253 research outputs found
The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen
BACKGROUND: The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic Ī²-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway. RESULTS: Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95Ā Mb contained a 70Ā kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58Eu(T), than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate. CONCLUSIONS: The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen
Linalool isomerase, a membrane-anchored enzyme in the anaerobic monoterpene degradation in Thauera linaloolentis 47Lol
Background: Thauera linaloolentis 47Lol uses the tertiary monoterpene alcohol (R,S)-linalool as sole carbon and energy source under denitrifying conditions. The conversion of linalool to geraniol had been observed in carbon-excess cultures, suggesting the presence of a 3,1-hydroxyl-Delta(1)-Delta(2)-mutase (linalool isomerase) as responsible enzyme. To date, only a single enzyme catalyzing such a reaction is described: the linalool dehydratase/isomerase (Ldi) from Castellaniella defragrans 65Phen acting only on (S)-linalool. Results: The linalool isomerase activity was located in the inner membrane. It was enriched by subcellular fractionation and sucrose gradient centrifugation. MALDI-ToF MS analysis of the enriched protein identified the corresponding gene named lis that codes for the protein in the strain with the highest similarity to the Ldi. Linalool isomerase is predicted to have four transmembrane helices at the N-terminal domain and a cytosolic domain. Enzyme activity required a reductant for activation. A specific activity of 3.42 +/- 0.28 nkat mg * protein(-1) and a k(M) value of 455 +/- 124 mu M were determined for the thermodynamically favored isomerization of geraniol to both linalool isomers at optimal conditions of pH 8 and 35 degrees C. Conclusion: The linalool isomerase from T. linaloolentis 47Lol represents a second member of the enzyme class 5.4.4.4, next to the linalool dehydratase/isomerase from C. defragrans 65Phen. Besides considerable amino acid sequence similarity both enzymes share common characteristics with respect to substrate affinity, pH and temperature optima, but differ in the dehydratase activity and the turnover of linalool isomers
Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors
Detergents enable the purification of membrane proteins and are indispensable reagents instructural biology. Even though a large variety of detergents have been developed in the lastcentury, the challenge remains to identify guidelines that allowfine-tuning of detergents forindividual applications in membrane protein research. Addressing this challenge, here weintroduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS)reveals that the modular OGD architecture offers the ability to control protein purificationand to preserve interactions with native membrane lipids during purification. In addition to abroad range of bacterial membrane proteins, OGDs also enable the purification and analysisof a functional G-protein coupled receptor (GPCR). Moreover, given the modular design ofthese detergents, we anticipatefine-tuning of their properties for specific applications instructural biology. Seen from a broader perspective, this represents a significant advance forthe investigation of membrane proteins and their interactions with lipids
The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen
Background: The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic beta-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway. Results: Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58Eu(T), than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH: ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate. Conclusions: The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen
Functional characterization of polysaccharide utilization loci in the marine Bacteroidetes 'Gramella forsetii' KT0803
Members of the phylum Bacteroidetes are abundant in many marine ecosystems and are known to have a pivotal role in the mineralization of complex organic substrates such as polysaccharides and proteins. We studied the decomposition of the algal glycans laminarin and alginate by 'Gramella forsetii' KT0803, a bacteroidetal isolate from North Sea surface waters. A combined application of isotope labeling, subcellular protein fractionation and quantitative proteomics revealed two large polysaccharide utilization loci (PULs) that were specifically induced, one by alginate and the other by laminarin. These regulons comprised genes of surface-exposed proteins such as oligomer transporters, substrate-binding proteins, carbohydrate-active enzymes and hypothetical proteins. Besides, several glycan-specific TonB-dependent receptors and SusD-like substrate-binding proteins were expressed also in the absence of polysaccharide substrates, suggesting an anticipatory sensing function. Genes for the utilization of the beta-1,3-glucan laminarin were found to be co-regulated with genes for glucose and alpha-1,4-glucan utilization, which was not the case for the non-glucan alginate. Strong syntenies of the PULs of 'G. forsetii' with similar loci in other Bacteroidetes indicate that the specific response mechanisms of 'G. forsetii' to changes in polysaccharide availability likely apply to other Bacteroidetes. Our results can thus contribute to an improved understanding of the ecological niches of marine Bacteroidetes and their roles in the polysaccharide decomposition part of carbon cycling in marine ecosystems
Comparative proteomics of related symbiotic mussel species reveals high variability of host-symbiont interactions.
