Publications

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developing a genetic manipulation system for the antarctic archaeon, halorubrum lacusprofundi: investigating acetamidase gene function.no systems have been reported for genetic manipulation of cold-adapted archaea. halorubrum lacusprofundi is an important member of deep lake, antarctica (~10% of the population), and is amendable to laboratory cultivation. here we report the development of a shuttle-vector and targeted gene-knockout system for this species. to investigate the function of acetamidase/formamidase genes, a class of genes not experimentally studied in archaea, the acetamidase gene, amd3, was disrupted. the wild-type ...201627708407
a synthetic ecology perspective: how well does behavior of model organisms in the laboratory predict microbial activities in natural habitats?in this perspective article, we question how well model organisms, the ones that are easy to cultivate in the laboratory and that show robust growth and biomass accumulation, reflect the dynamics and interactions of microbial communities observed in nature. today's -omics toolbox allows assessing the genomic potential of microbes in natural environments in a high-throughput fashion and at a strain-level resolution. however, understanding of the details of microbial activities and of the mechanis ...201627379075
structure of type iil restriction-modification enzyme mmei in complex with dna has implications for engineering new specificities.the creation of restriction enzymes with programmable dna-binding and -cleavage specificities has long been a goal of modern biology. the recently discovered type iil mmei family of restriction-and-modification (rm) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. however, a lack of structural information on type iil enzymes has limited the repertoire that can be rationally engineered. we report here a crystal structure of mmei ...201627082731
nicotine dehydrogenase complexed with 6-hydroxypseudooxynicotine oxidase involved in the hybrid nicotine-degrading pathway in agrobacterium tumefaciens s33.nicotine, a major toxic alkaloid in tobacco wastes, is degraded by bacteria, mainly via pyridine and pyrrolidine pathways. previously, we discovered a new hybrid of the pyridine and pyrrolidine pathways in agrobacterium tumefaciens s33 and characterized its key enzyme 6-hydroxy-3-succinoylpyridine (hsp) hydroxylase. here, we purified the nicotine dehydrogenase initializing the nicotine degradation from the strain and found that it forms a complex with a novel 6-hydroxypseudooxynicotine oxidase. ...201626729714
time-dependent transcriptional changes in axenic giardia duodenalis trophozoites.giardia duodenalis is the most common gastrointestinal protozoan parasite of humans and a significant contributor to the global burden of both diarrheal disease and post-infectious chronic disorders. although g. duodenalis can be cultured axenically, significant gaps exist in our understanding of the molecular biology and metabolism of this pathogen. the present study employed rna sequencing to characterize the mrna transcriptome of g. duodenalis trophozoites in axenic culture, at log (48 h of g ...201526636323
the "speedy" synthesis of atom-specific (15)n imino/amido-labeled rna.although numerous reports on the synthesis of atom-specific (15)n-labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for rna solid-phase synthesis is still lacking. this situation represents a severe bottleneck for nmr spectroscopic investigations on functional rnas. here, we present optimized procedures to speed up the synthesis of (15)n(1) adenosine and (15)n(1) guanosine amidites, which are the much needed counterparts of the more straightforward-to-achiev ...201526237536
the ecology of pelagic freshwater methylotrophs assessed by a high-resolution monitoring and isolation campaign.methylotrophic planktonic bacteria fulfill a particular role in the carbon cycle of lakes via the turnover of single-carbon compounds. we studied two planktonic freshwater lineages (ld28 and prd01a001b) affiliated with methylophilaceae (betaproteobacteria) in lake zurich, switzerland, by a combination of molecular and cultivation-based approaches. their spatio-temporal distribution was monitored at high resolution (n=992 samples) for 4 consecutive years. ld28 methylotrophs constituted up to 11 × ...201525942006
c₁-pathways in methyloversatilis universalis fam5: genome wide gene expression and mutagenesis studies.methyloversatilis universalis fam5 utilizes single carbon compounds such as methanol or methylamine as a sole source of carbon and energy. expression profiling reveals distinct sets of genes altered during growth on methylamine vs methanol. as expected, all genes for the n-methylglutamate pathway were induced during growth on methylamine. among other functions responding to the aminated source of c₁-carbon, are a heme-containing amine dehydrogenase (qhp), a distant homologue of formaldehyde acti ...201527682085
sip metagenomics identifies uncultivated methylophilaceae as dimethylsulphide degrading bacteria in soil and lake sediment.dimethylsulphide (dms) has an important role in the global sulphur cycle and atmospheric chemistry. microorganisms using dms as sole carbon, sulphur or energy source, contribute to the cycling of dms in a wide variety of ecosystems. the diversity of microbial populations degrading dms in terrestrial environments is poorly understood. based on cultivation studies, a wide range of bacteria isolated from terrestrial ecosystems were shown to be able to degrade dms, yet it remains unknown whether any ...201525822481
draft genome sequences of five new strains of methylophilaceae isolated from lake washington sediment.we sequenced the genomes of five new methylophilaceae strains isolated from lake washington sediment. we used the new sequences to sort these new strains into specific methylophilaceae ecotypes, including one novel ecotype. the new genomes expand the known diversity of methylophilaceae and provide new models for studying the ecology of methylotrophy.201525657279
endonuclease specificity and sequence dependence of type iis restriction enzymes.restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. despite this, systematic functional categorization of cleavage performance has largely been lacking. we established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. we coupled this to massively parallel sequencing in order to provide sensitive and accurat ...201525629514
phylogenetic study of methanol oxidizers from chilika-lake sediments using genomic and metagenomic approaches.group-wise diversity of sediment methylotrophs of chilika lake (lat. 19°28'-19°54'n; long. 85°06'-85°35'e) odisha, india at various identified sites was studied. both the culturable and unculturable (metagenome) methylotrophs were investigated in the lake sediments employing both mxaf and 16s rrna genes as markers. ardra profiling, 16s rrna gene sequencing, page profiling of haeiii, ecori restricted mxaf gene and the mxaf gene sequences using culture-dependent approach revealed the relatedness o ...201525805901
