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MBio Feb 2022Polyhydroxyalkanoates (PHAs) are polyesters produced by numerous microorganisms for energy and carbon storage. Simultaneous synthesis and degradation of PHA drives a...
Polyhydroxyalkanoates (PHAs) are polyesters produced by numerous microorganisms for energy and carbon storage. Simultaneous synthesis and degradation of PHA drives a dynamic cycle linked to the central carbon metabolism, which modulates numerous and diverse bacterial processes, such as stress endurance, pathogenesis, and persistence. Here, we analyze the role of the PHA cycle in conferring robustness to the model bacterium P. putida KT2440. To assess the effect of this cycle in the cell, we began by constructing a PHA depolymerase (PhaZ) mutant strain that had its PHA cycle blocked. We then restored the flux through the cycle in the context of an engineered library of P. putida strains harboring differential levels of PhaZ. High-throughput phenotyping analyses of this collection of strains revealed significant changes in response to PHA cycle performance impacting cell number and size, PHA accumulation, and production of extracellular ()-hydroxyalkanoic acids. To understand the metabolic changes at the system level due to PHA turnover, we contextualized these physiological data using the genome-scale metabolic model JN1411. Model-based predictions suggest successive metabolic steady states during the growth curve and an important carbon flux rerouting driven by the activity of the PHA cycle. Overall, we demonstrate that modulating the activity of the PHA cycle gives us control over the carbon metabolism of P. putida, which in turn will give us the ability to tailor cellular mechanisms driving stress tolerance, e.g., defenses against oxidative stress, and any potential biotechnological applications. Despite large research efforts devoted to understanding the flexible metabolism of Pseudomonas beyond the role of key regulatory players, the metabolic basis powering the dynamic control of its biological fitness under disturbance conditions remains largely unknown. Among other metabolic hubs, the so-called PHA cycle, involving simultaneous synthesis and degradation of PHAs, is emerging as a pivotal metabolic trait powering metabolic robustness and resilience in this bacterial group. Here, we provide evidence suggesting that metabolic states in Pseudomonas can be anticipated, controlled, and engineered by tailoring the flux through the PHA cycle. Overall, our study suggests that the PHA cycle is a promising metabolic target toward achieving control over bacterial metabolic robustness. This is likely to open up a broad range of applications in areas as diverse as pathogenesis and biotechnology.
Topics: Pseudomonas putida; Polyhydroxyalkanoates; Biotechnology; Carbon
PubMed: 35038900
DOI: 10.1128/mbio.01794-21 -
Scientific Reports Apr 2023Biodesulfurization (BDS) was employed in this study to degrade dibenzothiophene (DBT) which accounts for 70% of the sulfur compounds in diesel using a synthetic and...
Biodesulfurization (BDS) was employed in this study to degrade dibenzothiophene (DBT) which accounts for 70% of the sulfur compounds in diesel using a synthetic and typical South African diesel in the aqueous and biphasic medium. Two Pseudomonas sp. bacteria namely Pseudomonas aeruginosa and Pseudomonas putida were used as biocatalysts. The desulfurization pathways of DBT by the two bacteria were determined by gas chromatography (GC)/mass spectrometry (MS) and High-Performance Liquid Chromatography (HPLC). Both organisms were found to produce 2-hydroxy biphenyl, the desulfurized product of DBT. Results showed BDS performance of 67.53% and 50.02%, by Pseudomonas aeruginosa and Pseudomonas putida, respectively for 500 ppm initial DBT concentration. In order to study the desulfurization of diesel oils obtained from an oil refinery, resting cells studies by Pseudomonas aeruginosa were carried out which showed a decrease of about 30% and 70.54% DBT removal for 5200 ppm in hydrodesulfurization (HDS) feed diesel and 120 ppm in HDS outlet diesel, respectively. Pseudomonas aeruginosa and Pseudomonas putida selectively degraded DBT to form 2-HBP. Application of these bacteria for the desulfurization of diesel showed promising potential for decreasing the sulfur content of South African diesel oil.
Topics: Pseudomonas; Petroleum; Thiophenes; Sulfur Compounds; Gasoline; Pseudomonas putida; Pseudomonas aeruginosa; Biodegradation, Environmental
PubMed: 37055435
DOI: 10.1038/s41598-023-31951-8 -
Microbial Biotechnology Oct 2022The extracellular 373-kDa PehA heme peroxidase of Pseudomonas putida KT2440 has two enzymatic domains which depend on heme cofactor for their peroxidase activity. A null...