Deep-sea Bathymodiolus mussels and their chemoautotrophic symbionts are well-studied representatives of mutualistic host-microbe associations. However, how host-symbiont interactions vary on the molecular level between related host and symbiont species remains unclear. Therefore, we compared the host and symbiont metaproteomes of Pacific B. thermophilus, hosting a thiotrophic symbiont, and Atlantic B. azoricus, containing two symbionts, a thiotroph and a methanotroph. We identified common strategies of metabolic support between hosts and symbionts, such as the oxidation of sulfide by the host, which provides a thiosulfate reservoir for the thiotrophic symbionts, and a cycling mechanism that could supply the host with symbiont-derived amino acids. However, expression levels of these processes differed substantially between both symbioses. Backed up by genomic comparisons, our results furthermore revealed an exceptionally large repertoire of attachment-related proteins in the B. thermophilus symbiont. These findings imply that host-microbe interactions can be quite variable, even between closely related systems
Sequential Monte Carlo multiple testing
Motivation: In molecular biology, as in many other scientific fields, the scale of analyses is ever increasing. Often, complex Monte Carlo simulation is required, sometimes within a large-scale multiple testing setting. The resulting computational costs may be prohibitively high
Aquatic adaptation of a laterally acquired pectin degradation pathway in marine gammaproteobacteria
Mobile genomic islands distribute functional traits between microbes and habitats, yet it remains unclear how their proteins adapt to new environments. Here we used a comparative phylogenomic and proteomic approach to show that the marine bacterium Pseudoalteromonas haloplanktis ANT/505 acquired a genomic island with a functional pathway for pectin catabolism. Bioinformatics and biochemical experiments revealed that this pathway encodes a series of carbohydrate-active enzymes including two multimodular pectate lyases, PelA and PelB. PelA is a large enzyme with a polysaccharide lyase family 1 (PL1) domain and a carbohydrate esterase family 8 domain, and PelB contains a PL1 domain and two carbohydrate-binding domains of family 13. Comparative phylogenomic analyses indicate that the pathway was most likely acquired from terrestrial microbes, yet we observed multi-modular orthologues only in marine bacteria. Proteomic experiments showed that P. haloplanktis ANT/505 secretes both pectate lyases into the environment in the presence of pectin. These multi-modular enzymes may therefore represent a marine innovation that enhances physical interaction with pectins to reduce loss of substrate and enzymes by diffusion. Our results revealed that marine bacteria can catabolize pectin, and highlight enzyme fusion as a potential adaptation that may facilitate microbial consumption of polymeric substrates in aquatic environments
Individual Physiological Adaptations Enable Selected Bacterial Taxa To Prevail during Long-Term Incubations
Enclosure experiments are frequently used to investigate the impact of
changing environmental conditions on microbial assemblages. Yet, how the incuba-
tion itself challenges complex bacterial communities is thus far unknown. In this
study, metaproteomic profiling, 16S rRNA gene analyses, and cell counts were com-
bined to evaluate bacterial communities derived from marine, mesohaline, and oli-
gohaline conditions after long-term batch incubations. Early in the experiment, the
three bacterial communities were highly diverse and differed significantly in their
compositions. Manipulation of the enclosures with terrigenous dissolved organic car-
bon resulted in notable differences compared to the control enclosures at this early
phase of the experiment. However, after 55 days, bacterial communities in the ma-
nipulated and the control enclosures under marine and mesohaline conditions were
all dominated by gammaproteobacterium Spongiibacter. In the oligohaline enclo-
sures, actinobacterial cluster I of the hgc group (hgc-I) remained abundant in the late
phase of the incubation. Metaproteome analyses suggested that the ability to use
outer membrane-based internal energy stores, in addition to the previously de-
scribed grazing resistance, may enable the gammaproteobacterium Spongiibacter to
prevail in long-time incubations. Under oligohaline conditions, the utilization of ex-
ternal recalcitrant carbon appeared to be more important (hgc-I). Enclosure experi-
ments with complex natural microbial communities are important tools to investi-
gate the effects of manipulations. However, species-specific properties, such as
individual carbon storage strategies, can cause manipulation-independent effects
and need to be considered when interpreting results from enclosures.This study was financially supported by the SAW-funded ATKiM project, which provided funds to D. P. R. Herlemann, C. Meeske, K. JĆ¼rgens, S. Markert, and T. Schweder. D. P. R. Herlemann was also supported by the European Regional Develop- ment Fund/Estonian Research Council funded Mobilitas Plus Top Researcher grant MOBTT24. We thank the crew and captain of the RV Meteor (M86, M87) for support during the research cruise. The computations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) at the PDC Centre for High Performance Computing (PDC-HPC) and Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX). We thank Jana Matulla for excellent technical assis- tance and Stephan Fuchs for his help and advice in MS database construction. We also thank Stefan E. Heiden for valuable help with the CDD BLAST analyses.This study was financially supported by the SAW-funded ATKiM project, which
provided funds to D. P. R. Herlemann, C. Meeske, K. JĆ¼rgens, S. Markert, and T.
Schweder. D. P. R. Herlemann was also supported by the European Regional Develop-
ment Fund/Estonian Research Council funded Mobilitas Plus Top Researcher grant
MOBTT24.
We thank the crew and captain of the RV Meteor (M86, M87) for support during the
research cruise. The computations were performed on resources provided by the
Swedish National Infrastructure for Computing (SNIC) at the PDC Centre for High
Performance Computing (PDC-HPC) and Uppsala Multidisciplinary Center for Advanced
Computational Science (UPPMAX). We thank Jana Matulla for excellent technical assis-
tance and Stephan Fuchs for his help and advice in MS database construction. We also
thank Stefan E. Heiden for valuable help with the CDD BLAST analyses
Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors
Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids
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