methane-fed microbial microcosms show differential community dynamics and pinpoint taxa involved in communal response.we report observations on the dynamics of bacterial communities in response to methane stimulus in laboratory microcosm incubations prepared with lake sediment samples. we first measured taxonomic compositions of long-term enrichment cultures and determined that, although dominated by methylococcaceae types, these cultures also contained accompanying types belonging to a limited number of bacterial taxa, methylotrophs and non-methylotrophs. we then followed the short-term community dynamics, in ...201525333464
methane-fed microbial microcosms show differential community dynamics and pinpoint taxa involved in communal response.we report observations on the dynamics of bacterial communities in response to methane stimulus in laboratory microcosm incubations prepared with lake sediment samples. we first measured taxonomic compositions of long-term enrichment cultures and determined that, although dominated by methylococcaceae types, these cultures also contained accompanying types belonging to a limited number of bacterial taxa, methylotrophs and non-methylotrophs. we then followed the short-term community dynamics, in ...201525333464
structure and protein-protein interactions of methanol dehydrogenase from methylococcus capsulatus (bath).in the initial steps of their metabolic pathway, methanotrophic bacteria oxidize methane to methanol with methane monooxygenases (mmos) and methanol to formaldehyde with methanol dehydrogenases (mdhs). several lines of evidence suggest that the membrane-bound or particulate mmo (pmmo) and mdh interact to form a metabolic supercomplex. to further investigate the possible existence of such a supercomplex, native mdh from methylococcus capsulatus (bath) has been purified and characterized by size e ...201425185034
the expanded diversity of methylophilaceae from lake washington through cultivation and genomic sequencing of novel ecotypes.we describe five novel methylophilaceae ecotypes from a single ecological niche in lake washington, usa, and compare them to three previously described ecotypes, in terms of their phenotype and genome sequence divergence. two of the ecotypes appear to represent novel genera within the methylophilaceae. genome-based metabolic reconstruction highlights metabolic versatility of methylophilaceae with respect to methylotrophy and nitrogen metabolism, different ecotypes possessing different combinatio ...201425058595
crystallization and preliminary crystallographic analysis of histamine dehydrogenase from natrinema gari bcc 24369.histamine dehydrogenase (hadh) catalyzes the oxidative deamination of histamine, resulting in the production of imidazole acetaldehyde and an ammonium ion. the enzyme isolated from the newly identified halophilic archaeon natrinema gari bcc 24369 is significantly different from the previously described protein from nocardioides simplex. this newly identified hadh comprises three subunits with molecular weights of 49.0, 24.7 and 23.9 kda, respectively, and is optimally active under high-salt cond ...201425005094
bacterial community dynamics during the early stages of biofilm formation in a chlorinated experimental drinking water distribution system: implications for drinking water discolouration.to characterize bacterial communities during the early stages of biofilm formation and their role in water discolouration in a fully representative, chlorinated, experimental drinking water distribution systems (dwds).201424712449
studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (etf) and butyryl-coa dehydrogenase (bcd) of acidaminococcus fermentans.electron bifurcation is a fundamental strategy of energy coupling originally discovered in the q-cycle of many organisms. recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. it enables anaerobic bacteria and archaea to reduce the low-potential [4fe-4s] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. here we characterize the bifurcating electr ...201424379410
studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (etf) and butyryl-coa dehydrogenase (bcd) of acidaminococcus fermentans.electron bifurcation is a fundamental strategy of energy coupling originally discovered in the q-cycle of many organisms. recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. it enables anaerobic bacteria and archaea to reduce the low-potential [4fe-4s] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. here we characterize the bifurcating electr ...201424379410
comparative transcriptomics in three methylophilaceae species uncover different strategies for environmental adaptation.we carried out whole transcriptome analysis of three species of methylophilaceae, methylotenera mobilis, methylotenera versatilis and methylovorus glucosotrophus, in order to determine which metabolic pathways are actively transcribed in cultures grown in laboratory on c1 substrates and how metabolism changes under semi-in situ conditions. comparative analyses of the transcriptomes were used to probe the metabolic strategies utilized by each of the organisms in the environment. our analysis of t ...201323904993
the methanol dehydrogenase gene, mxaf, as a functional and phylogenetic marker for proteobacterial methanotrophs in natural environments.the mxaf gene, coding for the large (α) subunit of methanol dehydrogenase, is highly conserved among distantly related methylotrophic species in the alpha-, beta- and gammaproteobacteria. it is ubiquitous in methanotrophs, in contrast to other methanotroph-specific genes such as the pmoa and mmox genes, which are absent in some methanotrophic proteobacterial genera. this study examined the potential for using the mxaf gene as a functional and phylogenetic marker for methanotrophs. mxaf and 16s r ...201323451130
c1 metabolism in corynebacterium glutamicum: an endogenous pathway for oxidation of methanol to carbon dioxide.methanol is considered an interesting carbon source in "bio-based" microbial production processes. since corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies of the response of this organism to methanol. the c. glutamicum wild type was able to convert (13)c-labeled methanol to (13)co2. analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be upregulated, ...201324014532
community shifts of actively growing lake bacteria after n-acetyl-glucosamine addition: improving the brdu-facs method.in aquatic environments, community dynamics of bacteria, especially actively growing bacteria (agb), are tightly linked with dissolved organic matter (dom) quantity and quality. we analyzed the community dynamics of dna-synthesizing and accordingly agb by linking an improved bromodeoxyuridine immunocytochemistry approach with fluorescence-activated cell sorting (brdu-facs). facs-sorted cells of even oligotrophic ecosystems in winter were characterized by 16s rrna gene analysis. in incubation exp ...201323985742
community shifts of actively growing lake bacteria after n-acetyl-glucosamine addition: improving the brdu-facs method.in aquatic environments, community dynamics of bacteria, especially actively growing bacteria (agb), are tightly linked with dissolved organic matter (dom) quantity and quality. we analyzed the community dynamics of dna-synthesizing and accordingly agb by linking an improved bromodeoxyuridine immunocytochemistry approach with fluorescence-activated cell sorting (brdu-facs). facs-sorted cells of even oligotrophic ecosystems in winter were characterized by 16s rrna gene analysis. in incubation exp ...201323985742
