Stable isotope-assisted metabolite profiling reveals new insights into L-tryptophan chemotrophic metabolism of Rubrivivax benzoatilyticus.

Anoxygenic photosynthetic bacteria (APB) are metabolically versatile, capable of surviving with an extended range of carbon and nitrogen sources. This group of phototrophic bacteria have remarkable metabolic plasticity in utilizing an array of organic compounds as carbon source/electron donors and nitrogen sources with sophisticated growth modes. Rubrivivax benzoatilyticus JA2 is one such photosynthetic bacterium utilizes L-tryptophan as nitrogen source under phototrophic growth mode and produces an array of indolic compounds of biotechnological significance. However, chemotrophic L-tryptophan metabolism is largely unexplored and studying L-tryptophan metabolism under chemotrophic mode would provide new insights into metabolic potential of strain JA2. In the present study, we employed stable-isotopes assisted metabolite profiling to unravel the L-tryptophan catabolism in Rubrivivax benzoatilyticus strain JA2 under chemotrophic (dark aerobic) conditions. Utilization of L-tryptophan as a nitrogen source for growth and simultaneous production of indole derivatives was observed in strain JA2. Liquid chromatography mass spectrometry (LC-MS) analysis of exo-metabolite profiling of carbon labeled L-tryptophan (13C11) fed cultures of strain JA2 revealed at least seventy labeled metabolites. Of these, only fourteen metabolites were confirmed using standards, while sixteen were putative and forty metabolites remained unidentified. L-tryptophan chemotrophic catabolism revealed multiple catabolic pathways and distinct differential catabolism of L-tryptophan under chemotropic state as compared to photo-catabolism of L-tryptophan in strain JA2.

Tryptophan, a non-canonical melanin precursor: New L-tryptophan based melanin production by Rubrivivax benzoatilyticus JA2.

Melanins are chemically diverse ubiquitous pigments found across the life forms synthesized via different biochemical pathways mainly from L-tyrosine or acetyl CoA. Though few reports suggest the possibility of tryptophan-based melanin synthesis, however, such tryptophan-based melanin and its biosynthesis remained a biochemical riddle. Here we report tryptophan-based melanin production by bacterium, Rubrivivax benzoatilyticus JA2. Aerobic cultures of strain JA2 produced brown pigment when grown on L-tryptophan-containing media. Purified pigment showed typical physico-chemical properties of melanin. Further, extensive spectroscopic studies revealed that pigment is an amorphous, indole-type polymer with stable free radical centers. Further, hydrolysis of the brown pigment revealed the presence of indole moiety, confirming the indolic nature of the pigment. Demonstration of in vitro and in vivo pigment synthesis directly from L-tryptophan or hydroxytryptophan confirms tryptophan-based melanin synthesis in strain JA2. Interestingly, canonical melanin biosynthetic inhibitors did not affect the pigment synthesis indicating possible non-canonical tryptophan-based melanin biosynthesis in strain JA2. Further, the exometabolite profiling and precursor feeding studies suggests that L-tryptophan converted to hydroxytryptophan/hydroxyindoles and their subsequent polymerization lead to the formation of melanin. The current study sheds light on biosynthetic diversity of melanins and L-tryptophan can be a potential precursor for melanin synthesis in life forms.

New insights into aniline toxicity: Aniline exposure triggers envelope stress and extracellular polymeric substance formation in Rubrivivax benzoatilyticus JA2.

Aniline is a major environmental pollutant of serious concern due to its toxicity. Although microbial metabolism of aniline is well-studied, its toxic effects and physiological responses of microorganisms to aniline are largely unexplored. Rubrivivax benzoatilyticus JA2, an aniline non-degrading bacterium, tolerates high concentrations of aniline and produces extracellular polymeric substance(EPS). Surprisingly, strain JA2 forms EPS only when exposed to aniline and other toxic compounds like organic solvents and heavy metals indicating that EPS formation is coupled to cell toxicity. Further, extensive reanalysis of the previous proteomic data of aniline exposed cells revealed up-regulation of envelope stress response(ESR) proteins such as periplasmic protein folding, envelope integrity, transmembrane complex, and cell-wall remodelling proteins. In silico analysis and molecular modeling of three highly up-regulated proteins revealed that these proteins were homologous to CpxARP proteins of ESR signalling pathway. Furthermore, EPS formation to known ESR activators(Triton-X-100, EDTA) suggests that envelope stress possibly regulating the EPS production. The present study suggests that aniline triggers envelope stress; to counter this strain JA2 activates ESR pathway and EPS production. Our study revealed the hitherto unknown toxic effects of aniline as an acute envelope stressor thus toxicity of aniline may be more profound to life-forms than previously thought.

