Potential DNA slippage structures acquired during evolutionary divergence of Acinetobacter calcoaceticus chromosomal benABC and Pseudomonas putida TOL pWW0 plasmid xylXYZ, genes encoding benzoate dioxygenases.

The xylXYZ DNA region is carried on the TOL pWW0 plasmid in Pseudomonas putida and encodes a benzoate dioxygenase with broad substrate specificity. The DNA sequence of the region is presented and compared with benABC, the chromosomal region encoding the benzoate dioxygenase of Acinetobacter calcoaceticus. Corresponding genes from the two biological sources share common ancestry: comparison of aligned XylX-BenA, XylY-BenB, and XylZ-BenC amino acid sequences revealed respective identities of 58.3, 61.3, and 53%. The aligned genes have diverged to assume G+C contents that differ by 14.0 to 14.9%. Usage of the unusual arginine codons AGA and AGG appears to have been selected in the P. putida xylX gene as it diverged from the ancestor it shared with A. calcoaceticus benA. Homologous A. calcoaceticus and P. putida genes exhibit different patterns of DNA sequence repetition, and analysis of one such pattern suggests that mutations creating different DNA slippage structures made a significant contribution to the evolutionary divergence of xylX.     

DNA sequence determination of the TOL plasmid (pWW0) xylGFJ genes of Pseudomonas putida: implications for the evolution of aromatic catabolism

The meta operon of the Pseudomonas putida TOL plasmid (pWWO) encodes all enzymes of a meta-cleavage pathway for the metabolism of benzoic acids to Krebs-cycle intermediates. We have determined and analysed the nucleic acid sequence of a 3442 bp region of the meta operon containing the xyl-GFJ genes whose products are involved in the post meta-ring fission transformation of catechols. Homology analysis of the xylGFJ gene products revealed evidence of biochemical relatedness, suggested enzymatic mechanisms, and permitted us to propose evolutionary events which may have generated the current variety of aromatic degradative pathways. The xylG gene, which specifies 2-hydroxymuconic semialdehyde dehydrogenase (HMSD), was found to encode a protein of 51.7 kDa. The predicted protein sequence exhibits significant homology to eukaryotic aldehyde dehydrogenases (ADHs) and to the products of two other Pseudomonas catabolic genes, i.e. xylC and alkH. Expansion of the ADH superfamily to include these prokaryotic enzymes permitted a broader analysis of functionally critical ADH residues and phylogenetic relationships among superfamily members. The importance of three regions of these enzymes previously thought to be critical to ADH activity was reinforced by this analysis. However glutamine-487, also thought to be critical, is less well conserved. The revised ADH phylogeny proposed here suggests early catabolic ADH divergence with subsequent interkingdom gene exchange. The xylF gene, which specifies 2-hydroxymuconic semialdehyde hydrolase (HMSH), was delineated by N-terminal sequence analysis of the purified gene product and is shown to encode a protein of 30.6 kDa. Homology analysis revealed sequence similarity to a chromosomally encoded serine hydrolase, especially in the region of the previously identified active-site serine residue, suggesting that HMSH may also possess a serine hydrolytic enzymatic mechanism. Likewise, the xylJ gene, which specifies 2-hydroxy-pent-2,4-dienoate hydratase (HPH), was delineated by N-terminal sequence analysis of purified HPH, and was found to encode a 23.9 kDa protein. Sequence comparisons revealed that both HMSH and HPH have analogues in the tod gene cluster, which specifies a toluene/benzene degradative pathway. Although the newly identified todF and todJ genes had been at least partially sequenced (Zylstra and Gibson, 1989), the open reading frames had not been positively identified. The presence of todJ provides strong evidence that the reactions following ring fission in the tod pathway are identical to those of the TOL pathway.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proline catabolism by Pseudomonas putida: cloning, characterization, and expression of the put genes in the presence of root exudates

Pseudomonas putida KT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. A P. putida mutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5-Km mutagenesis. The mini-Tn5 insertion was located at the putA gene, which is adjacent to and divergent from the putP gene. The putA gene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein of Escherichia coli, Salmonella enterica serovar Typhimurium, Rhodobacter capsulatus, and several Rhizobium strains. The central part of P. putida PutA showed homology to the proline dehydrogenase of Saccharomyces cerevisiae and Drosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases. This suggests that in P. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. The putP gene was homologous to the putP genes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In a P. putida putA-deficient background, expression of both putA and putP genes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced the P. putida put genes, as determined by using fusions of the put promoters to 'lacZ. The induction ratio for the putA promoter (about 20-fold) was 6-fold higher than the induction ratio for the putP promoter.     

