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Phenotype fingerprinting suggests the involvement of single-genotype consortia in degradation of aromatic compounds by Rhodopseudomonas palustris.

ABSTRACT: Anaerobic degradation of complex organic compounds by microorganisms is crucial for development of innovative biotechnologies for bioethanol production and for efficient degradation of environmental pollutants. In natural environments, the degradation is usually accomplished by syntrophic consortia comprised of different bacterial species. This strategy allows consortium organisms to reduce efforts required for maintenance of the redox homeostasis at each syntrophic level. Cellular mechanisms that maintain the redox homeostasis during the degradation of aromatic compounds by one organism are not fully understood. Here we present a hypothesis that the metabolically versatile phototrophic bacterium Rhodopseudomonas palustris forms its own syntrophic consortia, when it grows anaerobically on p-coumarate or benzoate as a sole carbon source. We have revealed the consortia from large-scale measurements of mRNA and protein expressions under p-coumarate, benzoate and succinate degrading conditions using a novel computational approach referred as phenotype fingerprinting. In this approach, marker genes for known R. palustris phenotypes are employed to determine the relative expression levels of genes and proteins in aromatics versus non-aromatics degrading condition. Subpopulations of the consortia are inferred from the expression of phenotypes and known metabolic modes of the R. palustris growth. We find that p-coumarate degrading conditions may lead to at least three R. palustris subpopulations utilizing p-coumarate, benzoate, and CO2 and H2. Benzoate degrading conditions may also produce at least three subpopulations utilizing benzoate, CO2 and H2, and N2 and formate. Communication among syntrophs and inter-syntrophic dynamics in each consortium are indicated by up-regulation of transporters and genes involved in the curli formation and chemotaxis. The N2-fixing subpopulation in the benzoate degrading consortium has preferential activation of the vanadium nitrogenase over the molybdenum nitrogenase. This subpopulation in the consortium was confirmed in an independent experiment by consumption of dissolved nitrogen gas under the benzoate degrading conditions.

SUBMITTER: Karpinets TV

PROVIDER: S-EPMC2643473 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Phenotype fingerprinting suggests the involvement of single-genotype consortia in degradation of aromatic compounds by Rhodopseudomonas palustris.

Karpinets Tatiana V TV Pelletier Dale A DA Pan Chongle C Uberbacher Edward C EC Melnichenko Galina V GV Hettich Robert L RL Samatova Nagiza F NF

PloS one 20090226 2

Anaerobic degradation of complex organic compounds by microorganisms is crucial for development of innovative biotechnologies for bioethanol production and for efficient degradation of environmental pollutants. In natural environments, the degradation is usually accomplished by syntrophic consortia comprised of different bacterial species. This strategy allows consortium organisms to reduce efforts required for maintenance of the redox homeostasis at each syntrophic level. Cellular mechanisms th ...[more]

PMID: 19242537

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Project description:The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris strain CGA009 uses the three-carbon dicarboxylic acid malonate as the sole carbon source under phototrophic conditions. However, this bacterium grows extremely slowly on this compound and does not have operons for the two pathways for malonate degradation that have been detected in other bacteria. Many bacteria grow on a spectrum of carbon sources, some of which are classified as poor growth substrates because they support low growth rates. This trait is rarely addressed in the literature, but slow growth is potentially useful in biotechnological applications where it is imperative for bacteria to divert cellular resources to value-added products rather than to growth. This prompted us to explore the genetic and physiological basis for the slow growth of R. palustris with malonate as a carbon source. There are two unlinked genes annotated as encoding a malonyl coenzyme A (malonyl-CoA) synthetase (MatB) and a malonyl-CoA decarboxylase (MatA) in the genome of R. palustris, which we verified as having the predicted functions. Additionally, two tripartite ATP-independent periplasmic transporters (TRAP systems) encoded by rpa2047 to rpa2049 and rpa2541 to rpa2543 were needed for optimal growth on malonate. Most of these genes were expressed constitutively during growth on several carbon sources, including malonate. Our data indicate that R. palustris uses a piecemeal approach to growing on malonate. The data also raise the possibility that this bacterium will evolve to use malonate efficiently if confronted with an appropriate selection pressure.IMPORTANCE There is interest in understanding how bacteria metabolize malonate because this three-carbon dicarboxylic acid can serve as a building block in bioengineering applications to generate useful compounds that have an odd number of carbons. We found that the phototrophic bacterium Rhodopseudomonas palustris grows extremely slowly on malonate. We identified two enzymes and two TRAP transporters involved in the uptake and metabolism of malonate, but some of these elements are apparently not very efficient. R. palustris cells growing with malonate have the potential to be excellent biocatalysts, because cells would be able to divert cellular resources to the production of value-added compounds instead of using them to support rapid growth. In addition, our results suggest that R. palustris is a candidate for directed evolution studies to improve growth on malonate and to observe the kinds of genetic adaptations that occur to make a metabolic pathway operate more efficiently.

