Madan Junghare | University of Konstanz, Germany (original) (raw)

Papers by Madan Junghare

BMC Microbiology

Background Environmental contamination from synthetic plastics and their additives is a widesprea... more Background Environmental contamination from synthetic plastics and their additives is a widespread problem. Phthalate esters are a class of refractory synthetic organic compounds which are widely used in plastics, coatings, and for several industrial applications such as packaging, pharmaceuticals, and/or paints. They are released into the environment during production, use and disposal, and some of them are potential mutagens and carcinogens. Isophthalate (1,3-benzenedicarboxylic acid) is a synthetic chemical that is globally produced at a million-ton scale for industrial applications and is considered a priority pollutant. Here we describe the biochemical characterization of an enzyme involved in anaerobic degradation of isophthalate by the syntrophically fermenting bacterium Syntrophorhabdus aromaticivorans strain UI that activate isophthalate to isophthalyl-CoA followed by its decarboxylation to benzoyl-CoA. Results Isophthalate:Coenzyme A ligase (IPCL, AMP-forming) that activat...

Bergey's Manual of Systematics of Archaea and Bacteria

Phthalic acid esters (phthalates) are used as additives in various plastics and industrial applic... more Phthalic acid esters (phthalates) are used as additives in various plastics and industrial applications. They are produced worldwide in huge amounts causing major pollution in the environment. Biodegradation of phthalates from the environment is an important route for their removal. In our previous work, we showed that Azoarcus sp. strain PA01 catabolizes o-phthalate via the anaerobic benzoyl-CoA pathway that involved two putative enzymes: the succinyl-CoA:o-phthalate CoA-transferase activates o-phthalate to o-phthalyl-CoA which is subsequently decarboxylated to benzoyl-CoA by o-phthalyl-CoA decarboxylase. In this work, we provide the information on the enzymes involved in the promising step of anoxic decarboxylation of o-phthalate to benzoyl-CoA. We have identified that there are two proteins are involved in decarboxylation step, of which only one does the actual decarboxylation but other one is essential. o-Phthalyl-CoA decarboxylase (PhtDa and PhtDb) encoded by the two genes PA01...

Gel electrophoresis of genomic DNA isolated from GluBS11T cells grown with gluconate. (TIF 159 kb)

Putative transporters identified in the draft genome of A. acetethylicum GluBS11T. (DOCX 14 kb)

IMG annotated functions of selected putative key enzymes involved in the metabolic pathways ident... more IMG annotated functions of selected putative key enzymes involved in the metabolic pathways identified in the draft genome sequence of A. acetethylicum strain GluBS11T. (DOCX 15 kb)

