scholarly journals Clostridium acidurici Electron-Bifurcating Formate Dehydrogenase

2013 ◽  
Vol 79 (19) ◽  
pp. 6176-6179 ◽  
Author(s):  
Shuning Wang ◽  
Haiyan Huang ◽  
Jörg Kahnt ◽  
Rudolf K. Thauer
Keyword(s):  
Uric Acid ◽  
Gene Cluster ◽  
Formate Dehydrogenase ◽  
Specific Activity ◽  
Enzyme Complex ◽  
Content Type ◽  
Cell Extracts

ABSTRACTCell extracts of uric acid-grownClostridium aciduricicatalyzed the coupled reduction of NAD+and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene clusterhylCBA-fdhF2.

scholarly journals Identification of a Formate-Dependent Uric Acid Degradation Pathway inEscherichia coli

2019 ◽  
Vol 201 (11) ◽  
Author(s):  
Yumi Iwadate ◽  
Jun-ichi Kato
Keyword(s):  
Escherichia Coli ◽  
Uric Acid ◽  
Formate Dehydrogenase ◽  
Sole Source ◽  
Degradation Pathway ◽  
Anaerobic Conditions ◽  
Content Type ◽  
Purine Catabolism ◽  
E Coli ◽  
Acid Degradation

ABSTRACTPurine is a nitrogen-containing compound that is abundant in nature. In organisms that utilize purine as a nitrogen source, purine is converted to uric acid, which is then converted to allantoin. Allantoin is then converted to ammonia. InEscherichia coli, neither urate-degrading activity nor a gene encoding an enzyme homologous to the known urate-degrading enzymes had previously been found. Here, we demonstrate urate-degrading activity inE. coli. We first identifiedaegAas anE. coligene involved in oxidative stress tolerance. An examination of gene expression revealed that bothaegAand its paralogygfTare expressed under both microaerobic and anaerobic conditions. TheygfTgene is localized within a chromosomal gene cluster presumably involved in purine catabolism. Accordingly, the expression ofygfTincreased in the presence of exogenous uric acid, suggesting thatygfTis involved in urate degradation. Examination of the change of uric acid levels in the growth medium with time revealed urate-degrading activity under microaerobic and anaerobic conditions in the wild-type strain but not in theaegA ygfTdouble-deletion mutant. Furthermore, AegA- and YgfT-dependent urate-degrading activity was detected only in the presence of formate and formate dehydrogenase H. Collectively, these observations indicate the presence of urate-degrading activity inE. colithat is operational under microaerobic and anaerobic conditions. The activity requires formate, formate dehydrogenase H, and eitheraegAorygfT. We also identified other putative genes which are involved not only in formate-dependent but also in formate-independent urate degradation and may function in the regulation or cofactor synthesis in purine catabolism.IMPORTANCEThe metabolic pathway of uric acid degradation to date has been elucidated only in aerobic environments and is not understood in anaerobic and microaerobic environments. In the current study, we showed thatEscherichia coli, a facultative anaerobic organism, uses uric acid as a sole source of nitrogen under anaerobic and microaerobic conditions. We also showed that formate, formate dehydrogenase H, and either AegA or YgfT are involved in uric acid degradation. We propose that formate may act as an electron donor for a uric acid-degrading enzyme in this bacterium.

scholarly journals LaoABCR, a Novel System for Oxidation of Long-Chain Alcohols Derived from SDS and Alkane Degradation inPseudomonas aeruginosa

2018 ◽  
Vol 84 (13) ◽  
pp. e00626-18 ◽  
Author(s):  
Gianna Panasia ◽  
Bodo Philipp
Keyword(s):  
Gene Cluster ◽  
Distribution Systems ◽  
Sodium Dodecyl ◽  
Constitutive Expression ◽  
Opportunistic Pathogen ◽  
Alkane Degradation ◽  
Content Type ◽  
Cell Extracts ◽  
Growth Substrates ◽  
Long Chain