The extracellular 373-kDa PehA heme peroxidase of Pseudomonas putida KT2440 has two enzymatic domains which depend on heme cofactor for their peroxidase activity. A null pehA mutant was generated to examine the impact of PehA in rhizosphere colonization competence and the induction of plant systemic resistance (ISR). This mutant was not markedly hampered in colonization efficiency. However, increase in pehA dosage enhanced colonization fitness about 30 fold in the root and 900 fold in the root apex. In vitro assays with purified His-tagged enzymatic domains of PehA indicated that heme-dependent peroxidase activity was required for the enhancement of root tip colonization. Evaluation of live/dead cells confirmed that overexpression of pehA had a positive effect on bacterial cell viability. Following root colonization of rice plants by KT2440 strain, the incidence of rice blast caused by Magnaporthe oryzae was reduced by 65% and the severity of this disease was also diminished in comparison to non-treated plants. An increase in the pehA dosage was also beneficial for the control of rice blast as compared with gene inactivation. The results suggest that PehA helps P. putida to cope with the plant-imposed oxidative stress leading to enhanced colonization ability and concomitant ISR-elicitation.
Topics: Antioxidants; Heme; Peroxidases; Plant Diseases; Plant Roots; Pseudomonas putida
PubMed: 35986900
DOI: 10.1111/1751-7915.14123 -
Microbial Biotechnology Jan 2020Using agricultural wastes as a substrate for biotechnological processes is of great interest in industrial biotechnology. A prerequisite for using these wastes is the...
Using agricultural wastes as a substrate for biotechnological processes is of great interest in industrial biotechnology. A prerequisite for using these wastes is the ability of the industrially relevant microorganisms to metabolize the sugars present therein. Therefore, many metabolic engineering approaches are directed towards widening the substrate spectrum of the workhorses of industrial biotechnology like Escherichia coli, yeast or Pseudomonas putida. For instance, neither xylose or arabinose from cellulosic residues, nor sucrose, the main sugar in waste molasses, can be metabolized by most E. coli and P. putida wild types. We evaluated a new, so far uncharacterized gene cluster for sucrose metabolism from Pseudomonas protegens Pf-5 and showed that it enables P. putida to grow on sucrose as the sole carbon and energy source. Even when integrated into the genome of P. putida, the resulting strain grew on sucrose at rates similar to the rate of the wild type on glucose - making it the fastest growing, plasmid-free P. putida strain known so far using sucrose as substrate. Next, we elucidated the role of the porin, an orthologue of the sucrose porin ScrY, in the gene cluster and found that in P. putida, a porin is needed for sucrose transport across the outer membrane. Consequently, native porins were not sufficient to allow unlimited growth on sucrose. Therefore, we concluded that the outer membrane can be a considerable barrier for substrate transport, depending on strain, genotype and culture conditions, all of which should be taken into account in metabolic engineering approaches. We additionally showed the potential of the engineered P. putida strains by growing them on molasses with efficiencies twice as high as obtained with the wild-type P. putida. This can be seen as a further step towards the production of low-value chemicals and biofuels with P. putida from alternative and more affordable substrates in the future.
Topics: Escherichia coli; Metabolic Engineering; Porins; Pseudomonas; Pseudomonas putida; Sucrose
PubMed: 29808622
DOI: 10.1111/1751-7915.13283 -
Applied and Environmental Microbiology Apr 2022The second messenger cyclic di-GMP (c-di-GMP) is a key molecule that controls different physiological and behavioral processes in many bacteria, including...
