scholarly journals Cloning of Two Gene Clusters Involved in the Catabolism of 2,4-Dinitrophenol by Paraburkholderia sp. Strain KU-46 and Characterization of the Initial DnpAB Enzymes and a Two-Component Monooxygenase DnpC1C2

2019 ◽  
Author(s):  
Taisei Yamamoto ◽  
Yaxuan Liu ◽  
Nozomi Kohaya ◽  
Yoshie Hasegawa ◽  
Peter C.K. Lau ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Tricarboxylic Acid Cycle ◽  
Gene Clusters ◽  
Degradation Pathway ◽  
Pcr Analysis ◽  
Gram Negative Bacteria ◽  
Protein Database ◽  
Gene Encoding ◽  
Gram Negative ◽  
Two Component

AbstractBesides an industrial pollutant, 2,4-dinitrophenol (DNP) has been used illegally as a weight loss drug that had claimed human lives. Little is known about the metabolism of DNP, particularly among Gram-negative bacteria. In this study, two non-contiguous genetic loci of Paraburkholderia (formerly Burkholderia) sp. strain KU-46 genome were identified and four key initial genes (dnpA, dnpB, and dnpC1C2) were characterized to provide molecular and biochemical evidence for the degradation of DNP via the formation of 4-nitrophenol (NP), a pathway that is unique among DNP utilizing bacteria. Reverse transcription PCR analysis indicated that the dnpA gene encoding the initial hydride transferase (28 kDa), and the dnpB gene encoding a nitrite-eliminating enzyme (33 kDa), are inducible by DNP and the two genes are organized in an operon. Purified DnpA and DnpB from overexpression clones in Escherichia coli effected the transformation of DNP to NP via the formation of hydride-Meisenheimer complex of DNP. The function of DnpB appears new since all homologs of DnpB sequences in the protein database are annotated as putative nitrate ABC transporter substrate-binding proteins. The gene cluster responsible for the degradation of DNP after NP formation was designated dnpC1C2DXFER. DnpC1 and DnpC2 were functionally characterized as the respective FAD reductase and oxygenase components of the two-component NP monooxygenase. Both NP and 4-nitrocatechol were shown to be substrates, producing hydroquinone and hydroxyquinol, respectively. Elucidation of the hqdA1A2BCD gene cluster allows the delineation of the final degradation pathway of hydroquinone to ß-ketoadipate prior to its entry to the tricarboxylic acid cycle.ImportanceThis study fills a gap in our knowledge and understanding of the genetic basis and biochemical pathway for the degradation of 2,4-dinitrophenol (DNP) in Gram-negative bacteria, represented by the prototypical Paraburkholderia sp. strain KU-46 that metabolizes DNP through the formation of 4-nitrophenol, a pathway unseen by other DNP utilizers. The newly cloned genes could serve as DNA probes in biomonitoring as well as finding application in new biocatalyst development to access green chemicals. By and large, knowledge of the diverse strategies used by microorganisms to degrade DNP will contribute to the development of bioremediation solutions since DNP is an industrial pollutant used widely in the chemical industry for the synthesis of pesticides, insecticides, sulfur dyes, wood preservatives, and explosives, etc. (119 words)

scholarly journals The fluorene catabolic linear plasmid in Terrabacter sp. strain DBF63 carries the β-ketoadipate pathway genes, pcaRHGBDCFIJ, also found in proteobacteria

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3713-3722 ◽  
Author(s):  
Hiroshi Habe ◽  
Jin-Sung Chung ◽  
Ayako Ishida ◽  
Kano Kasuga ◽  
Kazuki Ide ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Tricarboxylic Acid Cycle ◽  
Bacterial Species ◽  
Linear Plasmid ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Gene Encoding ◽  
Positive Trait ◽  
Tricarboxylic Acid Cycle Intermediates

