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Scientific Reports Aug 2017Aspartate family amino acids (AFAAs) have important commercial values due to their wide spectrum of applications. Most if not all AFAAs are produced under aerobic...
Aspartate family amino acids (AFAAs) have important commercial values due to their wide spectrum of applications. Most if not all AFAAs are produced under aerobic conditions which is energy-intensive. To establish a cost-effective anaerobic process for production of AFAAs, it holds great promise to develop a new pathway enabling the conversion of oxoloacetate into aspartate through direct amination which is catalyzed by aspartate dehydrogenase (AspDH). Compared with the well studied aspartate aminotransferase and aspartate ammonia-lyase, only a few AspDHs are characterized till date, and failure to reproduce the high activity of AspDH from Rastonia eutropha documented in the literature encouraged us to screen and characterize novel AspDHs from different origins. Interestingly, the AspDHs from Klebsiella pneumoniae 34618 (KpnAspDH) and Delftia sp. Cs1-4 (DelAspDH) showed successful soluble expression. KpnAspDH and DelAspDH containing C-terminal hexa-histidine tags were purified and characterized for their catalytic properties. Notably, in addition to its high reductive amination activity, DelAspDH exhibited considerable stability as compared to the other source of AspDHs. This work thus provides novel enzyme resource for engineering strains capable of producing AFAAs under anaerobic conditions.
Topics: Amination; Amino Acid Oxidoreductases; Anaerobiosis; Aspartic Acid; Bacterial Proteins; Delftia; Enzyme Stability; Industrial Microbiology; Kinetics; Klebsiella pneumoniae; Recombinant Proteins; Substrate Specificity
PubMed: 28801651
DOI: 10.1038/s41598-017-05522-7 -
Journal of Applied Microbiology May 2016The aim of this study was to develop a multiplex PCR (mPCR) assay for rapid, sensitive and simultaneous detection of six important rice pathogens: Xanthomonas oryzae pv....
AIMS
The aim of this study was to develop a multiplex PCR (mPCR) assay for rapid, sensitive and simultaneous detection of six important rice pathogens: Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Pseudomonas fuscovaginae, Burkholderia glumae, Burkholderia gladioli and Acidovorax avenae subsp. avenae.
METHODS AND RESULTS
Specific primers were designed through a bioinformatics pipeline. Sensitivity of detection was established using both traditional PCR and quantitative real-time PCR on isolated DNA and on bacterial cells both in vitro and in simulated diseased seeds and the parameters were optimized for an mPCR assay. A total of 150 bacterial strains were tested for specificity. The mPCR assay accurately predicted the presence of pathogens among 44 symptomatic and asymptomatic rice seed, sheath and leaf samples.
CONCLUSIONS
This study confirmed that this mPCR assay is a rapid, reliable and simple tool for the simultaneous detection of six important rice bacterial pathogens.
SIGNIFICANCE AND IMPACT OF THE STUDY
This study is the first report of a method allowing simultaneous detection of six major rice pathogens. The ability to use crude extracts from plants without bacterial isolation or DNA extraction enhances the value of this mPCR technology for rapid detection and aetiological/epidemiological studies.
Topics: Burkholderia; Comamonadaceae; DNA, Bacterial; Multiplex Polymerase Chain Reaction; Oryza; Plant Diseases; Plant Leaves; Pseudomonas; Seeds; Xanthomonas
PubMed: 26864896
DOI: 10.1111/jam.13094 -
Structure (London, England : 1993) Apr 2019Electron cryotomography enables 3D visualization of cells in a near-native state at molecular resolution. The produced cellular tomograms contain detailed information...
Electron cryotomography enables 3D visualization of cells in a near-native state at molecular resolution. The produced cellular tomograms contain detailed information about a plethora of macromolecular complexes, their structures, abundances, and specific spatial locations in the cell. However, extracting this information in a systematic way is very challenging, and current methods usually rely on individual templates of known structures. Here, we propose a framework called "Multi-Pattern Pursuit" for de novo discovery of different complexes from highly heterogeneous sets of particles extracted from entire cellular tomograms without using information of known structures. These initially detected structures can then serve as input for more targeted refinement efforts. Our tests on simulated and experimental tomograms show that our automated method is a promising tool for supporting large-scale template-free visual proteomics analysis.