crystallization and preliminary x-ray crystallographic analysis of mxaj, a component of the methanol-oxidizing system operon from the marine bacterium methylophaga aminisulfidivorans mpt.the methanol-oxidizing system (mox) is essential for methylotrophic bacteria to extract energy during the oxidoreduction reaction and consists of a series of electron-transfer proteins encoded by the mox operon. one of the key enzymes is the α₂β₂ methanol dehydrogenase complex (type i mdh), which converts methanol to formaldehyde during the 2e⁻ transfer through the prosthetic group pyrroloquinoline quinone. mxaj, a product of mxaj of the mox operon, is a component of the mdh complex and enhances ...201323908039
gradients in microbial methanol uptake: productive coastal upwelling waters to oligotrophic gyres in the atlantic ocean.methanol biogeochemistry and its importance as a carbon source in seawater is relatively unexplored. we report the first microbial methanol carbon assimilation rates (k) in productive coastal upwelling waters of up to 0.117±0.002 d(-1) (~10 nmol l(-1 )d(-1)). on average, coastal upwelling waters were 11 times greater than open ocean northern temperate (nt) waters, eight times greater than gyre waters and four times greater than equatorial upwelling (eu) waters; suggesting that all upwelling wate ...201323178665
gradients in microbial methanol uptake: productive coastal upwelling waters to oligotrophic gyres in the atlantic ocean.methanol biogeochemistry and its importance as a carbon source in seawater is relatively unexplored. we report the first microbial methanol carbon assimilation rates (k) in productive coastal upwelling waters of up to 0.117±0.002 d(-1) (~10 nmol l(-1 )d(-1)). on average, coastal upwelling waters were 11 times greater than open ocean northern temperate (nt) waters, eight times greater than gyre waters and four times greater than equatorial upwelling (eu) waters; suggesting that all upwelling wate ...201323178665
bacteria in crude oil survived autoclaving and stimulated differentially by exogenous bacteria.autoclaving of crude oil is often used to evaluate the hydrocarbon-degrading abilities of bacteria. this may be potentially useful for bioaugmentation and microbial enhanced oil recovery (meor). however, it is not entirely clear if "endogenous" bacteria (e.g., spores) in/on crude oil survive the autoclaving process, or influence subsequent evaluation of the hydrocarbon-degradation abilities of the "exogenous" bacterial strains. to test this, we inoculated autoclaved crude oil medium with six exo ...201223028421
novel dehalogenase mechanism for 2,3-dichloro-1-propanol utilization in pseudomonas putida strain mc4.a pseudomonas putida strain (mc4) that can utilize 2,3-dichloro-1-propanol (dcp) and several aliphatic haloacids and haloalcohols as sole carbon and energy source for growth was isolated from contaminated soil. degradation of dcp was found to start with oxidation and concomitant dehalogenation catalyzed by a 72-kda monomeric protein (dppa) that was isolated from cell lysate. the dppa gene was cloned from a cosmid library and appeared to encode a protein equipped with a signal peptide and that po ...201222752160
electrochemical characterization of escherichia coli adaptive response protein aidb.when exposed to known dna-damaging alkylating agents, escherichia coli cells increase production of four dna repair enzymes: ada, alka, alkb, and aidb. the role of three enzymes (ada, alka, and alkb) in repairing dna lesions has been well characterized, while the function of aidb is poorly understood. aidb has a distinct cofactor that is potentially related to the elusive role of aidb in adaptive response: a redox active flavin adenine dinucleotide (fad). in this study, we report the thermodynam ...201223443126
an orfan no more: the bacteriophage t4 39.2 gene product, nwgi, modulates groel chaperone function.bacteriophages are the most abundant biological entities in our biosphere, characterized by their hyperplasticity, mosaic composition, and the many unknown functions (orfans) encoded by their immense genetic repertoire. these genes are potentially maintained by the bacteriophage to allow efficient propagation on hosts encountered in nature. to test this hypothesis, we devised a selection to identify bacteriophage-encoded gene(s) that modulate the host escherichia coli groel/groes chaperone machi ...201222234860
the effect of nitrogen enrichment on c(1)-cycling microorganisms and methane flux in salt marsh sediments.methane (ch(4)) flux from ecosystems is driven by c(1)-cycling microorganisms - the methanogens and the methylotrophs. little is understood about what regulates these communities, complicating predictions about how global change drivers such as nitrogen enrichment will affect methane cycling. using a nitrogen addition gradient experiment in three southern california salt marshes, we show that sediment ch(4) flux increased linearly with increasing nitrogen addition (1.23 μg ch(4) m(-2) day(-1) fo ...201222470369
genome sequence of strain himb624, a cultured representative from the om43 clade of marine betaproteobacteria. 201222675594
analysis of bacterial core communities in the central baltic by comparative rna-dna-based fingerprinting provides links to structure-function relationships.understanding structure-function links of microbial communities is a central theme of microbial ecology since its beginning. to this end, we studied the spatial variability of the bacterioplankton community structure and composition across the central baltic sea at four stations, which were up to 450 km apart and at a depth profile representative for the central part (gotland deep, 235 m). bacterial community structure was followed by 16s ribosomal rna (rrna)- and 16s rrna gene-based fingerprint ...201121697960
analysis and manipulation of aspartate pathway genes for l-lysine overproduction from methanol by bacillus methanolicus.we investigated the regulation and roles of six aspartate pathway genes in l-lysine overproduction in bacillus methanolicus: dapg, encoding aspartokinase i (aki); lysc, encoding akii; yclm, encoding akiii; asd, encoding aspartate semialdehyde dehydrogenase; dapa, encoding dihydrodipicolinate synthase; and lysa, encoding meso-diaminopimelate decarboxylase. analysis of the wild-type strain revealed that in vivo lysc transcription was repressed 5-fold by l-lysine and induced 2-fold by dl-methionine ...201121724876
purification, crystallization and preliminary x-ray crystallographic analysis of a methanol dehydrogenase from the marine bacterium methylophaga aminisulfidivorans mp(t).methylophaga aminisulfidivorans mp(t) is a marine methylotrophic bacterium that utilizes c(1) compounds such as methanol as a carbon and energy source. the released electron from oxidation flows through a methanol-oxidizing system (mox) consisting of a series of electron-transfer proteins encoded by the mox operon. one of the key enzymes in the pathway is methanol dehydrogenase (mdh), which contains the prosthetic group pyrroloquinoline quinone (pqq) and converts methanol to formaldehyde in the ...201121505255
analysis of bacterial core communities in the central baltic by comparative rna-dna-based fingerprinting provides links to structure-function relationships.understanding structure-function links of microbial communities is a central theme of microbial ecology since its beginning. to this end, we studied the spatial variability of the bacterioplankton community structure and composition across the central baltic sea at four stations, which were up to 450 km apart and at a depth profile representative for the central part (gotland deep, 235 m). bacterial community structure was followed by 16s ribosomal rna (rrna)- and 16s rrna gene-based fingerprint ...201121697960