Draft genome sequence of a nitrate-reducing,

Anaerobic phthalate degradation was assumed to proceed through initial decarboxylation of phthala... more Anaerobic phthalate degradation was assumed to proceed through initial decarboxylation of phthalate (ortho) to benzoate. However, the intermediates and enzymes involved in anaerobic phthalate decarboxylation were largely unknown. The aim of this dissertation was to investigate the biochemistry of anaerobic phthalate degradation, especially the steps and enzymes involved in the decarboxylation of phthalate to benzoate. In particular, the study focused on the enrichment and isolation of anaerobic phthalate-degrading bacteria under nitrate-reducing, sulfate-reducing and fermenting conditions.Strain PA01 was isolated and purified from an enrichment culture that degrades phthalate coupled to nitrate reduction. 16S rRNA gene sequencing suggested that strain PA01 is a member of the genus Azoarcus that is known for aromatic compound degradation. Azoarcus sp. strain PA01 could degrade a wide variety of aromatic compounds, including phthalate and benzoate coupled to nitrate reduction. No growth was observed with isophthalate or terephthalate. To gain detailed insights into the biochemistry of phthalate degradation, strain PA01 was genome sequenced. The draft genome of strain PA01 possesses the gene clusters for degradation of aromatic compounds, i.e. for benzoate degradation. Differential two-dimensional protein profiling of phthalate- versus benzoate-grown cells identified the specific proteins induced with phthalate.The phthalate-induced protein-coding genes were found to constitute a single gene cluster in the genome of Azoarcus sp. strain PA01. Phthalate-induced proteins included a transporter, two CoA-transferases, and UbiX-like/UbiD-like decarboxylases. It was concluded that o-phthalate is first activated to o-phthalyl-CoA by a succinyl-CoA dependent succinyl-CoA:o-phthalate CoA-transferase (PhtSa and PhtSb), and is subsequently decarboxylated to benzoyl-CoA by an o-phthalyl-CoA decarboxylase (PhtDa and PhtDb). In vitro enzyme assays with cell-free extracts of phthalate-grown cells of Azoarcus sp. strain PA01 demonstrated the formation of o-phthalyl-CoA, specifically with o-phthalate and succinyl-CoA as the CoA donor, and established its subsequent decarboxylation to benzoyl-CoA using LC-MS analysis. Neither free CoA nor acetyl-CoA served as the CoA donor. Isophthalyl-CoA and terephthalyl-CoA were not decarboxylated. Phylogenetic analysis of phthalate-induced PhtSa and PhtSb proteins of strain PA01 revealed that they shared high sequence homology to the known enzyme succinyl-CoA:(R)-benzylsuccinate CoA-transferase involved in toluene activation in denitrifying T. aromatica. PhtDa and PhtDb proteins showed high similarity to the recently discovered enzyme family of UbiD-like and UbiX-like decarboxylases that function in ubiquinone synthesis in a wide range of bacteria.Furthermore, cloning and heterologous expression of the PhtDa and PhtDb proteins in host E. coli verified that these proteins together indeed decarboxylate phthalyl-CoA to benzoyl-CoA. PhtDb is a 8 | P a g e flavin mononucleotide (FMN)-binding protein homologous to known FMN-binding UbiX-like of E. coli, which itself does not possess decarboxylase activity. Instead, it generates a modified-FMN cofactor that is required by PhtDa for decarboxylase activity. Multiple sequence alignments and structure modelling of both proteins suggested that only PhtDb has a binding site for a FMN ligand. This strongly indicates that PhtDb bound FMN plays an essential role in the decarboxylation of o-phthalyl-CoA. Further, it is assumed that FMN functions as a potential electron shuttle between the phthalate ring and the enzyme complex (PhtDa and PhtDb) for facilitating the anaerobic phthalate decarboxylation. Additionally, structural modelling based on known structures of UbiX/UbiD-like enzymes suggested that PhtDb (22 kDa) forms a dodecamer and PhtDa (60 kDa) a homodimer that together build an enzyme complex of about 400 kDa. Native gel analysis of cell-free extract from Azoarcus sp. strain PA01 showed a protein band with an approximate molecular size of 380-400 kDa from which only PhtDa and PhtDb proteins were identified by MS analysis. These results were further supported by native gel analysis of recombinant the PhtDa and PhtDb proteins together which showed a single protein band of molecular size in the same range (380 - 400 kDa).A mixed culture (KOPA) degrading phthalate was enriched under sulfate-reducing conditions. Isolation and purification of bacteria from the mixed culture resulted in the identification of a novel benzoate-degrading bacterium Desulfoprunum benzoelyticum gen. nov., sp. nov. D. benzoelyticum could not degrade phthalate, but bacterial community analysis of the KOPA culture revealed that, it is a predominant bacterium in the enrichment culture. Other potential phthalate degraders include members of the family Desulfobulbaceae. The enrichment culture could also simultaneously be adapted for benzoate utilization, indicating that phthalate…