Topics: Bacterial Proteins; Bdellovibrio bacteriovorus; Chaperonin 60; Comamonadaceae; Cryoelectron Microscopy; Data Mining; Electron Microscope Tomography; Firmicutes; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Proteomics
PubMed: 30744995
DOI: 10.1016/j.str.2019.01.005 -
TheScientificWorldJournal 2019is a legume cover crop, found chiefly in the wet zone of Sri Lanka. Nitrogen fixation is performed by nodular inhabitants of this cover crop, comparable to the...
is a legume cover crop, found chiefly in the wet zone of Sri Lanka. Nitrogen fixation is performed by nodular inhabitants of this cover crop, comparable to the nodule-dwelling bacteria of most other legume plants. We isolated a bacterium (Sub1) from , of coccobacillus cell shape, that showed nodulation, when assessed by hydroponics, showing nodules as early as 3 weeks after reinfection. When a fragment from the genome of this bacterium was amplified using a pair of primers, it yielded an amplicon of 360 bp that, when sequenced, helped us identify the bacterium, as belonging to a species of at 99% sequence identity. When we constructed a phylogenetic tree with neighboring sequences, we encountered sequences of forming a monophyletic cluster, which too contained a single species. The genus is a bacterium that, so far, has not been associated with legume nodules. Sub1 secreted a pair of enzymes to the extracellular medium to degrade cellulose and milk proteins. The Sub1 bacterium showed biofilm formation and secreted into the extracellular medium, indole acetic acid. Sub1 also showed a "bulls eye" swarming pattern for the chemoattractant proline, while showing no significant chemotaxis movement, for naringenin, quercetin, and glutamate. Sub1 too possesses the basic genetic foundation ( and ) to produce a molybdenum-dependent nitrogenase enzyme. We finally show that this rare nonrhizobial bacterium is able to impact, positively, nodulation and shoot length of plants, demonstrating that this beta-proteobacterium can abet the biological vigor of this legume cover crop.
Topics: Comamonadaceae; Nitrogen Fixation; Phylogeny; Pueraria; Root Nodules, Plant
PubMed: 31057340
DOI: 10.1155/2019/9782684 -
Scientific Reports Mar 2018Colonic diverticula are protrusions of the mucosa through weak areas of the colonic musculature. The etiology of diverticulosis is poorly understood, but could be...
Colonic diverticula are protrusions of the mucosa through weak areas of the colonic musculature. The etiology of diverticulosis is poorly understood, but could be related to gut bacteria. Using mucosal biopsies from the sigmoid colon of 226 subjects with and 309 subjects without diverticula during first-time screening colonoscopy, we assessed whether individuals with incidental colonic diverticulosis have alternations in the adherent bacterial communities in the sigmoid colon. We found little evidence of substantial associations between the microbial community and diverticulosis among cases and controls. Comparisons of bacterial abundances across all taxonomic levels showed differences for phylum Proteobacteria (p = 0.038) and family Comamonadaceae (p = 0.035). The r-squared values measuring the strength of these associations were very weak, however, with values ~2%. There was a similarly small association between the abundance of each taxa and total diverticula counts. Cases with proximal only diverticula and distal only diverticula likewise showed little difference in overall microbiota profiles. This large study suggests little association between diverticula and the mucosal microbiota overall, or by diverticula number and location. We conclude that the mucosal adherent microbiota community composition is unlikely to play a substantial role in development of diverticulosis.
