Pseudomonas putida KT2440, a paradigm organism in biodegradation and a good competitive colonizer of the maize rhizosphere, was the subject of studies undertaken to establish the genetic determinants important for its rhizospheric lifestyle. By using in vivo expression technology (IVET) to positively select single cell survival, we identified 28 rap genes (root-activated promoters) preferentially expressed in the maize rhizosphere. The IVET system had two components: a mutant affected in aspartate-␤-semialdehyde dehydrogenase (asd), which was unable to survive in the rhizosphere, and plasmid pOR1, which carries a promoterless asd gene. pOR1-borne transcriptional fusions of the rap promoters to the essential gene asd, which were integrated into the chromosome at the original position of the corresponding rap gene, were active and allowed growth of the asd strain in the rhizosphere. The fact that five of the rap genes identified in the course of this work had been formerly characterized as being related to root colonization reinforced the IVET approach. Up to nine rap genes encoded proteins either of unknown function or that had been assigned an unspecific role based on conservation of the protein family domains. Rhizosphere-induced fusions included genes with probable functions in the cell envelope, chemotaxis and motility, transport, secretion, DNA metabolism and defense mechanism, regulation, energy metabolism, stress, detoxification, and protein synthesis.The rhizosphere is a densely populated area in which plant roots interact with soilborne microorganisms, including bacteria, fungi, and invertebrates, feeding on an abundant source of organic material (43). Many colonization traits and genes have been identified by random mutagenesis of good competitive root-colonizing bacteria (mainly Pseudomonas fluorescens and Pseudomonas chlororaphis) and through screening for gain or loss of competitive root tip colonization ability (see reference 28 for a recent review). Often these genes have been found to be unimportant for growth in the laboratory, and their role in bacterial fitness has required competition studies involving wild-type and mutant strains.Information on gene expression in the rhizosphere is, however, limited and partial. Previous results obtained by our group have shown that utilization of the imino acid proline by Pseudomonas putida KT2440, which involves uptake and catabolism, was induced by maize root exudates (50). In addition, an aminotransferase involved in the catabolism of lysine was also identified in a screen to select for maize root exudateinduced genes by using a promoter probe transposon (15).The most extensive study so far based on positive selection of bacterial traits as a consequence of their contribution to root colonization is that of Rainey (38). In that work, the in vitro expression technology (IVET) approach was used to select P. fluorescens genes activated during sugar cane root colonization.The study was partial, since only 10% of the genome was analyzed.Our work represents an ...

During the ripening of sweet pepper (Capsicum annuum L.) fruits, in a genetically controlled scenario, enormous metabolic changes occur that affect the physiology of most cell compartments. Peroxisomal catalase gene expression decreases after pepper fruit ripening, while the enzyme is also susceptible to undergo post-translational modifications (nitration, S-nitrosation, and oxidation) promoted by reactive oxygen and nitrogen species (ROS/RNS). Unlike most plant catalases, the pepper fruit enzyme acts as a homodimer, with an atypical native molecular mass of 125 to 135 kDa and an isoelectric point of 7.4, which is higher than that of most plant catalases. These data suggest that ROS/RNS could be essential to modulate the role of catalase in maintaining basic cellular peroxisomal functions during pepper fruit ripening when nitro-oxidative stress occurs. Using catalase from bovine liver as a model and biotin-switch labeling, in-gel trypsin digestion, and nanoliquid chromatography coupled with mass spectrometry, it was found that Cys377 from the bovine enzyme could potentially undergo S-nitrosation. To our knowledge, this is the first report of a cysteine residue from catalase that can be post-translationally modified by S-nitrosation, which makes it especially important to find the target points where the enzyme can be modulated under either physiological or adverse conditions.

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The tmoABCDEF genes encode the toluene-4-monooxygenase from Pseudomonas mendocina KR1. Upstream from the tmoA gene an open reading frame, tmoX, encoding a protein 83% identical to TodX (todX being the initial gene in the todXFC1C2BADEGIH operon from Pseudomonas putida DOT-T1E) was found. The tmoX gene is also the initial gene in the tmoXABCDEF gene cluster. The transcription initiation point from the tmoX promoter was mapped, and the sequence upstream revealed striking identity with the promoter of the tod operon of P. putida. The tod operon is regulated by a two-component signal transduction system encoded by the todST genes. Two novel genes from P. mendocina KR1, tmoST, were rescued by complementation of a P. putida DOT-T1E todST knockout mutant, whose gene products shared about 85% identity with TodS-TodT. We show that transcription from P tmoX and P todX can be mediated by TmoS-TmoT or TodS-TodT, in the presence of toluene, revealing cross-regulation between these two catabolic pathways.The first step in the metabolism of toluene in Pseudomonas mendocina KR1 is carried out by toluene-4-monooxygenase (T4MO), encoded by the tmoABCDEF gene cluster (38, 39), which is responsible for the oxidation of toluene to p-cresol (35,36). Two other independently regulated gene clusters are known to be required for the metabolism of toluene in P. mendocina KR1 (37). One encodes p-cresol methylhydroxylase and p-hydroxybenzaldehyde dehydrogenase, which catalyze the successive oxidation of the methyl group of p-cresol to the corresponding acid, p-hydroxybenzoate; the other encodes phydroxybenzoate hydroxylase for forming protocatechuate, which is channeled to the ␤-ketoadipate route through an ortho-cleavage pathway (12). Although T4MO activity is known to be inducible by toluene, chlorinated solvents, and alkanes (6,19), no information regarding the regulators involved in the expression of the tmo genes was available when this work was envisaged. To learn about the transcriptional control of the tmo genes, we first sequenced the region upstream of the tmoABCDEF genes in pMC4 (2) by the dideoxy sequencing method with the ABI Prism dRhodamine terminator kit (Applied Biosystems). A 1,362-nucleotide-long open reading frame 27 bp upstream of tmoA was found, whose deduced amino acid sequence shared significant homology with outer membrane proteins encoded in different toluene degradation operons from a number of gram-negative bacteria that use toluene as the sole C source (i.e., 83% identity with TodX of Pseudomonas putida F1 [34] and and 48% identity with TbuX of Ralstonia pickettii [13]). The initial gene in the tmo cluster was called tmoX. Identification of the transcription initiation point of the tmoX promoter and time course induction in response to different effectors.To analyze the expression of the tmo genes, total RNA was isolated from P. mendocina KR1 grown on citrate in the presence and in the absence of toluene or pcresol, the substrate and the hydroxylated product of the T4MO, respectively. A 23-mer oligonucleotide (...