Genes, 2021

Among other attributes, the Betaproteobacterial genus Azoarcus has biotechnological importance fo... more Among other attributes, the Betaproteobacterial genus Azoarcus has biotechnological importance for plant growth-promotion and remediation of petroleum waste-polluted water and soils. It comprises at least two phylogenetically distinct groups. The “plant-associated” group includes strains that are isolated from the rhizosphere or root interior of the C4 plant Kallar Grass, but also strains from soil and/or water; all are considered to be obligate aerobes and all are diazotrophic. The other group (now partly incorporated into the new genus Aromatoleum) comprises a diverse range of species and strains that live in water or soil that is contaminated with petroleum and/or aromatic compounds; all are facultative or obligate anaerobes. Some are diazotrophs. A comparative genome analysis of 32 genomes from 30 Azoarcus-Aromatoleum strains was performed in order to delineate generic boundaries more precisely than the single gene, 16S rRNA, that has been commonly used in bacterial taxonomy. Th...

Environmental Microbiology Reports, 2019

This article has been accepted for publication and undergone full peer review but has not been th... more This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as

Environmental Microbiology, 2019

The complete degradation of the xenobiotic and environmentally harmful phthalate esters is initia... more The complete degradation of the xenobiotic and environmentally harmful phthalate esters is initiated by hydrolysis to alcohols and o-phthalate (phthalate) by esterases. While further catabolism of phthalate has been studied in aerobic and denitrifying microorganisms, the degradation in obligately anaerobic bacteria has remained obscure. Here, we demonstrate a previously overseen growth of the δ-proteobacterium Desulfosarcina cetonica with phthalate/sulphate as only carbon and energy sources. Differential proteome and CoA ester pool analyses together with in vitro enzyme assays identified the genes, enzymes and metabolites involved in phthalate uptake and degradation in D. cetonica. Phthalate is initially activated to the shortlived phthaloyl-CoA by an ATP-dependent phthalate CoA ligase (PCL) followed by decarboxylation to the central intermediate benzoyl-CoA by an UbiD-like phthaloyl-CoA decarboxylase (PCD) containing a prenylated flavin cofactor. Genome/metagenome analyses predicted phthalate degradation capacity also in the sulphate-reducing Desulfobacula toluolica, strain NaphS2, and other δ-proteobacteria. Our results suggest that phthalate degradation proceeds in all anaerobic bacteria via the labile phthaloyl-CoA that is captured and decarboxylated by highly abundant PCDs. In contrast, two alternative strategies have been established for the formation of phthaloyl-CoA, the possibly most unstable CoA ester in biology.

The ISME Journal, 2019

Syntrophorhabdus aromaticivorans is a syntrophically fermenting bacterium that can degrade isopht... more Syntrophorhabdus aromaticivorans is a syntrophically fermenting bacterium that can degrade isophthalate (3carboxybenzoate). It is a xenobiotic compound which has accumulated in the environment for more than 50 years due to its global industrial usage and can cause negative effects on the environment. Isophthalate degradation by the strictly anaerobic S. aromaticivorans was investigated to advance our understanding of the degradation of xenobiotics introduced into nature, and to identify enzymes that might have ecological significance for bioremediation. Differential proteome analysis of isophthalate-vs benzoate-grown cells revealed over 400 differentially expressed proteins of which only four were unique to isophthalate-grown cells. The isophthalate-induced proteins include a phenylacetate:CoA ligase, a UbiD-like decarboxylase, a UbiX-like flavin prenyltransferase, and a hypothetical protein. These proteins are encoded by genes forming a single gene cluster that putatively codes for anaerobic conversion of isophthalate to benzoyl-CoA. Subsequently, benzoyl-CoA is metabolized by the enzymes of the anaerobic benzoate degradation pathway that were identified in the proteomic analysis. In vitro enzyme assays with cell-free extracts of isophthalate-grown cells indicated that isophthalate is activated to isophthalyl-CoA by an ATP-dependent isophthalate:CoA ligase (IPCL), and subsequently decarboxylated to benzoyl-CoA by a UbiD family isophthalyl-CoA decarboxylase (IPCD) that requires a prenylated flavin mononucleotide (prFMN) cofactor supplied by UbiX to effect decarboxylation. Phylogenetic analysis revealed that IPCD is a novel member of the functionally diverse UbiD family (de)carboxylases. Homologs of the IPCD encoding genes are found in several other bacteria, such as aromatic compound-degrading denitrifiers, marine sulfate-reducers, and methanogenic communities in a terephthalate-degrading reactor. These results suggest that metabolic strategies adapted for degradation of isophthalate and other phthalate are conserved between microorganisms that are involved in the anaerobic degradation of environmentally relevant aromatic compounds.

