The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor

ABSTRACTThe growth of the soil bacteriumPseudomonas putidaKT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy and flow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inactive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional Φ(glpD-gfp) fusion (a proxy of the glycerol-3-phosphate [G3P] dehydrogenase activity) linked the macroscopic phenotype to the expression of theglpgenes. Either deletingglpR(encoding the G3P-responsive transcriptional repressor that controls the expression of theglpFKRDgene cluster) or altering G3P formation (by overexpressingglpK, encoding glycerol kinase) abolished the bimodalglpDexpression. These manipulations eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision ofglpRintransrestored the phenotypes lost in theΔglpRmutant. The prolonged nongrowth regime ofP. putidaon glycerol could thus be traced to the regulatory device controlling the transcription of theglpgenes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional response, a layout fostering the observed time-dependent shift between two alternative physiological states.IMPORTANCEPhenotypic variation is a widespread attribute of prokaryotes that leads,inter alia, to the emergence of persistent bacteria, i.e., live but nongrowing members within a genetically clonal population. Persistence allows a fraction of cells to avoid the killing caused by conditions or agents that destroy most growing bacteria (e.g., some antibiotics). Known molecular mechanisms underlying the phenomenon include genetic changes, epigenetic variations, and feedback-based multistability. We show that a prolonged nongrowing state of the bacterial population can be brought about by a distinct regulatory architecture of metabolic genes when cells face specific nutrients (e.g., glycerol).Pseudomonas putidamay have adopted the resulting carbon source-dependent metabolic bet hedging as an advantageous trait for exploring new chemical and nutritional landscapes. Defeating such naturally occurring adaptive features of environmental bacteria is instrumental in improving the performance of these microorganisms as whole-cell catalysts in a bioreactor setup.

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Time-Course Proteomic Analysis of Pseudomonas putida KT2440 during Mcl-Polyhydroxyalkanoate Synthesis under Nitrogen Deficiency

Polymers ◽

10.3390/polym11050748 ◽

2019 ◽

Vol 11 (5) ◽

pp. 748 ◽

Cited By ~ 4

Author(s):

Justyna Możejko-Ciesielska ◽

Agnieszka Mostek

Keyword(s):

Pseudomonas Putida ◽

Proteomic Analysis ◽

Cellular Response ◽

Time Course ◽

Molecular Mechanisms ◽

Nitrogen Deficiency ◽

Growth Stages ◽

Bacterial Cells ◽

Pseudomonas Putida Kt2440 ◽

Wide Range

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) have gained great attention as a new green alternative to petrochemical-derived polymers. Due to their outstanding material properties they can be used in a wide range of applications. Pseudomonas putida KT2440 is a metabolically versatile producer of mcl-polyhydroxyalkanoates. Although the metabolism of polyhydroxyalkanoate synthesis by this bacterium has been extensively studied, the comparative proteome analysis from three growth stages of Pseudomonas putida KT2440 cultured with oleic acid during mcl-PHA synthesis has not yet been reported. Therefore; the aim of the study was to compare the proteome of Pseudomonas putida KT2440 at different time points of its cultivation using the 2D difference gel electrophoresis (2D-DIGE) technique. The analyses showed that low levels of a nitrogen source were beneficial for mcl-PHA synthesis. Proteomic analysis revealed that the proteins associated with carbon metabolism were affected by nitrogen starvation and mcl-PHA synthesis. Furthermore, the induction of proteins involved in nitrogen metabolism, ribosome synthesis, and transport was observed, which may be the cellular response to stress related to nitrogen deficiency and mcl-PHA content in bacterial cells. To sum up; this study enabled the investigators to acquire a better knowledge of the molecular mechanisms underlying the induction of polyhydroxyalkanoate synthesis and accumulation in Pseudomonas putida KT2440 that could lead to improved strategies for PHAs in industrial production.

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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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Study of pH Changes in Media during Bacterial Growth of Several Environmental Strains

Proceedings ◽

10.3390/proceedings2201297 ◽

2018 ◽

Vol 2 (20) ◽

pp. 1297 ◽

Cited By ~ 10

Author(s):

Rubén Sánchez-Clemente ◽

María Isabel Igeño ◽

Ana G. Población ◽

María Isabel Guijo ◽

Faustino Merchán ◽

...

