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

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.

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Stability of a Pseudomonas putida KT2440 Bacteriophage-Carried Genomic Island and Its Impact on Rhizosphere Fitness

Applied and Environmental Microbiology ◽

10.1128/aem.00901-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 6963-6974 ◽

Cited By ~ 10

Author(s):

Jose M. Quesada ◽

María Isabel Soriano ◽

Manuel Espinosa-Urgel

Keyword(s):

Pseudomonas Putida ◽

Genomic Island ◽

Mobile Element ◽

Direct Repeat ◽

Promoter Sequence ◽

Repeat Sequence ◽

Genomic Islands ◽

Pseudomonas Putida Kt2440 ◽

Bacterial Populations ◽

Content Type

ABSTRACTThe stability of seven genomic islands ofPseudomonas putidaKT2440 with predicted potential for mobilization was studied in bacterial populations associated with the rhizosphere of corn plants by multiplex PCR. DNA rearrangements were detected for only one of them (GI28), which was lost at high frequency. This genomic island of 39.4 kb, with 53 open reading frames, shows the characteristic organization of genes belonging to tailed phages. We present evidence indicating that it corresponds to the lysogenic state of a functional bacteriophage that we have designated Pspu28. Integrated and rarely excised forms of Pspu28 coexist in KT2440 populations. Pspu28 is self-transmissible, and an excisionase is essential for its removal from the bacterial chromosome. The excised Pspu28 forms a circular element that can integrate into the chromosome at a specific location,attsites containing a 17-bp direct repeat sequence. Excision/insertion of Pspu28 alters the promoter sequence and changes the expression level of PP_1531, which encodes a predicted arsenate reductase. Finally, we show that the presence of Pspu28 in the lysogenic state has a negative effect on bacterial fitness in the rhizosphere under conditions of intraspecific competition, thus explaining why clones having lost this mobile element are recovered from that environment.

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Mechanisms of Resistance to Chloramphenicol in Pseudomonas putida KT2440

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05398-11 ◽

2011 ◽

Vol 56 (2) ◽

pp. 1001-1009 ◽

Cited By ~ 50

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.

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HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866

Applied and Environmental Microbiology ◽

10.1128/aem.03105-15 ◽

2015 ◽

Vol 82 (2) ◽

pp. 724-731 ◽

Cited By ~ 6

Author(s):

Hong-Jun Chao ◽

Yan-Fei Chen ◽

Ti Fang ◽

Ying Xu ◽

Wei E. Huang ◽

...

Keyword(s):

Pseudomonas Putida ◽

Environmental Protection Agency ◽

High Performance ◽

Specific Activity ◽

Gene Knockout ◽

Transcriptional Analysis ◽

Ring Cleavage ◽

Protection Agency ◽

Content Type ◽

Priority Pollutant

ABSTRACTIn addition to growing onp-cresol,Pseudomonas putidaNCIMB 9866 is the only reported strain capable of aerobically growing on 2,4-xylenol, which is listed as a priority pollutant by the U.S. Environmental Protection Agency. Several enzymes involved in the oxidation of thepara-methyl group, as well as the corresponding genes, have previously been reported. The enzyme catalyzing oxidation of the catabolic intermediate 4-hydroxyisophthalate to the ring cleavage substrate protocatechuate was also purified from strain NCIMB 9866, but its genetic determinant is still unavailable. In this study, the genehipH, encoding 4-hydroxyisophthalate hydroxylase, from strain NCIMB 9866 was cloned by transposon mutagenesis. Purified recombinant HipH-His6was found to be a dimer protein with a molecular mass of approximately 110 kDa. HipH-His6catalyzed the hydroxylation of 4-hydroxyisophthalate to protocatechuate with a specific activity of 1.54 U mg−1and showed apparentKmvalues of 11.40 ± 3.05 μM for 4-hydroxyisophthalate with NADPH and 11.23 ± 2.43 μM with NADH and similarKmvalues for NADPH and NADH (64.31 ± 13.16 and 72.76 ± 12.06 μM, respectively). The identity of protocatechuate generated from 4-hydroxyisophthalate hydroxylation by HipH-His6has also been confirmed by high-performance liquid chromatography and mass spectrometry. Gene transcriptional analysis, gene knockout, and complementation indicated thathipHis essential for 2,4-xylenol catabolism but not forp-cresol catabolism in this strain. This fills a gap in our understanding of the gene that encodes a critical step in 2,4-xylenol catabolism and also provides another example of biochemical and genetic diversity of microbial catabolism of structurally similar compounds.

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The putative phosphate transporter PitB (PP1373) is involved in tellurite uptake in Pseudomonas putida KT2440

Microbiology ◽

10.1099/mic.0.001002 ◽

2020 ◽

Author(s):

Rafael Montenegro ◽

Sofía Vieto ◽

Daniela Wicki-Emmenegger ◽

Felipe Vásquez-Castro ◽

Carolina Coronado-Ruiz ◽

...

