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The Journal of Biological Chemistry Dec 2021Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase...
Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDO from Comamonas testosteroni KF1 and found that it had an apparent k/K for phthalate of 0.58 ± 0.09 μMs, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDO larger than that of other characterized ROs. Complexes of PDO with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.
Topics: Bacterial Proteins; Catalysis; Comamonas testosteroni; Crystallography, X-Ray; Oxygenases; Protein Domains; Substrate Specificity
PubMed: 34800435
DOI: 10.1016/j.jbc.2021.101416 -
International Journal of Molecular... Aug 2020Bacterial fruit blotch (BFB), caused by , seriously affects watermelon and other cucurbit crops, resulting in significant economic losses. However, the pathogenicity...
Bacterial fruit blotch (BFB), caused by , seriously affects watermelon and other cucurbit crops, resulting in significant economic losses. However, the pathogenicity mechanism of is not well understood. Plant pathogenic bacteria often suppress the plant immune response by secreting effector proteins. Thus, identifying effector proteins and determining their functions may improve our understanding of the underlying pathogenetic mechanisms. In this study, a novel effector, AopN, which is localized on the cell membrane of was identified. The functional analysis revealed that AopN significantly inhibited the flg22-induced reactive oxygen species burst. AopN induced a programmed cell death (PCD) response. Unlike its homologous protein, the ability of AopN to induce PCD was dependent on two motifs of unknown functions (including DUP4129 and Cpta_toxin), but was not dependent on LXXLL domain. More importantly, the virulence of the mutant of in significantly decreased, indicating that it was a core effector. Further analysis revealed that AopN interacted with watermelon ClHIPP and ClLTP, which responds to strain Aac5 infection at the transcription level. Collectively, these findings indicate that AopN suppresses plant immunity and activates the effector-triggered immunity pathway.
Topics: Amino Acid Motifs; Apoptosis; Bacterial Proteins; Cell Membrane; Citrullus; Comamonadaceae; Green Fluorescent Proteins; Host-Pathogen Interactions; Plant Cells; Plant Diseases; Plant Proteins; Reactive Oxygen Species; Nicotiana; Two-Hybrid System Techniques; Virulence
PubMed: 32842656
DOI: 10.3390/ijms21176050 -
Applied and Environmental Microbiology Jul 2005The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765,...
The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.
Topics: Comamonadaceae; Comamonas; Crystallization; Dioxygenases; Kinetics; Nitrobenzenes; Structure-Activity Relationship; Toluene
PubMed: 16000792
DOI: 10.1128/AEM.71.7.3806-3814.2005 -
Biophysical Journal Mar 2018Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste...
Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 Å resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic degradation.
Topics: Amino Acid Sequence; Catalytic Domain; Comamonadaceae; Hydrolases; Molecular Docking Simulation; Polyethylene Terephthalates; Protein Binding
PubMed: 29590588
DOI: 10.1016/j.bpj.2018.02.005 -
Genes Feb 2020Bacterial fruit blotch (BFB) causes losses in melon marketable yield. However, until now, there has been no information about the genetic loci responsible for resistance...
Bacterial fruit blotch (BFB) causes losses in melon marketable yield. However, until now, there has been no information about the genetic loci responsible for resistance to the disease or their pattern of inheritance. We determined the inheritance pattern of BFB resistance from a segregating population of 491 F individuals raised by crossing BFB-resistant (PI 353814) and susceptible (PI 614596) parental accessions. All F plants were resistant to strain KACC18782, and F plants segregated with a 3:1 ratio for resistant and susceptible phenotypes, respectively, in a seedling bioassay experiment, indicating that BFB resistance is controlled by a monogenic dominant gene. In an investigation of 57 putative disease-resistance related genes across the melon genome, only the MELO3C022157 gene (encoding TIR-NBS-LRR domain), showing polymorphism between resistant and susceptible parents, revealed as a good candidate for further investigation. Cloning, sequencing and quantitative RT-PCR expression of the polymorphic gene MELO3C022157 located on chromosome 9 revealed multiple insertion/deletions (InDels) and single nucleotide polymorphisms (SNPs), of which the SNP AT in the second exon of the gene caused loss of the LRR domain and truncated protein in the susceptible accession. The InDel marker MB157-2, based on the large (504 bp) insertion in the first intron of the susceptible accession, was able to distinguish resistant and susceptible accessions among 491 F and 22 landraces/inbred accessions with 98.17% and 100% detection accuracy, respectively. This novel PCR-based, co-dominant InDel marker represents a practical tool for marker-assisted breeding aimed at developing BFB-resistant melon accessions.