Topics: Aged; Aged, 80 and over; Bacteria; Biopsy; Case-Control Studies; Colon, Sigmoid; Colonoscopy; Comamonadaceae; Diverticulosis, Colonic; Female; Gastrointestinal Microbiome; Humans; Intestinal Mucosa; Male; Middle Aged; Proteobacteria; Severity of Illness Index
PubMed: 29563543
DOI: 10.1038/s41598-018-23023-z -
Journal of Bacteriology Apr 2016Bacteriophytochrome photoreceptors (BphPs) and their cognate response regulators make up two-component signal transduction systems which direct bacteria to mount...
UNLABELLED
Bacteriophytochrome photoreceptors (BphPs) and their cognate response regulators make up two-component signal transduction systems which direct bacteria to mount phenotypic responses to changes in environmental light quality. Most of these systems utilize single-domain response regulators to transduce signals through unknown pathways and mechanisms. Here we describe the photocycle and autophosphorylation kinetics of RtBphP1, a red light-regulated histidine kinase from the desert bacterium Ramlibacter tataouinensis RtBphP1 undergoes red to far-red photoconversion with rapid thermal reversion to the dark state. RtBphP1 is autophosphorylated in the dark; this activity is inhibited under red light. The RtBphP1 cognate response regulator, the R. tataouinensis bacteriophytochrome response regulator (RtBRR), and a homolog, AtBRR from Agrobacterium tumefaciens, crystallize unexpectedly as arm-in-arm dimers, reliant on a conserved hydrophobic motif, hFWAhL (where h is a hydrophobic M, V, L, or I residue). RtBRR and AtBRR dimerize distinctly from four structurally characterized phytochrome response regulators found in photosynthetic organisms and from all other receiver domain homodimers in the Protein Data Bank. A unique cacodylate-zinc-histidine tag metal organic framework yielded single-wavelength anomalous diffraction phases and may be of general interest. Examination of the effect of the BRR stoichiometry on signal transduction showed that phosphorylated RtBRR is accumulated more efficiently than the engineered monomeric RtBRR (RtBRRmon) in phosphotransfer reactions. Thus, we conclude that arm-in-arm dimers are a relevant signaling intermediate in this class of two-component regulatory systems.
IMPORTANCE
BphP histidine kinases and their cognate response regulators comprise widespread red light-sensing two-component systems. Much work on BphPs has focused on structural understanding of light sensing and on enhancing the natural infrared fluorescence of these proteins, rather than on signal transduction or the resultant phenotypes. To begin to address this knowledge gap, we solved the crystal structures of two single-domain response regulators encoded by a region immediately downstream of that encoding BphPs. We observed a previously unknown arm-in-arm dimer linkage. Monomerization via deletion of the C-terminal dimerization motif had an inhibitory effect on net response regulator phosphorylation, underlining the importance of these unusual dimers for signal transduction.
Topics: Amino Acid Sequence; Bacterial Proteins; Comamonadaceae; Gene Expression Regulation, Bacterial; Light; Models, Molecular; Molecular Sequence Data; Protein Conformation; Signal Transduction
PubMed: 26833410
DOI: 10.1128/JB.00872-15 -
Applied and Environmental Microbiology Jan 2020Cyanuric acid is an industrial chemical produced during the biodegradation of -triazine pesticides. The biodegradation of cyanuric acid has been elucidated using a...