crystallization and preliminary crystallographic analysis of the type iil restriction enzyme mmei in complex with dna.type iil restriction enzymes have rejuvenated the search for user-specified dna binding and cutting. by aligning and contrasting the highly comparable amino-acid sequences yet diverse recognition specificities across the family of enzymes, amino acids involved in dna binding have been identified and mutated to produce alternative binding specificities. to date, the specificity of mmei (a type iil restriction enzyme) has successfully been altered at positions 3, 4 and 6 of the asymmetric tccrac ( ...201122102043
methylotrophy in a lake: from metagenomics to single organism physiology.this review provides a brief summary of on-going studies in lake washington (seattle, usa) directed at understanding of the content and activities of microbial communities involved in methylotrophy. one of the findings from culture-independent approaches, including functional metagenomics, is the prominent presence of methylotenera species in the site and their inferred activity in c1 metabolism, highlighting the local environmental importance of this group. comparative analyses of individual ge ...201121622781
Biochemical and Mutational Studies of the Bacillus cereus CECT 5050T Formamidase Support the Existence of a C-E-E-K Tetrad in Several Members of the Nitrilase Superfamily.Formamidases (EC 3.5.1.49) are poorly characterized proteins. In spite of this scarce knowledge, ammonia has been described as playing a central role in the pathogenesis of human pathogens such as Helicobacter pylori, for which formamidase has been shown to participate in the nitrogen metabolic pathway. Sequence analysis has revealed that at least two different groups of formamidases are classified as EC 3.5.1.49: on the one hand, the derivatives of the FmdA-AmdA superfamily, which are the best ...201121705545
application of the bacteriophage mu-driven system for the integration/amplification of target genes in the chromosomes of engineered gram-negative bacteria--mini review.the advantages of phage mu transposition-based systems for the chromosomal editing of plasmid-less strains are reviewed. the cis and trans requirements for mu phage-mediated transposition, which include the l/r ends of the mu dna, the transposition factors mua and mub, and the cis/trans functioning of the e element as an enhancer, are presented. mini-mu(lr)/(ler) units are mu derivatives that lack most of the mu genes but contain the l/r ends or a properly arranged e element in cis to the l/r en ...201121698377
crystal structure of histamine dehydrogenase from nocardioides simplex.histamine dehydrogenase (hadh) isolated from nocardioides simplex catalyzes the oxidative deamination of histamine to imidazole acetaldehyde. hadh is highly specific for histamine, and we are interested in understanding the recognition mode of histamine in its active site. we describe the first crystal structure of a recombinant form of hadh (hadh) to 2.7-a resolution. hadh is a homodimer, where each 76-kda subunit contains an iron-sulfur cluster ([4fe-4s](2+)) and a 6-s-cysteinyl flavin mononuc ...201020538584
impact of the n5-proximal asn on the thermodynamic and kinetic stability of the semiquinone radical in photolyase.flavoproteins can dramatically adjust the thermodynamics and kinetics of electron transfer at their flavin cofactor. a versatile regulatory tool is proton transfer. here, we demonstrate the significance of proton-coupled electron transfer to redox tuning and semiquinone (sq) stability in photolyases (pls) and cryptochromes (crys). these light-responsive proteins share homologous overall architectures and fad-binding pockets, yet they have evolved divergent functions that include dna repair, phot ...201021131361
impact of the n5-proximal asn on the thermodynamic and kinetic stability of the semiquinone radical in photolyase.flavoproteins can dramatically adjust the thermodynamics and kinetics of electron transfer at their flavin cofactor. a versatile regulatory tool is proton transfer. here, we demonstrate the significance of proton-coupled electron transfer to redox tuning and semiquinone (sq) stability in photolyases (pls) and cryptochromes (crys). these light-responsive proteins share homologous overall architectures and fad-binding pockets, yet they have evolved divergent functions that include dna repair, phot ...201021131361
aromatic amino acid auxotrophs constructed by recombinant marker exchange in methylophilus methylotrophus as1 cells expressing the arop-encoded transporter of escherichia coli.the isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolic engineering of obligate methylotrophs, remains a rather complicated task. we describe a novel method of constructing mutants of the bacterium methylophilus methylotrophus as1 that are auxotrophic for aromatic amino acids. the procedure begins with the mu-driven integration of the escherichia coli gene arop, which encodes the common aromatic amino acid transporter, into the genome of m. ...201019880640
aromatic amino acid auxotrophs constructed by recombinant marker exchange in methylophilus methylotrophus as1 cells expressing the arop-encoded transporter of escherichia coli.the isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolic engineering of obligate methylotrophs, remains a rather complicated task. we describe a novel method of constructing mutants of the bacterium methylophilus methylotrophus as1 that are auxotrophic for aromatic amino acids. the procedure begins with the mu-driven integration of the escherichia coli gene arop, which encodes the common aromatic amino acid transporter, into the genome of m. ...201019880640
occurrence and sequence of sphaeroides heme protein and diheme cytochrome c in purple photosynthetic bacteria in the family rhodobacteraceae.sphaeroides heme protein (shp) was discovered in the purple photosynthetic bacterium, rhodobacter sphaeroides, and is the only known c-type heme protein that binds oxygen. although initially not believed to be widespread among the photosynthetic bacteria, the gene has now been found in more than 40 species of proteobacteria and generally appears to be regulated. rb. sphaeroides is exceptional in not having regulatory genes associated with the operon. we have thus analyzed additional purple bacte ...201020587053
growth of wildtype and mutant e. coli strains in minimal media for optimal production of nucleic acids for preparing labeled nucleotides.since rnas lie at the center of most cellular processes, there is a need for synthesizing large amounts of rnas made from stable isotope-labeled nucleotides to advance the study of their structure and dynamics by nuclear magnetic resonance (nmr) spectroscopy. a particularly effective means of obtaining labeled nucleotides is to harvest these nucleotides from bacteria grown in defined minimal media supplemented with 15nh4cl and various carbon sources. given the high cost of carbon precursors requ ...201020730533