Precursor-feeding and altered-growth conditions reveal novel blue pigment production by Rubrivivax benzoatilyticus JA2.

OBJECTIVE: To explore the secondary metabolite biosynthetic potential of Rubrivivax benzoatilyticus JA2 using a new metabolite mining strategy. RESULTS: Combination of precursor-feeding and altered growth conditions were used to mine new biomolecules. Strain JA2 utilised L-phenylalanine as sole source of nitrogen and showed pigments production only under phenylalanine-amended aerobic cultures. Stable isotope based precursor feeding studies indicated the blue pigment consists of 4-phenyl rings derived from L-phenylalanine. The purified blue pigment displayed characteristic visible-absorption and pH-dependent color variations. Precursor-feeding under altered growth conditions activated the plausible novel aromatic pigment production in strain JA2. CONCLUSION: Our approach unraveled the previously unknown pigment synthesis in strain JA2 and demonstrated the potential of mining strategy in discovering the hidden secondary metabolite repertoire in microorganisms.

iTRAQ-based quantitative proteomics reveals insights into metabolic and molecular responses of glucose-grown cells of Rubrivivax benzoatilyticus JA2.

Anoxygenic photosynthetic bacteria thrive under diverse habitats utilising an extended range of inorganic/organic compounds under different growth modes. Although they display incredible metabolic flexibility, their responses and adaptations to changing carbon regimes is largely unexplored. In the present study, we employed iTRAQ-based global proteomic profiling and physiological studies to uncover the adaptive strategies of a phototrophic bacterium, Rubrivivax benzoatilyticus JA2 to glucose. Strain JA2 displayed altered growth rates, reduced cell size and progressive loss of pigmentation when grown on glucose compared to malate under photoheterotrophic condition. A ten-fold increase in the saturated to unsaturated fatty acid ratio of glucose-grown cells indicates a possible membrane adaptation. Proteomic profiling revealed extensive metabolic remodelling in the glucose-grown cells wherein signal-transduction, selective-transcription, DNA-repair, transport and protein quality control processes were up-regulated to cope with the changing milieu. Proteins involved in DNA replication, translation, electron-transport, photosynthetic machinery were down-regulated possibly to conserve the energy. Glycolysis/gluconeogenesis, TCA cycle and pigment biosynthesis were also down-regulated. The cell has activated alternative energy metabolic pathways viz., fatty acid ß-oxidation, glyoxylate, acetate-switch and Entner-Doudoroff pathways. Overall, the present study deciphered the molecular/metabolic events associated with glucose-grown cells of strain JA2 and also unraveled how a carbon source modulates the metabolic phenotypes. SIGNIFICANCE: Anoxygenic photosynthetic bacteria (APB) exhibit incredible metabolic flexibility leading to diverse phenotypes. They thrive under diverse habitat using an array of inorganic/organic compounds as carbon sources, yet their metabolic adaptation to varying carbon regime is mostly unexplored. Present study uncovered the proteomic insights of the cellular responses of strain JA2 to changing carbon sources viz. malate and glucose under photoheterotrophic conditions. Our study suggests that carbon source can also determine the metabolic fate of the cells and reshape the energy dynamics of APB. Here, for the first time study highlighted the plausible carbon source (glucose) mediated regulation of photosynthesis in APB. The study sheds light on the plausible cellular events and adaptive metabolic strategies employed by strain JA2 in presence of non-preferred carbon source. It also revealed new insights into the metabolic plasticity of APB to the changing milieu.

