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 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 New Genome-to-Genome Comparison Approach for Large-Scale Revisiting of Current Microbial Taxonomy

2019 ◽  
Vol 7 (6) ◽  
pp. 161 ◽  
Author(s):  
Ming-Hsin Tsai ◽  
Yen-Yi Liu ◽  
Von-Wun Soo ◽  
Chih-Chieh Chen
Keyword(s):  
Microbial Diversity ◽  
Large Scale ◽  
Gene Selection ◽  
Marker Gene ◽  
Genome Comparison ◽  
Marker Genes ◽  
Species Classification ◽  
Genome Wide ◽  
A Genome ◽  
Comparison Approach

Microbial diversity has always presented taxonomic challenges. With the popularity of next-generation sequencing technology, more unculturable bacteria have been sequenced, facilitating the discovery of additional new species and complicated current microbial classification. The major challenge is to assign appropriate taxonomic names. Hence, assessing the consistency between taxonomy and genomic relatedness is critical. We proposed and applied a genome comparison approach to a large-scale survey to investigate the distribution of genomic differences among microorganisms. The approach applies a genome-wide criterion, homologous coverage ratio (HCR), for describing the homology between species. The survey included 7861 microbial genomes that excluded plasmids, and 1220 pairs of genera exhibited ambiguous classification. In this study, we also compared the performance of HCR and average nucleotide identity (ANI). The results indicated that HCR and ANI analyses yield comparable results, but a few examples suggested that HCR has a superior clustering effect. In addition, we used the Genome Taxonomy Database (GTDB), the gold standard for taxonomy, to validate our analysis. The GTDB offers 120 ubiquitous single-copy proteins as marker genes for species classification. We determined that the analysis of the GTDB still results in classification boundary blur between some genera and that the marker gene-based approach has limitations. Although the choice of marker genes has been quite rigorous, the bias of marker gene selection remains unavoidable. Therefore, methods based on genomic alignment should be considered for use for species classification in order to avoid the bias of marker gene selection. On the basis of our observations of microbial diversity, microbial classification should be re-examined using genome-wide comparisons.

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 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 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 High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440

2020 ◽  
Vol 8 ◽  
Author(s):  
Yuqian Gao ◽  
Thomas L. Fillmore ◽  
Nathalie Munoz ◽  
Gayle J. Bentley ◽  
Christopher W. Johnson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Systems Biology ◽  
Pseudomonas Putida ◽  
High Throughput ◽  
High Speed ◽  
Large Scale ◽  
Protein Quantification ◽  
Selected Reaction Monitoring ◽  
Targeted Proteomics ◽  
Pseudomonas Putida Kt2440

Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, accuracy, and reproducibility. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large sample sets with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. Recent advancements in mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM, thereby creating opportunities for applying the high speed of microflow LC-SRM without losing peptide multiplexing power or sacrificing sensitivity. Here, we studied the performance of microflow LC-SRM relative to nanoflow LC-SRM by monitoring 339 peptides representing 132 enzymes in Pseudomonas putida KT2440 grown on various carbon sources. The results from the two LC-SRM platforms are highly correlated. In addition, the response curve study of 248 peptides demonstrates that microflow LC-SRM has comparable sensitivity for the majority of detected peptides and better mass spectrometry signal and chromatography stability than nanoflow LC-SRM.

Growth phase-dependent expression of the Pseudomonas putida KT2440 transcriptional machinery analysed with a genome-wide DNA microarray

2006 ◽  
Vol 8 (1) ◽  
pp. 165-177 ◽  
Author(s):  
Luis Yuste ◽  
Ana B. Hervas ◽  
Ines Canosa ◽  
Raquel Tobes ◽  
Jose Ignacio Jimenez ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Dna Microarray ◽  
Growth Phase ◽  
Pseudomonas Putida Kt2440 ◽  
Genome Wide ◽  
A Genome ◽  
Transcriptional Machinery

scholarly journals Arabidopsis Genes Essential for Seedling Viability: Isolation of Insertional Mutants and Molecular Cloning

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1765-1778
Author(s):  
Gregory J Budziszewski ◽  
Sharon Potter Lewis ◽  
Lyn Wegrich Glover ◽  
Jennifer Reineke ◽  
Gary Jones ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Translocation ◽  
Gene Families ◽  
Mutant Phenotype ◽  
Lethal Mutant ◽  
A Genome ◽  
Genes Encoding ◽  
High Level ◽  
Mutant Lines ◽  
Genome Scale

Abstract We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening ~38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

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