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Control of expression of divergent Pseudomonas putida put promoters for proline catabolism.

ABSTRACT: Pseudomonas putida KT2440 uses proline as the sole C and N source. Utilization of this amino acid involves its uptake, which is mediated by the PutP protein, and its conversion into glutamate, mediated by the PutA protein. Sequence analysis revealed that the putA and putP genes are transcribed divergently. Expression from the putP and putA genes was analyzed at the mRNA level in different host backgrounds in the absence and presence of proline. Expression from the put promoters was induced by proline. The transcription initiation points of the putP and putA genes were precisely mapped via primer extension, and sequence analysis of the upstream DNA region showed well-separated promoters for these two genes. The PutA protein acts as a repressor of put gene expression in P. putida because expression from the put promoters is constitutive in a host background with a knockout putA gene. This regulatory activity is independent of the catabolic activity of PutA, because we show that a point mutation (Glu896-->Lys) that prevents catalytic activity allowed the protein to retain its regulatory activity. Expression from the put promoters in the presence of proline in a putA-proficient background requires a positive regulatory protein, still unidentified, whose expression seems to be sigma(54) dependent because the put genes were not expressed in a sigma(54)-deficient background. Expression of the putA and putP genes was equally high in the presence of proline in sigma(38)- and ihf-deficient P. putida backgrounds.

SUBMITTER: Vilchez S

PROVIDER: S-EPMC92447 | biostudies-literature | 2000 Dec

REPOSITORIES: biostudies-literature

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Control of expression of divergent Pseudomonas putida put promoters for proline catabolism.

Vílchez S S Manzanera M M Ramos J L JL

Applied and environmental microbiology 20001201 12

Pseudomonas putida KT2440 uses proline as the sole C and N source. Utilization of this amino acid involves its uptake, which is mediated by the PutP protein, and its conversion into glutamate, mediated by the PutA protein. Sequence analysis revealed that the putA and putP genes are transcribed divergently. Expression from the putP and putA genes was analyzed at the mRNA level in different host backgrounds in the absence and presence of proline. Expression from the put promoters was induced by pr ...[more]

PMID: 11097893

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Project description:Pseudomonas alloputida KT2440 (previously misclassified as P. putida KT2440 based on 16S rRNA gene homology) has emerged as an ideal host strain for plan t biomass valorization. However, P. alloputida KT2440 is unable to natively utilize abundant pentose sugars (e.g., xylose and arabinose) in hydrolysate streams, which may account for up to 25% of lignocellulosic biomass. In the last decades, microbes have been engineered to utilize the pentose sugars. However, most of the engineered strains were either slow-growing or displayed phenotypes that could not be replicated. In this work, we successfully isolated five Pseudomonas species with the native capability to utilize glucose, xylose and p-coumarate as a sole carbon source. These isolates were in two clusters; one set of isolates (M2 and M5) and the second set of isolates (BP6 and BP7) showed 85.6% and 96.2% ANI, respectively, to P. alloputida KT24440. BP8 showed 84.6% ANI to P. putida KT2440 and does not belong to any neighboring type strains indicating a new species. Notably, the isolates showed robust growth solely on xylose and higher growth rates (m, 0.36-0.49 h-1) when compared to only known xylose-utilizing Pseudomonas taiwanenesis VLB120 (m, 0.28 h-1) as a control. Unexpectedly, among five isolates, M2 and M5 grew solely on arabinose as well. Comprehensive analysis of genomics, transcriptomics and proteomics revealed the isolates utilize xylose and arabinose via Weimberg pathway (xylD-xylX-xylA) and oxidative pathway (araD-araX-araA), respectively. Furthermore, a preliminary result demonstrated the production of flaviolin solely on xylose and arabinose in the isolate, showing noteworthy potential to be an alternative host for lignocellulosic feedstocks into valuable products. This is the first report on isolating Pseudomonas strains natively capable of utilizing all of the major carbon sources in lignocellulosic biomass, and leading to higher consumption of available substrates and therefore maximizing the product yield.

Project description:In this study, we show that glucose catabolism in Pseudomonas putida occurs through the simultaneous operation of three pathways that converge at the level of 6-phosphogluconate, which is metabolized by the Edd and Eda Entner/Doudoroff enzymes to central metabolites. When glucose enters the periplasmic space through specific OprB porins, it can either be internalized into the cytoplasm or be oxidized to gluconate. Glucose is transported to the cytoplasm in a process mediated by an ABC uptake system encoded by open reading frames PP1015 to PP1018 and is then phosphorylated by glucokinase (encoded by the glk gene) and converted by glucose-6-phosphate dehydrogenase (encoded by the zwf genes) to 6-phosphogluconate. Gluconate in the periplasm can be transported into the cytoplasm and subsequently phosphorylated by gluconokinase to 6-phosphogluconate or oxidized to 2-ketogluconate, which is transported to the cytoplasm, and subsequently phosphorylated and reduced to 6-phosphogluconate. In the wild-type strain, glucose was consumed at a rate of around 6 mmol g(-1) h(-1), which allowed a growth rate of 0.58 h(-1) and a biomass yield of 0.44 g/g carbon used. Flux analysis of (13)C-labeled glucose revealed that, in the Krebs cycle, most of the oxalacetate fraction was produced by the pyruvate shunt rather than by the direct oxidation of malate by malate dehydrogenase. Enzymatic and microarray assays revealed that the enzymes, regulators, and transport systems of the three peripheral glucose pathways were induced in response to glucose in the outer medium. We generated a series of isogenic mutants in one or more of the steps of all three pathways and found that, although all three functioned simultaneously, the glucokinase pathway and the 2-ketogluconate loop were quantitatively more important than the direct phosphorylation of gluconate. In physical terms, glucose catabolism genes were organized in a series of clusters scattered along the chromosome. Within each of the clusters, genes encoding porins, transporters, enzymes, and regulators formed operons, suggesting that genes in each cluster coevolved. The glk gene encoding glucokinase was located in an operon with the edd gene, whereas the zwf-1 gene, encoding glucose-6-phosphate dehydrogenase, formed an operon with the eda gene. Therefore, the enzymes of the glucokinase pathway and those of the Entner-Doudoroff pathway are physically linked and induced simultaneously. It can therefore be concluded that the glucokinase pathway is a sine qua non condition for P. putida to grow with glucose.

Dataset Information

Control of expression of divergent Pseudomonas putida put promoters for proline catabolism.

Publications

Control of expression of divergent Pseudomonas putida put promoters for proline catabolism.

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