Keyword(s):

Carbon Source ◽

Pseudomonas Putida ◽

Bacterial Growth ◽

Extracellular Ph ◽

Cellular Growth ◽

Exponential Growth Phase ◽

Extracellular Medium ◽

Pseudomonas Putida Kt2440 ◽

E Coli ◽

Initial Ph

The effect of pH on bacterial cell-growth and the evolution of extracellular pH triggered by bacterial growth has been monitored for three bacterial strains, Escherichia coli ATCC 25922 and Pseudomonas putida KT2440 as reference strains, and Pseudomonas pseudoalcaligenes CECT 5344 because of its capacity to assimilate cyanide as the sole nitrogen source under alkaline conditions. In a first instance, the influence of the initial pH in the growth curve has been texted in LB-medium adjusted to pH 6, 7 and 8, for E. coli and P. putida, and 7.5, 8.25 and 9 for P. pseudoalcaligenes. Although the initial pH were different, the pH of the extracellular medium at the end of the stationary phase converged to a certain pH that is specific for each bacterium. Similar experiments were carried out in minimal medium with glucose as the carbon source. In this case, the pHs of the culture of both Pseudomonadaceae strains were almost constant, whereas it suddenly dropped during the exponential growth phase of E. coli. When the initial pH was 6 the extracellular pH fell sharply to 4.5, which irreversibly prevented further cellular growth. Nevertheless, at higher initial pH values subsequent cellular growth of E. coli restored the medium to the initial pHs values. Finally, in all cases the evolution of the pH has been shown to depend on the carbon source used. Among the sources used, cellular growth with glucose or glycerol did not affect the extracellular pH, whereas citrate caused the alkalinization of the media. This phenotype is in concordance with computational predictions, at least in the case of the genome-scale metabolic model of Pseudomonas putida KT2440.

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Molecular Mechanisms of Chlorhexidine Tolerance in Burkholderia cenocepacia Biofilms

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01571-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 1912-1919 ◽

Cited By ~ 50

Author(s):

Tom Coenye ◽

Heleen Van Acker ◽

Elke Peeters ◽

Andrea Sass ◽

Silvia Buroni ◽

...

Keyword(s):

Burkholderia Cepacia ◽

Molecular Mechanisms ◽

Antimicrobial Agents ◽

Efflux Pump ◽

Transcriptional Response ◽

Regulatory Elements ◽

Burkholderia Cenocepacia ◽

Phenotypic Analysis ◽

Content Type ◽

Sessile Cells

ABSTRACTThe high tolerance of biofilm-grownBurkholderia cepaciacomplex bacteria against antimicrobial agents presents considerable problems for the treatment of infected cystic fibrosis patients and the implementation of infection control guidelines. In the present study, we analyzed the tolerance of planktonic and sessileBurkholderia cenocepaciaJ2315 cultures and examined the transcriptional response of sessile cells to treatment with chlorhexidine. At low (0.0005%) and high (0.05%) concentrations, chlorhexidine had a similar effect on both populations, but at intermediate concentrations (0.015%) the antimicrobial activity was more pronounced in planktonic cultures. The exposure of sessile cells to chlorhexidine resulted in an upregulation of the transcription of 469 (6.56%) and the downregulation of 257 (3.59%) protein-coding genes. A major group of upregulated genes in the treated biofilms encoded membrane-related and regulatory proteins. In addition, several genes coding for drug resistance determinants also were upregulated. The phenotypic analysis of RND (resistance-nodulation-division) efflux pump mutants suggests the presence of lifestyle-specific chlorhexidine tolerance mechanisms; efflux system RND-4 (BCAL2820-BCAL2822) was more responsible for chlorhexidine tolerance in planktonic cells, while other systems (RND-3 [BCAL1672-BCAL1676] and RND-9 [BCAM1945-BCAM1947]) were linked to resistance in sessile cells. After sessile cell exposure, multiple genes encoding chemotaxis and motility-related proteins were upregulated in concert with the downregulation of an adhesin-encoding gene (BCAM2143), suggesting that sessile cells tried to escape the biofilm. We also observed the differential expression of 19 genes carying putative small RNA molecules, indicating a novel role for these regulatory elements in chlorhexidine tolerance.

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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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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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Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida

Applied and Environmental Microbiology ◽

10.1128/aem.03236-16 ◽

2017 ◽

Vol 83 (7) ◽

Cited By ~ 19

Author(s):

Klara Bojanovič ◽

Isotta D'Arrigo ◽

Katherine S. Long

Keyword(s):

Gene Expression ◽

Stress Response ◽

Pseudomonas Putida ◽

Cellular Response ◽

Functional Characterization ◽

Transcriptional Response ◽

Stress Conditions ◽

Differentially Expressed ◽

Transcriptional Responses ◽

Content Type

ABSTRACTBacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response ofPseudomonas putidaKT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through whichP. putidaresponds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings.IMPORTANCEThis study maps the complete transcriptional response ofP. putidaKT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one condition, suggesting their involvement in adaptation to stress conditions and identifying interesting candidates for further functional characterization.