Characterization of genes involved in the initial reactions of 4-chloronitrobenzene degradation in Pseudomonas putida ZWL73

The genes encoding enzymes involved in the initial reactions during degradation of 4-chloronitrobenzene (4CNB) were characterized from the 4CNB utilizer Pseudomonas putida ZWL73, in which a partial reductive pathway was adopted. A DNA fragment containing genes coding for chloronitrobenzene nitroreductase (CnbA) and hydroxylaminobenzene mutase (CnbB) were PCR-amplified and subsequently sequenced. These two genes were actively expressed in Escherichia coli, and recombinant E. coli cells catalyzed the conversion of 4CNB to 2-amino-5-chlorophenol, which is the ring-cleavage substrate in the degradation of 4CNB. Phylogenetic analyses on sequences of chloronitrobenzene nitroreductase and hydroxylaminobenzene mutase revealed that these two enzymes are closely related to the functionally identified nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas strains JS45 and HS12. The nitroreductase from strain ZWL73 showed a higher specific activity toward 4CNB than nitrobenzene (approximately at a ratio of 1.6:1 for the recombinant or 2:1 for the wild type), which is in contrast to the case where the nitroreductase from nitrobenzene utilizers Pseudomonas pseudoalcaligenes JS45 with an apparently lower specific activity against 4CNB than nitrobenzene (0.16:1) [Kadiyala et al. Appl Environ Microbiol 69:6520–6526, 2003]. This suggests that the nitroreductase from 4-chloronitrobenzene utilizer P. putida ZWL73 may have evolved to prefer chloronitrobenzene to nitrobenzene as its substrate.Y.X. and J.-F.W. equally contributed to this work.     

Cloning and characterization of the pnb genes, encoding enzymes for 4-nitrobenzoate catabolism in Pseudomonas putida TW3

Pseudomonas putida strain TW3 is able to metabolize 4-nitrotoluene via 4-nitrobenzoate (4NBen) and 3, 4-dihydroxybenzoic acid (protocatechuate [PCA]) to central metabolites. We have cloned, sequenced, and characterized a 6-kbp fragment of TW3 DNA which contains five genes, two of which encode the enzymes involved in the catabolism of 4NBen to PCA. In order, they encode a 4NBen reductase (PnbA) which is responsible for catalyzing the direct reduction of 4NBen to 4-hydroxylaminobenzoate with the oxidation of 2 mol of NADH per mol of 4NBen, a reductase-like enzyme (Orf1) which appears to have no function in the pathway, a regulator protein (PnbR) of the LysR family, a 4-hydroxylaminobenzoate lyase (PnbB) which catalyzes the conversion of 4-hydroxylaminobenzoate to PCA and ammonium, and a second lyase-like enzyme (Orf2) which is closely associated with pnbB but appears to have no function in the pathway. The central pnbR gene is transcribed in the opposite direction to the other four genes. These genes complete the characterization of the whole pathway of 4-nitrotoluene catabolism to the ring cleavage substrate PCA in P. putida strain TW3.     

Loss of the toluene-xylene catabolic genes of TOL plasmid pWW0 during growth of Pseudomonas putida on benzoate is due to a selective growth advantage of 'cured' segregants

During growth on benzoate-minimal medium Pseudomonas putida mt-2 (PaW1) segregates derivative ('cured') strains which have lost the ability to use the pathway encoded by its resident catabolic plasmid pWW0. Experiments with two plasmids identical to pWW0 but each with an insert of Tn401, which confers resistance to carbenicillin, suggested that the 'benzoate curing' occurs far more frequently by the specific deletion of the 39 kbp region carrying the catabolic genes than by total plasmid loss. This effect was not pH-dependent, and was not produced during growth on other weak organic acids, such as succinate or propionate, or when benzoate was present in the medium with an alternative, preferentially used carbon source such as succinate. Growth on benzoate did not cause loss from strain PaW174 of the plasmid pWW0174, a derivative of pWW0 which has deleted the 39 kbp region but carries Tn401. Similarly the naphthalene-catabolic plasmid pWW60-1, of the same incompatibility group as pWW0, was not lost from PaW701 during growth on benzoate. Competition between wild-type PaW1 and PaW174, which has the 'cured' phenotype, showed that the latter has a distinct growth advantage on benzoate over the wild-type even when initially present as only 1% of the population: when PaW174 was seeded at lower cell ratios, spontaneously 'cured' derivatives of PaW1 took over the culture after 60-80 generations, indicating that they are present in PaW1 cultures at frequencies between 10(-2) and 10(-3). We conclude that the progressive takeover of populations of PaW1 only occurs when benzoate is present as the sole growth source and that neither benzoate, nor other weak acids, affect plasmid segregation or deletion events: a sufficient explanation is that the 'cured' segregants grow faster than the wild-type using the chromosomally determined beta-ketoadipate pathway.     

p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate.

