Biomedical subjects

Julia A Vorholt

Publications and source records attributed to Julia A Vorholt.

At least 19 recordsLinked to original sources

PubMed2025

The Gfr Uptake System Provides a Context-Dependent Fitness Advantage to Salmonella Typhimurium SL1344 During the Initial Gut Colonization Phase.

Salmonella enterica serovar Typhimurium (S. Tm) is a major cause of foodborne diarrhea. However, in healthy individuals, the microbiota typically restricts the growth of incoming pathogens, a protective mechanism termed colonization resistance (CR). To circumvent CR, Salmonella strains can utilize private nutrients that remain untapped by the resident microbiota. However, the metabolic pathways and environmental niches promoting pathogen growth are still not completely understood. Here, we investigate the significance of the gfr operon in gut colonization of S. Tm, which is essential for the utilization of fructoselysine (FL) and glucoselysine (GL). These Amadori compounds are present in heated foods with high protein and carbohydrate contents. We detected FL in both mouse chow and the intestinal tract of mice and showed that gfr mutants are attenuated during the initial phase of colonization in the murine model. Experiments in gnotobiotic mice and competition experiments with Escherichia coli suggest that gfr-dependent fitness advantage is context-dependent. We conclude that dietary Amadori products like FL can support S. Tm gut colonization, depending on the metabolic capacities of the microbiota.

Salmonella typhimurium↗

PubMed2024

Salmonella Typhimurium screen identifies shifts in mixed-acid fermentation during gut colonization.

How enteric pathogens adapt their metabolism to a dynamic gut environment is not yet fully understood. To investigate how Salmonella enterica Typhimurium (S.Tm) colonizes the gut, we conducted an in vivo transposon mutagenesis screen in a gnotobiotic mouse model. Our data implicate mixed-acid fermentation in efficient gut-luminal growth and energy conservation throughout infection. During initial growth, the pathogen utilizes acetate fermentation and fumarate respiration. After the onset of gut inflammation, hexoses appear to become limiting, as indicated by carbohydrate analytics and the increased need for gluconeogenesis. In response, S.Tm adapts by ramping up ethanol fermentation for redox balancing and supplying the TCA cycle with α-ketoglutarate for additional energy. Our findings illustrate how S.Tm flexibly adapts mixed fermentation and its use of the TCA cycle to thrive in the changing gut environment. Similar metabolic wiring in other pathogenic Enterobacteriaceae may suggest a broadly conserved mechanism for gut colonization.

Animals↗

PubMed2016

Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking.

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry (MS) techniques are well-suited to high-throughput characterization of NP, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social Molecular Networking (GNPS; http://gnps.ucsd.edu), an open-access knowledge base for community-wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS, crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of 'living data' through continuous reanalysis of deposited data.

Biological Products↗

PubMed2005

Methylobacterium extorquens AM1 produces a novel type of acyl-homoserine lactone with a double unsaturated side chain under methylotrophic growth conditions.

Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain (N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.

4-Butyrolactone↗

PubMed2005

A genomic view of methane oxidation by aerobic bacteria and anaerobic archaea.

Recent sequencing of the genome and proteomic analysis of a model aerobic methanotrophic bacterium, Methylococcus capsulatus (Bath) has revealed a highly versatile metabolic potential. In parallel, environmental genomics has provided glimpses into anaerobic methane oxidation by certain archaea, further supporting the hypothesis of reverse methanogenesis.

Anaerobiosis↗

PubMed2005

How an enzyme binds the C1 carrier tetrahydromethanopterin. Structure of the tetrahydromethanopterin-dependent formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1.

Tetrahydromethanopterin (H4 MPT) is a tetrahydrofolate analogue involved as a C1 carrier in the metabolism of various groups of microorganisms. How H4MPT is bound to the respective C1 unit converting enzymes remained elusive. We describe here the structure of the homopentameric formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1 established at 2.0 angstrom without and at 1.9 angstrom with methylene-H4MPT bound. Methylene-H4MPT is bound in an "S"-shaped conformation into the cleft formed between two adjacent subunits. Coenzyme binding is accompanied by side chain rearrangements up to 5 angstrom and leads to a rigidification of the C-terminal arm, a formation of a new hydrophobic cluster, and an inversion of the amide side chain of Gln88. Methylene-H4MPT in Fae shows a characteristic kink between the tetrahydropyrazine and the imidazolidine rings of 70 degrees that is more pronounced than that reported for free methylene-H4MPT in solution (50 degrees). Fae is an essential enzyme for energy metabolism and formaldehyde detoxification of this bacterium and catalyzes the formation of methylene-H4MPT from H4MPT and formaldehyde. The molecular mechanism ofthis reaction involving His22 as acid catalyst is discussed.

Bacterial Proteins↗

PubMed2005

Methylotrophic metabolism is advantageous for Methylobacterium extorquens during colonization of Medicago truncatula under competitive conditions.

