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Starting from mature vegetable compost, four bacterial strains were selected using a lignin-rich medium. 16S ribosomal RNA identification of the isolates showed high score similarity with Pseudomonas spp. for three out of four isolates. Further characterization of growth on mixtures of six selected lignin model compounds (vanillin, vanillate, 4-hydroxybenzoate, p -coumarate, benzoate, and ferulate) was carried out with three of the Pseudomonas isolates and in addition with the strain Pseudomonas putida KT2440 from a culture collection. The specific growth rates on benzoate, p -coumarate, and 4-hydroxybenzoate were considerably higher (0.26–0.27 h −1 ) than those on ferulate and vanillate (0.21 and 0.22 h −1 ), as were the uptake rates. There was no direct growth of P. putida KT2440 on vanillin, but instead, vanillin was rapidly converted into vanillate at a rate of 4.87 mmol (g CDW  h) −1 after which the accumulated vanillate was taken up. The growth curve reflected a diauxic growth when mixtures of the model compounds were used as carbon source. Vanillin, 4-hydroxybenzoate, and benzoate were preferentially consumed first, whereas ferulate was always the last substrate to be taken in. These results contribute to a better understanding of the aromatic metabolism of P. putida in terms of growth and uptake rates, which will be helpful for the utilization of these bacteria as cell factories for upgrading lignin-derived mixtures of aromatic molecules.

Peptidoglycan is the major structural constituent of the bacterial cell wall, forming a meshwork outside the cytoplasmic membrane that maintains cell shape and prevents lysis. In Gram-negative bacteria, peptidoglycan is located in the periplasm, where it is protected from exogenous lytic enzymes by the outer membrane. Here we show that the type VI secretion system of Pseudomonas aeruginosa breaches this barrier to deliver two effector proteins, Tse1 and Tse3, to the periplasm of recipient cells. In this compartment, the effectors hydrolyse peptidoglycan, thereby providing a fitness advantage for P. aeruginosa cells in competition with other bacteria. To protect itself from lysis by Tse1 and Tse3, P. aeruginosa uses specific periplasmically localized immunity proteins. The requirement for these immunity proteins depends on intercellular self-intoxication through an active type VI secretion system, indicating a mechanism for export whereby effectors do not access donor cell periplasm in transit. Duelling bacterial pathogens The type VI secretion system (T6SS) is a protein-export machine that is present in about one-quarter of all sequenced bacteria. Bacteria can use this system to deliver toxic effector proteins in a contact-dependent manner to other bacterial cells. However, what these proteins do once their destination is reached has remained largely unknown. It is now shown that the opportunistic human pathogen Pseudomonas aeruginosa uses its T6SS to kill competing Gram-negative bacteria by injecting them with two peptidoglycan-degradative enzymes, the effector proteins Tse1 and Tse3. P. aeruginosa protects itself from these effectors by expressing immunity proteins that bind the toxins.

The oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs). Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) that are often functionally redundant. Here we report the first description and characterization of a lanthanide-dependent PQQ-ADH (PedH) in a nonmethylotrophic bacterium based on the use of purified enzymes from the soil-dwelling model organism Pseudomonas putida KT2440. PedH (PP_2679) exhibits enzyme activity on a range of substrates similar to that of its Ca 2+ -dependent counterpart PedE (PP_2674), including linear and aromatic primary and secondary alcohols, as well as aldehydes, but only in the presence of lanthanide ions, including La 3+ , Ce 3+ , Pr 3+ , Sm 3+ , or Nd 3+ . Reporter assays revealed that PedH not only has a catalytic function but is also involved in the transcriptional regulation of pedE and pedH , most likely acting as a sensory module. Notably, the underlying regulatory network is responsive to as little as 1 to 10 nM lanthanum, a concentration assumed to be of ecological relevance. The present study further demonstrates that the PQQ-dependent oxidation system is crucial for efficient growth with a variety of volatile alcohols. From these results, we conclude that functional redundancy and inverse regulation of PedE and PedH represent an adaptive strategy of P. putida KT2440 to optimize growth with volatile alcohols in response to the availability of different lanthanides. IMPORTANCE Because of their low bioavailability, lanthanides have long been considered biologically inert. In recent years, however, the identification of lanthanides as a cofactor in methylotrophic bacteria has attracted tremendous interest among various biological fields. The present study reveals that one of the two PQQ-ADHs produced by the model organism P. putida KT2440 also utilizes lanthanides as a cofactor, thus expanding the scope of lanthanide-employing bacteria beyond the methylotrophs. Similar to the system described in methylotrophic bacteria, a complex regulatory network is involved in lanthanide-responsive switching between the two PQQ-ADHs encoded by P. putida KT2440. We further show that the functional production of at least one of the enzymes is crucial for efficient growth with several volatile alcohols. Overall, our study provides a novel understanding of the redundancy of PQQ-ADHs observed in many organisms and further highlights the importance of lanthanides for bacterial metabolism, particularly in soil environments. Because of their low bioavailability, lanthanides have long been considered biologically inert. In recent years, however, the identification of lanthanides as a cofactor in methylotrophic bacteria has attracted tremendous interest among various biological fields. The present study reveals that one of the two PQQ-ADHs produced by the model organism P. putida KT2440 also utilizes lanthanides as a cofactor, thus expanding the scope of lanthanide-employing bacteria beyond the methylotrophs. Similar to the system described in methylotrophic bacteria, a complex regulatory network is involved in lanthanide-responsive switching between the two PQQ-ADHs encoded by P. putida KT2440. We further show that the functional production of at least one of the enzymes is crucial for efficient growth with several volatile alcohols. Overall, our study provides a novel understanding of the redundancy of PQQ-ADHs observed in many organisms and further highlights the importance of lanthanides for bacterial metabolism, particularly in soil environments.

