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

2015 ◽  
Vol 81 (8) ◽  
pp. 2869-2880 ◽  
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.

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

2012 ◽  
Vol 78 (19) ◽  
pp. 6963-6974 ◽  
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.

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

Microbiology ◽  
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.

scholarly journals Genetic and Functional Characterization of Cyclic Lipopeptide White-Line-Inducing Principle (WLIP) Production by Rice Rhizosphere Isolate Pseudomonas putida RW10S2

2012 ◽  
Vol 78 (14) ◽  
pp. 4826-4834 ◽  
Author(s):  
Hassan Rokni-Zadeh ◽  
Wen Li ◽  
Aminael Sanchez-Rodriguez ◽  
Davy Sinnaeve ◽  
Jef Rozenski ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Functional Characterization ◽  
Inhibitory Effect ◽  
White Line ◽  
Line Production ◽  
Content Type ◽  
Rice Rhizosphere ◽  
Cyclic Lipopeptide ◽  
Genes Encoding ◽  
Dependent Growth

ABSTRACTThe secondary metabolite mediating the GacS-dependent growth-inhibitory effect exerted by the rice rhizosphere isolatePseudomonas putidaRW10S2 on phytopathogenicXanthomonasspecies was identified as white-line-inducing principle (WLIP), a member of the viscosin group of cyclic lipononadepsipeptides. WLIP producers are commonly referred to by the taxonomically invalid name “Pseudomonas reactans,” based on their capacity to reveal the presence of a nearby colony ofPseudomonas tolaasiiby inducing the formation of a visible precipitate (“white line”) in agar medium between both colonies. This phenomenon is attributed to the interaction of WLIP with a cyclic lipopeptide of a distinct structural group, the fungitoxic tolaasin, and has found application as a diagnostic tool to identify tolaasin-producing bacteria pathogenic to mushrooms. The genes encoding the WLIP nonribosomal peptide synthetases WlpA, WlpB, and WlpC were identified in two separate genomic clusters (wlpR-wlpAandwlpBC) with an operon organization similar to that of the viscosin, massetolide, and entolysin biosynthetic systems. Expression ofwlpRis dependent ongacS, and the encoded regulator of the LuxR family (WlpR) activates transcription of the biosynthetic genes and the linked export genes, which is not controlled by the RW10S2 quorum-sensing system PmrR/PmrI. In addition to linking the known phenotypes of white line production and hemolytic activity of a WLIP producer with WLIP biosynthesis, additional properties of ecological relevance conferred by WLIP production were identified, namely, antagonism againstXanthomonasand involvement in swarming and biofilm formation.

scholarly journals The Bacteroides thetaiotaomicron Protein Bacteroides Host Factor A Participates in Integration of the Integrative Conjugative Element CTnDOT into the Chromosome

2015 ◽  
Vol 197 (8) ◽  
pp. 1339-1349 ◽  
Author(s):  
Kenneth Ringwald ◽  
Jeffrey Gardner
Keyword(s):  
Antibiotic Resistance Genes ◽  
Host Factor ◽  
Host Cells ◽  
Recombination Reaction ◽  
Host Chromosome ◽  
Content Type ◽  
Integrative And Conjugative Elements ◽  
Integrative Conjugative Element ◽  
Associated Proteins ◽  
A Site

ABSTRACTCTnDOT is a conjugative transposon found inBacteroidesspecies. It encodes multiple antibiotic resistances and is stimulated to transfer by exposure to tetracycline. CTnDOT integration into the host chromosome requires IntDOT and a previously unknown host factor. We have identified a protein, designated BHFa (Bacteroideshost factor A), that participates in integrative recombination. BHFa is the first host factor identified for a site-specific recombination reaction in the CTnDOT family of integrative and conjugative elements. Based on the amino acid sequence of BHFa, the ability to bind specifically to 4 sites in theattDOTDNA, and its activity in the integration reaction, BHFa is a member of the IHF/HU family of nucleoid-associated proteins. Other DNA bending proteins that bind DNA nonspecifically can substitute for BHFa in the integration reaction.IMPORTANCEBacteroidesspecies are normal members of the human colonic microbiota. These species can harbor and spread self-transmissible genetic elements (integrative conjugative elements [ICEs]) that contain antibiotic resistance genes. This work describes the role of a protein, BHFa, and its importance in the integration reaction required for the element CTnDOT to persist inBacteroideshost cells.

scholarly journals Response of Pseudomonas putida KT2440 to Increased NADH and ATP Demand

2011 ◽  
Vol 77 (18) ◽  
pp. 6597-6605 ◽  
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.

scholarly journals Eisosome Ultrastructure and Evolution in Fungi, Microalgae, and Lichens

2015 ◽  
Vol 14 (10) ◽  
pp. 1017-1042 ◽  
Author(s):  
Jae-Hyeok Lee ◽  
John E. Heuser ◽  
Robyn Roth ◽  
Ursula Goodenough
Keyword(s):  
Coiled Coil ◽  
Lipid Phase ◽  
Content Type ◽  
Bar Domain ◽  
Bar Domain Proteins ◽  
Genes Encoding ◽  
Conserved Genes ◽  
Associated Proteins ◽  
Target Membrane ◽  
Positively Charged