Project description:While lignin represents a major fraction of the carbon in plant biomass, biological strategies to convert the components of this heterogeneous polymer into products of industrial and biotechnological value are lacking. Syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) is a by-product of lignin degradation, appearing in lignocellulosic hydrolysates, deconstructed lignin streams, and other agricultural products. Rhodopseudomonas palustris CGA009 is a known degrader of phenolic compounds under photoheterotrophic conditions via the benzoyl coenzyme A (CoA) degradation (BAD) pathway. However, R. palustris CGA009 is reported to be unable to metabolize meta-methoxylated phenolics, such as syringic acid. We isolated a strain of R. palustris (strain SA008.1.07), adapted from CGA009, which can grow on syringic acid under photoheterotrophic conditions, utilizing it as a sole source of organic carbon and reducing power. An SA008.1.07 mutant with an inactive benzoyl-CoA reductase structural gene was able to grow on syringic acid, demonstrating that the metabolism of this aromatic compound is not through the BAD pathway. Comparative gene expression analyses of SA008.1.07 implicated the involvement of products of the vanARB operon (rpa3619, rpa3620, rpa3621), which has been described as catalyzing aerobic aromatic ring demethylation in other bacteria, in anaerobic syringic acid degradation. In addition, experiments with a vanARB deletion mutant demonstrated the involvement of the vanARB operon in anaerobic syringic acid degradation. These observations provide new insights into the anaerobic degradation of meta-methoxylated and other aromatics by R. palustrisIMPORTANCE Lignin is the most abundant aromatic polymer on Earth and a resource that could eventually substitute for fossil fuels as a source of aromatic compounds for industrial and biotechnological applications. Engineering microorganisms for the production of aromatic-based biochemicals requires detailed knowledge of the metabolic pathways for the degradation of aromatics that are present in lignin. Our isolation and analysis of a Rhodopseudomonas palustris strain capable of syringic acid degradation reveal a previously unknown metabolic route for aromatic degradation in R. palustris This study highlights several key features of this pathway and sets the stage for a more complete understanding of the microbial metabolic repertoire required to metabolize aromatic compounds from lignin and other renewable sources.

Project description:The degradation of lignin-derived aromatic compounds such as benzoate has been extensively studied in Rhodopseudomonas palustris, and the chemistry underpinning the conversion of benzoate to acetyl coenzyme A (acetyl-CoA) is well understood. Here we characterize the last unknown gene, badL, of the bad (benzoic acid degradation) cluster. BadL function is required for growth under photoheterotrophic conditions with benzoate as the organic carbon source (i.e., light plus anoxia). On the basis of bioinformatics and in vivo and in vitro data, we show that BadL, a Gcn5-related N-acetyltransferase (GNAT) (PF00583), acetylates aminobenzoates to yield acetamidobenzoates. The latter relieved repression of the badDEFGAB operon by binding to BadM, triggering the synthesis of enzymes that activate and dearomatize the benzene ring. We also show that acetamidobenzoates are required for the expression of genes encoding the photosynthetic reaction center light-harvesting complexes through a BadM-independent mechanism. The effect of acetamidobenzoates on pigment synthesis is new and different than their effect on the catabolism of benzoate.IMPORTANCE This work shows that the BadL protein of Rhodopseudomonas palustris has N-acetyltransferase activity and that this activity is required for the catabolism of benzoate under photosynthetic conditions in this bacterium. R. palustris occupies lignin-rich habitats, making its benzoate-degrading capability critical for the recycling of this important, energy-rich biopolymer. This work identifies the product of the BadL enzyme as acetamidobenzoates, which were needed to derepress genes encoding benzoate-degrading enzymes and proteins of the photosynthetic apparatus responsible for the generation of the proton motive force under anoxia in the presence of light. In short, acetamidobenzoates potentially coordinate the use of benzoate as a source of reducing power and carbon with the generation of a light-driven proton motive force that fuels ATP synthesis, motility, transport, and many other processes in the metabolically versatile bacterium R. palustris.