Facultative methylotrophic bacteria of the genus Methylobacterium are commonly found in association with plants. Inoculation experiments were performed to study the importance of methylotrophic metabolism for colonization of the model legume Medicago truncatula. Competition experiments with Methylobacterium extorquens wild-type strain AM1 and methylotrophy mutants revealed that the ability to use methanol as a carbon and energy source provides a selective advantage during colonization of M. truncatula. Differences in the fitness of mutants defective in different stages of methylotrophic metabolism were found; whereas approximately 25% of the mutant incapable of oxidizing methanol to formaldehyde (deficient in methanol dehydrogenase) was recovered, 10% or less of the mutants incapable of oxidizing formaldehyde to CO2 (defective in biosynthesis of the cofactor tetrahydromethanopterin) was recovered. Interestingly, impaired fitness of the mutant strains compared with the wild type was found on leaves and roots. Single-inoculation experiments showed, however, that mutants with defects in methylotrophy were capable of plant colonization at the wild-type level, indicating that methanol is not the only carbon source that is accessible to Methylobacterium while it is associated with plants. Fluorescence microscopy with a green fluorescent protein-labeled derivative of M. extorquens AM1 revealed that the majority of the bacterial cells on leaves were on the surface and that the cells were most abundant on the lower, abaxial side. However, bacterial cells were also found in the intercellular spaces inside the leaves, especially in the epidermal cell layer and immediately underneath this layer.

Alcohol Oxidoreductases↗

PubMed2005

MtdC, a novel class of methylene tetrahydromethanopterin dehydrogenases.

Novel methylene tetrahydromethanopterin (H4MPT) dehydrogenase enzymes, named MtdC, were purified after expressing in Escherichia coli genes from, respectively, Gemmata sp. strain Wa1-1 and environmental DNA originating from unidentified microbial species. The MtdC enzymes were shown to possess high affinities for methylene-H4MPT and NADP but low affinities for methylene tetrahydrofolate or NAD. The substrate range and the kinetic properties revealed by MtdC enzymes distinguish them from the previously characterized bacterial methylene-H4MPT dehydrogenases, MtdA and MtdB. While revealing higher sequence similarity to MtdA enzymes, MtdC enzymes appear to fulfill a function homologous to the function of MtdB, as part of the H4MPT-linked pathway for formaldehyde oxidation/detoxification.

Bacteria↗

PubMed2004

A dynamic zinc redox switch.

The crystal structures of glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans, which catalyzes the formation of S-hydroxymethylglutathione from formaldehyde and glutathione, and its complex with glutathione (Gfa-GTT) have been determined. Gfa has a new fold with two zinc-sulfur centers, one that is structural (zinc tetracoordinated) and one catalytic (zinc apparently tricoordinated). In Gfa-GTT, the catalytic zinc is displaced due to disulfide bond formation of glutathione with one of the zinc-coordinating cysteines. Soaking crystals of Gfa-GTT with formaldehyde restores the holoenzyme. Accordingly, the displaced zinc forms a complex by scavenging formaldehyde and glutathione. The activation of formaldehyde and of glutathione in this zinc complex favors the final nucleophilic addition, followed by relocation of zinc in the catalytic site. Therefore, the structures of Gfa and Gfa-GTT draw the critical association between a dynamic zinc redox switch and a nucleophilic addition as a new facet of the redox activity of zinc-sulfur sites.

Carbon-Sulfur Ligases↗

PubMed2004

The enigmatic planctomycetes may hold a key to the origins of methanogenesis and methylotrophy.

Methanogenesis and methane oxidation are the major biological processes affecting the global cycling of the powerful greenhouse gas methane. To carry out the two alternative bioconversions, Nature has cleverly recycled key reactions for the C1 transfers between the oxidation levels of formaldehyde and formate, and these involve analogous enzyme systems and common specialized cofactors, methanopterin and methanofuran. Until recently, the distribution of these functions has been limited to methanogenic archaea and methylotrophic proteobacteria, and their evolutionary history remained obscure. Single interdomain lateral transfer of the respective genes has been suggested to play a role. Here we show that genes for C1 transfer reactions linked to methanopterin and methanofuran are also present in diverse representatives of the enigmatic bacterial clade, the Planctomycetes. Phylogenetic analysis places the planctomycete sequences as distantly from their archaeal counterparts as from their proteobacterial counterparts, suggesting novel scenarios for the evolution of the C1 transfer functions in both methanogens and methylotrophs. This finding suggests a possible role for Planctomycetes in the evolution of the methane cycle on Earth.

Bacteria↗

PubMed2004

Comparison of the proteome of Methylobacterium extorquens AM1 grown under methylotrophic and nonmethylotrophic conditions.

Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium that is capable of growing in the presence of methanol as the sole carbon and energy source, but is also able to grow on a limited number of C(2), C(3), and C(4) compounds, for example succinate. This study provides a proteomic view of the cellular adaptation of M. extorquens AM1 to growth on methanol and succinate, respectively. Cytosolic proteins were separated by two-dimensional gel electrophoresis employing overlapping pH ranges and visualized by silver nitrate or fluorescence staining. A proteomic reference map containing 229 different proteins identified by peptide mass fingerprinting of tryptic fragments was established. Comparative proteome profiling of methanol- and succinate-grown cells led to the identification of 68 proteins that are induced under methylotrophic growth conditions in comparison to growth on succinate. This group includes most proteins known to be directly involved in methanol oxidation to CO(2) and in assimilation of one carbon units by the serine cycle as well as 18 proteins without any assigned function and two proteins with a predicted regulatory function. Furthermore, the proteome analysis revealed putative isoenzymes for formaldehyde-activating enzyme Fae, malyl-CoA lyase, malate-dehydrogenase, and fumarase, that need to be characterized functionally in future studies.

Bacterial Proteins↗

PubMed2004

Multiple formate dehydrogenase enzymes in the facultative methylotroph Methylobacterium extorquens AM1 are dispensable for growth on methanol.

Formate dehydrogenase has traditionally been assumed to play an essential role in energy generation during growth on C(1) compounds. However, this assumption has not yet been experimentally tested in methylotrophic bacteria. In this study, a whole-genome analysis approach was used to identify three different formate dehydrogenase systems in the facultative methylotroph Methylobacterium extorquens AM1 whose expression is affected by either molybdenum or tungsten. A complete set of single, double, and triple mutants was generated, and their phenotypes were analyzed. The growth phenotypes of the mutants suggest that any one of the three formate dehydrogenases is sufficient to sustain growth of M. extorquens AM1 on formate, while surprisingly, none is required for growth on methanol or methylamine. Nuclear magnetic resonance analysis of the fate of [(13)C]methanol revealed that while cells of wild-type M. extorquens AM1 as well as cells of all the single and the double mutants continuously produced [(13)C]bicarbonate and (13)CO(2), cells of the triple mutant accumulated [(13)C]formate instead. Further studies of the triple mutant showed that formate was not produced quantitatively and was consumed later in growth. These results demonstrated that all three formate dehydrogenase systems must be inactivated in order to disrupt the formate-oxidizing capacity of the organism but that an alternative formate-consuming capacity exists in the triple mutant.

Bacterial Proteins↗

PubMed2003

The tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1: purification and properties.

NAD-dependent formate dehydrogenase (FDH1) was isolated from the alpha-proteobacterium Methylobacterium extorquens AM1 under oxic conditions. The enzyme was found to be a heterodimer of two subunits (alpha1beta1) of 107 and 61 kDa, respectively. The purified enzyme contained per mol enzyme approximately 5 mol nonheme iron and acid-labile sulfur, 0.6 mol noncovalently bound FMN, and approximately 1.8 mol tungsten. The genes encoding the two subunits of FDH1 were identified on the M. extorquens AM1 chromosome next to each other in the order fdh1B, fdh1A. Sequence comparisons revealed that the alpha-subunit harbours putative binding motifs for the molybdopterin cofactor and at least one iron-sulfur cluster. Sequence identity was highest to the catalytic subunits of the tungsten- and selenocysteine-containing formate dehydrogenases characterized from Eubacterium acidaminophilum and Moorella thermoacetica (Clostridium thermoaceticum). The beta-subunit of FDH1 contains putative motifs for binding FMN and NAD, as well as an iron-sulfur cluster binding motif. The beta-subunit appears to be a fusion protein with its N-terminal domain related to NuoE-like subunits and its C-terminal domain related to NuoF-like subunits of known NADH-ubiquinone oxidoreductases.

Amino Acid Sequence↗

PubMed2003

Purification of the formate-tetrahydrofolate ligase from Methylobacterium extorquens AM1 and demonstration of its requirement for methylotrophic growth.

The serine cycle methylotroph Methylobacterium extorquens AM1 contains two pterin-dependent pathways for C(1) transfers, the tetrahydrofolate (H(4)F) pathway and the tetrahydromethanopterin (H(4)MPT) pathway, and both are required for growth on C(1) compounds. With the exception of formate-tetrahydrofolate ligase (FtfL, alternatively termed formyl-H(4)F synthetase), all of the genes encoding the enzymes comprising these two pathways have been identified, and the corresponding gene products have been purified and characterized. We present here the purification and characterization of FtfL from M. extorquens AM1 and the confirmation that this enzyme is encoded by an ftfL homolog identified previously through transposon mutagenesis. Phenotypic analyses of the ftfL mutant strain demonstrated that FtfL activity is required for growth on C(1) compounds. Unlike mutants defective for the H(4)MPT pathway, the ftfL mutant strain does not exhibit phenotypes indicative of defective formaldehyde oxidation. Furthermore, the ftfL mutant strain remained competent for wild-type conversion of [(14)C]methanol to [(14)C]CO(2). Collectively, these data confirm our previous presumptions that the H(4)F pathway is not the key formaldehyde oxidation pathway in M. extorquens AM1. Rather, our data suggest an alternative model for the role of the H(4)F pathway in this organism in which it functions to convert formate to methylene H(4)F for assimilatory metabolism.