Terrabacter sp. strain DBF63 is capable of degrading fluorene (FN) to tricarboxylic acid cycle intermediates via phthalate and protocatechuate. Genes were identified for the protocatechuate branch of the β-ketoadipate pathway (pcaR, pcaHGBDCFIJ) by sequence analysis of a 70 kb DNA region of the FN-catabolic linear plasmid pDBF1. RT-PCR analysis of RNA from DBF63 cells grown with FN, dibenzofuran, and protocatechuate indicated that the pcaHGBDCFIJ operon was expressed during both FN and protocatechuate degradation in strain DBF63. The gene encoding β-ketoadipate enol-lactone hydrolase (pcaD) was not fused to the next gene, which encodes γ-carboxymuconolactone decarboxylase (pcaC), in strain DBF63, even though the presence of the pcaL gene (the fusion of pcaD and pcaC) within a pca gene cluster has been thought to be a Gram-positive trait. Quantitative RT-PCR analysis revealed that pcaD mRNA levels increased sharply in response to protocatechuate, and a biotransformation experiment with cis,cis-muconate using Escherichia coli carrying both catBC and pcaD indicated that PcaD exhibited β-ketoadipate enol-lactone hydrolase activity. The location of the pca gene cluster on the linear plasmid, and the insertion sequences around the pca gene cluster suggest that the ecologically important β-ketoadipate pathway genes, usually located chromosomally, may be spread widely among bacterial species via horizontal transfer or transposition events.

scholarly journals Relationship of Yersinia pseudotuberculosis O Antigens IA, IIA, and IVB: the IIA Gene Cluster Was Derived from That of IVB

2002 ◽  
Vol 70 (6) ◽  
pp. 3271-3276 ◽  
Author(s):  
Elvia Pacinelli ◽  
Lei Wang ◽  
Peter R. Reeves
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Yersinia Pseudotuberculosis ◽  
Gene Clusters ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
O Antigen ◽  
O Antigens ◽  
Relationship Of ◽  
Six Genes

ABSTRACT O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria and is highly polymorphic. In this study, we obtained sequences of the O-antigen gene clusters for the Yersinia pseudotuberculosis antigens IA, IIA, and IVB. We propose that the IIA gene cluster was derived from the IVB cluster, one of the very few cases in which a parent gene cluster is identified, and that the IA gene cluster could be a hybrid of the IVB and IB gene clusters. All three O antigens contain 6-deoxy-d-mannoheptose, and we identified six genes for the biosynthetic pathway for the precursor of this sugar, GDP-6-deoxy-d-mannoheptose.

scholarly journals Relationships among the O-Antigen Gene Clusters ofSalmonella enterica Groups B, D1, D2, and D3

1998 ◽  
Vol 180 (4) ◽  
pp. 1002-1007 ◽  
Author(s):  
Heather Curd ◽  
Dan Liu ◽  
Peter R. Reeves
Keyword(s):  
Cell Wall ◽  
Gene Cluster ◽  
Salmonella Enterica ◽  
Sequence Similarity ◽  
Gene Clusters ◽  
Gram Negative Bacteria ◽  
Polymerase Gene ◽  
Gram Negative ◽  
Antigen Gene ◽  
O Antigen

ABSTRACT The O antigen is an important cell wall antigen of gram-negative bacteria, and the genes responsible for its biosynthesis are located in a gene cluster. We have cloned and sequenced the DNA segment unique to the O-antigen gene cluster of Salmonella enterica group D3. This segment includes a novel O-antigen polymerase gene (wzy D3). The polymerase gives α(1→6) linkages but has no detectable sequence similarity to that of group D2, which confers the same linkage. We find the remnant of a D3-likewzy gene in the O-antigen gene clusters of groups D1 and B and suggest that this is the original wzy gene of these O-antigen gene clusters.

scholarly journals The Erwinia chrysanthemi 3937 PhoQ Sensor Kinase Regulates Several Virulence Determinants

2006 ◽  
Vol 188 (8) ◽  
pp. 3088-3098 ◽  
Author(s):  
Balakrishnan Venkatesh ◽  
Lavanya Babujee ◽  
Hui Liu ◽  
Pete Hedley ◽  
Takashi Fujikawa ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Regulatory System ◽  
Soft Rot ◽  
Erwinia Chrysanthemi ◽  
Pcr Analysis ◽  
Sensor Kinase ◽  
Virulence Determinants ◽  
Gene Encoding ◽  
Reverse Transcription Pcr ◽  
Two Component