a conserved active-site threonine is important for both sugar and flavin oxidations of pyranose 2-oxidase.pyranose 2-oxidase (p2o) catalyzes the oxidation by o(2) of d-glucose and several aldopyranoses to yield the 2-ketoaldoses and h(2)o(2). based on crystal structures, in one rotamer conformation, the threonine hydroxyl of thr(169) forms h-bonds to the flavin-n5/o4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the p2o.sugar complex. transient kinetics of wild-type (wt) and thr(169) --> s/n/g/a replacement variants show that d-glc binds to t169s, t169n, ...201020089849
superpose3d: a local structural comparison program that allows for user-defined structure representations.local structural comparison methods can be used to find structural similarities involving functional protein patches such as enzyme active sites and ligand binding sites. the outcome of such analyses is critically dependent on the representation used to describe the structure. indeed different categories of functional sites may require the comparison program to focus on different characteristics of the protein residues. we have therefore developed superpose3d, a novel structural comparison softw ...201020700534
modular architecture of nucleotide-binding pockets.recently, modularity has emerged as a general attribute of complex biological systems. this is probably because modular systems lend themselves readily to optimization via random mutation followed by natural selection. although they are not traditionally considered to evolve by this process, biological ligands are also modular, being composed of recurring chemical fragments, and moreover they exhibit similarities reminiscent of mutations (e.g. the few atoms differentiating adenine and guanine). ...201020185567
analysis of the electrochemistry of hemes with e(m)s spanning 800 mv.the free energy of heme reduction in different proteins is found to vary over more than 18 kcal/mol. it is a challenge to determine how proteins manage to achieve this enormous range of e(m)s with a single type of redox cofactor. proteins containing 141 unique hemes of a-, b-, and c-type, with bis-his, his-met, and aquo-his ligation were calculated using multi-conformation continuum electrostatics (mcce). the experimental e(m)s range over 800 mv from -350 mv in cytochrome c(3) to 450 mv in cytoc ...200919003997
cloning and analysis of a bifunctional methyltransferase/restriction endonuclease tspgwi, the prototype of a thermus sp. enzyme family.restriction-modification systems are a diverse class of enzymes. they are classified into four major types: i, ii, iii and iv. we have previously proposed the existence of a thermus sp. enzyme family, which belongs to type ii restriction endonucleases (reases), however, it features also some characteristics of types i and iii. members include related thermophilic endonucleases: tspgwi, taqii, tspdti, and tth111ii.200919480701
the mmei family: type ii restriction-modification enzymes that employ single-strand modification for host protection.the type ii restriction endonucleases form one of the largest families of biochemically-characterized proteins. these endonucleases typically share little sequence similarity, except among isoschizomers that recognize the same sequence. mmei is an unusual type ii restriction endonuclease that combines endonuclease and methyltransferase activities in a single polypeptide. mmei cuts dna 20 bases from its recognition sequence and modifies just one dna strand for host protection. using mmei as query ...200919578066
functional and shunt states of bacteriorhodopsin resolved by 250 ghz dynamic nuclear polarization-enhanced solid-state nmr.observation and structural studies of reaction intermediates of proteins are challenging because of the mixtures of states usually present at low concentrations. here, we use a 250 ghz gyrotron (cyclotron resonance maser) and cryogenic temperatures to perform high-frequency dynamic nuclear polarization (dnp) nmr experiments that enhance sensitivity in magic-angle spinning nmr spectra of cryo-trapped photocycle intermediates of bacteriorhodopsin (br) by a factor of approximately 90. multidimensio ...200919474298
functional analysis of mmei from methanol utilizer methylophilus methylotrophus, a subtype iic restriction-modification enzyme related to type i enzymes.mmei from methylophilus methylotrophus belongs to the type ii restriction-modification enzymes. it recognizes an asymmetric dna sequence, 5'-tccrac-3' (r indicates g or a), and cuts both strands at fixed positions downstream of the specific site. this particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). we have shown previously that the endonucleolytic activity of mmei is strongly dependent on the presence of s-ad ...200918997032
oligomeric structure and functional characterization of the urea transporter from actinobacillus pleuropneumoniae.urea transporters (uts) facilitate urea permeation across cell membranes in prokaryotes and eukaryotes. bacteria use urea as a means to survive in acidic environments and/or as a nitrogen source. the ut from actinobacillus pleuropneumoniae, aput, the pathogen that causes porcine pleurisy and pneumonia, was expressed in escherichia coli and purified. analysis of the recombinant protein using cross-linking and blue-native gel electrophoresis established that aput is a dimer in detergent solution. ...200919361419
functional characterization of the re-face loop spanning residues 536-541 and its interactions with the cofactor in the flavin mononucleotide-binding domain of flavocytochrome p450 from bacillus megaterium.flavocytochrome p450bm-3, a bacterial monooxygenase, contains a flavin mononucleotide-binding domain bearing a strong structural homology to the bacterial flavodoxin. the flavin mononucleotide (fmn) serves as the one-electron donor to the heme iron, but in contrast to the electron transfer mechanism of mammalian cytochrome p450 reductase, the fmn semiquinone state is not thermodynamically stable and appears transiently as the anionic rather than the neutral form. a unique loop region comprised o ...200919432415
functional analysis of mmei from methanol utilizer methylophilus methylotrophus, a subtype iic restriction-modification enzyme related to type i enzymes.mmei from methylophilus methylotrophus belongs to the type ii restriction-modification enzymes. it recognizes an asymmetric dna sequence, 5'-tccrac-3' (r indicates g or a), and cuts both strands at fixed positions downstream of the specific site. this particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). we have shown previously that the endonucleolytic activity of mmei is strongly dependent on the presence of s-ad ...200918997032
quantitative rrna-targeted solution-based hybridization assay using peptide nucleic acid molecular beacons.the potential of a solution-based hybridization assay using peptide nucleic acid (pna) molecular beacon (mb) probes to quantify 16s rrna of specific populations in rna extracts of environmental samples was evaluated by designing pna mb probes for the genera dechloromonas and dechlorosoma. in a kinetic study with 16s rrna from pure cultures, the hybridization of pna mb to target 16s rrna exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to no ...200818820054
characterization of a novel methanol dehydrogenase in representatives of burkholderiales: implications for environmental detection of methylotrophy and evidence for convergent evolution.some members of burkholderiales are able to grow on methanol but lack the genes (mxafi) responsible for the well-characterized two-subunit pyrroloquinoline quinone-dependent quinoprotein methanol dehydrogenase that is widespread in methylotrophic proteobacteria. here, we characterized novel, mono-subunit enzymes responsible for methanol oxidation in four strains, methyloversatilis universalis fam5, methylibium petroleiphilum pm1, and unclassified burkholderiales strains rz18-153 and fam1. the en ...200818390659