Cyanuric acid is an industrial chemical produced during the biodegradation of -triazine pesticides. The biodegradation of cyanuric acid has been elucidated using a single model system, sp. strain ADP, in which cyanuric acid hydrolase (AtzD) opens the -triazine ring and AtzEG deaminates the ring-opened product. A significant question remains as to whether the metabolic pathway found in sp. ADP is the exception or the rule in bacterial genomes globally. Here, we show that most bacteria utilize a different pathway, metabolizing cyanuric acid via biuret. The new pathway was determined by reconstituting the pathway with purified enzymes and by mining more than 250,000 genomes and metagenomes. We isolated soil bacteria that grow on cyanuric acid as a sole nitrogen source and showed that the genome from a strain had a canonical cyanuric acid hydrolase gene but different flanking genes. The flanking gene encoded an enzyme that we show catalyzed the decarboxylation of the cyanuric acid hydrolase product, carboxybiuret. The reaction generated biuret, a pathway intermediate further transformed by biuret hydrolase (BiuH). The prevalence of the newly defined pathway was determined by cooccurrence analysis of cyanuric acid hydrolase genes and flanking genes. Here, we show the biuret pathway was more than 1 order of magnitude more prevalent than the original sp. ADP pathway. Mining a database of over 40,000 bacterial isolates with precise geospatial metadata showed that bacteria with concurrent cyanuric acid and biuret hydrolase genes were distributed throughout the United States. Cyanuric acid is produced naturally as a contaminant in urea fertilizer, and it is used as a chlorine stabilizer in swimming pools. Cyanuric acid-degrading bacteria are used commercially in removing cyanuric acid from pool water when it exceeds desired levels. The total volume of cyanuric acid produced annually exceeds 200 million kilograms, most of which enters the natural environment. In this context, it is important to have a global understanding of cyanuric acid biodegradation by microbial communities in natural and engineered systems. Current knowledge of cyanuric acid metabolism largely derives from studies on the enzymes from a single model organism, sp. ADP. In this study, we obtained and studied new microbes and discovered a previously unknown cyanuric acid degradation pathway. The new pathway identified here was found to be much more prevalent than the pathway previously established for sp. ADP. In addition, the types of environment, taxonomic prevalences, and geospatial distributions of the different cyanuric acid degradation pathways are described here.
Topics: Biodegradation, Environmental; Biuret; Comamonas; Environmental Pollutants; Herbaspirillum; Pseudomonas; Triazines
PubMed: 31676480
DOI: 10.1128/AEM.01964-19 -
BMC Microbiology Oct 2020Numerous studies have reported the health-promoting effects of exopolysaccharides (EPSs) in in vitro models; however, a functional evaluation of EPSs will provide...
BACKGROUND
Numerous studies have reported the health-promoting effects of exopolysaccharides (EPSs) in in vitro models; however, a functional evaluation of EPSs will provide additional knowledge of EPS-microbe interactions by in vivo intestinal microbial model. In the present study, high-throughput amplicon sequencing, short-chain fatty acid (SCFAs) and intestinal inflammation evaluation were performed to explore the potential benefits of exopolysaccharides (EPSs) and EPS-producing Lactobacillus (HNUB20 group) using the healthy zebrafish (Danio rerio) model.
RESULTS
The results based on microbial taxonomic analysis revealed that the abundance of four genera, Ochrobactrum, Sediminibacterium, Sphingomonas and Sphingobium, were increased in the control group in comparison to HNUB20 group. Pelomonas spp. levels were significantly higher and that of the genera Lactobacillus and Brachybacterium were significantly decreased in EPS group compared with control group. PICRUSt based functional prediction of gut microbiota metabolic pathways indicated that significantly lower abundance was found for transcription, and membrane transport, whereas folding, sorting and degradation and energy metabolism had significantly higher abundance after HNUB20 treatment. Two metabolic pathways, including metabolism and endocrine functions, were more abundant in the EPS group than control group. Similar to the HNUB20 group, transcription was also decreased in the EPS group compared with the control group. However, SCFAs and immune indexes indicated EPS and HNUB20 performed limited efficacy in the healthy zebrafish.
CONCLUSIONS
The present intestinal microbial model-based study indicated that EPSs and high-yield EPS-producing Lactobacillus can shake the structure of intestinal microbiota, but cannot change SCFAs presence and intestinal inflammation.
Topics: Actinobacteria; Animals; Bacteroidetes; Comamonadaceae; Fatty Acids, Volatile; Female; Gastrointestinal Microbiome; Intestines; Lactobacillus; Male; Metabolic Networks and Pathways; Ochrobactrum; Polysaccharides, Bacterial; Sphingomonadaceae; Sphingomonas; Transcription, Genetic; Zebrafish
PubMed: 33023493
DOI: 10.1186/s12866-020-01990-6 -
Applied Microbiology and Biotechnology Jun 2017The surfactant sodium lauryl ether sulfate (SLES) is widely used in the composition of detergents and frequently ends up in wastewater treatment plants (WWTPs). While...