ABSTRACTThe opportunistic pathogenPseudomonas aeruginosastrain PAO1 is able to use a variety of organic pollutants as growth substrates, including the anionic detergent sodium dodecyl sulfate (SDS) and long-chain alkanes. While the enzymes initiating SDS and alkane degradation are well known, the subsequent enzymatic steps for degradation of the derived primary long-chain alcohols have not yet been identified. By evaluating genes specifically induced during growth with SDS, a gene cluster encoding a putative alcohol dehydrogenase (PA0364/LaoA), a probable inner membrane protein (PA0365/LaoB), and a presumable aldehyde dehydrogenase (PA0366/LaoC) was identified and designated the Lao (long-chain-alcohol/aldehyde-oxidation) system. Growth experiments with deletion mutants with SDS, 1-dodecanol, and alkanes revealed that LaoA and LaoB are involved in the degradation of primary long-chain alcohols. Moreover, detection of 1-dodecanol oxidation in cell extracts by activity staining revealed an interdependency of LaoA and LaoB for efficient 1-dodecanol oxidation. Anin silicoanalysis yielded no well-characterized homologue proteins for LaoA and LaoB. Furthermore, a gene adjacent to thelaogene cluster encodes a putative transcriptional regulator (PA0367/LaoR). AlaoRdeletion mutant exhibited constitutive expression of LaoA and LaoB, indicating that LaoR is a repressor for the expression oflaoABC. Taken together, these results showed that the proteins LaoA and LaoB constitute a novel oxidation system for long-chain alcohols derived from pollutants.IMPORTANCEThe versatile and highly adaptive bacteriumPseudomonas aeruginosa is able to colonize a variety of habitats, including anthropogenic environments, where it is often challenged with toxic compounds. Its ability to degrade such compounds and to use them as growth substrates can significantly enhance spreading of this opportunistic pathogen in hygienic settings, such as clinics or water distribution systems. Thus, knowledge about the metabolism ofP. aeruginosacan contribute to novel approaches for preventing its growth and reducing nosocomial infections. As the Lao system is important for the degradation of two different classes of pollutants, the identification of these novel enzymes can be a useful contribution for developing effective antibacterial strategies.

scholarly journals Molecular Basis and Phylogenetic Implications of Deoxycylindrospermopsin Biosynthesis in the Cyanobacterium Raphidiopsis curvata

2012 ◽  
Vol 78 (7) ◽  
pp. 2256-2263 ◽  
Author(s):  
Yongguang Jiang ◽  
Peng Xiao ◽  
Gongliang Yu ◽  
Tomoharu Sano ◽  
Qianqian Pan ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Phylogenetic Trees ◽  
Purifying Selection ◽  
Neutral Evolution ◽  
Cylindrospermopsis Raciborskii ◽  
Insertion Mutation ◽  
Biosynthesis Gene Cluster ◽  
Content Type ◽  
Cell Extracts ◽  
Phylogenetic Implications

ABSTRACTNew insights into the distribution and biochemistry of the cyanotoxin cylindrospermopsin (CYN) have been provided by the recent determination of its biosynthesis gene cluster (cyr) in several cyanobacterial species.Raphidiopsis curvataCHAB1150 isolated from China was analyzed for CYN analogues. Only 7-deoxy-CYN was detected in the cell extracts. Thecyrgene cluster ofR. curvataCHAB1150 was sequenced, and thecyrgenes of this strain were found to have extremely high similarities (96% to 100%) to those from other nostocalean species. These species includeCylindrospermopsis raciborskiiAWT205,Aphanizomenonsp. strain 10E6, andAphanizomenon ovalisporumILC-146. Insertion mutation was identified within thecyrIgene, and transcripts ofcyrIand another functional genecyrJwere detected inR. curvataCHAB1150. General congruence between the phylogenetic trees based on bothcyrand 16Srrnwas displayed. Neutral evolution was found on the whole sequences of thecyrgenes, and 0 to 89 negative selected codons were detected in each gene. Therefore, the function of CyrI is to catalyze the oxygenation of 7-deoxy-CYN in CYN biosynthesis. The transcripts of the mutatedcyrIgene may result from polycistronic transcription. The high conservation of thecyrgenes may be ascribed to purifying selection and horizontal gene transfer.

scholarly journals Glyoxylate Metabolism Is a Key Feature of the Metabolic Degradation of 1,4-Dioxane by Pseudonocardia dioxanivorans Strain CB1190