a comparison of random sequence reads versus 16s rdna sequences for estimating the biodiversity of a metagenomic library.the construction of metagenomic libraries has permitted the study of microorganisms resistant to isolation and the analysis of 16s rdna sequences has been used for over two decades to examine bacterial biodiversity. here, we show that the analysis of random sequence reads (rsrs) instead of 16s is a suitable shortcut to estimate the biodiversity of a bacterial community from metagenomic libraries. we generated 10,010 rsrs from a metagenomic library of microorganisms found in human faecal samples. ...200818682527
expression, purification, crystallization and preliminary x-ray studies of histamine dehydrogenase from nocardioides simplex.histamine dehydrogenase (hadh) from nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and an ammonium ion. hadh is functionally related to trimethylamine dehydrogenase (tmadh), but hadh has strict substrate specificity towards histamine. hadh is a homodimer, with each 76 kda subunit containing two redox cofactors: a [4fe-4s] cluster and an unusual covalently bound flavin mononucleotide, 6-s-cysteinyl-fmn. in order to understand the substrate ...200818765904
mmei: a minimal type ii restriction-modification system that only modifies one dna strand for host protection.mmei is an unusual type ii restriction enzyme that is useful for generating long sequence tags. we have cloned the mmei restriction-modification (r-m) system and found it to consist of a single protein having both endonuclease and dna methyltransferase activities. the protein comprises an amino-terminal endonuclease domain, a central dna methyltransferase domain and c-terminal dna recognition domain. the endonuclease cuts the two dna strands at one site simultaneously, with enzyme bound at two s ...200818931376
key labeling technologies to tackle sizeable problems in rna structural biology.the ability to adopt complex three-dimensional (3d) structures that can rapidly interconvert between multiple functional states (folding and dynamics) is vital for the proper functioning of rnas. consequently, rna structure and dynamics necessarily determine their biological function. in the post-genomic era, it is clear that rnas comprise a larger proportion (>50%) of the transcribed genome compared to proteins (< or =2%). yet the determination of the 3d structures of rnas lags considerably beh ...200819325801
relative axial ligand orientation in bis(imidazole)iron(ii) porphyrinates: are "picket fence" derivatives different?the synthesis of three new bis(imidazole)-ligated iron(ii) picket fence porphyrin derivatives, [fe(tpivpp)(1-rim) 2] 1-rim = 1-methyl-, 1-ethyl-, or 1-vinylimidazole) are reported. x-ray structure determinations reveal that the steric requirements of the four alpha,alpha,alpha,alpha-o-pivalamidophenyl groups lead to very restricted rotation of the imidazole ligand on the picket side of the porphyrin plane; the crowding leads to an imidazole plane orientation eclipsing an iron-porphyrin nitrogen ...200818351735
insights into the mechanism of flavoprotein-catalyzed amine oxidation from nitrogen isotope effects on the reaction of n-methyltryptophan oxidase.the mechanism of n-methyltryptophan oxidase, a flavin-dependent amine oxidase from escherichia coli, was studied using a combination of kinetic isotope effects and theoretical calculations. the 15(kcat/km) kinetic isotope effect for sarcosine oxidation is ph-dependent with a limiting value of 0.994-0.995 at high ph. density functional theory calculations on model systems were used to interpret these isotope effects. the isotope effects are inconsistent with proposed mechanisms involving covalent ...200717542620
dissection of the caffeate respiratory chain in the acetogen acetobacterium woodii: identification of an rnf-type nadh dehydrogenase as a potential coupling site.the anaerobic acetogenic bacterium acetobacterium woodii couples caffeate reduction with electrons derived from hydrogen to the synthesis of atp by a chemiosmotic mechanism with sodium ions as coupling ions, a process referred to as caffeate respiration. we addressed the nature of the hitherto unknown enzymatic activities involved in this process and their cellular localization. cell extract of a. woodii catalyzes h(2)-dependent caffeate reduction. this reaction is strictly atp dependent but can ...200717873051
clonothrix fusca roze 1896, a filamentous, sheathed, methanotrophic gamma-proteobacterium.crenothrix polyspora cohn 1870 and clonothrix fusca roze 1896 are two filamentous, sheathed microorganisms exhibiting complex morphological differentiation, whose phylogeny and physiology have been obscure for a long time due to the inability to cultivate them. very recently, dna sequencing data from uncultured c. polyspora-enriched material have suggested that crenothrix is a methane-oxidizing gamma-proteobacterium (39). in contrast, the possible ecological function of c. fusca, originally cons ...200717416684
protein aq_1862 from the hyperthermophilic bacterium aquifex aeolicus is a porin and contains two conductance pathways of different selectivity.the "hypothetical protein" aq_1862 was isolated from the membrane fraction of aquifex aeolicus and identified as the major porin. in experiments with one conducting unit (molecule) a conductance of 1.4 ns was observed in 0.1 m kcl at ph 7.5. this stable (basic) conductance was superimposed by conductance fluctuations of approximately 0.25 ns. because both events were always observed simultaneously, it is suggested that they are caused by the same molecular entity. nonetheless they show very diff ...200717586565
construction of stably maintained non-mobilizable derivatives of rsf1010 lacking all known elements essential for mobilization.rsf1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. rsf1010-derived plasmid vectors are widely used in both basic research and industrial applications. in the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing dna fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. previously, several mutations significantly decreasin ...200718028554
composition and dynamics of bacterial communities of a drinking water supply system as assessed by rna- and dna-based 16s rrna gene fingerprinting.bacterial community dynamics of a whole drinking water supply system (dwss) were studied from source to tap. raw water for this dwss is provided by two reservoirs with different water characteristics in the harz mountains of northern germany. samples were taken after different steps of treatment of raw water (i.e., flocculation, sand filtration, and chlorination) and at different points along the supply system to the tap. rna and dna were extracted from the sampled water. the 16s rrna or its gen ...200616517632
