scholarly journals Elimination of Manganese(II,III) Oxidation in Pseudomonas putida GB-1 by a Double Knockout of Two Putative Multicopper Oxidase Genes

2012 ◽  
Vol 79 (1) ◽  
pp. 357-366 ◽  
Author(s):  
Kati Geszvain ◽  
James K. McCarthy ◽  
Bradley M. Tebo
Keyword(s):  
Pseudomonas Putida ◽  
Multicopper Oxidase ◽  
Genome Comparison ◽  
Double Knockout ◽  
Oxidase Gene ◽  
Content Type ◽  
Heme Peroxidase ◽  
A Genome ◽  
Oxidation Phenotype ◽  
Encoding Genes

ABSTRACTBacterial manganese(II) oxidation impacts the redox cycling of Mn, other elements, and compounds in the environment; therefore, it is important to understand the mechanisms of and enzymes responsible for Mn(II) oxidation. In several Mn(II)-oxidizing organisms, the identified Mn(II) oxidase belongs to either the multicopper oxidase (MCO) or the heme peroxidase family of proteins. However, the identity of the oxidase inPseudomonas putidaGB-1 has long remained unknown. To identify theP. putidaGB-1 oxidase, we searched its genome and found several homologues of known or suspected Mn(II) oxidase-encoding genes (mnxG,mofA,moxA, andmopA). To narrow this list, we assumed that the Mn(II) oxidase gene would be conserved among Mn(II)-oxidizing pseudomonads but not in nonoxidizers and performed a genome comparison to 11Pseudomonasspecies. We further assumed that the oxidase gene would be regulated by MnxR, a transcription factor required for Mn(II) oxidation. Two loci met all these criteria: PputGB1_2447, which encodes an MCO homologous to MnxG, and PputGB1_2665, which encodes an MCO with very low homology to MofA. In-frame deletions of each locus resulted in strains that retained some ability to oxidize Mn(II) or Mn(III); loss of oxidation was attained only upon deletion of both genes. These results suggest that PputGB1_2447 and PputGB1_2665 encode two MCOs that are independently capable of oxidizing both Mn(II) and Mn(III). The purpose of this redundancy is unclear; however, differences in oxidation phenotype for the single mutants suggest specialization in function for the two enzymes.

scholarly journals Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1

2016 ◽  
Vol 82 (13) ◽  
pp. 3774-3782 ◽  
Author(s):  
Kati Geszvain ◽  
Logan Smesrud ◽  
Bradley M. Tebo
Keyword(s):  
Pseudomonas Putida ◽  
Oxidase Activity ◽  
Sequence Similarity ◽  
Preliminary Investigation ◽  
Model Organism ◽  
Multicopper Oxidase ◽  
Content Type ◽  
Regulatory Pathways ◽  
Heme Peroxidase ◽  
Oxidase Enzyme

ABSTRACTThe oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacteriumPseudomonas putidaGB-1, two Mn(II) oxidase genes, namedmnxGandmcoA, were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity inP. putidaGB-1. A strain from which both of the Mn(II) oxidase genes andfleQwere deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the ΔfleQstrain,P. putidaGB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion offleQunmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes.IMPORTANCEThe identity of the Mn(II) oxidase enzyme inPseudomonas putidaGB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate thatP. putidaGB-1 employs both the multicopper oxidase- and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes inP. putidaGB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the deletion of FleQ, a regulator involved in both flagellum synthesis and biofilm synthesis inPseudomonas aeruginosa. Therefore, these results are also an important step toward understanding the regulation of Mn(II) oxidation.

scholarly journals Genome Sequencing Identifies Two Nearly Unchanged Strains of Persistent Listeria monocytogenes Isolated at Two Different Fish Processing Plants Sampled 6 Years Apart

2013 ◽  
Vol 79 (9) ◽  
pp. 2944-2951 ◽  
Author(s):  
Anne Holch ◽  
Kristen Webb ◽  
Oksana Lukjancenko ◽  
David Ussery ◽  
Benjamin M. Rosenthal ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Random Amplified Polymorphic Dna ◽  
The United States ◽  
Genome Comparison ◽  
Physiological Factors ◽  
Content Type ◽  
Human Pathogenic Bacterium ◽  
A Genome ◽  
Genome Analyses

