Physical map of the DNA of bacteriophage tf of Pseudomonas putida

A physical map has been constructed for P. putida bacteriophage tf DNA containing single-strand breaks (nicks). Localization of cleavage sites for EcoRI, HindIII, HpaI ClaI, BamHI, SalI, XbaI and XhoI restriction endonucleases was determined. Position of single-strand breaks was mapped by electrophoretic analysis of denatured tf DNA and electron microscopy of partially denatured DNA samples. The tf genome is characterized by the presence of two classes of nicks differing in the frequency of their presence in population of bacteriophage DNA molecules.     

Transformation of Pseudomonas putida with chromosomal DNA

The $\text{in vitro}$ deiodination of [¹²⁵I]-4-iodobiphenyl, [¹²⁵I]-4-iodonitrobenzene and [¹²⁵I]-4-iodoaniline was investigated. No deiodination was detected in rat thyroid homogenates. However, at least three biodeiodination mechanisms were indicated for substrates in rat liver subcellar fractions. Microsomal dehalogenation occurred to a minor extent with increased dehalogenation taking place in the cytosol fraction. The cytosol deiodination was extensive for 4-iodonitrobenzene and was mediated by glutathione. A second cytosol deiodination mechanism, not mediated by glutathione, was evident when 4-iodobiphenyl was the substrate. This soluble enzyme system could be enhanced by Arochlor 1254 or 4-iodobiphenyl pretreatment.     

Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440

Analysis of the catabolic potential of Pseudomonas putida KT2440 against a wide range of natural aromatic compounds and sequence comparisons with the entire genome of this microorganism predicted the existence of at least four main pathways for the catabolism of central aromatic intermediates, that is, the protocatechuate (pca genes) and catechol (cat genes) branches of the beta-ketoadipate pathway, the homogentisate pathway (hmg/fah/mai genes) and the phenylacetate pathway (pha genes). Two additional gene clusters that might be involved in the catabolism of N-heterocyclic aromatic compounds (nic cluster) and in a central meta-cleavage pathway (pcm genes) were also identified. Furthermore, the genes encoding the peripheral pathways for the catabolism of p-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compounds (fcs, ech, vdh, cal, van, acd and acs), phenylalanine and tyrosine (phh, hpd) and n-phenylalkanoic acids (fad) were mapped in the chromosome of P. putida KT2440. Although a repetitive extragenic palindromic (REP) element is usually associated with the gene clusters, a supraoperonic clustering of catabolic genes that channel different aromatic compounds into a common central pathway (catabolic island) was not observed in P. putida KT2440. The global view on the mineralization of aromatic compounds by P. putida KT2440 will facilitate the rational manipulation of this strain for improving biodegradation/biotransformation processes, and reveals this bacterium as a useful model system for studying biochemical, genetic, evolutionary and ecological aspects of the catabolism of aromatic compounds.     

外源质粒电击转入Pseudomonas putida的条件研究

以自行筛选的恶臭假单胞菌（Pseudomonas putida）(命名为Rs198,Genbank登录号为FJ788425）为受体菌,将具有卡那霉素抗性标记的大肠杆菌假单胞菌穿梭质粒PDSK519通过电转化法导入到受体菌中,对细胞生长状态、电转化温度、质粒DNA及感受态细胞浓度、电击电压及电转化介质给予转化效率的影响进行研究。结果表明,在细胞生长至OD600为0.5左右时收集菌体,在低温条件下制备浓度为 4.6×1012/ml 的感受态细胞,以0.3mol/L的蔗糖为电转化介质,在13kV/cm的场强下电击能获得较高的转化效率,最高可达1.3×107个转化子/μ g DNA。为构建恶臭假单胞的遗传转化系统,利用基因工程手段为该菌的进一步研究奠定了理论基础。     

Bacteriophages of Pseudomonas putida containing single-stranded canonical DNA breaks

It was shown that bacteriophage tf as well as bacteriophages phi p4/40, phi p25/42, phi p23/40 and phi p6/40, which are specific to different P. putida strains, contain the single strand breaks in their DNA. The breaks are localized in one strand of DNA molecules and are repairable with T4 DNA ligase. Bacteriophage tf has no detectable DNA homology with phi p4/40, phi p25/42, phi p23/40 and phi p6/40 bacteriophages. All the phages studied have no relation with other known Pseudomonas phages. Bacteriophages phi p4/40 and phi p25/42 share the extensive DNA homology.     

