scholarly journals Development of a Method for Markerless Gene Deletion in Pseudomonas putida

2011 ◽  
Vol 77 (15) ◽  
pp. 5549-5552 ◽  
Author(s):  
Nadja Graf ◽  
Josef Altenbuchner
Keyword(s):  
Pseudomonas Putida ◽  
Gene Deletion ◽  
Protein Gene ◽  
Surface Adhesion ◽  
Content Type ◽  
Adhesion Protein ◽  
Uracil Phosphoribosyltransferase ◽  
Flagellum Biosynthesis

ABSTRACTWe developed a negative counterselection system forPseudomonas putidabased on uracil phosphoribosyltransferase (UPRTase) and sensitivity against the antimetabolite 5-fluorouracil (5-FU). We constructed aP. putidastrain that is resistant to 5-FU and constructed vectors for the deletion of the surface adhesion protein gene, the flagellum biosynthesis operon, and two endonuclease genes. The genes were efficiently disrupted and left a markerless chromosomal in-frame deletion.

scholarly journals Dechlorination of Chloral Hydrate Is Influenced by the Biofilm Adhesin Protein LapA in Pseudomonas putida LF54

2013 ◽  
Vol 79 (13) ◽  
pp. 4166-4169
Author(s):  
Wanjun Zhang ◽  
Huhe ◽  
Yuanbai Pan ◽  
Masanori Toyofuku ◽  
Nobuhiko Nomura ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Biofilm Formation ◽  
Chloral Hydrate ◽  
Insertion Mutagenesis ◽  
Surface Adhesion ◽  
Content Type ◽  
Adhesion Protein ◽  
Transposon Insertion ◽  
Adhesin Protein ◽  
First Time

ABSTRACTLapA is the largest surface adhesion protein ofPseudomonas putidathat initiates biofilm formation. Here, by using transposon insertion mutagenesis and a conditionallapAmutant, we demonstrate for the first time that LapA influences chloral hydrate (CH) dechlorination inP. putidaLF54.

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.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽  
2021 ◽  
Vol 167 (3) ◽  
Author(s):  
Sathi Mallick ◽  
Shanti Kiran ◽  
Tapas Kumar Maiti ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Species ◽  
Pentose Phosphate ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

scholarly journals Complete Genome Sequence of Pseudomonas putida Strain TS312, Harboring an HdtS-Type N-Acyl-Homoserine Lactone Synthase, Isolated from a Paper Mill

2020 ◽  
Vol 9 (13) ◽  
Author(s):  
Ayaka Hosoe ◽  
Toshikazu Suenaga ◽  
Takumi Sugi ◽  
Taro Iizumi ◽  
Naohiro Nagai ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Paper Mill ◽  
Homoserine Lactone ◽  
Acyl Homoserine Lactone ◽  
Gene Encoding ◽  
Content Type ◽  
Engineered Systems

We report the complete genome sequence of Pseudomonas putida strain TS312, in the class of Gammaproteobacteria. The strain, isolated from a paper mill, harbors the hdtS gene, encoding N-acyl-homoserine lactone synthase. Deciphering the genome contributes to revealing the mechanisms of quorum sensing and associated biofilm formation in engineered systems.

scholarly journals Complete Genome Sequence of Pseudomonas putida BS3701, a Promising Polycyclic Aromatic Hydrocarbon-Degrading Strain for Bioremediation Technologies

2020 ◽  
Vol 9 (40) ◽  
Author(s):  
Andrey Filonov ◽  
Yanina Delegan ◽  
Irina Puntus ◽  
Leonid Valentovich ◽  
Artur Akhremchuk ◽  
...  
Keyword(s):  
Polycyclic Aromatic Hydrocarbons ◽  
Polycyclic Aromatic Hydrocarbon ◽  
Aromatic Hydrocarbon ◽  
Pseudomonas Putida ◽  
Russian Federation ◽  
Aromatic Hydrocarbons ◽  
Complete Genome Sequence ◽  
Circular Chromosome ◽  
Content Type ◽  
Polycyclic Aromatic

ABSTRACT The strain Pseudomonas putida BS3701 was isolated from soil contaminated with coke by-product waste (Moscow Region, Russian Federation). It is capable of degrading crude oil and polycyclic aromatic hydrocarbons (PAHs). The P. putida BS3701 genome consists of a 6,337,358-bp circular chromosome and two circular plasmids (pBS1141 with 107,388 bp and pBS1142 with 54,501 bp).

scholarly journals First Description of KPC-2-Producing Pseudomonas putida in Brazil

2012 ◽  
Vol 56 (4) ◽  
pp. 2205-2206 ◽  
Author(s):  
Anna C. S. Almeida ◽  
Marinalda A. Vilela ◽  
Felipe L. S. Cavalcanti ◽  
Willames M. B. S. Martins ◽  
Marcos A. Morais ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Pseudomonas Putida ◽  
Polymyxin B ◽  
Class 1 Integron ◽  
Content Type ◽  
First Case ◽  
Class 1

ABSTRACTThis work reports the identification of the first case of a KΡC-2-producingPseudomonas putidaisolate (PP36) in Brazil. The PP36 isolate was resistant to all the antimicrobials tested except polymyxin B. In addition to the discoveredblaKPC-2gene, genetic analysis showed the presence of a class 1 integron containing thedhfrXVb gene and the new allelearr-6, which codes for resistance to rifampin. These elements were found in an IncFI 65-kb plasmid.