Topics: Chromosome Mapping; Comamonadaceae; Cucumis melo; Disease Resistance; Fruit; INDEL Mutation; Inheritance Patterns; Phenotype; Plant Diseases; Polymorphism, Single Nucleotide
PubMed: 32093120
DOI: 10.3390/genes11020220 -
Molecular Microbiology Sep 2019Transmembrane chemoreceptors are widely present in Bacteria and Archaea. They play a critical role in sensing various signals outside and transmitting to the cell...
Transmembrane chemoreceptors are widely present in Bacteria and Archaea. They play a critical role in sensing various signals outside and transmitting to the cell interior. Here, we report the structure of the periplasmic ligand-binding domain (LBD) of the transmembrane chemoreceptor MCP2201, which governs chemotaxis to citrate and other organic compounds in Comamonas testosteroni. The apo-form LBD crystal revealed a typical four-helix bundle homodimer, similar to previously well-studied chemoreceptors such as Tar and Tsr of Escherichia coli. However, the citrate-bound LBD revealed a four-helix bundle homotrimer that had not been observed in bacterial chemoreceptor LBDs. This homotrimer was further confirmed with size-exclusion chromatography, analytical ultracentrifugation and cross-linking experiments. The physiological importance of the homotrimer for chemotaxis was demonstrated with site-directed mutations of key amino acid residues in C. testosteroni mutants.
Topics: Bacterial Proteins; Chemotaxis; Citric Acid; Comamonas testosteroni; Dimerization; Ligands; Methyl-Accepting Chemotaxis Proteins; Protein Binding; Protein Conformation; Protein Conformation, alpha-Helical; Protein Domains
PubMed: 31177588
DOI: 10.1111/mmi.14326 -
Scientific Reports Oct 2016In recent years, use of the DNA-intercalating dye propidium monoazide (PMA) in real-time PCR has been reported as a novel method to detect viable bacteria in different...
In recent years, use of the DNA-intercalating dye propidium monoazide (PMA) in real-time PCR has been reported as a novel method to detect viable bacteria in different types of samples, such as food, environmental, and microbiological samples. In this study, viable cells of Acidovorax citrulli, the causal agent of bacterial seedling blight and fruit blotch, were selectively detected and differentiated from dead cells by real-time fluorescent polymerase chain reaction amplification after the bacterial solution was treated with the DNA-binding dye PMA. The primers and TaqMan probe were based on the A. citrulli genome (Aave_1909, Gene ID: 4669443) and were highly specific for A. citrulli. The detection threshold of this assay was 10 colony-forming units per mL (CFU/mL) in pure cell suspensions containing viable and dead cells and infected watermelon seeds. Application of this assay enables the selective detection of viable cells of A. citrulli and facilitates monitoring of the pathogen in watermelon and melon seeds.
Topics: Azides; Citrullus; Comamonadaceae; Food Safety; Propidium; Real-Time Polymerase Chain Reaction; Seeds
PubMed: 27739469
DOI: 10.1038/srep35457 -
PloS One 2016The Caatinga is a semi-arid biome in northeast Brazil. The Paraguaçú River is located in the Caatinga biome, and part of its course is protected by the National Park...
The Caatinga is a semi-arid biome in northeast Brazil. The Paraguaçú River is located in the Caatinga biome, and part of its course is protected by the National Park of Chapada Diamantina (PNCD). In this study we evaluated the effect of PNCD protection on the water quality and microbial community diversity of this river by analyzing water samples obtained from points located inside and outside the PNCD in both wet and dry seasons. Results of water quality analysis showed higher levels of silicate, ammonia, particulate organic carbon, and nitrite in samples from the unprotected area compared with those from protected areas. Pyrosequencing of the 16S rRNA genes revealed that Burkholderiales was abundant in samples from all three sites during both seasons and was represented primarily by the genus Polynucleobacter and members of the Comamonadaceae family (e.g., genus Limnohabitans). During the dry season, the unprotected area showed a higher abundance of Flavobacterium sp. and Arthrobacter sp., which are frequently associated with the presence and/or degradation of arsenic and pesticide compounds. In addition, genes that appear to be related to agricultural impacts on the environment, as well as those involved in arsenic and cadmium resistance, copper homeostasis, and propanediol utilization, were detected in the unprotected areas by metagenomic sequencing. Although PNCD protection improves water quality, agricultural activities around the park may affect water quality within the park and may account for the presence of bacteria capable of pesticide degradation and assimilation, evidencing possible anthropogenic impacts on the Caatinga.