ABSTRACTListeria monocytogenesis a food-borne human-pathogenic bacterium that can cause infections with a high mortality rate. It has a remarkable ability to persist in food processing facilities. Here we report the genome sequences for twoL. monocytogenesstrains (N53-1 and La111) that were isolated 6 years apart from two different Danish fish processers. Both strains are of serotype 1/2a and belong to a highly persistent DNA subtype (random amplified polymorphic DNA [RAPD] type 9). We demonstrate usingin silicoanalyses that both strains belong to the multilocus sequence typing (MLST) type ST121 that has been isolated as a persistent subtype in several European countries. The purpose of this study was to use genome analyses to identify genes or proteins that could contribute to persistence. In a genome comparison, the two persistent strains were extremely similar and collectively differed from the reference lineage II strain, EGD-e. Also, they differed markedly from a lineage I strain (F2365). On the proteome level, the two strains were almost identical, with a predicted protein homology of 99.94%, differing at only 2 proteins. No single-nucleotide polymorphism (SNP) differences were seen between the two strains; in contrast, N53-1 and La111 differed from the EGD-e reference strain by 3,942 and 3,471 SNPs, respectively. We included a persistentL. monocytogenesstrain from the United States (F6854) in our comparisons. Compared to nonpersistent strains, all three persistent strains were distinguished by two genome deletions: one, of 2,472 bp, typically contains the gene forinlF, and the other, of 3,017 bp, includes three genes potentially related to bacteriocin production and transport (lmo2774,lmo2775, and the 3′-terminal part oflmo2776). Further studies of highly persistent strains are required to determine if the absence of these genes promotes persistence. While the genome comparison did not point to a clear physiological explanation of the persistent phenotype, the remarkable similarity between the two strains indicates that subtypes with specific traits are selected for in the food processing environment and that particular genetic and physiological factors are responsible for the persistent phenotype.

scholarly journals Mechanisms of Resistance to Chloramphenicol in Pseudomonas putida KT2440

2011 ◽  
Vol 56 (2) ◽  
pp. 1001-1009 ◽  
Author(s):  
Matilde Fernández ◽  
Susana Conde ◽  
Jesús de la Torre ◽  
Carlos Molina-Santiago ◽  
Juan-Luis Ramos ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Efflux Pump ◽  
Protein Biosynthesis ◽  
Cellular Stress Response ◽  
Knockout Mutant ◽  
Pseudomonas Putida Kt2440 ◽  
Altered Expression ◽  
Content Type ◽  
Reading Frame ◽  
A Genome

ABSTRACTPseudomonas putidaKT2440 is a chloramphenicol-resistant bacterium that is able to grow in the presence of this antibiotic at a concentration of up to 25 μg/ml. Transcriptomic analyses revealed that the expression profile of 102 genes changed in response to this concentration of chloramphenicol in the culture medium. The genes that showed altered expression include those involved in general metabolism, cellular stress response, gene regulation, efflux pump transporters, and protein biosynthesis. Analysis of a genome-wide collection of mutants showed that survival of a knockout mutant in the TtgABC resistance-nodulation-division (RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline (PQQ) were compromised in the presence of chloramphenicol. The analysis also revealed that an ABC extrusion system (PP2669/PP2668/PP2667) and the AgmR regulator (PP2665) were needed for full resistance toward chloramphenicol. Transcriptional arrays revealed that AgmR controls the expression of thepqqgenes and the operon encoding the ABC extrusion pump from the promoter upstream of open reading frame (ORF) PP2669.

scholarly journals Development of a Markerless Knockout Method for Actinobacillus succinogenes

2014 ◽  
Vol 80 (10) ◽  
pp. 3053-3061 ◽  
Author(s):  
Rajasi V. Joshi ◽  
Bryan D. Schindler ◽  
Nikolas R. McPherson ◽  
Kanupriya Tiwari ◽  
Claire Vieille
Keyword(s):  
High Efficiency ◽  
Direct Selection ◽  
Selection Marker ◽  
Actinobacillus Succinogenes ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Pyruvate Formate Lyase ◽  
Content Type ◽  
Knockout Mutants ◽  
Encoding Genes

ABSTRACTActinobacillus succinogenesis one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method forA. succinogenesusing natural transformation or electroporation. TheEscherichia coliisocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use ofA. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. TheSaccharomyces cerevisiaeflippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressingflpwas cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.

scholarly journals A Rational Design of Pseudomonas putida KT2440 capable of Anaerobic Respiration

2020 ◽  
Author(s):  
Linde F. C. Kampers ◽  
Jasper J. Koehorst ◽  
Ruben G. A. van Heck ◽  
Maria Suarez-Diez ◽  
Alfons J. M. Stams ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Rational Design ◽  
Large Scale ◽  
Anaerobic Fermentation ◽  
Anaerobic Respiration ◽  
Genome Comparison ◽  
Pseudomonas Putida Kt2440 ◽  
Design Data ◽  
Atp Production ◽  
A Genome