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Proteomic Response of Pseudomonas putida KT2440 to Dual Carbon-Phosphorus Limitation during mcl-PHAs Synthesis

Biomolecules ◽

10.3390/biom9120796 ◽

2019 ◽

Vol 9 (12) ◽

pp. 796 ◽

Cited By ~ 5

Author(s):

Justyna Możejko-Ciesielska ◽

Luísa S. Serafim

Keyword(s):

Pseudomonas Putida ◽

Time Course ◽

Molecular Mechanisms ◽

Phosphorus Limitation ◽

Growth Conditions ◽

Pseudomonas Putida Kt2440 ◽

Medium Chain Length ◽

Metabolic Activities ◽

Proteomic Responses ◽

Phosphate Regulon

Pseudomonas putida KT2440, one of the best characterized pseudomonads, is a metabolically versatile producer of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) that serves as a model bacterium for molecular studies. The synthesis of mcl-PHAs is of great interest due to their commercial potential. Carbon and phosphorus are the essential nutrients for growth and their limitation can trigger mcl-PHAs’ production in microorganisms. However, the specific molecular mechanisms that drive this synthesis in Pseudomonas species under unfavorable growth conditions remain poorly understood. Therefore, the proteomic responses of Pseudomonas putida KT2440 to the limited carbon and phosphorus levels in the different growth phases during mcl-PHAs synthesis were investigated. The data indicated that biopolymers’ production was associated with the cell growth of P. putida KT2440 under carbon- and phosphorus-limiting conditions. The protein expression pattern changed during mcl-PHAs synthesis and accumulation, and during the different physiological states of the microorganism. The data suggested that the majority of metabolic activities ceased under carbon and phosphorus limitation. The abundance of polyhydroxyalkanoate granule-associated protein (PhaF) involved in PHA synthesis increased significantly at 24 and 48 h of the cultivations. The activation of proteins belonging to the phosphate regulon was also detected. Moreover, these results indicated changes in the protein profiles related to amino acids metabolism, replication, transcription, translation, stress response mechanisms, transport or signal transduction. The presented data allowed the investigation of time-course proteome alterations in response to carbon and phosphorus limitation, and PHAs synthesis. This study provided information about proteins that can be potential targets in improving the efficiency of mcl-PHAs synthesis.

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Molecular Deceleration Regulates Toxicant Release to Prevent Cell Damage in Pseudomonas putida S16 (DSM 28022)

mBio ◽

10.1128/mbio.02012-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Hongzhi Tang ◽

Kunzhi Zhang ◽

Haiyang Hu ◽

Geng Wu ◽

Weiwei Wang ◽

...

Keyword(s):

Crystal Structure ◽

Pseudomonas Putida ◽

Molecular Mechanisms ◽

Cell Damage ◽

Amino Acid Residues ◽

Amine Oxidases ◽

Content Type ◽

Toxic Product ◽

Nicotine Degradation ◽

Cytotoxic Compounds

ABSTRACT The underlying molecular mechanisms of flavin-dependent amine oxidases remain relatively poorly understood, even though many of these enzymes have been reported. The nicotine oxidoreductase NicA2 is a crucial enzyme for the first step of nicotine degradation in Pseudomonas putida S16 (DSM 28022). Here, we present the crystal structure of a ternary complex comprising NicA2 residues 21 to 482, flavin adenine dinucleotide (FAD), and nicotine at 2.25 Å resolution. Unlike other, related structures, NicA2 does not have an associated diacyl glycerophospholipid, wraps its substrate more tightly, and has an intriguing exit passage in which nine bulky amino acid residues occlude the release of its toxic product, pseudooxynicotine (PN). The replacement of these bulky residues by amino acids with small side chains effectively increases the catalytic turnover rate of NicA2. Our results indicate that the passage in wild-type NicA2 effectively controls the rate of PN release and thus prevents its rapid intracellular accumulation. It gives ample time for PN to be converted to less-harmful substances by downstream enzymes such as pseudooxynicotine amine oxidase (Pnao) before its accumulation causes cell damage or even death. The temporal metabolic regulation mode revealed in this study may shed light on the production of cytotoxic compounds. IMPORTANCE Flavin-dependent amine oxidases have received extensive attention because of their importance in drug metabolism, Parkinson’s disease, and neurotransmitter catabolism. However, the underlying molecular mechanisms remain relatively poorly understood. Here, combining the crystal structure of NicA2 (an enzyme in the first step of the bacterial nicotine degradation pathway in Pseudomonas putida S16 (DSM 28022)), biochemical analysis, and mutant construction, we found an intriguing exit passage in which bulky amino acid residues occlude the release of the toxic product of NicA2, in contrast to other, related structures. The selective product exportation register for NicA2 has proven to be beneficial to cell growth. Those seeking to produce cytotoxic compounds could greatly benefit from the use of such an export register mechanism.

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