Metabolically versatile free-living bacteria have global regulation systems that allow cells to selectively assimilate a preferred compound among a mixture of several potential carbon sources. This process is known as carbon catabolite repression (CCR). CCR optimizes metabolism, improving the ability of bacteria to compete in their natural habitats. This review summarizes the regulatory mechanisms responsible for CCR in the bacteria of the genus Pseudomonas, which can live in many different habitats. Although the information available is still limited, the molecular mechanisms responsible for CCR in Pseudomonas are clearly different from those of Enterobacteriaceae or Firmicutes. An understanding of the molecular mechanisms underlying CCR is important to know how metabolism is regulated and how bacteria degrade compounds in the environment. This is particularly relevant for compounds that are degraded slowly and accumulate, creating environmental problems. CCR has a major impact on the genes involved in the transport and metabolism of nonpreferred carbon sources, but also affects the expression of virulence factors in several bacterial species, genes that are frequently directed to allow the bacterium to gain access to new sources of nutrients. Finally, CCR has implications in the optimization of biotechnological processes such as biotransformations or bioremediation strategies.

Carbon catabolite repression in Pseudomonas : optimizing metabolic versatility and interactions with the environment

Benzoxazinoids, such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are secondary metabolites in grasses. In addition to their function in plant defence against pests and diseases above-ground, benzoxazinoids (BXs) have also been implicated in defence below-ground, where they can exert allelochemical or antimicrobial activities. We have studied the impact of BXs on the interaction between maize and Pseudomonas putida KT2440, a competitive coloniser of the maize rhizosphere with plant-beneficial traits. Chromatographic analyses revealed that DIMBOA is the main BX compound in root exudates of maize. In vitro analysis of DIMBOA stability indicated that KT2440 tolerance of DIMBOA is based on metabolism-dependent breakdown of this BX compound. Transcriptome analysis of DIMBOA-exposed P. putida identified increased transcription of genes controlling benzoate catabolism and chemotaxis. Chemotaxis assays confirmed motility of P. putida towards DIMBOA. Moreover, colonisation essays in soil with Green Fluorescent Protein (GFP)-expressing P. putida showed that DIMBOA-producing roots of wild-type maize attract significantly higher numbers of P. putida cells than roots of the DIMBOA-deficient bx1 mutant. Our results demonstrate a central role for DIMBOA as a below-ground semiochemical for recruitment of plant-beneficial rhizobacteria during the relatively young and vulnerable growth stages of maize.

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Benzoxazinoids in Root Exudates of Maize Attract Pseudomonas putida to the Rhizosphere

Many agricultural uses of bacteria require the establishment of efficient bacterial populations in the rhizosphere, for which colonization of plant seeds often constitutes a critical first step. Pseudomonas putida KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by P. putida KT2440, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Eight different mutants were isolated and characterized. While all of them showed reduced attachment to seeds, only two had strong defects in their adhesion to abiotic surfaces (glass and different plastics). Sequences of the loci affected in all eight mutants were obtained. None of the isolated genes had previously been described in P. putida, although four of them showed clear similarities with genes of known functions in other organisms. They corresponded to putative surface and membrane proteins, including a calcium-binding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump. One other showed limited similarities with surface proteins, while the remaining three presented no obvious similarities with known genes, indicating that this study has disclosed novel functions.

Genetic Analysis of Functions Involved in Adhesion of Pseudomonas putida to Seeds

Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation

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.

Convergent Peripheral Pathways Catalyze Initial Glucose Catabolism in Pseudomonas putida: Genomic and Flux Analysis

Pseudomonas putida KT2440 is a root colonizer of potential interest for the rhizoremediation of pollutants and the biological control of pests. The short- and long-term survival of this strain, as well as the possible eAects of its introduction on diAerent populations of indigenous soil bacteria, were tested in soil under greenhouse and field conditions. The greenhouse studies showed that inoculated P. putida KT2440 was able to establish itself after 3 d in nonvegetated soils at a density of 822 10 3 CFU g ˇ1 soil. The introduction of this strain had no significant eAect on the number of several soil bacteria including those that were resistant to tetracycline; those that utilized p-hydroxyphenylacetic acid as the sole C-source, and total fluorescent pseudomonads. In four independent field assays in nonplanted soils, the numbers of P. putida KT2440 decreased during 50 d from an initial density of 1 10 6 CFU g ˇ1 soil to approximately 221 10 2 CFU g ˇ1 soil. Thereafter, the number of cells was below detection limits (i.e. <10 2 CFU g ˇ1 soil), although they were still present because they could be recovered using selective enrichment from the soil for up to 200 d after the beginning of the experiment. This suggested that P. putida was maintained at a low cell density long after inoculation. In contrast, when P. putida KT2440 was introduced in the soil as a coating of corn (Zea mays) or broad bean (Vicia faba) seeds, the bacteria established at high cell densities in the rhizosphere (10 4 ‐10 5 CFU g ˇ1 soil in corn; 10 6 ‐10 7 CFU g ˇ1 soil in broad beans) during the growth of the crops over 12 to 16 weeks. The numbers of P. putida in the bulk soil after 2 weeks were 1 to 2 orders of magnitude below those in the rhizosphere. During the field assays, the population of p-hydroxyphenylacetic acid users was also monitored in the rhizosphere and the bulk soil. No significant seasonal variations were found. # 2000 Elsevier Science Ltd. All rights reserved.

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Juan M. Garcı́a

Papers

Survival of Pseudomonas putida KT2440 in soil and in the rhizosphere of plants under greenhouse and environmental conditions.