Alleles↗

PubMed2002

Generation of formate by the formyltransferase/hydrolase complex (Fhc) from Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 possesses a formyltransferase (Ftr) complex that is essential for growth in the presence of methanol and involved in formaldehyde oxidation to CO(2). One of the subunits of the complex carries the catalytic site for transfer of the formyl group from tetrahydromethanopterin to methanofuran (MFR). We now found via nuclear magnetic resonance-based studies that the Ftr complex also catalyzes the hydrolysis of formyl-MFR and generates formate. The enzyme was therefore renamed Ftr/hydrolase complex (Fhc). FhcA shares a sequence pattern with amidohydrolases and is assumed to be the catalytic site where the hydrolysis takes place.

Catalytic Domain↗

PubMed2002

Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria.

Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane. These compounds are metabolized via the cytotoxin formaldehyde. The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO(2) for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria. Principally, two main types of pathways for the conversion of formaldehyde to CO(2) have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C(1) unit conversion bound to the cofactors tetrahydrofolate (H(4)F), tetrahydromethanopterin (H(4)MPT), glutathione (GSH), or mycothiol (MySH). The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C(1) carrier, the oxidation of the cofactor-bound C(1) unit and its conversion to formate, and the oxidation of formate to CO(2). However, the H(4)MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea. The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria. The pathways are in part also used by other organisms to provide C(1) units for biosynthetic reactions (e.g., H(4)F-dependent enzymes) or detoxification of formaldehyde (e.g., GSH-dependent enzymes).

Cysteine↗

PubMed2002

Purification and characterization of the methylene tetrahydromethanopterin dehydrogenase MtdB and the methylene tetrahydrofolate dehydrogenase FolD from Hyphomicrobium zavarzinii ZV580.

Recently, it has been shown that heterotrophic methylotrophic Proteobacteria contain tetrahydrofolate (H(4)F)- and tetrahydromethanopterin (H(4)MPT)-dependent enzymes. Here we report on the purification of two methylene tetrahydropterin dehydrogenases from the methylotroph Hyphomicrobium zavarzinii ZV580. Both dehydrogenases are composed of one type of subunit of 31 kDa. One of the dehydrogenases is NAD(P)-dependent and specific for methylene H(4)MPT (specific activity: 680 U/mg). Its N-terminal amino acid sequence showed sequence identity to NAD(P)-dependent methylene H(4)MPT dehydrogenase MtdB from Methylobacterium extorquens AM1. The second dehydrogenase is specific for NADP and methylene H(4)F (specific activity: 180 U/mg) and also exhibits methenyl H(4)F cyclohydrolase activity. Via N-terminal amino acid sequencing this dehydrogenase was identified as belonging to the classical bifunctional methylene H(4)F dehydrogenases/cyclohydrolases (FolD) found in many bacteria and eukarya. Apparently, the occurrence of methylene tetrahydrofolate and methylene tetrahydromethanopterin dehydrogenases is not uniform among different methylotrophic alpha-Proteobacteria. For example, FolD was not found in M. extorquens AM1, and the NADP-dependent methylene H(4)MPT dehydrogenase MtdA was present in the bacterium that also shows H(4)F activity.

Amino Acid Sequence↗

PubMed2002

Structure of methylene-tetrahydromethanopterin dehydrogenase from methylobacterium extorquens AM1.

NADP-dependent methylene-H(4)MPT dehydrogenase, MtdA, from Methylobacterium extorquens AM1 catalyzes the dehydrogenation of methylene-tetrahydromethanopterin and methylene-tetrahydrofolate with NADP(+) as cosubstrate. The X-ray structure of MtdA with and without NADP bound was established at 1.9 A resolution. The enzyme is present as a homotrimer. The alpha,beta fold of the monomer is related to that of methylene-H(4)F dehydrogenases, suggesting a common evolutionary origin. The position of the active site is located within a large crevice built up by the two domains of one subunit and one domain of a second subunit. Methylene-H(4)MPT could be modeled into the cleft, and crucial active site residues such as Phe18, Lys256, His260, and Thr102 were identified. The molecular basis of the different substrate specificities and different catalytic demands of MtdA compared to methylene-H(4)F dehydrogenases are discussed.

Amino Acid Sequence↗