primer extension enrichment reaction (peer): a new subtraction method for identification of genetic differences between biological specimens.we developed a conceptually new subtraction strategy for the detection and isolation of target dna and/or rna from complex nucleic acid mixtures, called primer extension enrichment reaction (peer). peer uses adapters and class iis restriction enzymes to generate tagged oligonucleotides from dsdna fragments derived from specimens containing an unknown target ('tester'). subtraction is achieved by selectively disabling these oligonucleotides by extension reaction using ddntps and a double stranded ...200616790564
functional annotation by identification of local surface similarities: a novel tool for structural genomics.protein function is often dependent on subsets of solvent-exposed residues that may exist in a similar three-dimensional configuration in non homologous proteins thus having different order and/or spacing in the sequence. hence, functional annotation by means of sequence or fold similarity is not adequate for such cases.200516076399
soluble expression of recombinant proteins in the cytoplasm of escherichia coli.pure, soluble and functional proteins are of high demand in modern biotechnology. natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing. eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its i ...200515629064
characterization of a bifunctional puta homologue from bradyrhizobium japonicum and identification of an active site residue that modulates proline reduction of the flavin adenine dinucleotide cofactor.puta is a bifunctional flavoenzyme in bacteria that catalyzes the four-electron oxidation of proline to glutamate. in certain prokaryotes such as escherichia coli, puta is also a transcriptional repressor of the proline utilization (put) genes and thus is trifunctional. in this work, we have begun to assess differences between bifunctional and trifunctional puta enzymes by examining the puta protein from bradyrhizobium japonicum (bjputa). primary structure analysis of bjputa shows it lacks the d ...200515966737
specific detection, isolation, and characterization of selected, previously uncultured members of the freshwater bacterioplankton community.high-throughput cultivation was combined with rapid and group-specific phylogenetic fingerprinting in order to recover representatives of three freshwater bacterioplankton communities. a total of 570 bacterial cultures were obtained by employing the most probable number and microdrop techniques. the majority of the cultured bacteria were closely related to previously uncultured bacteria and grouped with the alpha-proteobacteria, beta-proteobacteria, actinobacteria, firmicutes, or flavobacteria-c ...200516204504
kinetic isotope effects and ligand binding in pqq-dependent methanol dehydrogenase.the reaction of pqq (2,7,9-tricarboxypyrroloquinoline quinone)-dependent mdh (methanol dehydrogenase) from methylophilus methylotrophus has been studied under steady-state conditions in the presence of an alternative activator [gee (glycine ethyl ester)] and compared with similar reactions performed with ammonium (used more generally as an activator in steady-state analysis of mdh). studies of initial velocity with methanol (protiated methanol, c1h3o1h) and [2h]methanol (deuteriated methanol, c2 ...200515617516
l-selective amidase with extremely broad substrate specificity from ochrobactrum anthropi ncimb 40321.an industrially attractive l-specific amidase was purified to homogeneity from ochrobactrum anthropi ncimb 40321 wild-type cells. the purified amidase displayed maximum initial activity between ph 6 and 8.5 and was fully stable for at least 1 h up to 60 degrees c. the purified enzyme was strongly inhibited by the metal-chelating compounds edta and 1,10-phenanthroline. the activity of the edta-treated enzyme could be restored by the addition of zn2+ (to 80%), mn2+ (to 400%), and mg2+ (to 560%). s ...200516332774
investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rrna analysis, and fluorescent in situ hybridization-microautoradiography.the acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (sip). [13c]acetate was used in sip to label the dna of the denitrifiers. the [13c]dna fraction that was extracted was subjected to a full-cycle rrna analysis. the dominant 16s rrna gene phylotypes in the 13c library were closely related to the bacterial families comamonadaceae and rhodocyclaceae in the class betaproteobacteria. seven oligonuc ...200516332863
ligand orientation control in low-spin six-coordinate (porphinato)iron(ii) species.the synthesis of a low-spin six-coordinate iron(ii) porphyrinate in which the two axial ligands are forced to have a relative perpendicular orientation has been successfully accomplished for the first time. the reaction of four-coordinate (tetramesitylporphinato)iron(ii) with 2-methylimidazole leads to the preparation of [fe(tmp)(2-mehim)(2)] which cocrystallizes with five-coordinate [fe(tmp)(2-mehim)]. the six-coordinate complex accommodates the sterically crowded pair of imidazoles with a stro ...200515934765
microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria.traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. new approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environm ...200516332866
kinetics of dithionite-dependent reduction of cytochrome p450 3a4: heterogeneity of the enzyme caused by its oligomerization.to explore the basis of apparent conformational heterogeneity of cytochrome p450 3a4 (cyp3a4), the kinetics of dithionite-dependent reduction was studied in solution, in proteoliposomes, and in nanodiscs. in cyp3a4 oligomers in solution the kinetics obeys a three-exponential equation with similar amplitudes of each of the phases. addition of substrate (bromocriptine) displaces the phase distribution toward the slow phase at the expense of the fast one, while the middle phase remains unaffected. ...200516229479
determination of enzyme mechanisms by molecular dynamics: studies on quinoproteins, methanol dehydrogenase, and soluble glucose dehydrogenase.molecular dynamics (md) simulations have been carried out to study the enzymatic mechanisms of quinoproteins, methanol dehydrogenase (mdh), and soluble glucose dehydrogenase (sgdh). the mechanisms of reduction of the orthoquinone cofactor (pqq) of mdh and sgdh involve concerted base-catalyzed proton abstraction from the hydroxyl moiety of methanol or from the 1-hydroxyl of glucose, and hydride equivalent transfer from the substrate to the quinone carbonyl carbon c5 of pqq. the products of methan ...200415273299
pcogr: phylogenetic cog ranking as an online tool to judge the specificity of cogs with respect to freely definable groups of organisms.the rapidly increasing number of completely sequenced genomes led to the establishment of the cog-database which, based on sequence homologies, assigns similar proteins from different organisms to clusters of orthologous groups (cogs). there are several bioinformatic studies that made use of this database to determine (hyper)thermophile-specific proteins by searching for cogs containing (almost) exclusively proteins from (hyper)thermophilic genomes. however, public software to perform individual ...200415488147
flow sorting of marine bacterioplankton after fluorescence in situ hybridization.we describe an approach to sort cells from coastal north sea bacterioplankton by flow cytometry after in situ hybridization with rrna-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (card-fish). in a sample from spring 2003 >90% of the cells were detected by card-fish with a bacterial probe (eub338). approximately 30% of the microbial assemblage was affiliated with the cytophaga-flavobacterium lineage of the bacteroidetes (cfb group) ( ...200415466568