Topics: Agriculture; Ammonia; Arthrobacter; Brazil; Burkholderiaceae; Carbon; Comamonadaceae; Ecosystem; Flavobacterium; High-Throughput Nucleotide Sequencing; Metagenome; Microbial Consortia; Nitrites; Particulate Matter; Phylogeny; RNA, Ribosomal, 16S; Rivers; Seasons; Silicates; Water Quality
PubMed: 26881432
DOI: 10.1371/journal.pone.0148296 -
Applied and Environmental Microbiology May 2020Most freshwater bacterial communities are characterized by a few dominant taxa that are often ubiquitous across freshwater biomes worldwide. Our understanding of the...
Most freshwater bacterial communities are characterized by a few dominant taxa that are often ubiquitous across freshwater biomes worldwide. Our understanding of the genomic diversity within these taxonomic groups is limited to a subset of taxa. Here, we investigated the genomic diversity that enables , a freshwater genus key in funneling carbon from primary producers to higher trophic levels, to achieve abundance and ubiquity. We reconstructed eight putative metagenome-assembled genomes (MAGs) from stations located along broad environmental gradients existing in Lake Michigan, part of Earth's largest surface freshwater system. strain inference analysis resolved a total of 23 strains from these MAGs, which strongly partitioned into two habitat-specific clusters with cooccurring strains from different lineages. The largest number of strains belonged to the abundant LimB lineage, for which robust strain delineation had not previously been achieved. Our data show that temperature and nutrient levels may be important environmental parameters associated with microdiversification within the genus. In addition, strains predominant in low- and high-phosphorus conditions had larger genomic divergence than strains abundant under different temperatures. Comparative genomics and gene expression analysis yielded evidence for the ability of LimB populations to exhibit cellular motility and chemotaxis, a phenotype not yet associated with available isolates. Our findings broaden historical marker gene-based surveys of microdiversification and provide evidence of genome diversity and its functional implications across freshwater gradients. is an important bacterial taxonomic group for cycling carbon in freshwater ecosystems worldwide. Here, we examined the genomic diversity of different lineages. We focused on the LimB lineage of this genus, which is globally distributed and often abundant, and its abundance has shown to be largely invariant to environmental change. Our data show that the LimB lineage is actually comprised of multiple cooccurring populations for which the composition and genomic characteristics are associated with variations in temperature and nutrient levels. The gene expression profiles of this lineage suggest the importance of chemotaxis and motility, traits that had not yet been associated with the genus, in adapting to environmental conditions.
Topics: Comamonadaceae; Gene Expression; Genes, Bacterial; Genetic Variation; Lakes; Michigan; Microbiota; Nutrients; Temperature
PubMed: 32169939
DOI: 10.1128/AEM.00140-20 -
Scientific Reports Jul 2019Microbial communities have not been studied using molecular approaches at high elevations on the African continent. Here we describe the diversity of microbial...
Microbial communities have not been studied using molecular approaches at high elevations on the African continent. Here we describe the diversity of microbial communities from ice and periglacial soils from near the summit of Mt. Kilimanjaro by using both Illumina and Sanger sequencing of 16S and 18S rRNA genes. Ice and periglacial soils contain unexpectedly diverse and rich assemblages of Bacteria and Eukarya indicating that there may be high rates of dispersal to the top of this tropical mountain and/or that the habitat is more conducive to microbial life than was previously thought. Most bacterial OTUs are cosmopolitan and an analysis of isolation by geographic distance patterns of the genus Polaromonas emphasized the importance of global Aeolian transport in the assembly of bacterial communities on Kilimanjaro. The eukaryotic communities were less diverse than the bacterial communities and showed more evidence of dispersal limitations and apparent endemism. Cercozoa dominated the 18S communities, including a high abundance of testate amoebae and a high diversity of endemic OTUs within the Vampyrellida. These results argue for more intense study of this unique high-elevation "island of the cryosphere" before the glaciers of Kilimanjaro disappear forever.
Topics: Biodiversity; Comamonadaceae; Ecosystem; Eukaryota; Ice Cover; Microbiota; RNA, Ribosomal, 16S; RNA, Ribosomal, 18S; Soil Microbiology; Tanzania
PubMed: 31337772
DOI: 10.1038/s41598-019-46521-0