Abstract Pseudomonas putida was metabolically engineered to produce medium chain length polyhydroxyalkanoate (mcl-PHA) from acetate, a promising carbon source to achieve cost-effective microbial processes. As acetate is known to be harmful to cell growth, P. putida KT2440 was screened from three Pseudomonas strains (P. putida KT2440, P. putida NBRC14164, and P. aeruginosa PH1) as the host with the highest tolerance to 10 g/L of acetate in the medium. Subsequently, P. putida KT2440 was engineered by amplifying the acetate assimilation pathway, including overexpression of the acs (encoding acetyl-CoA synthetase) route and construction of the ackA-pta (encoding acetate kinase-phosphotransacetylase) pathway. The acs overexpressing P. putida KT2440 showed a remarkable increase of mcl-PHA titer (+ 92%), mcl-PHA yield (+ 50%), and cellular mcl-PHA content (+ 43%) compared with the wild-type P. putida KT2440, which indicated that acetate could be a potential substrate for biochemical production of mcl-PHA by engineered P. putida.

Salinity stress negatively affects the growth and yield of crops worldwide. Onion ( Allium cepa L.) is moderately sensitive to salinity. Beneficial microorganisms can potentially confer salinity tolerance. This study investigated the effects of endomycorrhizal fungi (M), Pseudomonas putida (Ps) and their combination (MPs) on onion growth under control (0 ppm), moderate (2000 ppm) and high (4000 ppm) NaCl salinity levels. A pot experiment was conducted with sandy loam soil and onion cultivar Giza 20. Results showed that salinity reduced growth attributes, leaf pigments, biomass and bulb yield while increasing oxidative stress markers. However, individual or combined inoculations significantly increased plant height, bulb diameter and biomass production compared to uninoculated plants under saline conditions. MPs treatment provided the highest stimulation, followed by Pseudomonas and mycorrhizae alone. Overall, dual microbial inoculation showed synergistic interaction, conferring maximum benefits for onion growth, bulbing through integrated physiological and biochemical processes under salinity. Bulb yield showed 3.5, 36 and 83% increase over control at 0, 2000 and 4000 ppm salinity, respectively. In conclusion, combined application of mycorrhizal-Pseudomonas inoculations (MPs) effectively mitigate salinity stress. This approach serves as a promising biotechnology for ensuring sustainable onion productivity under saline conditions.

Phototrophic organisms worldwide produce estimated 10 gigatons of sulfoquinovose (SQ) per year; hence, complete degradation of SQ by bacteria is an important part of the biogeochemical sulfur cycle. Here, we show that Pseudomonas putida SQ1 catabolizes SQ to 3-sulfolactate (SL) in analogy to the Entner–Doudoroff pathway for glucose-6-phosphate, involving five newly discovered reactions, enzymes, and genes, and three newly discovered organosulfur intermediates. The SL can be mineralized by other bacteria, thus closing the sulfur cycle within a bacterial community. The genes for the SQ Entner–Doudoroff pathway can be found in genomes of a wide range of Proteobacteria, which shows that SQ utilization is a widespread and important, but still underrecognized, trait of bacteria in all environments where SQ is produced and degraded. Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid SQ-diacylglycerol, and SQ comprises a major proportion of the organosulfur in nature, where it is degraded by bacteria. A first degradation pathway for SQ has been demonstrated recently, a “sulfoglycolytic” pathway, in addition to the classical glycolytic (Embden–Meyerhof) pathway in Escherichia coli K-12; half of the carbon of SQ is abstracted as dihydroxyacetonephosphate (DHAP) and used for growth, whereas a C 3 -organosulfonate, 2,3-dihydroxypropane sulfonate (DHPS), is excreted. The environmental isolate Pseudomonas putida SQ1 is also able to use SQ for growth, and excretes a different C 3 -organosulfonate, 3-sulfolactate (SL). In this study, we revealed the catabolic pathway for SQ in P. putida SQ1 through differential proteomics and transcriptional analyses, by in vitro reconstitution of the complete pathway by five heterologously produced enzymes, and by identification of all four organosulfonate intermediates. The pathway follows a reaction sequence analogous to the Entner–Doudoroff pathway for glucose-6-phosphate: It involves an NAD + -dependent SQ dehydrogenase, 6-deoxy-6-sulfogluconolactone (SGL) lactonase, 6-deoxy-6-sulfogluconate (SG) dehydratase, and 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase. The aldolase reaction yields pyruvate, which supports growth of P. putida , and 3-sulfolactaldehyde (SLA), which is oxidized to SL by an NAD(P) + -dependent SLA dehydrogenase. All five enzymes are encoded in a single gene cluster that includes, for example, genes for transport and regulation. Homologous gene clusters were found in genomes of other P. putida strains, in other gamma-Proteobacteria, and in beta- and alpha-Proteobacteria, for example, in genomes of Enterobacteria, Vibrio , and Halomonas species, and in typical soil bacteria, such as Burkholderia , Herbaspirillum , and Rhizobium .