ABSTRACTEisosomes are among the few remaining eukaryotic cellular differentations that lack a defined function(s). These trough-shaped invaginations of the plasma membrane have largely been studied inSaccharomyces cerevisiae, in which their associated proteins, including two BAR domain proteins, have been identified, and homologues have been found throughout the fungal radiation. Using quick-freeze deep-etch electron microscopy to generate high-resolution replicas of membrane fracture faces without the use of chemical fixation, we report that eisosomes are also present in a subset of red and green microalgae as well as in the cysts of the ciliateEuplotes. Eisosome assembly is closely correlated with both the presence and the nature of cell walls. Microalgal eisosomes vary extensively in topology and internal organization. Unlike fungi, their convex fracture faces can carry lineage-specific arrays of intramembranous particles, and their concave fracture faces usually display fine striations, also seen in fungi, that are pitched at lineage-specific angles and, in some cases, adopt a broad-banded patterning. The conserved genes that encode fungal eisosome-associated proteins are not found in sequenced algal genomes, but we identified genes encoding two algal lineage-specific families of predicted BAR domain proteins, called Green-BAR and Red-BAR, that are candidate eisosome organizers. We propose a model for eisosome formation wherein (i) positively charged recognition patches first establish contact with target membrane regions and (ii) a (partial) unwinding of the coiled-coil conformation of the BAR domains then allows interactions between the hydrophobic faces of their amphipathic helices and the lipid phase of the inner membrane leaflet, generating the striated patterns.

scholarly journals MvaT Family Proteins Encoded on IncP-7 Plasmid pCAR1 and the Host Chromosome Regulate the Host Transcriptome Cooperatively but Differently

2015 ◽  
Vol 82 (3) ◽  
pp. 832-842 ◽  
Author(s):  
Choong-Soo Yun ◽  
Yurika Takahashi ◽  
Masaki Shintani ◽  
Toshiharu Takeda ◽  
Chiho Suzuki-Minakuchi ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Conjugative Plasmid ◽  
Homologous Gene ◽  
Early Stationary Phase ◽  
Host Chromosome ◽  
Content Type ◽  
Homologous Genes ◽  
Genes Encoding ◽  
Plasmid Carriage

ABSTRACTMvaT proteins are members of the H-NS family of proteins in pseudomonads. The IncP-7 conjugative plasmid pCAR1 carries anmvaT-homologous gene,pmr. InPseudomonas putidaKT2440 bearing pCAR1,pmrand the chromosomally carried homologous genes,turAandturB, are transcribed at high levels, and Pmr interacts with TurA and TurBin vitro. In the present study, we clarified how the three MvaT proteins regulate the transcriptome ofP. putidaKT2440(pCAR1). Analyses performed by a modified chromatin immunoprecipitation assay with microarray technology (ChIP-chip) suggested that the binding regions of Pmr, TurA, and TurB in theP. putidaKT2440(pCAR1) genome are almost identical; nevertheless, transcriptomic analyses using mutants with deletions of the genes encoding the MvaT proteins during the log and early stationary growth phases clearly suggested that their regulons were different. Indeed, significant regulon dissimilarity was found between Pmr and the other two proteins. Transcription of a larger number of genes was affected by Pmr deletion during early stationary phase than during log phase, suggesting that Pmr ameliorates the effects of pCAR1 on host fitness more effectively during the early stationary phase. Alternatively, the similarity of the TurA and TurB regulons implied that they might play complementary roles as global transcriptional regulators in response to plasmid carriage.

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

2014 ◽  
Vol 80 (16) ◽  
pp. 4911-4919 ◽  
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.

scholarly journals Carbon Source-Dependent Inducible Metabolism of Veratryl Alcohol and Ferulic Acid in Pseudomonas putida CSV86

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Karishma Mohan ◽  
Prashant S. Phale
Keyword(s):  
Carbon Source ◽  
Ferulic Acid ◽  
Pseudomonas Putida ◽  
Vanillic Acid ◽  
Veratryl Alcohol ◽  
Veratric Acid ◽  
Suitable Candidate ◽  
Content Type ◽  
Metabolic Intermediates ◽  
Genes Encoding

ABSTRACT Pseudomonas putida CSV86 degrades lignin-derived metabolic intermediates, viz., veratryl alcohol, ferulic acid, vanillin, and vanillic acid, as the sole sources of carbon and energy. Strain CSV86 also degraded lignin sulfonate. Cell respiration, enzyme activity, biotransformation, and high-pressure liquid chromatography (HPLC) analyses suggest that veratryl alcohol and ferulic acid are metabolized to vanillic acid by two distinct carbon source-dependent inducible pathways. Vanillic acid was further metabolized to protocatechuic acid and entered the central carbon pathway via the β-ketoadipate route after ortho ring cleavage. Genes encoding putative enzymes involved in the degradation were found to be present at fer, ver, and van loci. The transcriptional analysis suggests a carbon source-dependent cotranscription of these loci, substantiating the metabolic studies. Biochemical and quantitative real-time (qRT)-PCR studies revealed the presence of two distinct O-demethylases, viz., VerAB and VanAB, involved in the oxidative demethylation of veratric acid and vanillic acid, respectively. This report describes the various steps involved in metabolizing lignin-derived aromatic compounds at the biochemical level and identifies the genes involved in degrading veratric acid and the arrangement of phenylpropanoid metabolic genes as three distinct inducible transcription units/operons. This study provides insight into the bacterial degradation of lignin-derived aromatics and the potential of P. putida CSV86 as a suitable candidate for producing valuable products. IMPORTANCE Pseudomonas putida CSV86 metabolizes lignin and its metabolic intermediates as a carbon source. Strain CSV86 displays a unique property of preferential utilization of aromatics, including for phenylpropanoids over glucose. This report unravels veratryl alcohol metabolism and genes encoding veratric acid O-demethylase, hitherto unknown in pseudomonads, thereby providing new insight into the metabolic pathway and gene pool for lignin degradation in bacteria. The biochemical and genetic characterization of phenylpropanoid metabolism makes it a prospective system for its application in producing valuable products, such as vanillin and vanillic acid, from lignocellulose. This study supports the immense potential of P. putida CSV86 as a suitable candidate for bioremediation and biorefinery.

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