Standards in genomic sciences, 2017

Anaerobium acetethylicum strain GluBS11(T) belongs to the family Lachnospiraceae within the order... more Anaerobium acetethylicum strain GluBS11(T) belongs to the family Lachnospiraceae within the order Clostridiales. It is a Gram-positive, non-motile and strictly anaerobic bacterium isolated from biogas slurry that was originally enriched with gluconate as carbon source (Patil, et al., Int J Syst Evol Microbiol 65:3289-3296, 2015). Here we describe the draft genome sequence of strain GluBS11(T) and provide a detailed insight into its physiological and metabolic features. The draft genome sequence generated 4,609,043 bp, distributed among 105 scaffolds assembled using the SPAdes genome assembler method. It comprises in total 4,132 genes, of which 4,008 were predicted to be protein coding genes, 124 RNA genes and 867 pseudogenes. The G + C content was 43.51 mol %. The annotated genome of strain GluBS11(T) contains putative genes coding for the pentose phosphate pathway, the Embden-Meyerhoff-Parnas pathway, the Entner-Doudoroff pathway and the tricarboxylic acid cycle. The genome reveale...

Microbial Biotechnology, 2016

Growth of biodiesel industries resulted in increased coproduction of crude glycerol which is ther... more Growth of biodiesel industries resulted in increased coproduction of crude glycerol which is therefore becoming a waste product instead of a valuable 'coproduct'. Glycerol can be used for the production of valuable chemicals, e.g. biofuels, to reduce glycerol waste disposal. In this study, a novel bacterial strain is described which converts glycerol mainly to ethanol and hydrogen with very little amounts of acetate, formate and 1,2-propanediol as coproducts. The bacterium offers certain advantages over previously studied glycerol-fermenting microorganisms. Anaerobium acetethylicum during growth with glycerol produces very little side products and grows in the presence of maximum glycerol concentrations up to 1500 mM and in the complete absence of complex organic supplements such as yeast extract or tryptone. The highest observed growth rate of 0.116 h À1 is similar to that of other glycerol degraders, and the maximum concentration of ethanol that can be tolerated was found to be about 60 mM (2.8 g l À1) and further growth was likely inhibited due to ethanol toxicity. Proteome analysis as well as enzyme assays performed in cell-free extracts demonstrated that glycerol is degraded via glyceraldehyde-3phosphate, which is further metabolized through the lower part of glycolysis leading to formation of mainly ethanol and hydrogen. In conclusion, fermentation of glycerol to ethanol and hydrogen by this bacterium represents a remarkable option to add value to the biodiesel industries by utilization of surplus glycerol.