ABSTRACT The PhoPQ two-component system regulates virulence factors in Erwinia chrysanthemi, a pectinolytic enterobacterium that causes soft rot in several plant species. We characterized the effect of a mutation in phoQ, the gene encoding the sensor kinase PhoQ of the PhoPQ two-component regulatory system, on the global transcriptional profile of E. chrysanthemi using cDNA microarrays and further confirmed our results by quantitative reverse transcription-PCR analysis. Our results indicate that a mutation in phoQ affects transcription of at least 40 genes, even in the absence of inducing conditions. Enhanced expression of several genes involved in iron metabolism was observed in the mutant, including that of the acs operon that is involved in achromobactin biosynthesis and transport. This siderophore is required for full virulence of E. chrysanthemi, and its expression is governed by the global repressor protein Fur. Changes in gene expression were also observed for membrane transporters, stress-related genes, toxins, and transcriptional regulators. Our results indicate that the PhoPQ system governs the expression of several additional virulence factors and may also be involved in interactions with other regulatory systems.

scholarly journals The Gene Cluster forpara-Nitrophenol Catabolism Is Responsible for 2-Chloro-4-Nitrophenol Degradation in Burkholderia sp. Strain SJ98

2014 ◽  
Vol 80 (19) ◽  
pp. 6212-6222 ◽  
Author(s):  
Jun Min ◽  
Jun-Jie Zhang ◽  
Ning-Yi Zhou
Keyword(s):  
Gene Cluster ◽  
Gene Knockout ◽  
Gene Clusters ◽  
Sole Source ◽  
Pcr Analysis ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Biochemical Analyses ◽  
Nitrophenol Degradation

ABSTRACTBurkholderiasp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) orpara-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of thepnpgenes in thepnpABA1CDEFcluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activityin vitroin the conversion of 2C4NP to CBQ. Genetic analyses indicated thatpnpAplays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels.

scholarly journals Regulatory Roles of the GacS/GacA Two-Component System in Plant-Associated and Other Gram-Negative Bacteria

2001 ◽  
Vol 14 (12) ◽  
pp. 1351-1363 ◽  
Author(s):  
Stephan Heeb ◽  
Dieter Haas
Keyword(s):  
Rna Binding ◽  
Enteric Bacteria ◽  
Plant Diseases ◽  
Gram Negative Bacteria ◽  
Two Component System ◽  
Component System ◽  
Gram Negative ◽  
Ecological Fitness ◽  
Two Component ◽  
Sensor Kinases

The sensor kinase GacS and the response regulator GacA are members of a two-component system that is present in a wide variety of Gram-negative bacteria and has been studied mainly in enteric bacteria and fluorescent pseudomonads. The GacS/GacA system controls the production of secondary metabolites and extracellular enzymes involved in pathogenicity to plants and animals, biocontrol of soilborne plant diseases, ecological fitness, or tolerance to stress. A current model proposes that GacS senses a still-unknown signal and activates, via a phosphorelay mechanism, the GacA transcription regulator, which in turn triggers the expression of target genes. The GacS protein belongs to the unorthodox sensor kinases, characterized by an autophosphorylation, a receiver, and an output domain. The periplasmic loop domain of GacS is poorly conserved in diverse bacteria. Thus, a common signal interacting with this domain would be unexpected. Based on a comparison with the transcriptional regulator NarL, a secondary structure can be predicted for the GacA sensor kinases. Certain genes whose expression is regulated by the GacS/GacA system are regulated in parallel by the small RNA binding protein RsmA (CsrA) at a posttranscriptional level. It is suggested that the GacS/GacA system operates a switch between primary and secondary metabolism, with a major involvement of posttranscriptional control mechanisms.

scholarly journals Bacillus phage endolysin, lys46, bactericidal properties against Gram-negative bacteria

2020 ◽  
Author(s):  
Mohammad Reza Sarjoughian ◽  
Fereshte Rahmani ◽  
Shamsozoha Abolmaali ◽  
Shakiba Darvish Alipour Astaneh
Keyword(s):  
Bacillus Subtilis ◽  
Screening Program ◽  
Inhibitory Effect ◽  
Indicator Bacteria ◽  
Subtilis Strain ◽  
Gram Negative Bacteria ◽  
Gene Encoding ◽  
Gram Negative ◽  
Antibacterial Screening ◽  
Bacillus Phage

