Pseudomonas putida

About: Pseudomonas putida is a research topic. Over the lifetime, 6854 publications have been published within this topic receiving 230572 citations.

Metabolism of dibenzothiophene by a Beijerinckia species.

Abstract: Beijerinckia B8/36 when grown with succinate in the presence of dibenzothiophene, accumulated (+)-cis-1,2-dihydroxy-1,2-dihydrodibenzothiophene and dibenzothiophene-5-oxide in the culture medium. Each metabolite was isolated in crystalline form and characterized by a variety of chemical techniques, cis-Naphthalene dihydrodiol dehydrogenase, isolated from Pseudomonas putida, oxidized (+)-cis-1,2-dihydroxy-1,2-dihydrodibenzothiophene to a compound that was tentatively identified as 1,2-dihydroxydibenzothiophene. The same product was formed when crude cell extracts of the parent strain of Beijerinckia oxidized (+)-cis-1,2-dihydroxy-1,2-dihydrodibenzothiophene under anaerobic conditions. Further metabolism of 1,2-dihydroxydibenzothiophene by heat-treated cell extracts led to the formation of 4[2-(3-hydroxy)-thionaphthenyl]-2-oxo-3-butenoic acid. The latter compound was metabolized by crude cell extracts to 3-hydroxy-2-formylthionaphthene. Further degradation of this metabolite was not observed.

Antibiotic-dependent induction of Pseudomonas putida DOT-T1E TtgABC efflux pump is mediated by the drug binding repressor TtgR.

Abstract: Pseudomonas putida is well known for its metabolic capabilities, but recently, it has been shown to exhibit resistance to a wide range of antibiotics. In P. putida DOT-T1E, the TtgABC efflux pump, which has a broad substrate specificity, extrudes antibiotics such as ampicillin, carbenicillin, tetracycline, nalidixic acid, and chloramphenicol. We have analyzed the expression of the ttgABC efflux pump operon and its regulatory gene, ttgR, in response to several structurally unrelated antibiotics at the transcriptional level and investigated the role of the TtgR protein in this process. ttgABC and ttgR are expressed in vivo at a moderate basal level, which increases in the presence of hydrophobic antibiotics like chloramphenicol and tetracycline. In vitro experiments show that, in the absence of inducers, TtgR binds to a palindromic operator site which overlaps both ttgABC and ttgR promoters and dissociates from it in the presence of chloramphenicol and tetracycline. These results suggest that the TtgR repressor is able to bind to structurally different antibiotics, which allows induction of TtgABC multidrug efflux pump expression in response to these antimicrobial agents. This is the first case in which the expression of a drug transporter of the resistance-nodulation-division family has been shown to be regulated directly by antibiotics.

