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Applied and Environmental Microbiology Sep 2016In the plant-beneficial bacterium Pseudomonas putida KT2440, three genes have been identified that encode posttranscriptional regulators of the CsrA/RsmA family. Their...
UNLABELLED
In the plant-beneficial bacterium Pseudomonas putida KT2440, three genes have been identified that encode posttranscriptional regulators of the CsrA/RsmA family. Their regulatory roles in the motile and sessile lifestyles of P. putida have been investigated by generating single-, double-, and triple-null mutants and by overexpressing each protein (RsmA, RsmE, and RsmI) in different genetic backgrounds. The rsm triple mutant shows reduced swimming and swarming motilities and increased biofilm formation, whereas overexpression of RsmE or RsmI results in reduced bacterial attachment. However, biofilms formed on glass surfaces by the triple mutant are more labile than those of the wild-type strain and are easily detached from the surface, a phenomenon that is not observed on plastic surfaces. Analysis of the expression of adhesins and exopolysaccharides in the different genetic backgrounds suggests that the biofilm phenotypes are due to alterations in the composition of the extracellular matrix and in the timing of synthesis of its elements. We have also studied the expression patterns of Rsm proteins and obtained data that indicate the existence of autoregulation mechanisms.
IMPORTANCE
Proteins of the CsrA/RsmA family function as global regulators in different bacteria. More than one of these proteins is present in certain species. In this study, all of the RsmA homologs in P. putida are characterized and globally taken into account to investigate their roles in controlling bacterial lifestyles and the regulatory interactions among them. The results offer new perspectives on how biofilm formation is modulated in this environmentally relevant bacterium.
Topics: Bacterial Adhesion; Bacterial Proteins; Biofilms; Gene Deletion; Gene Expression; Gene Expression Regulation, Bacterial; Locomotion; Pseudomonas putida; Self-Control
PubMed: 27422830
DOI: 10.1128/AEM.01724-16 -
MBio Jun 2019Nicotine, a toxic and addictive alkaloid from tobacco, is an environmental pollutant in areas near cigarette production facilities. Over the last decade, our group has...
Nicotine, a toxic and addictive alkaloid from tobacco, is an environmental pollutant in areas near cigarette production facilities. Over the last decade, our group has studied, in depth, the pyrrolidine pathway of nicotine degradation in S16. However, little is known regarding whole mechanism(s) regulating transcription of the nicotine degradation pathway gene cluster. In the present study, we comprehensively elucidate an overall view of the NicR2-mediated two-step mechanism regulating 3-succinoyl-pyridine (SP) biotransformation, which involves the association of free NicR2 with two promoters and the dissociation of NicR2 from the NicR2-promoter complex. NicR2 can bind to another promoter, , and regulate expression of the nicotine-degrading genes in the middle of gene cluster, which are not controlled by the previously reported promoter. We identified the function of the inverted repeat bases on the two promoters responsible for NicR2 binding and found out that the -35/-10 motif for RNA polymerase is overlapped by the NicR2 binding site. We clarify the exact role of 6-hydroxy-3-succinoyl-pyridine (HSP), which acts as an antagonist and may prevent binding of free NicR2 to the promoters but cannot release NicR2 from the promoters. Finally, a regulatory model is proposed, which consists of three parts: the interaction between NicR2 and two promoters ( and ), the interaction between NicR2 and two effectors (HSP and SP), and the interaction between NicR2 and RNA polymerase. We report the entire process underlying the NicR2 regulatory mechanism from association between free NicR2 and two promoters to dissociation of the NicR2-promoter complex. NicR2 can bind to another promoter, , which controls expression of nicotine-degrading genes that are not controlled by the promoter. We identified specific nucleotides of the promoter responsible for NicR2 binding. HSP was further demonstrated as an antagonist, which prevents the binding of NicR2 to the and promoters, by locking NicR2 in the derepression conformation. The competition between NicR2 and RNA polymerase is essential to initiate transcription of nicotine-degrading genes. This study extends our understanding of molecular mechanisms in biodegradation of environmental pollutants and toxicants.
Topics: Biodegradation, Environmental; Gene Expression Regulation, Bacterial; Metabolic Networks and Pathways; Multigene Family; Nicotine; Promoter Regions, Genetic; Pseudomonas putida; Pyridines; Succinates
PubMed: 31164460
DOI: 10.1128/mBio.00602-19 -
Microbial Cell Factories Feb 2024Bio-upcycling of plastics is an emerging alternative process that focuses on extracting value from a wide range of plastic waste streams. Such streams are typically too...