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1.     

Characterization of five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products.

The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes which transform toluene and xylenes to benzoate and toluates. The genetic organization of the operon was characterized by cloning of the upper operon genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the upper operon contains at least five genes in the order of xylC-xylM-xylA-xylB-xylN. Between the promoter of the operon and xylC, there is a 1.7-kilobase-long space of DNA in which no gene function was identified. In contrast, most of the DNA between xylC and xylN consists of coding sequences. The xylC gene encodes the 57-kilodalton benzaldehyde dehydrogenase. The xylM and xylA genes encode 35- and 40-kilodalton polypeptides, respectively, which were shown by genetic complementation tests to be subunits of xylene oxygenase. The structural gene for benzyl alcohol dehydrogenase, xylB, encodes a 40-kilodalton polypeptide. The last gene of this operon is xylN, which synthesizes a 52-kilodalton polypeptide of unknown function.     

Production and study of Bacillus subtilis mutants for genes involved in nucleoside catabolism]

By means of selection for a low thymine requirement the mutants fo thymine auxotrophs for deoxyriboaldolase (dra) and phosphodeoxyribomutase (drm) genes were obtained. Besides the mutants for pyrimidinenucleoside phosphorylase gene (pdp) were olso isolated using selection on the fluorodeoxyuridine resistance. The latter enzyme provides for pyrimidine nucleosides catabolism (thymidine, uridine) in Bacilli, as well as the conversion of exogenous thymine to thymidine in thymine auxotrophs. The data obtained when studying the deo-enzymes activities in various types of the mutants and also under the condition of induction by thymidine and acetoaldehyde are in accordance with the assumption that deoxyriboso-5-phosphate is an inductor of the deo-enzymes in Bacillus subtilis. The genes dra and pdp were tightly linked as it had been shown by the transformation experiments; in contrast, no linkage was revealed between dra and drm or pdp and drm. A secondary mutation (adn), not linked with dra and blocking the ability of bacteria to catabolise adenosine (purine nucleoside phosphorylase activity remains constant) was found in some dra-mutants.     

p-Cumate catabolic pathway in Pseudomonas putida Fl: cloning and characterization of DNA carrying the cmt operon.

Pseudomonas putida F1 utilizes p-cumate (p-isopropylbenzoate) as a growth substrate by means of an eight-step catabolic pathway. A 35.75-kb DNA segment, within which the cmt operon encoding the catabolism of p-cumate is located, was cloned as four separate overlapping restriction fragments and mapped with restriction endonucleases. By examining enzyme activities in recombinant bacteria carrying these fragments and sub-cloned fragments, genes encoding most of the enzymes of the p-cumate pathway were located. Subsequent sequence analysis of 11,260 bp gave precise locations of the 12 genes of the cmt operon. The first three genes, cmtAaAbAc, and the sixth gene, cmtAd, encode the components of p-cumate 2,3-dioxygenase (ferredoxin reductase, large subunit of the terminal dioxygenase, small subunit of the terminal dioxygenase, and ferredoxin, respectively); these genes are separated by cmtC, which encodes 2,3-dihydroxy-p-cumate 3,4-dioxygenase, and cmtB, coding for 2,3-dihydroxy-2,3-dihydro-p-cumate dehydrogenase. The ring cleavage product, 2-hydroxy-3-carboxy-6-oxo-7-methylocta-2,4-dienoate, is acted on by a decarboxylase encoded by the seventh gene, cmtD, which is followed by a large open reading frame, cmtI, of unknown function. The next four genes, cmtEFHG, encode 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase, 2-hydroxypenta-2,4-dienoate hydratase, 4-hydroxy-2-oxovalerate aldolase, and acetaldehyde dehydrogenase, respectively, which transform the decarboxylation product to amphibolic intermediates. The deduced amino acid sequences of all the cmt gene products except CmtD and CmtI have a recognizable but low level of identity with amino acid sequences of enzymes catalyzing analogous reactions in other catabolic pathways. This identity is highest for the last two enzymes of the pathway (4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase [acylating]), which have identities of 66 to 77% with the corresponding enzymes from other aromatic meta-cleavage pathways. Recombinant bacteria carrying certain restriction fragments bordering the cmt operon were found to transform indole to indigo. This reaction, known to be catalyzed by toluene 2,3-dioxygenase, led to the discovery that the tod operon, encoding the catabolism of toluene, is located 2.8 kb downstream from and in the same orientation as the cmt operon in P. putida F1.     