An Alkane-Responsive Expression System for the Production of Fine Chemicals

Abstract: Pseudomonas putida mt-2-derived xylene oxygenase is encoded in the catabolic TOL plasmid pWW0 upper pathway operon, and together with a set of other enzymes it forms a catalytic cluster which degrades toluene and xylene to (substituted) benzoic acids (18, 30, 40). The ability of xylene oxygenase to hydroxylate methyl substituents on substituted benzenes or their heteroaromatic equivalents has made it an important biotechnological enzyme (24, 56). This enzyme consists of a membrane-bound component, XylM, which carries out the oxygenation step (50), and a cytoplasmic NADH:acceptor reductase component, XylA, which supplies reducing equivalents to XylM (45). The potential of this system for biological production of fine chemicals has already been exploited; wild-type cells of P. putida mt-2 are used by Lonza to produce heteroaromatic acids on a commercial scale (24). Furthermore, xylene oxygenase is selective for the si-face of prochiral vinyl functions on aromatic ring systems, which leads to the formation of optically active epoxides, such as (S)-styrene oxide (55) (Fig. ​(Fig.1).1). Escherichia coli recombinants carrying the genes for xylene oxygenase have produced (S)-styrene oxide from inexpensive styrene in a 2-liter reactor (54). Unfortunately, so far the productivities displayed by such recombinants have been insufficient to commercially exploit their synthetic potential (17, 55). A number of observations have indicated that expressing the xylene oxygenase genes via the alk regulatory system of Pseudomonas oleovorans GPo1 might provide suitable biocatalysis strains for two-liquid-phase cultures. P. oleovorans GPo1 degrades medium-chain-length alkanes with a set of enzymes encoded by two alk gene clusters on the catabolic OCT plasmid (Fig. ​(Fig.2A)2A) (51). The first cluster contains the alkBFGHJKL operon, which contains all but one of the structural genes for conversion of alkanes to the corresponding alkanoic acids and coupling of these compounds to coenzyme A (Fig. ​(Fig.2B).2B). The second cluster contains the remaining structural gene, alkT, and the gene which encodes the regulatory protein AlkS (11). Expression of the genes in the first cluster is under control of alkBp, the alk promoter, and is initiated in the presence of functional AlkS and alkanes or other, structurally nonrelated inducers, such as dicyclopropylketone (DCPK) (16, 49). DCPK is water soluble and hence is a convenient inducer in aqueous cultures, while alkanes are useful inducers in two-liquid-phase cultures which contain an organic phase. Expression of the alk genes in E. coli W3110 via the alk regulatory system from the low-copy-number RK2 derivative pGEc47 led to accumulation of membrane-located AlkB until it accounted for up to 10% of the total cell protein (35). This indicated that AlkS, together with its cognate promoter alkBp (26), could be a powerful general system to direct synthesis of recombinant proteins. In addition, the alk regulatory system is not subject to catabolite repression in E. coli (46, 58), which allows convenient utilization of cheap carbon sources, such as glucose, in cultures of recombinant strains. Because of these attractive features of the alk regulatory system we developed its components into an expression system for general use. In this paper we describe this system and its potential for efficient synthesis of xylene oxygenase for biotransformation of styrene to (S)-styrene oxide at high enantiomeric excess in a two-liquid-phase biotransformation system. FIG. 1 Conversion of inexpensive styrene to (S)-styrene oxide by xylene monooxygenase. The chiral carbon atom is indicated by an asterisk. FIG. 2 (A) Organization and regulation of the alk genes on the OCT plasmid in P. oleovorans GPo1. The regulatory protein AlkS is activated by octane or DCPK and induces transcription from the alkBp promoter. The directions of transcription are indicated by arrows ...

Transcriptional control of the Pseudomonas putida TOL plasmid catabolic pathways

Abstract: TOL plasmid pWW0 of Pseudomonas putida contains two operons that specify a pathway for the degradation of aromatic hydrocarbons. The upper pathway operon encodes the enzymes for the oxidation of toluene/xylenes to benzoate/toluates, and the metacleavage pathway operon encodes the enzymes for the further oxidation of these compounds to Krebs cycle intermediates. Their expression is controlled by the gene products of two divergently transcribed regulatory genes, xyIR and xyIS. The XyIR protein, which belongs to the NtrC family of regulators, is expressed from two tandem promoters and autoregulates its synthesis. XyIR stimulates transcription from the xyIS gene promoter (Ps) and the upper pathway operon promoter (Pu) in the presence of pathway substrates. Both promoters are sigma 54 dependent, and Pu also requires the presence of integration host factor (IHF) for activation of transcription. Binding sites for XyIR and IHF in the Pu promoter and for XyIR in the Ps promoters have been defined. The XyIS protein, which belongs to the AraC family of regulators, stimulates transcription from the meta-cleavage pathway operon promoter (Pm) in the presence of benzoates. The effector binding pocket and DNA-binding region of XyIS have been defined through the isolation of mutants that exhibit altered effector specificity and modified transcriptional patterns, respectively. Expression of the meta-cleavage pathway operon is also induced by xylene-activated XyIR protein via a cascade regulatory system in which this protein, in combination with sigma 54, stimulates the expression from the xyIS promoter.(ABSTRACT TRUNCATED AT 250 WORDS)