The surfactant sodium lauryl ether sulfate (SLES) is widely used in the composition of detergents and frequently ends up in wastewater treatment plants (WWTPs). While aerobic SLES degradation is well studied, little is known about the fate of this compound in anoxic environments, such as denitrification tanks of WWTPs, nor about the bacteria involved in the anoxic biodegradation. Here, we used SLES as sole carbon and energy source, at concentrations ranging from 50 to 1000 mg L, to enrich and isolate nitrate-reducing bacteria from activated sludge of a WWTP with the anaerobic-anoxic-oxic (A/O) concept. In the 50 mg L enrichment, Comamonas (50%), Pseudomonas (24%), and Alicycliphilus (12%) were present at higher relative abundance, while Pseudomonas (53%) became dominant in the 1000 mg L enrichment. Aeromonas hydrophila strain S7, Pseudomonas stutzeri strain S8, and Pseudomonas nitroreducens strain S11 were isolated from the enriched cultures. Under denitrifying conditions, strains S8 and S11 degraded 500 mg L SLES in less than 1 day, while strain S7 required more than 6 days. Strains S8 and S11 also showed a remarkable resistance to SLES, being able to grow and reduce nitrate with SLES concentrations up to 40 g L. Strain S11 turned out to be the best anoxic SLES degrader, degrading up to 41% of 500 mg L. The comparison between SLES anoxic and oxic degradation by strain S11 revealed differences in SLES cleavage, degradation, and sulfate accumulation; both ester and ether cleavage were probably employed in SLES anoxic degradation by strain S11.
Topics: Aeromonas; Biodegradation, Environmental; Carbon; Comamonadaceae; Comamonas; Denitrification; Gram-Negative Bacteria; Oxidation-Reduction; Pseudomonas; Sewage; Sodium Dodecyl Sulfate; Surface-Active Agents
PubMed: 28299401
DOI: 10.1007/s00253-017-8212-x -
Nature Nov 2020Plants grow within a complex web of species that interact with each other and with the plant. These interactions are governed by a wide repertoire of chemical signals,...
Plants grow within a complex web of species that interact with each other and with the plant. These interactions are governed by a wide repertoire of chemical signals, and the resulting chemical landscape of the rhizosphere can strongly affect root health and development. Here, to understand how interactions between microorganisms influence root growth in Arabidopsis, we established a model system for interactions between plants, microorganisms and the environment. We inoculated seedlings with a 185-member bacterial synthetic community, manipulated the abiotic environment and measured bacterial colonization of the plant. This enabled us to classify the synthetic community into four modules of co-occurring strains. We deconstructed the synthetic community on the basis of these modules, and identified interactions between microorganisms that determine root phenotype. These interactions primarily involve a single bacterial genus (Variovorax), which completely reverses the severe inhibition of root growth that is induced by a wide diversity of bacterial strains as well as by the entire 185-member community. We demonstrate that Variovorax manipulates plant hormone levels to balance the effects of our ecologically realistic synthetic root community on root growth. We identify an auxin-degradation operon that is conserved in all available genomes of Variovorax and is necessary and sufficient for the reversion of root growth inhibition. Therefore, metabolic signal interference shapes bacteria-plant communication networks and is essential for maintaining the stereotypic developmental programme of the root. Optimizing the feedbacks that shape chemical interaction networks in the rhizosphere provides a promising ecological strategy for developing more resilient and productive crops.
Topics: Arabidopsis; Comamonadaceae; Ethylenes; Indoleacetic Acids; Microbiota; Operon; Plant Growth Regulators; Plant Roots; Rhizosphere; Signal Transduction
PubMed: 32999461
DOI: 10.1038/s41586-020-2778-7