Spontaneous deletions in the TOL plasmid pWW20 which give rise to the B3 regulatory mutants of Pseudomonas putida MT20

The size of the TOL plasmid pWW20 from Pseudomonas putida MT20, as measured by analysis of agarose electrophoresis gels after restriction endonuclease hydrolysis, was 270-280 kilobase pairs (kb). During growth on benzoate, MT20 segregates strains carrying mutations in the plasmid regulatory gene xylS; these so-called B3 strains retain the ability to grow on m-xylene (Mxy+) but do not grow on its metabolite m-toluate (Mtol-) and have also lost the ability to transfer the plasmid (Tra-). Analysis of restriction digests of plasmid DNA from seven such segregants, independently isolated, showed that pWW20 had undergone extensive deletions of 90-100 kb. All the deleted plasmids had lost a common core of DNA, of about 72-80 kb, but in class A mutants the deletion extended at one end of this core and in class B mutants at the other end. Class A and B mutants also differed in their rate of growth on m-xylene as a result of differences in the level of expression of their plasmid-coded catabolic enzymes. This suggests that an additional gene, involved in regulating levels of gene expression, is located in the region uniquely deleted in the class B mutants.     

Roles of CatR and cis,cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida.

In Pseudomonas putida, the catBC operon encodes enzymes involved in benzoate degradation. Previous studies have determined that these enzymes are induced when P. putida is grown in the presence of benzoate. Induction of the enzymes of the catBC operon requires an intermediate of benzoate degradation, cis,cis-muconate, and a regulatory protein, CatR. It has been determined that CatR binds to a 27-bp region of the catBC promoter in the presence or absence of inducer. We have called this the repression binding site. In this study, we used a gel shift assay to demonstrate that the inducer, cis,cis-muconate, increases the affinity of CatR for the catBC promoter region by 20-fold. Furthermore, in the absence of cis,cis-muconate, CatR forms two complexes in the gel shift assay. The inducer cis,cis-muconate confers specificity primarily for the formation of complex 2. DNase I footprinting showed that an additional 27 bp of the catBC promoter region is protected by CatR in the presence of cis,cis-muconate. We have named this second binding site the activation binding site. Methylation interference footprinting determined that in the presence or absence of inducer, five G nucleotides of the catBC promoter region were necessary for CatR interaction with the repression binding site, while a single G residue was important for CatR interaction with the activation binding site in the presence of cis,cis-muconate. Using polymerase chain reaction-generated constructs, we found that the binding of CatR to the repression binding site is independent of the activation binding site. However, binding of CatR to the activation binding site required an intact repression binding site.     

Isolation and analysis of genes involved in siderophore biosynthesis in plant-growth-stimulating Pseudomonas putida WCS358

The plant-growth-stimulating Pseudomonas putida WCS358 was mutagenized with transposon Tn5. The resulting mutant colony bank was screened for mutants defective in the biosynthesis of the fluorescent siderophore. A total of 28 mutants, divided into six different classes, were isolated that were nonfluorescent or defective in iron acquisition or both. These different types of mutants together with the probable overall structure of the siderophore, i.e., a small peptide chain attached to a fluorescing group, suggest a biosynthetic pathway in which the synthesis of the fluorescing group is preceded by the synthesis of the peptide part. A gene colony bank of P. putida WCS358 was constructed with the broad-host-range cosmid vector pLAFR1. This genomic library, established in Escherichia coli, was mobilized into the 28 individual mutants, screening for transconjugants restored in fluorescence or growth under iron-limiting conditions or both. A total of 13 cosmids were found to complement 13 distinct mutants. The complementation analysis revealed that at least five gene clusters, with a minimum of seven genes, are needed for siderophore biosynthesis. Some of these genes seem to be arranged in an operon-like structure.     

Isolation and characterization of Pseudomonas putida mutants affected in arginine, ornithine and citrulline catabolism: function of the arginine oxidase and arginine succinyltransferase pathways.