Keyword(s):

Pseudomonas Putida ◽

Type Strain ◽

Type Species ◽

Wild Type Strain ◽

Phosphate Transporter ◽

Transport Systems ◽

Wild Type ◽

Pseudomonas Putida Kt2440 ◽

Content Type ◽

Link Type

Tellurium oxyanions are chemical species of great toxicity and their presence in the environment has increased because of mining industries and photovoltaic and electronic waste. Recovery strategies for this metalloid that are based on micro-organisms are of interest, but further studies of the transport systems and enzymes responsible for implementing tellurium transformations are required because many mechanisms remain unknown. Here, we investigated the involvement in tellurite uptake of the putative phosphate transporter PitB (PP1373) in soil bacterium Pseudomonas putida KT2440. For this purpose, through a method based on the CRISPR/Cas9 system, we generated a strain deficient in the pitB gene and characterized its phenotype on exposing it to varied concentrations of tellurite. Growth curves and transmission electronic microscopy experiments for the wild-type and ΔpitB strains showed that both were able to internalize tellurite into the cytoplasm and reduce the oxyanion to black nano-sized and rod-shaped tellurium particles, although the ΔpitB strain showed an increased resistance to the tellurite toxic effects. At a concentration of 100 μM tellurite, where the biomass formation of the wild-type strain decreased by half, we observed a greater ability of ΔpitB to reduce this oxyanion with respect to the wild-type strain (~38 vs ~16 %), which is related to the greater biomass production of ΔpitB and not to a greater consumption of tellurite per cell. The phenotype of the mutant was restored on over-expressing pitB in trans. In summary, our results indicate that PitB is one of several transporters responsible for tellurite uptake in P. putida KT2440.

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Effect of Soybean Coumestrol on Bradyrhizobium japonicum Nodulation Ability, Biofilm Formation, and Transcriptional Profile

Applied and Environmental Microbiology ◽

10.1128/aem.07336-11 ◽

2012 ◽

Vol 78 (8) ◽

pp. 2896-2903 ◽

Cited By ~ 29

Author(s):

Hae-In Lee ◽

Jin-Hwan Lee ◽

Ki-Hun Park ◽

Dipen Sangurdekar ◽

Woo-Suk Chang

Keyword(s):

Bradyrhizobium Japonicum ◽

Biofilm Formation ◽

Transcriptional Analysis ◽

Plant Metabolites ◽

Content Type ◽

Secondary Plant Metabolites ◽

Reverse Transcription Pcr ◽

Genome Wide ◽

A Genome ◽

Soybean Nodulation

ABSTRACTFlavonoids, secondary plant metabolites which mainly have a polyphenolic structure, play an important role in plant-microbe communications for nitrogen-fixing symbiosis. Among 10 polyphenolic compounds isolated from soybean roots in our previous study, coumestrol showed the highest antioxidant activity. In this study, its effect on the soybean nodulation was tested. The soybean symbiontBradyrhizobium japonicumUSDA110 pretreated with 20 μM coumestrol enhanced soybean nodulation by increasing the number of nodules 1.7-fold compared to the control. We also tested the effect of coumestrol onB. japonicumbiofilm formation. At a concentration of 2 μM, coumestrol caused a higher degree of biofilm formation than two major soybean isoflavonoids, genistein and daidzein, although no biofilm formation was observed at a concentration of 20 μM each compound. A genome-wide transcriptional analysis was performed to obtain a comprehensive snapshot of theB. japonicumresponse to coumestrol. When the bacterium was incubated in 20 μM coumestrol for 24 h, a total of 371 genes (139 upregulated and 232 downregulated) were differentially expressed at a 2-fold cutoff with aqvalue of less than 5%. No commonnodgene induction was found in the microarray data. However, quantitative reverse transcription-PCR (qRT-PCR) data showed that incubation for 12 h resulted in a moderate induction (ca. 2-fold) ofnodD1andnodABC, indicating that soybean coumestrol is a weak inducer of commonnodgenes. In addition, disruption ofnfeD(bll4952) affected the soybean nodulation by an approximate 30% reduction in the average number of nodules.

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Effects of Three Different Nucleoid-Associated Proteins Encoded on IncP-7 Plasmid pCAR1 on Host Pseudomonas putida KT2440

Applied and Environmental Microbiology ◽

10.1128/aem.00023-15 ◽

2015 ◽

Vol 81 (8) ◽

pp. 2869-2880 ◽

Cited By ~ 14

Author(s):

Chiho Suzuki-Minakuchi ◽

Ryusuke Hirotani ◽

Masaki Shintani ◽

Toshiharu Takeda ◽

Yurika Takahashi ◽

...