scholarly journals Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming ActinobacteriumCorynebacterium matruchotii

2018 ◽  
Vol 200 (9) ◽  
pp. e00783-17 ◽  
Author(s):  
Truc Thanh Luong ◽  
Reyhaneh Tirgar ◽  
Melissa E. Reardon-Robinson ◽  
Andrzej Joachimiak ◽  
Jerzy Osipiuk ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Gene Deletion ◽  
Bond Formation ◽  
Toxin Production ◽  
Content Type ◽  
Cellular Processes ◽  
Disulfide Oxidoreductase ◽  
New Gene ◽  
Resolution Structure

ABSTRACTThe actinobacteriumCorynebacterium matruchotiihas been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of theC. matruchotiigenome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to theCorynebacterium diphtheriaethiol-disulfide oxidoreductase MdbA (MdbACd). Crystallization studies uncovered that the 1.2-Å resolution structure ofC. matruchotiiMdbA (MdbACm) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machinein vitro, we demonstrated that MdbACmcatalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported thatmdbAis essential inC. matruchotii. Remarkably, heterologous expression of MdbACmin theC. diphtheriaeΔmdbAmutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbACmis a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding inC. matruchotiiby a mechanism that is conserved inActinobacteria.IMPORTANCEThe actinobacteriumCorynebacterium matruchotiihas been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of aC. matruchotiioxidoreductase that is highly homologous to theCorynebacterium diphtheriaethiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated thatC. matruchotiiMdbA catalyzes disulfide bond formationin vitro. Furthermore, a new gene deletion method revealed that deletion ofmdbAis lethal inC. matruchotii. Remarkably,C. matruchotiiMdbA can replaceC. diphtheriaeMdbA to maintain normal cell growth and morphology, toxin production, and pilus assembly. Overall, our studies support the hypothesis thatC. matruchotiiutilizes MdbA as a major oxidoreductase to catalyze oxidative protein folding.

scholarly journals Primary and Secondary Metabolic Effects of a Key Gene Deletion (ΔYPL062W) in Metabolically Engineered Terpenoid-ProducingSaccharomyces cerevisiae

2019 ◽  
Vol 85 (7) ◽  
Author(s):  
Yan Chen ◽  
Ying Wang ◽  
Ming Liu ◽  
Junze Qu ◽  
Mingdong Yao ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Gene Deletion ◽  
Core Promoter ◽  
Strain Improvement ◽  
Genetic Alterations ◽  
Metabolic Effects ◽  
Industrial Biotechnology ◽  
Cell Factory ◽  
Content Type ◽  
Genetic Function

ABSTRACTSaccharomyces cerevisiaeis an established cell factory for production of terpenoid pharmaceuticals and chemicals. Numerous studies have demonstrated that deletion or overexpression of off-pathway genes in yeast can improve terpenoid production. The deletion ofYPL062WinS. cerevisiae, in particular, has benefitted carotenoid production by channeling carbon toward carotenoid precursors acetyl coenzyme A (acetyl-CoA) and mevalonate. The genetic function ofYPL062Wand the molecular mechanisms for these benefits are unknown. In this study, we systematically examined this gene deletion to uncover the gene function and its molecular mechanism. RNA sequencing (RNA-seq) analysis uncovered thatYPL062Wdeletion upregulated the pyruvate dehydrogenase bypass, the mevalonate pathway, heterologous expression of galactose (GAL) promoter-regulated genes, energy metabolism, and membrane composition synthesis. Bioinformatics analysis and serial promoter deletion assay revealed thatYPL062Wfunctions as a core promoter forALD6and that the expression level ofALD6is negatively correlated to terpenoid productivity. We demonstrate that ΔYPL062Wincreases the production of all major terpenoid classes (C10, C15, C20, C30, and C40). Our study not only elucidated the biological function ofYPL062Wbut also provided a detailed methodology for understanding the mechanistic aspects of strain improvement.IMPORTANCEAlthough computational and reverse metabolic engineering approaches often lead to improved gene deletion mutants for cell factory engineering, the systems level effects of such gene deletions on the production phenotypes have not been extensively studied. Understanding the genetic and molecular function of such gene alterations on production strains will minimize the risk inherent in the development of large-scale fermentation processes, which is a daunting challenge in the field of industrial biotechnology. Therefore, we established a detailed experimental and systems biology approach to uncover the molecular mechanisms ofYPL062Wdeletion inS. cerevisiae, which is shown to improve the production of all terpenoid classes. This study redefines the genetic function ofYPL062W, demonstrates a strong correlation betweenYPL062Wand terpenoid production, and provides a useful modification for the creation of terpenoid production platform strains. Further, this study underscores the benefits of detailed and systematic characterization of the metabolic effects of genetic alterations on engineered biosynthetic factories.

scholarly journals A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Marta Tibúrcio ◽  
Annie S. P. Yang ◽  
Kazuhide Yahata ◽  
Pablo Suárez-Cortés ◽  
Hugo Belda ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Genetic Modification ◽  
Gene Deletion ◽  
Genetically Modify ◽  
Complex Life Cycle ◽  
Mosquito Vector ◽  
Parasite Line ◽  
Content Type ◽  
First Time

ABSTRACT Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1. IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.

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