Bio-upcycling of plastics is an emerging alternative process that focuses on extracting value from a wide range of plastic waste streams. Such streams are typically too contaminated to be effectively processed using traditional recycling technologies. Medium-chain-length (mcl) diols and dicarboxylates (DCA) are major products of chemically or enzymatically depolymerized plastics, such as polyesters or polyethers. In this study, we enabled the efficient metabolism of mcl-diols and -DCA in engineered Pseudomonas putida as a prerequisite for subsequent bio-upcycling. We identified the transcriptional regulator GcdR as target for enabling metabolism of uneven mcl-DCA such as pimelate, and uncovered amino acid substitutions that lead to an increased coupling between the heterologous β-oxidation of mcl-DCA and the native degradation of short-chain-length DCA. Adaptive laboratory evolution and subsequent reverse engineering unravelled two distinct pathways for mcl-diol metabolism in P. putida, namely via the hydroxy acid and subsequent native β-oxidation or via full oxidation to the dicarboxylic acid that is further metabolized by heterologous β-oxidation. Furthermore, we demonstrated the production of polyhydroxyalkanoates from mcl-diols and -DCA by a single strain combining all required metabolic features. Overall, this study provides a powerful platform strain for the bio-upcycling of complex plastic hydrolysates to polyhydroxyalkanoates and leads the path for future yield optimizations.
Topics: Pseudomonas putida; Polyhydroxyalkanoates; Polyesters; Carboxylic Acids; Oxidation-Reduction
PubMed: 38365718
DOI: 10.1186/s12934-024-02310-7 -
Journal of Bacteriology Sep 2020KT2440 retains three homologs (PplR1 to PplR3) of the LitR/CarH family, an adenosyl B-dependent light-sensitive MerR family transcriptional regulator. Transcriptome...
KT2440 retains three homologs (PplR1 to PplR3) of the LitR/CarH family, an adenosyl B-dependent light-sensitive MerR family transcriptional regulator. Transcriptome analysis revealed the existence of a number of photoinducible genes, including , (encoding DNA photolyase), (furan-containing fatty acid synthase), (GTP cyclohydrolase I), (cryptochrome-like protein), and multiple genes without annotated/known function. Transcriptional analysis by quantitative reverse transcription-PCR with knockout mutants of to showed that a triple knockout completely abolished the light-inducible transcription in , 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)TACANTGTA(C/T)A-3'. The disruption of B biosynthesis cluster did not affect the light-inducible transcription; however, disruption of (where LOV indicates "light, oxygen, or voltage") and , encoding blue light photoreceptors adjacently located to and , 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 / cognate pair homologs in spp. and related bacteria suggests that the response and adaptation to light are similarly regulated in the group of nonphototrophic bacteria. 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). 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 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.
Topics: Bacterial Proteins; Binding Sites; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Light; Operon; Photoreceptors, Microbial; Promoter Regions, Genetic; Pseudomonas putida
PubMed: 32967908
DOI: 10.1128/JB.00146-20 -
Microbial Biotechnology Mar 2024Medium-chain-length α,ω-diols (mcl-diols) play an important role in polymer production, traditionally depending on energy-intensive chemical processes. Microbial cell...
Medium-chain-length α,ω-diols (mcl-diols) play an important role in polymer production, traditionally depending on energy-intensive chemical processes. Microbial cell factories offer an alternative, but conventional strains like Escherichia coli and Saccharomyces cerevisiae face challenges in mcl-diol production due to the toxicity of intermediates such as alcohols and acids. Metabolic engineering and synthetic biology enable the engineering of non-model strains for such purposes with P. putida emerging as a promising microbial platform. This study reviews the advancement in diol production using P. putida and proposes a four-module approach for the sustainable production of diols. Despite progress, challenges persist, and this study discusses current obstacles and future opportunities for leveraging P. putida as a microbial cell factory for mcl-diol production. Furthermore, this study highlights the potential of using P. putida as an efficient chassis for diol synthesis.
Topics: Pseudomonas putida; Polyhydroxyalkanoates; Metabolic Engineering; Escherichia coli; Synthetic Biology
PubMed: 38528784
DOI: 10.1111/1751-7915.14423 -
Environmental Microbiology Aug 2022Indole-3-acetic acid (IAA) is the main naturally occurring auxin and is produced by organisms of all kingdoms of life. In addition to the regulation of plant growth and...