Abstract Pseudomonas putida KT2440 is a metabolically versatile, HV1-certified, genetically accessible, and thus interesting microbial chassis for biotechnological applications. However, its obligate aerobic nature hampers production of oxygen sensitive products and drives up costs in large scale fermentation. The inability to perform anaerobic fermentation has been attributed to insufficient ATP production and an inability to produce pyrimidines under these conditions. Addressing these bottlenecks enabled growth under micro-oxic conditions, but does not lead to growth or survival under anoxic conditions. Here, a data-driven approach was used to develop a rational design for aP. putida KT2440 derivative strain capable of anaerobic respiration. To come to the design, data derived from a genome comparison of 1628 Pseudomonas strains was combined with genome-scale metabolic modelling simulations and a transcriptome dataset of 47 samples representing 14 environmental conditions from the facultative anaerobe Pseudomonas aeruginosa. The results indicate that the implementation of anaerobic respiration inP. putida KT2440 would require at least 61 additional genes of known function, at least 8 genes encoding proteins of unknown function, and 3 externally added vitamins.

scholarly journals A metabolic and physiological design study of Pseudomonas putida KT2440 capable of anaerobic respiration

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Linde F. C. Kampers ◽  
Jasper J. Koehorst ◽  
Ruben J. A. van Heck ◽  
Maria Suarez-Diez ◽  
Alfons J. M. Stams ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Rational Design ◽  
Large Scale ◽  
Anaerobic Fermentation ◽  
Anaerobic Respiration ◽  
Genome Comparison ◽  
Pseudomonas Putida Kt2440 ◽  
Design Data ◽  
Atp Production ◽  
A Genome

Abstract Background Pseudomonas putida KT2440 is a metabolically versatile, HV1-certified, genetically accessible, and thus interesting microbial chassis for biotechnological applications. However, its obligate aerobic nature hampers production of oxygen sensitive products and drives up costs in large scale fermentation. The inability to perform anaerobic fermentation has been attributed to insufficient ATP production and an inability to produce pyrimidines under these conditions. Addressing these bottlenecks enabled growth under micro-oxic conditions but does not lead to growth or survival under anoxic conditions. Results Here, a data-driven approach was used to develop a rational design for a P. putida KT2440 derivative strain capable of anaerobic respiration. To come to the design, data derived from a genome comparison of 1628 Pseudomonas strains was combined with genome-scale metabolic modelling simulations and a transcriptome dataset of 47 samples representing 14 environmental conditions from the facultative anaerobe Pseudomonas aeruginosa. Conclusions The results indicate that the implementation of anaerobic respiration in P. putida KT2440 would require at least 49 additional genes of known function, at least 8 genes encoding proteins of unknown function, and 3 externally added vitamins.

scholarly journals Light Response of Pseudomonas putida KT2440 Mediated by Class II LitR, a Photosensor Homolog

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Satoru Sumi ◽  
Naotaka Mutaguchi ◽  
Teppei Ebuchi ◽  
Hiroaki Tsuchida ◽  
Takahiro Yamamoto ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Transcriptional Control ◽  
Fatty Acid Synthase ◽  
Direct Repeat ◽  
Class Ii ◽  
Wide Distribution ◽  
Transcriptional Analysis ◽  
Pseudomonas Putida Kt2440 ◽  
Content Type ◽  
A Genome