The second messenger cyclic di-GMP (c-di-GMP) is a key molecule that controls different physiological and behavioral processes in many bacteria, including motile-to-sessile lifestyle transitions. Although the external stimuli that modulate cellular c-di-GMP contents are not fully characterized, there is growing evidence that certain amino acids act as environmental cues for c-di-GMP turnover. In the plant-beneficial bacterium Pseudomonas putida KT2440, both arginine biosynthesis and uptake influence second messenger contents and the associated phenotypes. To further understand this connection, we have analyzed the role of ArgR, which in different bacteria is the master transcriptional regulator of arginine metabolism but had not been characterized in P. putida. The results show that ArgR controls arginine biosynthesis and transport, and an -null mutant grows poorly with arginine as the sole carbon or nitrogen source and also displays increased biofilm formation and reduced surface motility. Modulation of c-di-GMP levels by exogenous arginine requires ArgR. The expression of certain biofilm matrix components, namely, the adhesin LapF and the exopolysaccharide Pea, as well as the diguanylate cyclase CfcR is influenced by ArgR, likely through the alternative sigma factor RpoS. Our data indicate the existence of a regulatory feedback loop between ArgR and c-di-GMP mediated by FleQ. Identifying the molecular mechanisms by which metabolic and environmental signals influence the turnover of the second messenger c-di-GMP is key to understanding the regulation of bacterial lifestyles. The results presented here point at the transcriptional regulator ArgR as a central node linking arginine metabolism and c-di-GMP signaling and indicate the existence of a complex balancing mechanism that connects cellular arginine contents and second messenger levels, ultimately controlling the lifestyles of Pseudomonas putida.
Topics: Arginine; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas putida
PubMed: 35254100
DOI: 10.1128/aem.00064-22 -
Microbial Biotechnology Sep 2019Pseudomonas putida is rapidly becoming a workhorse for industrial production due to its metabolic versatility, genetic accessibility and stress-resistance properties....
Pseudomonas putida is rapidly becoming a workhorse for industrial production due to its metabolic versatility, genetic accessibility and stress-resistance properties. The P. putida strain KT2440 is often described as Generally Regarded as Safe, or GRAS, indicating the strain is safe to use as food additive. This description is incorrect. P. putida KT2440 is classified by the FDA as HV1 certified, indicating it is safe to use in a P1 or ML1 environment.
Topics: Food Microbiology; Food Safety; Industrial Microbiology; Pseudomonas putida; United States; United States Food and Drug Administration
PubMed: 31199068
DOI: 10.1111/1751-7915.13443 -
Microbial Biotechnology Jan 2020Targeted gene regulation is indispensable for reprogramming a cellular network to modulate a microbial phenotype. Here, we adopted the type II CRISPR interference...
Targeted gene regulation is indispensable for reprogramming a cellular network to modulate a microbial phenotype. Here, we adopted the type II CRISPR interference (CRISPRi) system for simple and efficient regulation of target genes in Pseudomonas putida KT2440. A single CRISPRi plasmid was generated to express a nuclease-deficient Cas9 gene and a designed single guide RNA, under control of l-rhamnose-inducible P and the constitutive Biobrick J23119 promoter respectively. Two target genes were selected to probe the CRISPRi-mediated gene regulation: exogenous green fluorescent protein on the multicopy plasmid and endogenous glpR on the P. putida KT2440 chromosome, encoding GlpR, a transcriptional regulator that represses expression of the glpFKRD gene cluster for glycerol utilization. The CRISPRi system successfully repressed the two target genes, as evidenced by a reduction in the fluorescence intensity and the lag phase of P. putida KT2440 cell growth on glycerol. Furthermore, CRISPRi-mediated repression of glpR improved both the cell growth and glycerol utilization, resulting in the enhanced production of mevalonate in an engineered P. putida KT2440 harbouring heterologous genes for the mevalonate pathway. CRISPRi is expected to become a robust tool to reprogram P. putida KT2440 for the development of microbial cell factories producing industrially valuable products.
Topics: Clustered Regularly Interspaced Short Palindromic Repeats; Gene Expression Regulation, Bacterial; Glycerol; Plasmids; Pseudomonas putida
PubMed: 30793496
DOI: 10.1111/1751-7915.13382 -
Environmental Microbiology Apr 2022Bacteria possess various regulatory mechanisms to detect and coordinate a response to elemental nutrient limitation. In pseudomonads, the two-component system regulators...