Keyword(s):

Pseudomonas Putida ◽

Structural Instability ◽

Metabolic Phenotype ◽

Pseudomonas Putida Kt2440 ◽

Host Chromosome ◽

Content Type ◽

Inorganic Ion ◽

Segregational Stability ◽

Genes Encoding ◽

Associated Proteins

ABSTRACTNucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption ofpmr,pnd, andphuwere assessed in hostPseudomonas putidaKT2440. Whenpmrandpndorpmrandphuwere simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation ofpmr, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype.

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Response of Pseudomonas putida KT2440 to Increased NADH and ATP Demand

Applied and Environmental Microbiology ◽

10.1128/aem.05588-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6597-6605 ◽

Cited By ~ 76

Author(s):

Birgitta E. Ebert ◽

Felix Kurth ◽

Marcel Grund ◽

Lars M. Blank ◽

Andreas Schmid

Keyword(s):

Pseudomonas Putida ◽

Atp Hydrolysis ◽

Nadh Oxidase ◽

Cell Metabolism ◽

Pseudomonas Putida Kt2440 ◽

Nadh Regeneration ◽

Regeneration Rate ◽

Content Type ◽

Oxidation Rates ◽

The Impact

ABSTRACTAdenosine phosphate and NAD cofactors play a vital role in the operation of cell metabolism, and their levels and ratios are carefully regulated in tight ranges. Perturbations of the consumption of these metabolites might have a great impact on cell metabolism and physiology. Here, we investigated the impact of increased ATP hydrolysis and NADH oxidation rates on the metabolism ofPseudomonas putidaKT2440 by titration of 2,4-dinitrophenol (DNP) and overproduction of a water-forming NADH oxidase, respectively. Both perturbations resulted in a reduction of the biomass yield and, as a consequence of the uncoupling of catabolic and anabolic activities, in an amplification of the net NADH regeneration rate. However, a stimulation of the specific carbon uptake rate was observed only whenP. putidawas challenged with very high 2,4-dinitrophenol concentrations and was comparatively unaffected by recombinant NADH oxidase activity. This behavior contrasts with the comparably sensitive performance described, for example, forEscherichia coliorSaccharomyces cerevisiae. The apparent robustness ofP. putidametabolism indicates that it possesses a certain buffering capacity and a high flexibility to adapt to and counteract different stresses without showing a distinct phenotype. These findings are important, e.g., for the development of whole-cell redox biocatalytic processes that impose equivalent burdens on the cell metabolism: stoichiometric consumption of (reduced) redox cofactors and increased energy expenditures, due to the toxicity of the biocatalytic compounds.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01850-12 ◽

2012 ◽

Vol 79 (1) ◽

pp. 357-366 ◽

Cited By ~ 57

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.

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Knockout of Extracytoplasmic Function Sigma Factor ECF-10 Affects Stress Resistance and Biofilm Formation in Pseudomonas putida KT2440

Applied and Environmental Microbiology ◽

10.1128/aem.01291-14 ◽

2014 ◽

Vol 80 (16) ◽

pp. 4911-4919 ◽

Cited By ~ 17

Author(s):

Beatrix Tettmann ◽

Andreas Dötsch ◽

Olivier Armant ◽

Christopher D. Fjell ◽

Joerg Overhage

Keyword(s):

Antibiotic Resistance ◽

Pseudomonas Putida ◽

Mutant Strain ◽

Stress Resistance ◽

Sigma Factor ◽

Biofilm Formation ◽

Efflux Pump ◽

Pseudomonas Putida Kt2440 ◽

Content Type ◽

Extracytoplasmic Function Sigma Factor

ABSTRACTPseudomonas putidais a Gram-negative soil bacterium which is well-known for its versatile lifestyle, controlled by a large repertoire of transcriptional regulators. Besides one- and two-component regulatory systems, the genome ofP. putidareveals 19 extracytoplasmic function (ECF) sigma factors involved in the adaptation to changing environmental conditions. In this study, we demonstrate that knockout of extracytoplasmic function sigma factor ECF-10, encoded by open reading frame PP4553, resulted in 2- to 4-fold increased antibiotic resistance to quinolone, β-lactam, sulfonamide, and chloramphenicol antibiotics. In addition, the ECF-10 mutant exhibited enhanced formation of biofilms after 24 h of incubation. Transcriptome analysis using Illumina sequencing technology resulted in the detection of 12 genes differentially expressed (>2-fold) in the ECF-10 knockout mutant strain compared to their levels of expression in wild-type cells. Among the upregulated genes werettgA,ttgB, andttgC, which code for the major multidrug efflux pump TtgABC inP. putidaKT2440. Investigation of an ECF-10 andttgAdouble-knockout strain and attgABC-overexpressing strain demonstrated the involvement of efflux pump TtgABC in the stress resistance and biofilm formation phenotypes of the ECF-10 mutant strain, indicating a new role for this efflux pump beyond simple antibiotic resistance inP. putidaKT2440.

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A Rational Design of Pseudomonas putida KT2440 capable of Anaerobic Respiration

10.21203/rs.3.rs-21150/v1 ◽

2020 ◽

Cited By ~ 1

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.

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