Pyomelanin production: Insights into the incomplete aerobic l-phenylalanine catabolism of a photosynthetic bacterium, Rubrivivax benzoatilyticus JA2.

Rubrivivax benzoatilyticus JA2 is a metabolically versatile bacterium, thrives on a wide array of organic compounds under different growth modes. Though genomic insights revealed the aromatic compound catabolic potential of strain JA2 under anaerobic/aerobic conditions, the studies are largely restricted to anaerobic metabolism. The previous study on phenylalanine metabolism in strain JA2 indicated melanin-like pigment production under aerobic conditions; however, characterization of pigment and its biosynthetic pathway is not explored. The current study aims at the characterization of pigment and elucidation of its biosynthetic pathway. Strain JA2 utilized l-phenylalanine as source of nitrogen under anaerobic/aerobic conditions but not as a carbon source. Strain JA2 produced a brown-pigment under phenylalanine-amended aerobic conditions. Spectroscopic and physicochemical analysis identified the purified brown-pigment as a melanin. Further, the genomic insights revealed the presence of a complete set of genes related to pyomelanin synthesis. Identification of key metabolites l-tyrosine, 4-hydroxyphenylpyruvic acid and homogentisic acid and their respective enzyme activities further supports the pyomelanin synthesis. Moreover, the precursors feeding, pathway specific inhibitor studies confirmed the pyomelanin synthesis in strain JA2. Our study revealed an incomplete catabolism of phenylalanine; absence of ring cleavage gene, homogentisate dioxygenase leading to homogentisate accumulation thereby pyomelanin synthesis in strain JA2.

Stable Isotope-Assisted Metabolic Profiling Reveals Growth Mode Dependent Differential Metabolism and Multiple Catabolic Pathways of l-Phenylalanine in Rubrivivax benzoatilyticus JA2.

Anoxygenic phototrophic bacteria are metabolically versatile and survive under different growth modes using diverse organic compounds, yet their metabolic diversity is largely unexplored. In the present study, we employed stable-isotope-assisted metabolic profiling to unravel the l-phenylalanine catabolism in Rubrivivax benzoatilyticus JA2 under varying growth modes. Strain JA2 grows under anaerobic and aerobic conditions by utilizing l-phenylalanine as a nitrogen source. Furthermore, ring-labeled 13C6-phenylalanine feeding followed by liquid chromatography-mass spectrometry exometabolite profiling revealed 60 labeled metabolic features (M + 6, M + 12, and M + 18) derived solely from l-phenylalanine, of which 11 were identified, 7 putatively identified, and 42 unidentified under anaerobic and aerobic conditions. However, labeled metabolites were significantly higher in aerobic compared to anaerobic conditions. Furthermore, detected metabolites and enzyme activities indicated multiple l-phenylalanine catabolic routes mainly Ehrlich, homogentisate-dependent melanin, benzenoid, and unidentified pathways operating under anaerobic and aerobic conditions in strain JA2. Interestingly, the study indicated l-phenylalanine-dependent and independent benzenoid biosynthesis in strain JA2 and a differential flux of l-phenylalanine to Ehrlich and benzenoid pathways under anaerobic and aerobic conditions. Additionally, unidentified labeled metabolites strongly suggest the presence of unknown phenylalanine catabolic routes in strain JA2. Overall, the study uncovered the l-phenylalanine catabolic diversity in strain JA2 and demonstrated the potential of stable isotope-assisted metabolomics in unraveling the hidden metabolic repertoire.

Genome sequence of the phototrophic betaproteobacterium Rubrivivax benzoatilyticus strain JA2T.

Herein we report the draft genome sequence of a phototrophic bacterium, Rubrivivax benzoatilyticus strain JA2(T), which apparently is the first genome sequence report of a phototrophic member belonging to the class Betaproteobacteria. The unique feature of this strain is its capability to synthesize carotenoids through both spirilloxanthin and spheroidenone pathways. Strain JA2(T) produces several novel secondary metabolites, and the genome insights help in understanding the unique machinery that the strain adapted.