Bacteria possess various regulatory mechanisms to detect and coordinate a response to elemental nutrient limitation. In pseudomonads, the two-component system regulators CbrAB, NtrBC and PhoBR, are responsible for regulating cellular response to carbon (C), nitrogen (N) and phosphorus (P) respectively. Phosphonates are reduced organophosphorus compounds produced by a broad range of biota and typified by a direct C-P bond. Numerous pseudomonads can use the environmentally abundant phosphonate species 2-aminoethylphosphonate (2AEP) as a source of C, N, or P, but only PhoBR has been shown to play a role in 2AEP utilization. On the other hand, utilization of 2AEP as a C and N source is considered substrate inducible. Here, using the plant-growth-promoting rhizobacterium Pseudomonas putida BIRD-1 we present evidence that 2AEP utilization is under dual regulation and only occurs upon depletion of C, N, or P, controlled by CbrAB, NtrBC, or PhoBR respectively. However, the presence of 2AEP was necessary for full gene expression, i.e. expression was substrate inducible. Mutation of a LysR-type regulator, termed AepR, upstream of the 2AEP transaminase-phosphonatase system (PhnWX), confirmed this dual regulatory mechanism. To our knowledge, this is the first study identifying coordination between global stress response and substrate-specific regulators in phosphonate metabolism.
Topics: Bacterial Proteins; Carbon; Gene Expression Regulation, Bacterial; Nitrogen; Organophosphonates; Phosphorus; Pseudomonas putida
PubMed: 35229442
DOI: 10.1111/1462-2920.15959 -
Frontiers in Bioengineering and... 2022The present study elaborates on the propionic acid (PA) production by the well-known microbial cell factory EM42 and its capacity to utilize biomass-derived levulinic...
The present study elaborates on the propionic acid (PA) production by the well-known microbial cell factory EM42 and its capacity to utilize biomass-derived levulinic acid (LA). Primarily, the EM42 strain was engineered to produce PA by deleting the methylcitrate synthase (PrpC) and propionyl-CoA synthase (PrpE) genes. Subsequently, a LA-inducible expression system was employed to express (encoding thioesterase) from and (encoding propionyl-CoA: succinate CoA transferase) from to improve the PA production by up to 10-fold under flask scale cultivation. The engineered EM42:ΔCE: was used to optimize the bioprocess to further improve the PA production titer. Moreover, the fed-batch fermentation performed under optimized conditions in a 5 L bioreactor resulted in the titer, productivity, and molar yield for PA production of 26.8 g/L, 0.3 g/L/h, and 83%, respectively. This study, thus, successfully explored the LA catabolic pathway of as an alternative route for the sustainable and industrial production of PA from LA.
PubMed: 36032729
DOI: 10.3389/fbioe.2022.939248 -
Frontiers in Bioscience (Landmark... Jan 2023wilt and blight are the most important diseases of chickpea. The current study was designed to investigate the individual and combined effect of salicylic acid (SA)...
BACKGROUND
wilt and blight are the most important diseases of chickpea. The current study was designed to investigate the individual and combined effect of salicylic acid (SA) with and to suppress wilt and promote growth of chickpea varieties: Thal-2006 and Punjab-2008.
METHODS
At the time of sowing, inoculum of was applied to the soil and the incidence of wilt was recorded after 60 days. The seeds were inoculated with and prior to sowing. Chickpea plants were treated with salicylic acid at seedling stage.
RESULTS
The combination of and SA significantly increased root length (166% and 145%), shoot height (50% and 47%) and shoot biomass (300% and 233%) in cv. Thal-2006 and cv. Punjab-2008, respectively, in infected plants. Similarly, the combined treatment of + SA, also enhanced the plant growth parameters of chickpea varieties. Maximum reduction in disease severity was observed in both + SA (90% and 84%) and + SA (79% and 77%) treatments in cv. Thal-2006 and Punjab-2008, respectively. Both + SA and + SA treatments resulted in increased leaf relative water and total protein content, peroxidase, superoxide dismutase, phenylalanine ammonia-lyase and polyphenol oxidase activities in both resistant (cv. Thal-2006) and susceptible (cv. Punjab-2008) cultivars. Both treatments also significantly reduced malondialdehyde (MDA) and proline content in cv. Thal-2006 and Punjab-2008. Cultivar Thal-2006 was more effective than cv. Punjab-2008.
CONCLUSIONS
The results suggested that, in combination, salicylic acid and may play an important role in controlling wilt diseases by inducing systemic resistance in chickpea.
Topics: Biomass; Cicer; Combined Modality Therapy; Fusarium; Malondialdehyde; Plant Diseases; Salicylic Acid; Pseudomonas; Agricultural Inoculants
PubMed: 36722276
DOI: 10.31083/j.fbl2801020