ABSTRACT Pseudomonas putida KT2440 retains three homologs (PplR1 to PplR3) of the LitR/CarH family, an adenosyl B12-dependent light-sensitive MerR family transcriptional regulator. Transcriptome analysis revealed the existence of a number of photoinducible genes, including pplR1, phrB (encoding DNA photolyase), ufaM (furan-containing fatty acid synthase), folE (GTP cyclohydrolase I), cryB (cryptochrome-like protein), and multiple genes without annotated/known function. Transcriptional analysis by quantitative reverse transcription-PCR with knockout mutants of pplR1 to pplR3 showed that a triple knockout completely abolished the light-inducible transcription in P. putida, which indicates the occurrence of ternary regulation of PplR proteins. A DNase I footprint assay showed that PplR1 protein specifically binds to the promoter regions of light-inducible genes, suggesting a consensus PplR1-binding direct repeat, 5′-T(G/A)TACAN12TGTA(C/T)A-3′. The disruption of B12 biosynthesis cluster did not affect the light-inducible transcription; however, disruption of ppSB1-LOV (where LOV indicates “light, oxygen, or voltage”) and ppSB2-LOV, encoding blue light photoreceptors adjacently located to pplR3 and pplR2, respectively, led to the complete loss of light-inducible transcription. Overall, the results suggest that the three PplRs and two PpSB-LOVs cooperatively regulate the light-inducible gene expression. The wide distribution of the pplR/ppSB-LOV cognate pair homologs in Pseudomonas spp. and related bacteria suggests that the response and adaptation to light are similarly regulated in the group of nonphototrophic bacteria. IMPORTANCE The LitR/CarH family is a new group of photosensor homologous to MerR-type transcriptional regulators. Proteins of this family are distributed to various nonphototrophic bacteria and grouped into at least five classes (I to V). Pseudomonas putida retaining three class II LitR proteins exhibited a genome-wide response to light. All three paralogs were functional and mediated photodependent activation of promoters directing the transcription of light-induced genes or operons. Two LOV (light, oxygen, or voltage) domain proteins, adjacently encoded by two litR genes, were also essential for the photodependent transcriptional control. Despite the difference in light-sensing mechanisms, the DNA binding consensus of class II LitR [T(G/A)TA(C/T)A] was the same as that of class I. This is the first study showing the actual involvement of class II LitR in light-induced transcription.

scholarly journals Draft Genome Sequence of Pseudomonas putida Strain GM4FR, an Endophytic Bacterium Isolated from Festuca rubra L

2017 ◽  
Vol 5 (13) ◽  
Author(s):  
Franziska Wemheuer ◽  
Jacqueline Hollensteiner ◽  
Anja Poehlein ◽  
Sandra Granzow ◽  
Rolf Daniel ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Draft Genome ◽  
Endophytic Bacterium ◽  
Plant Tissues ◽  
Festuca Rubra ◽  
Content Type ◽  
Protein Encoding ◽  
Plant Growth Promoting ◽  
Growth Promoting ◽  
Encoding Genes

ABSTRACT Pseudomonas putida GM4FR is an endophytic bacterium isolated from aerial plant tissues of Festuca rubra L. Functional annotation of the draft genome (7.1 Mb) revealed 6,272 predicted protein-encoding genes. The genome provides insights into the biocontrol and plant growth-promoting potential of P. putida GM4FR.

scholarly journals Grimontia sedimenti sp. nov., isolated from benthic sediments near coral reefs south of Kuwait

2019 ◽  
Vol 71 (3) ◽  
Author(s):  
Huda Mahmoud ◽  
Liny Jose ◽  
Susan Eapen
Keyword(s):  
Coral Reefs ◽  
Type Species ◽  
Phenotypic Analysis ◽  
Phenotypic Properties ◽  
Content Type ◽  
Link Type ◽  
A Genome ◽  
Protein Encoding ◽  
Benthic Sediments ◽  
Encoding Genes

A Gram-stain-negative, rod and rod-curved shaped motile bacterium designated strain S25T was obtained from benthic sediment collected near the Kubbar Island coral reefs south of Kuwait. Phenotypic analysis revealed that strain S25T was slightly halophilic, mesophilic and facultative anaerobic, fermenting d-glucose, d-ribose, d-mannose, d-mannitol, maltose, fructose, gentiobiose, cellobiose, melibiose, trehalose and sucrose. It was positive for oxidase and indole production and negative for arginine dihydrolase and lysine and ornithine decarboxylases. It contained C16 : 1  ω7c/C16 : 1  ω6c (summed feature 3), C18 : 1 ω7c (summed feature 8) and C16 : 0 as the major fatty acids. Strain S25T grew optimally at 30 °C and pH 8 in the presence of 3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA sequences revealed that strain S25T is related to species of the genus Grimontia , having 99.15 % similarity to ‘Grimontia indica’ AK16T, 99.08 % to Grimontia celer 96-237T and 98.66 % to Grimontia marina IMCC 5001T. The DNA G+C content was 48.8 mol% and the full genome analysis for the strain S25T showed that the bacterium has a genome size of 5 158 621 bp and contains 4730 predicted protein-encoding genes. The average nucleotide identity values between the S25T genome and the genomes of its nearest matches ranged between 81.39 and 94.16 %. The strain was distinguishable from the phylogenetically related genera through differences in several phenotypic properties. On the basis of the phenotypic, phylogenetic and genetic data, strain S25T represents a novel species in the genus Grimontia , for which the name Grimontia sedimenti sp. nov. is proposed. The type strain of Grimontia sedimenti is S25T (=DSM 28878T=LMG 28315T).

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.

Sign in / Sign up

Export Citation Format

Share Document