A 4-methoxybenzoate O-demethylase from Pseudomonas putida. A new type of monooxygenase system.

A strain of Pseudomonas putida grown on 4-methoxybenzoate as sole carbon source contains an enzyme system for the O-demethylation of this substrate. The enzyme system is purifiable and can be separated into two components: an NADH-dependent reductase and an iron-containing and acid-labile-sulfur-containing monooxygenase. The reductase, of molecular weight 42000 and containing two chromophores, an FMN and an iron-sulfur complex (EPR at g = 1.95), reduces both one-electron and two-electron acceptors (i.e., ferricyanide, 2,6-dichloroindophenol, cytochrome c, and cytochrome b5) at an optimum pH of 8.0. Increasing ionic strength affects these activities differently. The absolute spectrum of the oxidized displays distinct absorption peaks at 409 and 463 nm and a small shoulder between 538 and 554 nm. Treatment with dithionite or NADH reduces the absorbance throughout the visible range, yielding a spectrum with small maxima at 402 and 538 nm. Spectroscopic characteristics of the reductase indicate a tight coupling between its two chromophores. The iron-containing and acid-labile-sulfur-containing monooxygenase, which has a molecular weight of about 120000, contains an iron-sulfur chromophore with an EPR signal at g = 1.90. This protein is a dimer whose subunits each have a molecular weight of about 50000 and are perhaps identical. The optical absorption properties are somewhat unusual. In contrast to other iron-sulfur proteins, there is no significant peak near 415 nm in the absorption spectrum of the oxidized protein, but rather one at 455 nm. The presence of the substrate 4-methoxybenzoate increases both the NADH-dependent reductase. Hydroxylation can be achieved by the monooxygenase also in absence of the reductase with artifical reductants. This enzyme opens a new group of oxygenases within the classification scheme, i.e., iron-containing and labile-sulfur-containing monooxygenases. From the reported data, a scheme for the interaction of the isolated pigments and their relationship to various acceptors is proposed.     

An aerotaxis transducer gene from Pseudomonas putida

An aerotaxis gene, aer, was cloned from Pseudomonas putida PRS2000. A P. putida aer mutant displayed an altered aerotactic response in a capillary assay. Wild-type P. putida clustered at the air/liquid interface. In contrast, the aer mutant did not cluster at the interface, but instead formed a diffuse band at a distance from the meniscus. Wild-type aer, provided in trans, complemented the aer mutant to an aerotactic response that was stronger than wild-type. The P. putida Aer sequence is similar over its entire length to the aerotaxis (energy taxis) signal transducer protein, Aer, of Escherichia coli. The amino-terminus is similar to redox-sensing regulatory proteins, and the carboxy-terminus contains the highly conserved domain present in chemotactic transducers.     

Revealing oxidative pentose metabolism in new Pseudomonas putida isolates

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.     

Biosurfactant production by a new Pseudomonas putida strain

Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by the newly isolated and promising strain Pseudomonas putida 21BN. The biosurfactants were identified as rhamnolipids, the amphiphilic surface-active glycolipids usually secreted by Pseudomonas spp. Their production was observed when the strain was grown on soluble substrates, such as glucose or on poorly soluble substrates, such as hexadecane, reaching values of 1.2 g l(-1). When grown on hexadecane as the sole carbon source the biosurfactant lowered the surface tension of the medium to 29 mN m(-1) and formed stable and compact emulsions with emulsifying activity of 69%.     

A siderophore from Pseudomonas putida type A1: structural and biological characterization

A siderophore from a root-colonizing, plant-beneficial fluorescent Pseudomonas (P. putida type A1) isolated from chickpea rhizosphere was studied. Culture conditions required for optimal production of the chromophore by the organism were standardized. The compound was purified by gel filtration, ion exchange and RP-HPLC chromatographic procedures. The purified compound exhibited siderophore activity for P. putida and antifungal activity on phytopathogens, Fusarium oxysporum f. sp. ciceri and Helminthosporium oryzae. Growth inhibition of the pathogens was observed under iron-deficient conditions. Complete acid hydrolysis of the compound revealed that it is a peptide containing Asx, Thr, Glx, Val, His, Lys, Ser and Gly. Spectral analysis revealed that it contains hydroxyquinoline-based chromophore in addition to an aromatic residue and the molecular weight of the compound was 1.5 kDa. EPR analysis of the peptide-chromophore-iron complex showed that the compound binds to iron and the bound iron was in the Fe(3+) oxidation state having a high spin d(5) system. The peptide-chromophore-iron complex takes a turn structure in solution as shown by circular dichroism spectroscopy, a feature which was hitherto not known for other siderophores. The siderophore studied here is unique in this respect but otherwise strikingly similar to other pseudobactin-type siderophores of plant growth-promoting and plant-deleterious pseudomonads. The possible functional significance of the compound is discussed in relation to the secondary structure described earlier for siderophores.     