Background and Objectives: The great potential of bacteriophage for removing pathogen bacteria via targeting the cell wall is highly concerned. With a priority for overcoming drug-resistance, we screened against endolysins targeting Gram-negative bacteria to introduce a new antibacterial agent. This study was aimed to identify endolysins from the lysogenic phage of the Siphoviridea family in Bacillus subtilis. Materials and Methods: The Bacillus subtilis strain DDBCC46 was isolated from a preliminary antibacterial screening program. The endolysin (s) was extracted, concentrated with ammonium sulfate saturation, and their activity evaluated against the indicator bacteria. The phage particles were extracted from the bacteria using the minimum inhibition concentration of mitomycin C, followed by testing the phage inhibitory effect on the growth of indicator bacteria. The NCBI, Virus-Host DB, and EXPASY databases were used to obtain and confirm the sequences of the genes encoding PG hydrolases in Siphoviridea phages hosted in B. subtilis. Results: An 816 bp gene encoding an endolysin enzyme, was approved in the B. subtilis DDBCC 46, with specific primers of Bacillus phage SPP1. The purified-endolysin indicated antibacterial activity against Klebsiella pneumoniae, Salmonella typhimurium, Proteus (sp), and Escherichia coli. SDS-PAGE profiling followed by silica gel purification, led to introduceLys4630 as a therapeutic product and food preservative. Conclusion: lys4630 showed antibacterial effects on the common Gram-negative pathogens in clinics and food industries; E. coli, P. aeruginosa and Salmonella (sp).

scholarly journals A Pathway for Isethionate Dissimilation inBacillus krulwichiae

2019 ◽  
Vol 85 (15) ◽  
Author(s):  
Yang Tong ◽  
Yifeng Wei ◽  
Yiling Hu ◽  
Ee Lui Ang ◽  
Huimin Zhao ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Waste Product ◽  
Gene Clusters ◽  
Sulfur Cycle ◽  
Thiamine Pyrophosphate ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Sulfoacetaldehyde Acetyltransferase

ABSTRACTHydroxyethyl sulfonate (isethionate) is widely distributed in the environment as an industrial pollutant and as a product of microbial metabolism. It is used as a substrate for growth by metabolically diverse environmental bacteria. Aerobic pathways for isethionate dissimilation in Gram-negative bacteria involve the cytochromec-dependent oxidation of isethionate to sulfoacetaldehyde by a membrane-bound flavoenzyme (IseJ), followed by C-S cleavage by the thiamine pyrophosphate (TPP)-dependent enzyme sulfoacetaldehyde acetyltransferase (Xsc). Here, we report a bioinformatics analysis of Xsc-containing gene clusters in Gram-positive bacteria, which revealed the presence of an alternative isethionate dissimilation pathway involving the NAD+-dependent oxidation of isethionate by a cytosolic metal-dependent alcohol dehydrogenase (IseD). We describe the biochemical characterization of recombinant IseD from the haloalkaliphilic environmental bacteriumBacillus krulwichiaeAM31DTand demonstrate the growth of this bacterium using isethionate as its sole carbon source, with the excretion of sulfite as a waste product. The IseD-dependent pathway provides the only mechanism for isethionate dissimilation in Gram-positive species to date and suggests a role of the metabolically versatileBacilliin the mineralization of this ubiquitous organosulfur compound.IMPORTANCEIsethionate of biotic and industrial sources is prevalent. Dissimilation of isethionate under aerobic conditions is thus far only known in Gram-negative bacteria. Here, we report the discovery of a new pathway in Gram-positiveBacillus krulwichiae. Isethionate is oxidized by a cytosolic metal-dependent alcohol dehydrogenase (which we named IseD), with NAD+as the electron acceptor, generating sulfoacetaldehyde for subsequent cleavage by Xsc. This work highlights the diversity of organisms and pathways involved in the degradation of this ubiquitous organosulfonate. The new pathway that we discovered may play an important role in organosulfur mineralization and in the sulfur cycle in certain environments.

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