Environmental Microbiology, 2016

The pathway of anaerobic degradation of o-phthalate was studied in the nitrate-reducing bacterium... more The pathway of anaerobic degradation of o-phthalate was studied in the nitrate-reducing bacterium Azoarcus sp. strain PA01. Differential two-dimensional protein gel profiling allowed the identification of specifically induced proteins in o-phthalate-grown compared to benzoate-grown cells. The genes encoding o-phthalate-induced proteins were found in a 9.9 kb gene cluster in the genome of Azoarcus sp. strain PA01. The o-phthalate-induced gene cluster codes for proteins homologous to a dicarboxylic acid transporter, putative CoA-transferases and a UbiD-like decarboxylase that were assigned to be specifically involved in the initial steps of anaerobic o-phthalate degradation. We propose that o-phthalate is first activated to o-phthalyl-CoA by a putative succinyl-CoA-dependent succinyl-CoA:o-phthalate CoA-transferase, and o-phthalyl-CoA is subsequently decarboxylated to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase. Results from in vitro enzyme assays with cell-free extracts of o-phthalate-grown cells demonstrated the formation of o-phthalyl-CoA from o-phthalate and succinyl-CoA as CoA donor, and its subsequent decarboxylation to benzoyl-CoA. The putative succinyl-CoA:o-phthalate CoA-transferase showed high substrate specificity for o-phthalate and did not accept isophthalate, terephthalate or 3-fluoro-o-phthalate whereas the putative o-phthalyl-CoA decarboxylase converted fluoro-ophthalyl-CoA to fluoro-benzoyl-CoA. No decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA. Both enzyme activities were oxygen-insensitive and inducible only after growth with o-phthalate. Further degradation of benzoyl-CoA proceeds analogous to the well-established anaerobic benzoyl-CoA degradation pathway of nitrate-reducing bacteria.

International Journal of Hydrogen Energy, 2012

A mesophilic alkaline tolerant fermentative microbe was isolated from estuarine sediment samples ... more A mesophilic alkaline tolerant fermentative microbe was isolated from estuarine sediment samples and designated as Clostridium butyricum TM-9A, based on 16S rRNA gene sequence. Batch experiments were conducted for investigation of TM-9A strain for its growth and hydrogen productivity from glucose, in an iron containing basal solution supplemented with yeast extract as organic nitrogen source. Hydrogen production started to evolve when cell growth entered exponential phase and reached maximum production rate at late exponential phase. Maximum hydrogen production was observed at 37 C, initial pH of 8.0 in the presence of 1% glucose. Optimization of process parameters resulted in increase in hydrogen yield from 1.64 to 2.67 mol of H 2 /mol glucose. Molar yield of H 2 increased further from 2.67 to 3.1 mol of H 2 /mol of glucose with the decrease in hydrogen partial pressure, obtained by lowering the total pressure in the head space of the batch reactor. Acetate and butyrate were the measure volatile fatty acids generated during hydrogen fermentation. TM-9A strain produced hydrogen efficiently from a range of pentose and hexose sugars including di-, tri and poly-saccharides like;

Standards in Genomic Sciences, 2015

Azoarcus sp. strain PA01(T) belongs to the genus Azoarcus, of the family Rhodocyclaceae within th... more Azoarcus sp. strain PA01(T) belongs to the genus Azoarcus, of the family Rhodocyclaceae within the class Betaproteobacteria. It is a facultatively anaerobic, mesophilic, non-motile, Gram-stain negative, non-spore-forming, short rod-shaped bacterium that was isolated from a wastewater treatment plant in Constance, Germany. It is of interest because of its ability to degrade o-phthalate and a wide variety of aromatic compounds with nitrate as an electron acceptor. Elucidation of the o-phthalate degradation pathway may help to improve the treatment of phthalate-containing wastes in the future. Here, we describe the features of this organism, together with the draft genome sequence information and annotation. The draft genome consists of 4 contigs with 3,908,301 bp and an overall G + C content of 66.08 %. Out of 3,712 total genes predicted, 3,625 genes code for proteins and 87 genes for RNAs. The majority of the protein-encoding genes (83.51 %) were assigned a putative function while those remaining were annotated as hypothetical proteins.