2012 ◽  
Vol 78 (9) ◽  
pp. 3298-3308 ◽  
Author(s):  
Ariel Grostern ◽  
Christopher M. Sales ◽  
Wei-Qin Zhuang ◽  
Onur Erbilgin ◽  
Lisa Alvarez-Cohen
Keyword(s):  
Energy Source ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Glycolate Oxidase ◽  
Content Type ◽  
Cell Extracts ◽  
Expression Of Genes ◽  
Monooxygenase Gene ◽  
Glyoxylate Metabolism ◽  
Metabolic Degradation

ABSTRACTThe groundwater contaminant 1,4-dioxane (dioxane) is transformed by several monooxygenase-expressing microorganisms, but only a few of these, includingPseudonocardia dioxanivoransstrain CB1190, can metabolize the compound as a sole carbon and energy source. However, nothing is yet known about the genetic basis of dioxane metabolism. In this study, we used a microarray to study differential expression of genes in strain CB1190 grown on dioxane, glycolate (a previously identified intermediate of dioxane degradation), or pyruvate. Of eight multicomponent monooxygenase gene clusters carried by the strain CB1190 genome, only the monooxygenase gene cluster located on plasmid pPSED02 was upregulated with dioxane relative to pyruvate. Plasmid-borne genes for putative aldehyde dehydrogenases, an aldehyde reductase, and an alcohol oxidoreductase were also induced during growth with dioxane. With both dioxane and glycolate, a chromosomal gene cluster encoding a putative glycolate oxidase was upregulated, as were chromosomal genes related to glyoxylate metabolism through the glyoxylate carboligase pathway. Glyoxylate carboligase activity in cell extracts from cells pregrown with dioxane and inRhodococcus jostiistrain RHA1 cells expressing the putative strain CB1190 glyoxylate carboligase gene further demonstrated the role of glyoxylate metabolism in the degradation of dioxane. Finally, we used13C-labeled dioxane amino acid isotopomer analysis to provide additional evidence that metabolites of dioxane enter central metabolism as three-carbon compounds, likely as phosphoglycerate. The routing of dioxane metabolites via the glyoxylate carboligase pathway helps to explain how dioxane is metabolized as a sole carbon and energy source for strain CB1190.

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

scholarly journals A Novel CO-Responsive Transcriptional Regulator and Enhanced H2Production by an Engineered Thermococcus onnurineus NA1 Strain

2014 ◽  
Vol 81 (5) ◽  
pp. 1708-1714 ◽  
Author(s):  
Min-Sik Kim ◽  
Ae Ran Choi ◽  
Seong Hyuk Lee ◽  
Hae-Chang Jung ◽  
Seung Seob Bae ◽  
...  
Keyword(s):  
Production Rate ◽  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory System ◽  
Transcriptional Regulator ◽  
Bioreactor Culture ◽  
Wild Type ◽  
Content Type ◽  
Transcriptional Regulatory

ABSTRACTGenome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism inThermococcus onnurineusNA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes ofThermococcusspecies and “CandidatusKorarchaeum cryptofilum” OPF8. In-frame deletion of eithercorQorcorRcaused a severe impairment in CO-dependent growth and H2production. WhencorQandcorRdeletion mutants were complemented by introducing thecorQRgenes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integratedcorQR(ΔCorR/corQR↑) compared with those in the wild-type strain. In addition, the ΔCorR/corQR↑strain exhibited a much higher H2production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2production rate (191.9 mmol liter−1h−1) and the specific H2production rate (249.6 mmol g−1h−1) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that thecorQRgenes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2production.

scholarly journals Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17

2014 ◽  
Vol 58 (10) ◽  
pp. 6185-6196 ◽  
Author(s):  
Marius Spohn ◽  
Norbert Kirchner ◽  
Andreas Kulik ◽  
Angelika Jochim ◽  
Felix Wolf ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gene Cluster ◽  
High Performance ◽  
Pathogenic Bacteria ◽  
Genome Mining ◽  
Gene Clusters ◽  
Von Willebrand Disease ◽  
Biosynthesis Gene Cluster ◽  
Content Type ◽  
Bioinformatic Tools

ABSTRACTThe emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products isAmycolatopsis. However,Amycolatopsis japonicumdoes not produce an antibiotic under standard laboratory conditions. In contrast to mostAmycolatopsisstrains,A. japonicumis genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, thebbrgene fromAmycolatopsis balhimycina(bbrAba), intoA. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing ofA. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed thein silicoprediction that the recombinantA. japonicum/pRM4-bbrAbasynthesizes ristomycin A.