Background The implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. Ongoing efforts include the adoption of Pseudomonas putida KT2440 variants with a reduced genome as the functional chassis for biotechnological purposes. In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome). Results In this study, two previously constructed multiple-deletion P. putida strains were systematically evaluated as microbial cell factories for heterologous protein production and compared to the parental bacterium (strain KT2440) with regards to several industrially-relevant physiological traits. Energetic parameters were quantified at different controlled growth rates in continuous cultivations and both strains had a higher adenosine triphosphate content, increased adenylate energy charges, and diminished maintenance demands than the wild-type strain. Under all the conditions tested the mutants also grew faster, had enhanced biomass yields and showed higher viability, and displayed increased plasmid stability than the parental strain. In addition to small-scale shaken-flask cultivations, the performance of the genome-streamlined strains was evaluated in larger scale bioreactor batch cultivations taking a step towards industrial growth conditions. When the production of the green fluorescent protein (used as a model heterologous protein) was assessed in these cultures, the mutants reached a recombinant protein yield with respect to biomass up to 40% higher than that of P. putida KT2440. Conclusions The two streamlined-genome derivatives of P. putida KT2440 outcompeted the parental strain in every industrially-relevant trait assessed, particularly under the working conditions of a bioreactor. Our results demonstrate that these genome-streamlined bacteria are not only robust microbial cell factories on their own, but also a promising foundation for further biotechnological applications.

Soluble C 1 feedstocks, such as formate and methanol, have gained attention as sustainable substrates for biotechnology, with the potential to reduce greenhouse gas emissions and reliance on sugar-based resources. Despite their promise, the metabolic assimilation of these compounds remains uncharacterized in robust bacterial hosts beyond a few model species. Pseudomonas putida , known for its metabolic versatility and industrial relevance, has lacked the ability to grow solely on C 1 compounds. This study is a first-case example of strict synthetic formatotrophy and methylotrophy in any Pseudomonas species, enabling growth on formate and methanol as sole carbon and energy sources. Through pathway rewiring and adaptive laboratory evolution, key metabolic and regulatory adaptations were identified that enabled efficient C 1 assimilation. These findings not only expand the known capabilities of P. putida but also open directions for its deployment in carbon-efficient biomanufacturing. This study sets a precedent for leveraging non-model microorganisms in the development of scalable, carbon-efficient bioprocesses.

Pseudomonas putida NCIMB 9866 utilizes p -cresol or 2,4-xylenol as a sole carbon and energy source. Enzymes catalyzing the oxidation of the para -methyl group of p -cresol have been studied in detail. However, those responsible for the oxidation of the para -methyl group in 2,4-xylenol catabolism are still not reported. In this study, real-time quantitative PCR analysis indicated pchC - and pchF -encoded p -cresol methylhydroxylase (PCMH) and pchA -encoded p -hydroxybenzaldehyde dehydrogenase (PHBDD) in p -cresol catabolism were also likely involved in the catabolism of 2,4-xylenol. Enzyme activity assays and intermediate identification indicated that the PCMH and PHBDD catalyzed the oxidations of 2,4-xylenol to 4-hydroxy-3-methylbenzaldehyde and 4-hydroxy-3-methylbenzaldehyde to 4-hydroxy-3-methylbenzoic acid, respectively. Furthermore, the PCMH-encoding gene pchF was found to be necessary for the catabolism of 2,4-xylenol, whereas the PHBDD-encoding gene pchA was not essential for the catabolism by gene knockout and complementation. Analyses of the maximum specific growth rate ( μ m ) and specific activity of the gene-knockout strain to different intermediates revealed the presence of other enzyme(s) with PHBDD activity in strain 9866. However, PHBDD played a major role in the catabolism of 2,4-xylenol in contrast to the other enzyme(s).

Background Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa . In addition, separation of rhamnolipids from fatty acids is difficult and hence costly. Results Here, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum , and Escherichia coli. P. putida KT2440 expressing the rhlAB -genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C 10 :C 10 ). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/g glucose corresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation. Conclusions A functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_ rhlAB featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer P. aeruginosa .