Indole-3-acetic acid (IAA) is the main naturally occurring auxin and is produced by organisms of all kingdoms of life. In addition to the regulation of plant growth and development, IAA plays an important role in the interaction between plants and growth-promoting and phytopathogenic bacteria by regulating bacterial gene expression and physiology. We show here that an IAA metabolizing plant-associated Pseudomonas putida isolate exhibits chemotaxis to IAA that is independent of auxin metabolism. We found that IAA chemotaxis is based on the activity of the PcpI chemoreceptor and heterologous expression of pcpI conferred IAA taxis to different environmental and human pathogenic isolates of the Pseudomonas genus. Using ligand screening, microcalorimetry and quantitative chemotaxis assays, we found that PcpI failed to bind IAA directly, but recognized and mediated chemoattractions to various aromatic compounds, including the phytohormone salicylic acid. The expression of pcpI and its role in the interactions with plants was also investigated. PcpI extends the range of central signal molecules recognized by chemoreceptors. To our knowledge, this is the first report on a bacterial receptor that responds to two different phytohormones. Our study reinforces the multifunctional role of IAA and salicylic acid as intra- and inter-kingdom signal molecules.
Topics: Chemotaxis; Humans; Indoleacetic Acids; Plant Growth Regulators; Plants; Pseudomonas putida; Salicylic Acid
PubMed: 35088505
DOI: 10.1111/1462-2920.15920 -
Scientific Reports Apr 2018Integrative and conjugative elements (ICEs) comprise ubiquitous large mobile regions in prokaryotic chromosomes that transmit vertically to daughter cells and transfer...
Integrative and conjugative elements (ICEs) comprise ubiquitous large mobile regions in prokaryotic chromosomes that transmit vertically to daughter cells and transfer horizontally to distantly related lineages. Their evolutionary success originates in maximized combined ICE-host fitness trade-offs, but how the ICE impacts on the host metabolism and physiology is poorly understood. Here we investigate global changes in the host genetic network and physiology of Pseudomonas putida with or without an integrated ICEclc, a model ICE widely distributed in proteobacterial genomes. Genome-wide gene expression differences were analyzed by RNA-seq using exponentially growing or stationary phase-restimulated cultures on 3-chlorobenzoate, an aromatic compound metabolizable thanks to specific ICEclc-located genes. We found that the presence of ICEclc imposes a variety of changes in global pathways such as cell cycle and amino acid metabolism, which were more numerous in stationary-restimulated than exponential phase cells. Unexpectedly, ICEclc stimulates cellular motility and leads to more rapid growth on 3-chlorobenzoate than cells carrying only the integrated clc genes. ICEclc also concomitantly activates the P. putida Pspu28-prophage, but this in itself did not provoke measurable fitness effects. ICEclc thus interferes in a number of cellular pathways, inducing both direct benefits as well as indirect costs in P. putida.
Topics: Bacterial Proteins; Conjugation, Genetic; Gene Expression Regulation, Bacterial; Genome, Bacterial; Genomic Islands; Interspersed Repetitive Sequences; Prophages; Pseudomonas putida; Transcriptome
PubMed: 29615803
DOI: 10.1038/s41598-018-23858-6 -
Metabolic Engineering Sep 2022Anthranilate, an intermediate of the shikimate pathway, is a high-value aromatic compound widely used as a precursor in the production of dyes, fragrances, plastics and...
Anthranilate, an intermediate of the shikimate pathway, is a high-value aromatic compound widely used as a precursor in the production of dyes, fragrances, plastics and pharmaceuticals. Traditional strategies adopted for microbial anthranilate production rely on the implementation of auxotrophic strains-which requires aromatic amino acids or complex additives to be supplemented in the culture medium, negatively impacting production costs. In this work, we engineered the soil bacterium Pseudomonas putida for high-titer, glucose-dependent anthranilate production by repurposing elements of the Esa quorum sensing (QS) system of Pantoea stewartii. The P promoter mediated a self-regulated transcriptional response that effectively knocked-down the expression of the trpDC genes. Next, we harnessed the synthetic QS elements to engineer a growth-to-anthranilate production switch. The resulting plasmid-free P. putida strain produced the target compound at 3.8 ± 0.3 mM in shaken-flask cultures after 72 h-a titer >2-fold higher than anthranilate levels reported thus far. Our results highlight the value of dynamic flux regulation for the production of intermediate metabolites within highly-regulated routes (such as the shikimate pathway), thereby circumventing the need of expensive additives.