Characterization of a factor that stimulates hydrolysis of GTP bound to ras gene product p21 (GTPase-activating protein) and correlation of its activity to cell density

1988 ◽  
Vol 8 (10) ◽  
pp. 4169-4173
Author(s):  
M Hoshino ◽  
M Kawakita ◽  
S Hattori
Keyword(s):  
Gene Product ◽  
Cell Density ◽  
Cultured Cells ◽  
Specific Activity ◽  
Gtpase Activating Protein ◽  
Ras Gene ◽  
Cell Extracts ◽  
Mouse Tissues ◽  
Almost All ◽  
Hydrolysis Of

The postmicrosomal fraction of the extract from NIH 3T3 and BALB/c 3T3 cells stimulated the hydrolysis of GTP bound to H-ras gene product p21 by severalfold. The stimulation was observed with normal p21 but not with p21 with valine as the 12th residue. This specificity is similar to that of GTPase-activating protein (GAP) for N-ras p21 described by M. Trahey and F. McCormick (Science 238:542-545, 1987). Consistent with this specificity, analysis of p21-bound nucleotides in living cells revealed that almost all normal p21 bound GDP, whereas oncogenic mutant p21s bound both GTP and GDP. Similar activity was also found in various mouse tissues, with brain tissue showing the highest specific activity. When cell extracts were prepared from cultured cells, there was a linear relationship between GAP activity and cell density. These results suggest the factor is involved in the regulation of cell proliferation.

scholarly journals Characterization of a Signaling System inStreptococcus mitisThat Mediates Interspecies Communication withStreptococcus pneumoniae

2018 ◽  
Vol 85 (2) ◽  
Author(s):  
R. Junges ◽  
K. Sturød ◽  
G. Salvadori ◽  
H. A. Åmdal ◽  
T. Chen ◽  
...  
Keyword(s):  
Oral Cavity ◽  
Gene Cluster ◽  
Communication System ◽  
Target Genes ◽  
Genetic Mutation ◽  
Signal Molecules ◽  
Streptococcus Mitis ◽  
Futile Cycle ◽  
Content Type ◽  
Surface Polysaccharide

ABSTRACTStreptococcus mitisis found in the oral cavity and nasopharynx and forms a significant portion of the human microbiome. In this study,in silicoanalyses indicated the presence of an Rgg regulator and short hydrophobic peptide (Rgg/SHP) cell-to-cell communication system inS. mitis. Although Rgg presented greater similarity to a repressor inStreptococcus pyogenes, autoinducing assays and genetic mutation analysis revealed that inS. mitisRgg acts as an activator. Transcriptome analysis showed that in addition toshp, the system regulates two other downstream genes, comprising a segment of a putative lantibiotic gene cluster that is in a conjugative element locus in different members of the mitis group. Close comparison to a similar lantibiotic gene cluster inStreptococcus pneumoniaeindicated thatS. mitislacked the full set of genes. Despite the potential of SHP to trigger a futile cycle of autoinduction, growth was not significantly affected for therggmutant under normal or antibiotic stress conditions. TheS. mitisSHP was, however, fully functional in promoting cross-species communication and increasingS. pneumoniaesurface polysaccharide production, which in this species is regulated by Rgg/SHP. The activity of SHPs produced by both species was detected in cocultures using aS. mitisreporter strain. In competitive assays, a slight advantage was observed for therggmutants. We conclude that the Rgg/SHP system inS. mitisregulates the expression of its ownshpand activates an Rgg/SHP system inS. pneumoniaethat regulates surface polysaccharide synthesis. Fundamentally, cross-communication of such systems may have a role during multispecies interactions.IMPORTANCEBacteria secrete signal molecules into the environment which are sensed by other cells when the density reaches a certain threshold. In this study, we describe a communication system inStreptococcus mitis, a commensal species from the oral cavity, which we also found in several species and strains of streptococci from the mitis group. Further, we show that this system can promote cross-communication withS. pneumoniae, a closely related major human pathogen. Importantly, we show that this cross-communication can take place during coculture. While the genes regulated inS. mitisare likely part of a futile cycle of activation, the target genes inS. pneumoniaeare potentially involved in virulence. The understanding of such complex communication networks can provide important insights into the dynamics of bacterial communities.

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