Project description:The biodegradation of lignin, one of the most abundant carbon compounds on Earth, has important biotechnological applications in the derivation of useful products from lignocellulosic wastes. The purple photosynthetic bacterium Rhodopseudomonas palustris is able to grow photoheterotrophically under anaerobic conditions on a range of phenylpropeneoid lignin monomers, including coumarate, ferulate, caffeate, and cinnamate. RPA1789 (CouP) is the periplasmic binding-protein component of an ABC system (CouPSTU; RPA1789, RPA1791-1793), which has previously been implicated in the active transport of this class of aromatic substrate. Here, we show using both intrinsic tryptophan fluorescence and isothermal titration calorimetry that CouP binds a range of phenylpropeneoid ligands with K d values in the nanomolar range. The crystal structure of CouP with ferulate as the bound ligand shows H-bond interactions between the 4-OH group of the aromatic ring with His309 and Gln305. H-bonds are also made between the carboxyl group on the ferulate side chain and Arg197, Ser222, and Thr102. An additional transport system (TarPQM; RPA1782-1784), a member of the tripartite ATP-independent periplasmic (TRAP) transporter family, is encoded at the same locus as rpa1789 and several other genes involved in coumarate metabolism. We show that the periplasmic binding-protein of this system (TarP; RPA1782) also binds coumarate, ferulate, caffeate, and cinnamate with nanomolar K d values. Thus, we conclude that R. palustris uses two redundant but energetically distinct primary and secondary transporters that both employ high-affinity periplasmic binding-proteins to maximise the uptake of lignin-derived aromatic substrates from the environment. Our data provide a detailed thermodynamic and structural basis for understanding the interaction of lignin-derived aromatic substrates with proteins and will be of use in the further exploitation of the flexible metabolism of R. palustris for anaerobic aromatic biotransformations.

Project description:Rhodopseudomonas palustris, a nonsulphur purple photosynthetic bacteria, has been extensively investigated for its metabolic versatility including ability to produce hydrogen gas from sunlight and biomass. The availability of the finished genome sequences of six R. palustris strains (BisA53, BisB18, BisB5, CGA009, HaA2 and TIE-1) combined with online bioinformatics software for integrated analysis presents new opportunities to determine the genomic basis of metabolic versatility and ecological lifestyles of the bacteria species. The purpose of this investigation was to compare the functional annotations available for multiple R. palustris genomes to identify annotations that can be further investigated for strain-specific or uniquely shared phenotypic characteristics. A total of 2,355 protein family Pfam domain annotations were clustered based on presence or absence in the six genomes. The clustering process identified groups of functional annotations including those that could be verified as strain-specific or uniquely shared phenotypes. For example, genes encoding water/glycerol transport were present in the genome sequences of strains CGA009 and BisB5, but absent in strains BisA53, BisB18, HaA2 and TIE-1. Protein structural homology modeling predicted that the two orthologous 240 aa R. palustris aquaporins have water-specific transport function. Based on observations in other microbes, the presence of aquaporin in R. palustris strains may improve freeze tolerance in natural conditions of rapid freezing such as nitrogen fixation at low temperatures where access to liquid water is a limiting factor for nitrogenase activation. In the case of adaptive loss of aquaporin genes, strains may be better adapted to survive in conditions of high-sugar content such as fermentation of biomass for biohydrogen production. Finally, web-based resources were developed to allow for interactive, user-defined selection of the relationship between protein family annotations and the R. palustris genomes.

Dataset Information

Phenotype fingerprinting suggests the involvement of single-genotype consortia in degradation of aromatic compounds by Rhodopseudomonas palustris.

Publications

Phenotype fingerprinting suggests the involvement of single-genotype consortia in degradation of aromatic compounds by Rhodopseudomonas palustris.

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