integron diversity in heavy-metal-contaminated mine tailings and inferences about integron evolution.integrons are horizontal gene transfer (hgt) systems containing elements necessary for site-specific recombination and expression of foreign dna. the overall phylogenetic distribution of integrons and range of genes that can be transferred by integrons are unknown. this report contains an exploration of integrons in an environmental microbial community and an investigation of integron evolution. first, using culture-independent techniques, we explored the diversity of integrons and integron-tran ...200414766601
molecular basis of bacterial outer membrane permeability revisited.gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. this review summarizes the development in the field since ...200314665678
a mutant of paracoccus denitrificans with disrupted genes coding for cytochrome c550 and pseudoazurin establishes these two proteins as the in vivo electron donors to cytochrome cd1 nitrite reductase.in paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc(1) complex to the periplasmic nitrite reductase, cytochrome cd(1). the periplasmic protein cytochrome c(550) has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells. here, the hypothesis that cytochrome c(550) and pseudoazurin are alternative el ...200314563865
bacterioplankton community shifts in an arctic lake correlate with seasonal changes in organic matter source.seasonal shifts in bacterioplankton community composition in toolik lake, a tundra lake on the north slope of alaska, were related to shifts in the source (terrestrial versus phytoplankton) and lability of dissolved organic matter (dom). a shift in community composition, measured by denaturing gradient gel electrophoresis (dgge) of 16s rrna genes, occurred at 4 degrees c in near-surface waters beneath seasonal ice and snow cover in spring. this shift was associated with an annual peak in bacteri ...200312676708
two distinct alcohol dehydrogenases participate in butane metabolism by pseudomonas butanovora.the involvement of two primary alcohol dehydrogenases, bdh and boh, in butane utilization in pseudomonas butanovora (atcc 43655) was demonstrated. the genes coding for boh and bdh were isolated and characterized. the deduced amino acid sequence of boh suggests a 67-kda alcohol dehydrogenase containing pyrroloquinoline quinone (pqq) as cofactor and in the periplasm (29-residue leader sequence). the deduced amino acid sequence of bdh is consistent with a 70.9-kda, soluble, periplasmic (37-residue ...200211889098
cloning and heterologous expression of an enantioselective amidase from rhodococcus erythropolis strain mp50.the gene for an enantioselective amidase was cloned from rhodococcus erythropolis mp50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. the gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kda. the deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. the nucleotide sequence approximately 2.5 kb upstream and downstream of t ...200212089004
isolation and metabolic characteristics of previously uncultured members of the order aquificales in a subsurface gold mine.culture-dependent and -independent techniques were combined to characterize the physiological properties and the ecological impacts of culture-resistant phylotypes of thermophiles within the order aquificales from a subsurface hot aquifer of a japanese gold mine. thermophilic bacteria phylogenetically associated with previously uncultured phylotypes of aquificales were successfully isolated. 16s ribosomal dna clone analysis of the entire microbial dna assemblage and fluorescence in situ whole-ce ...200212039766
genomic signature tags (gsts): a system for profiling genomic dna.genomic signature tags (gsts) are the products of a method we have developed for identifying and quantitatively analyzing genomic dnas. the dna is initially fragmented with a type ii restriction enzyme. an oligonucleotide adaptor containing a recognition site for mmei, a type iis restriction enzyme, is then used to release 21-bp tags from fixed positions in the dna relative to the sites recognized by the fragmenting enzyme. these tags are pcr-amplified, purified, concatenated, and then cloned an ...200212421763
rhizobium leguminosarum has a second general amino acid permease with unusually broad substrate specificity and high similarity to branched-chain amino acid transporters (bra/liv) of the abc family.amino acid uptake by rhizobium leguminosarum is dominated by two abc transporters, the general amino acid permease (aap) and the branched-chain amino acid permease (bra(rl)). characterization of the solute specificity of bra(rl) shows it to be the second general amino acid permease of r. leguminosarum. although bra(rl) has high sequence identity to members of the family of hydrophobic amino acid transporters (haat), it transports a broad range of solutes, including acidic and basic polar amino a ...200212107123
amia is a negative regulator of acetamidase expression in mycobacterium smegmatis.the acetamidase of mycobacterium smegmatis is a highly inducible enzyme. expression of this enzyme is increased 100-fold when the substrate acetamide is present. the acetamidase gene is found immediately downstream of three open reading frames. two of these are proposed to be involved in regulation.200111570974
chloromethane utilization gene cluster from hyphomicrobium chloromethanicum strain cm2(t) and development of functional gene probes to detect halomethane-degrading bacteria.hyphomicrobium chloromethanicum cm2(t), an aerobic methylotrophic member of the alpha subclass of the class proteobacteria, can grow with chloromethane as the sole carbon and energy source. h. chloromethanicum possesses an inducible enzyme system for utilization of chloromethane, in which two polypeptides (67-kda cmua and 35-kda cmub) are expressed. previously, four genes, cmua, cmub, cmuc, and puru, were shown to be essential for growth of methylobacterium chloromethanicum on chloromethane. the ...200111133460
catalytic mechanism of quinoprotein methanol dehydrogenase: a theoretical and x-ray crystallographic investigation.the catalytic mechanism of the reductive half reaction of the quinoprotein methanol dehydrogenase (mdh) is believed to proceed either through a hemiketal intermediate or by direct transfer of a hydride ion from the substrate methyl group to the cofactor, pyrroloquinoline quinone (pqq). a crystal structure of the enzyme-substrate complex of a similar quinoprotein, glucose dehydrogenase, has recently been reported that strongly favors the hydride transfer mechanism in that enzyme. a theoretical an ...200111149955
multiphasic kinetics of transformation of 1,2,4-trichlorobenzene at nano- and micromolar concentrations by burkholderia sp. strain ps14.the transformation of 1,2,4-trichlorobenzene (1,2,4-tcb) at initial concentrations in nano- and micromolar ranges was studied in batch experiments with burkholderia sp. strain ps14. 1,2,4-tcb was metabolized from nano- and micromolar concentrations to below its detection limit of 0.5 nm. at low initial 1,2,4-tcb concentrations, a first-order relationship between specific transformation rate and substrate concentration was observed with a specific affinity (a(0)(a)) of 0.32 liter. mg (dry weight) ...200111472925
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