at a depth of 1.2 mm. Further down in the sediment the pH increased to about 7.8 and remained constant. Most-probable-number (MPN) counts of chemolithoautotrophic sulfur-oxidizing bacteria revealed nearly constant numbers along the vertical profile; the cell concentration ranged from 0.93 x 10(5) to 9.3 x 10(5) cells per g of sediment. A specific PCR was used to detect the presence of Thiomicrospira cells in the MPN count preparations and to determine their 16S rRNA sequences. The concentration of Thiomicr...
A gene of Thermoanaerobacterium thermosulfurigenes EM1 encoding a protein with similarity to the ... more A gene of Thermoanaerobacterium thermosulfurigenes EM1 encoding a protein with similarity to the maltose-binding protein of Escherichia coli was cloned and sequenced. It was located in the amy gene region of the chromosome downstream of the pullulanase-encoding amyB gene and upstream of amyDC, encoding membrane components of an ABC transport system, and the alpha-amylase gene amyA. The gene was designated amyE. Analysis of mRNA by Northern (RNA) blotting revealed that expression of the amy gene region is repressed during growth on glucose. Maximum levels of mRNA were detected with maltose as a substrate. An operon which was transcribed in the order amyBEDC was identified. However, an additional transcription start point was found in front of amyE. The amyA gene represented a monocistronic operon. Putative -35 and -10 promoter sites were deduced from the three transcription start sites of the amy gene region, and possible regulatory regions mediating induction by maltose and cataboli...
The complete pullulanase gene (amyB) from Thermoanaerobacterium thermosulfurigenes EM1 was cloned... more The complete pullulanase gene (amyB) from Thermoanaerobacterium thermosulfurigenes EM1 was cloned in Escherichia coli, and the nucleotide sequence was determined. The reading frame of amyB consisted of 5,586 bp encoding an exceptionally large enzyme of 205,991 Da. Sequence analysis revealed a composite structure of the pullulanase consisting of catalytic and noncatalytic domains. The N-terminal half of the protein contained a leader peptide of 35 amino acid residues and the catalytic domain, which included the four consensus regions of amylases. Comparison of the consensus regions of several pullulanases suggested that enzymes like pullulanase type II from T. thermosulfurigenes EM1 which hydrolyze alpha-1,4- and alpha-1,6-glycosidic linkages have specific amino acid sequences in the consensus regions. These are different from those of pullulanases type I which only cleave alpha-1,6 linkages. The C-terminal half, which is not necessary for enzymatic function, consisted of at least tw...
Extremophiles : life under extreme conditions, 2014
Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psyc... more Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psychrophilic aerobic bacteria on cellulose, xylan, chitin, and starch. A variety of species belonging to Alpha- and Gammaproteobacteria and to Flavobacteria were isolated from sediment depths between 12 and 42 mbsf. Metagenomic DNA purified from the pooled enrichments was sequenced and analyzed for phylogenetic composition and presence of genes encoding carbohydrate-active enzymes. More than 200 open reading frames coding for glycoside hydrolases were identified, and more than 60 of them relevant for enzymatic degradation of lignocellulose. Four genes encoding β-glucosidases with less than 52% identities to characterized enzymes were chosen for recombinant expression in Escherichia coli. In addition one endomannanase, two endoxylanases, and three β-xylosidases were produced recombinantly. All genes could be actively expressed. Functional analysis revealed discrepancies and additional variab...
ABSTRACT Die weiße Biotechnologie ist ein hochaktuelles, schnell wachsendes Feld in der angewandt... more ABSTRACT Die weiße Biotechnologie ist ein hochaktuelles, schnell wachsendes Feld in der angewandten Forschung. Die Bedeutung von Biokatalysatoren für Prozesse in der Chemikalienherstellung, der Lebensmittel- und der Textilindustrie sowie im Bereich der Bioraffinerie nimmt rapide zu. Während in der Vergangenheit zumeist Enzyme aus mesophilen Mikroorganismen als Alternative in traditionell chemischen Prozessen eingesetzt wurden, verschiebt sich der Fokus des Interesses zurzeit immer mehr zugunsten von Biokatalysatoren aus extremophilen Mikroben, welche als Extremozyme bezeichnet werden. Mikroorganismen, die beispielsweise aus besonders heißen, sauren oder salzigen Habitaten isoliert wurden, beherbergen einen großen Schatz an industriell nutzbaren Extremozymen. Diese zeigen zumeist optimale Aktivität unter extremen Umweltbedingungen und sind daher für biotechnologische Anwendungen besonders interessant.Extremozymes for biotechnological applicationsIndustrial biotechnology is a fast growing and proliferating field of research. Biocatalysis gradually replaces chemical processes and is widely used in textile or food industry or in the sustainable production of fine chemicals. Although currently most of the enzymes in industry are of mesophilic origin, the focus is changing towards more robust biocatalysts from extremophilic organisms. Research on extremophiles will progressively supply novel extremozymes for biotechnological applications. In particular (hyper-)thermophiles, acidophiles or salt-tolerant microorganisms are a rich source of industrial applicable and robust extremozymes with optimal activity under harsh conditions.
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