Topics: Glucose; Plasmids; Pseudomonas putida; Quorum Sensing; ortho-Aminobenzoates
PubMed: 35659519
DOI: 10.1016/j.ymben.2022.05.008 -
Responds to the Toxin GraT by Inducing Ribosome Biogenesis Factors and Repressing TCA Cycle Enzymes.Toxins Feb 2019The potentially self-poisonous toxin-antitoxin modules are widespread in bacterial chromosomes, but despite extensive studies, their biological importance remains poorly...
The potentially self-poisonous toxin-antitoxin modules are widespread in bacterial chromosomes, but despite extensive studies, their biological importance remains poorly understood. Here, we used whole-cell proteomics to study the cellular effects of the toxin GraT that is known to inhibit growth and ribosome maturation in a cold-dependent manner when the antitoxin gene is deleted from the genome. Proteomic analysis of wild-type and Δ strains at 30 °C and 25 °C, where the growth is differently affected by GraT, revealed two major responses to GraT at both temperatures. First, ribosome biogenesis factors, including the RNA helicase DeaD and RNase III, are upregulated in Δ. This likely serves to alleviate the ribosome biogenesis defect of the Δ strain. Secondly, proteome data indicated that GraT induces downregulation of central carbon metabolism, as suggested by the decreased levels of TCA cycle enzymes isocitrate dehydrogenase Idh, α-ketoglutarate dehydrogenase subunit SucA, and succinate-CoA ligase subunit SucD. Metabolomic analysis revealed remarkable GraT-dependent accumulation of oxaloacetate at 25 °C and a reduced amount of malate, another TCA intermediate. The accumulation of oxaloacetate is likely due to decreased flux through the TCA cycle but also indicates inhibition of anabolic pathways in GraT-affected bacteria. Thus, proteomic and metabolomic analysis of the Δ strain revealed that GraT-mediated stress triggers several responses that reprogram the cell physiology to alleviate the GraT-caused damage.
Topics: Antitoxins; Bacterial Proteins; Bacterial Toxins; Citric Acid Cycle; Metabolome; Proteome; Pseudomonas putida; Ribosomal Proteins
PubMed: 30744127
DOI: 10.3390/toxins11020103 -
Applied and Environmental Microbiology Nov 2019Although alcohols are toxic to many microorganisms, they are good carbon and energy sources for some bacteria, including many pseudomonads. However, most studies that...
Although alcohols are toxic to many microorganisms, they are good carbon and energy sources for some bacteria, including many pseudomonads. However, most studies that have examined chemosensory responses to alcohols have reported that alcohols are sensed as repellents, which is consistent with their toxic properties. In this study, we examined the chemotaxis of strain F1 to -alcohols with chain lengths of 1 to 12 carbons. F1 was attracted to all -alcohols that served as growth substrates (C to C) for the strain, and the responses were induced when cells were grown in the presence of alcohols. By assaying mutant strains lacking single or multiple methyl-accepting chemotaxis proteins, the receptor mediating the response to C to C alcohols was identified as McfP, the ortholog of the strain KT2440 receptor for C and C carboxylic acids. Besides being a requirement for the response to -alcohols, McfP was required for the response of F1 to pyruvate, l-lactate, acetate, and propionate, which are detected by the KT2440 receptor, and the medium- and long-chain carboxylic acids hexanoic acid and dodecanoic acid. β-Galactosidase assays of F1 carrying an transcriptional fusion showed that the gene is not induced in response to alcohols. Together, our results are consistent with the idea that the carboxylic acids generated from the oxidation of alcohols are the actual attractants sensed by McfP in F1, rather than the alcohols themselves. Alcohols, released as fermentation products and produced as intermediates in the catabolism of many organic compounds, including hydrocarbons and fatty acids, are common components of the microbial food web in soil and sediments. Although they serve as good carbon and energy sources for many soil bacteria, alcohols have primarily been reported to be repellents rather than attractants for motile bacteria. Little is known about how alcohols are sensed by microbes in the environment. We report here that catabolizable -alcohols with linear chains of up to 12 carbons serve as attractants for the soil bacterium , and rather than being detected directly, alcohols appear to be catabolized to acetate, which is then sensed by a specific cell-surface chemoreceptor protein.
Topics: Alcohols; Bacterial Proteins; Carboxylic Acids; Chemotaxis; Methyl-Accepting Chemotaxis Proteins; Pseudomonas putida
PubMed: 31471307
DOI: 10.1128/AEM.01625-19