International Journal of Systematic and Evolutionary Microbiology, 2015

A new type of a strictly anaerobic, mesophilic bacterium was enriched and isolated with gluconate... more A new type of a strictly anaerobic, mesophilic bacterium was enriched and isolated with gluconate as sole substrate from a methanogenic sludge collected from a biogas reactor. Cells of strain GluBS11T stained Gram-positive, non-motile, straight rods measuring 3 - 4.5 x 0.8 - 1.2 µm. The temperature range for growth was 15 - 37ºC, with optimal growth at 30ºC, the pH range was 6.5 to 8.5, with optimal growth at pH 7 and generation time 60 min. API rapid 32A reactions tested positive for α-galactosidase, α-glucosidase and β-glucosidase and negative for catalase and oxidase. A broad variety of utilized substrates include gluconate, glucose, fructose, maltose, sucrose, lactose, galactose, melezitose, melibiose, mannitol, erythritol, glycerol and esculin. Products of gluconate fermentation were ethanol, acetate, formate, H2 and CO2. Sulfate or nitrate did not serve as electron acceptor. Predominant cellular fatty acids (&amp;amp;amp;amp;amp;gt;10%) were C14:0, C16:0, C16:1 ω7c/15 iso-2-OH and C18:1 ω7c. The DNA G+C content of strain GluBS11T was 44.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequence data revealed that strain GluBS11T is a member of the subcluster XIVa within the order Clostridiales. The closest cultured relatives are Clostridium herbivorans (93.1%), Clostridium populeti (93.3%), Eubacterium uniforme (92.4%) and Clostridium polysacharolyticum (91.5%). Based on this 16S rRNA sequence divergence (&amp;amp;amp;amp;amp;gt;6.5%) as well as on chemotaxonomic and phenotypic differences to these taxa, strain GluBS11T is considered to represent a novel genus and species, for which the name Anaerobium acetethylicum gen. nov. sp. nov., is proposed. The type strain is GluBS11T (LMG28619T=KCTC15450T=DSM29698T).

International journal of systematic and evolutionary microbiology, 2015

A strictly anaerobic, mesophilic, sulfate-reducing bacterium, strain KoBa311(T), isolated from th... more A strictly anaerobic, mesophilic, sulfate-reducing bacterium, strain KoBa311(T), isolated from the wastewater treatment plant at Konstanz, Germany, was characterized phenotypically and phylogenetically. Cells were Gram-stain-negative, non-motile, oval to short rods, 3-5 µm long and 0.8-1.0 µm wide with rounded ends, dividing by binary fission and occurring singly or in pairs. The strain grew optimally in freshwater medium and the optimum temperature was 30 °C. Strain KoBa311(T) showed optimum growth at pH 7.3-7.6. Organic electron donors were oxidized completely to carbon dioxide concomitant with sulfate reduction to sulfide. At excess substrate supply, substrates were oxidized incompletely and acetate (mainly) and/or propionate accumulated. The strain utilized short-chain fatty acids, alcohols (except methanol) and benzoate. Sulfate and DMSO were used as terminal electron acceptors for growth. The genomic DNA G+C content was 52.3 mol% and the respiratory quinone was menaquinone MK-...

The present study reports hydrogen production potential by an alkaline-tolerant bacterium Clostri... more The present study reports hydrogen production potential by an alkaline-tolerant bacterium Clostridium butyricum strain TM-9A isolated from an estuarine river sediment sample and identified on the basis of 16S rRNA gene sequencing. Different process parameters such as initial pH, temperature and NaCl concentration affected the hydrogen production potential and growth of TM-9A strain in batch dark fermentation experiments. Glucose (10 g L -1 ) was used as substrate for an optimization study. TM-9A strain was able to tolerate up to 16 g L -1 of NaCl. Strain TM-9A produced maximum hydrogen, 57.8 mmol L -1 , at an initial pH 8 under mesophilic conditions, i.e. in the absence of NaCl. Acetic and butyric acid were the major soluble metabolites detected at 12.32 and 11.61 mmol L -1 , respectively. Hydrogen yield was 2.0-2.1 mol H 2 /mol glucose. Furthermore, the strain was also evaluated for its ability to utilize different carbohydrate-rich substrates like corn syrup (25.74 mmol L -1 ), molasses (23.44 mmol L -1 ) and starch (43.29 mmol L -1 ), sucrose (31.45 mmol L -1 ), and cellulose (4.16 mmol L -1 ), respectively for hydrogen production.