Pseudomonas putida mutants impaired in the utilization of arginine are affected in either the arginine succinyltransferase pathway, the arginine oxidase route, or both. However, mutants affected in one of the pathways still grow on arginine as sole carbon source. Analysis of the products excreted by both wild-type and mutant strains suggests that arginine is mainly channelled by the oxidase route. Proline non-utilizing mutants are also affected in ornithine utilization, confirming the role of proline as an intermediate in ornithine catabolism. Mutants affected in ornithine cyclodeaminase activity still grow on proline and become unable to use ornithine. Both proline non-utilizing mutants and ornithine-cyclodeaminase-minus mutants are unable to use citrulline. These results, together with induction of ornithine cyclodeaminase when wild-type P. putida is grown on citrulline, indicate that utilization of citrulline as a carbon source proceeds via proline with ornithine as an intermediate. Thus in P. putida, the aerobic catabolism of arginine on the one hand and citrulline and ornithine on the other proceed by quite different metabolic segments.     

Evolutionary differences in chromosomal locations of four early genes of the tryptophan pathway in fluorescent pseudomonads: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC.

Pseudomonas putida possesses seven structural genes for enzymes of the tryptophan pathway. All but one, trpG, which encodes the small (beta) subunit of anthranilate synthase, have been mapped on the circular chromosome. This report describes the cloning and sequencing of P. putida trpE, trpG, trpD, and trpC. In P. putida and Pseudomonas aeruginosa, DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also contains trpD and trpC. In P. putida, trpE is 2.2 kilobases upstream from the trpGDC cluster, whereas in P. aeruginosa, they are separated by at least 25 kilobases (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983). The DNA sequence in P. putida shows an open reading frame on the opposite strand between trpE and trpGDC; this putative gene was not characterized. Evidence is also presented for sequence similarities in the 5' untranslated regions of trpE and trpGDC in both pseudomonads; the function of these regions is unknown, but it is possible that they play some role in regulation of these genes, since all the genes respond to repression by tryptophan. The sequences of the anthranilate synthase genes in the fluorescent pseudomonads resemble those of p-aminobenzoate synthase genes of the enteric bacteria more closely than the anthranilate synthase genes of those organisms; however, no requirement for p-aminobenzoate was found in the Pseudomonas mutants created in this study.     

Cell envelope mutants of Pseudomonas putida: physiological characterization and analysis of their ability to survive in soil

To generate mutants with altered lipopolysaccharides (LPS) of the wild-type Pseudomonas putida KT2442, we used the mini-Tn 5luxAB -Km transposon. A mutant was found among luminescent colonies and selected as a negative clone in enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (mAb) 7.3B, which recognizes the O-antigen of P. putida LPS. The DNA region of the LPS mutant interrupted by the minitransposon insertion was cloned and sequenced. Comparison of the deduced amino acid sequence with protein sequence databases showed similarity to the O-antigen polymerase (Wzy) of Salmonella enterica (muenchen). The wild-type gene was rescued by polymerase chain reaction (PCR), cloned into a broad-host-range plasmid and used to carry out complementation assays. The cloned gene was able to restore the wild-type phenotype of the P. putida wzy mutant. We constructed an isogenic mutant of the luminescent wzy mutant to which an oprL mutation was transferred by homologous recombination with an oprL :: xylE cassette. The wzy mutants of P. putida were more sensitive to SDS, deoxycholate and EDTA than the corresponding parental strains. We analysed the ability of wzy , oprL and wzy oprL mutants of P. putida to colonize soil. In comparison with the wild-type strain, the ability of single mutants to colonize soil decreased; this characteristic was more evident for the double mutant, especially at high temperatures.     

The davDT operon of Pseudomonas putida, involved in lysine catabolism, is induced in response to the pathway intermediate delta-aminovaleric acid

Pseudomonas putida KT2440 is a soil microorganism that attaches to seeds and efficiently colonizes the plant's rhizosphere. Lysine is one of the major compounds in root exudates, and P. putida KT2440 uses this amino acid as a source of carbon, nitrogen, and energy. Lysine is channeled to delta-aminovaleric acid and then further degraded to glutaric acid via the action of the davDT gene products. We show that the davDT genes form an operon transcribed from a single sigma70-dependent promoter. The relatively high level of basal expression from the davD promoter increased about fourfold in response to the addition of exogenous lysine to the culture medium. However, the true inducer of this operon seems to be delta-aminovaleric acid because in a mutant unable to metabolize lysine to delta-aminovaleric acid, this compound, but not lysine, acted as an effector. Effective induction of the P. putida P(davD) promoter by exogenously added lysine requires efficient uptake of this amino acid, which seems to proceed by at least two uptake systems for basic amino acids that belong to the superfamily of ABC transporters. Mutants in these ABC uptake systems retained basal expression from the davD promoter but exhibited lower induction levels in response to exogenous lysine than the wild-type strain.