Structural and genetic characterization of glycosylation of type a flagellin in Pseudomonas aeruginosa

Type a flagellins from two strains of Pseudomonas aeruginosa, strains PAK and JJ692, were found to be glycosylated with unique glycan structures. In both cases, two sites of O-linked glycosylation were identified on each monomer, and these sites were localized to the central, surface-exposed domain of the monomer in the assembled filament. The PAK flagellin was modified with a heterogeneous glycan comprising up to 11 monosaccharide units that were O linked through a rhamnose residue to the protein backbone. The flagellin of JJ692 was less complex and had a single rhamnose substitution at each site. The role of the glycosylation island gene cluster in the production of each of these glycosyl moieties was investigated. These studies revealed that the orfA and orfN genes were required for attachment of the heterologous glycan and the proximal rhamnose residue, respectively.     

Development of a method for markerless gene deletion in Pseudomonas putida

We developed a negative counterselection system for Pseudomonas putida based on uracil phosphoribosyltransferase (UPRTase) and sensitivity against the antimetabolite 5-fluorouracil (5-FU). We constructed a P. putida strain that is resistant to 5-FU and constructed vectors for the deletion of the surface adhesion protein gene, the flagellum biosynthesis operon, and two endonuclease genes. The genes were efficiently disrupted and left a markerless chromosomal in-frame deletion.     

Genetic analysis of an incomplete mutS gene from Pseudomonas putida

We genetically characterized the Pseudomonas putida mutS gene and found that it encodes a smaller MutS protein than do the genes of other bacteria. This gene is able to function in the mutS mutants of Escherichia coli and Bacillus subtilis. A P. putida mutS mutant has a mutation frequency 1,000-fold greater than that of the wild-type strain.     

Phage-stable mutants of Pseudomonas putida: new types of stability

More than 170 phage-resistant mutants (PRM) of the first order of Pseudomonas putida strain PpG1 were obtained using newly isolated and previously described bacteriophages specific for this strain. According to the results of analysis of resistance of the mutants to each of 31 phages of PpG1 strain and 8 phages of the PpN strain, the PRM strains were distributed into 20 groups. In most cases, the reason for resistance is loss of absorption capacity of bacteria. However, no direct relation between the level of absorption and efficiency of phage plating was detected. It was shown that some of the PRM of P. putida PpG1 strains acquired the ability to maintain the growth of phages specific for the other P. putida strain, PpN. Frequencies of isolating mutants of various resistance types depend on the concrete phage used. In accordance with their absorption specificity, all phages were distributed into 23 groups, and a tridimensional formal scheme of receptor sites for these phages on the PpG1 strain was drawn. In the process of selection of the PpG1 clones resistant to non-lysogenizing mutant of temperate PP71 phage, a variant of this strain manifesting the phenomenon of "auto-plaquing" was found. These results support the mutational origin of this phenomenon in some cases.     

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.     

Competitiveness in root colonization by Pseudomonas putida requires the rpoS gene

The rpoS gene in Pseudomonas putida was essential for plant root colonization under competitive conditions from other microbes. The RpoS- mutant survived less well than the wild-type strain in culture medium, and unlike the wild-type, failed to colonize the roots in a peat matrix containing an established diverse microflora. The RpoS-deficient P. putida isolate was generated by insertion of a glucuronidase-npt cassette into the rpoS gene. The RpoS mutant had dose-dependent increased sensitivity to oxidative stress and produced Mn-superoxide dismutase activity earlier than the parent. While extracts from wild-type P. putida stationary-phase cells contained three isozymes of catalase (CatA, CatB, and CatC), the sigma38-deficient P. putida lacked CatB. These results are consistent with previous findings that CatB is induced in stationary-phase.     

Molecular cloning of the Pseudomonas putida glyoxalase I gene in Escherichia coli

The glyoxalase I gene of Pseudomonas putida was cloned onto a vector plasmid pBR 322 as a 7.5 kilobase Sau 3AI fragment of chromosomal DNA and the hybrid plasmid was designated pGI 318. The gene responsible for the glyoxalase I activity in pGI 318 was recloned in pBR 322 as a 2.2 kilobase Hin dIII fragment and was designated pGI 423. The P. putida glyoxalase I gene on pGI 318 and pGI 423 was highly expressed in E. coli cells and the glyoxalase I activity level was increased more than 150 fold in the pGI 423 bearing strain compared with that of E. coli cells without pGI 423. The E. coli transformants harboring pGI 318 or pGI 423 could grow normally in the presence of methylglyoxal, although the E. coli cells without plasmid were inhibited to grow and showed the extremely elongated cell shape.     

Chromium reduction in Pseudomonas putida

Reduction of hexavalent chromium (chromate) to less-toxic trivalent chromium was studied by using cell suspensions and cell-free supernatant fluids from Pseudomonas putida PRS2000. Chromate reductase activity was associated with soluble protein and not with the membrane fraction. The crude enzyme activity was heat labile and showed a Km of 40 microM CrO4(2-). Neither sulfate nor nitrate affected chromate reduction either in vitro or with intact cells.