DNA sequence of the tryptophan synthase genes of Pseudomonas putida

Genes encoding the 2 subunits of tryptophan synthase in Pseudomonas putida have been identified and cloned by their similarity to the corresponding genes in Pseudomonas aeruginosa. The deduced amino acid sequences were confirmed by comparison with regions ascertained earlier by protein sequencing. The Pseudomonas amino acid sequences are 85% identical for the beta subunit and 70% identical for the alpha subunit. These sequences are compared to those of Salmonella typhimurium, where the structure is known from X-ray crystallography. Although amino acid conservation drops to 54% and 36% for the beta and alpha subunits, only 3 single residue gaps are required to maintain alignment throughout and most of the residues identified as important for catalysis or cofactor binding are conserved. The 23 residues surrounding the beta chain lysine that enters into a Schiff base linkage with the pyridoxal phosphate cofactor are compared in 13 species, including representatives from the eukaryotic and both prokaryotic kingdoms; appreciable conservation is apparent. The approximately 100 base pairs separating the trpB gene from its divergently transcribed activator gene are similar in the 2 pseudomonads, but do not resemble those of any other bacterium or fungus studied to date.     

Chromosomal location of TOL plasmid DNA in Pseudomonas putida.

The soil isolate Pseudomonas putida MW1000 can grow on toluene and other hydrocarbons; in this respect it is similar to strains of Pseudomonas which carry the TOL plasmid. By conjugation experiments, the genes conferring these growth abilities have been shown to be located on the bacterial chromosome, linked to vil and catB. A 56-kilobase segment of the bacterial chromosome of MW strains carrying the TOL genes can transpose to the IncP-1 plasmid R18-18. Physical analysis of these TOL R18-18 hybrids has shown that the TOL segment is almost identical to the same region found in the TOL plasmid pWW0.     

Acetate inhibition of Pseudomonas putida

To study the effect of acetate inhibition on the parameters of yield and maintenance for bacterial growth, Pseudomonas putida ATCC 23467 was grown in a minimal salts medium with acetate as the sole carbon source with limiting and with excess quantities of urea in the feed medium. The behavior of the chemostat cultures under sole acetate limitation results in low residual acetate present in the fermentation broth. These cultures can be described satisfactorily using the equation q(s) = D/Y(g) + m, i.e., the acetate is consumed only for growth and maintenance,. Those cultures in which urea was limiting or where urea was present in large excess contained significant amounts of residual acetate in the broth. For these cultures it was necessary to add a third term for acetate inhibition to the above expression.     

Alpha-hydroxyglutarate oxidoreductase of Pseudomonas putida

Oxidation of d-alpha-hydroxyglutarate to alpha-ketoglutarate is catalyzed by d-alpha-hydroxyglutarate oxidoreductase, an inducible membrane-bound enzyme of the electron transport particle [ETP; a comminuted cytoplasmic membrane preparation with enzymic properties and chemical composition resembling beef heart mitochondrial ETP (1)] of Pseudomonas putida P2 (P2-ETP). Treatment of P2-ETP with a nonionic detergent yields a preparation with the sedimentation characteristics of a soluble enzyme, but which retains an intact electron transport chain. Oxygen acts solely as a terminal electron acceptor and may be replaced by ferricyanide, 2,6-dichlorophenol indophenol, or mammalian cytochrome c. The oxidoreductase is specific for the d-isomer (K(m) = 4.0 x 10(-4)m for dl-alpha-hydroxyglutarate) and is distinct both from l- and d-malate dehydrogenases. Spectral studies suggest that the carrier sequence is substrate --> flavine or nonheme iron --> cyt b --> [cyt c] --> oxygen.     

Stability of a Pseudomonas putida KT2440 Bacteriophage-Carried Genomic Island and Its Impact on Rhizosphere Fitness

The stability of seven genomic islands of Pseudomonas putida KT2440 with predicted potential for mobilization was studied in bacterial populations associated with the rhizosphere of corn plants by multiplex PCR. DNA rearrangements were detected for only one of them (GI28), which was lost at high frequency. This genomic island of 39.4 kb, with 53 open reading frames, shows the characteristic organization of genes belonging to tailed phages. We present evidence indicating that it corresponds to the lysogenic state of a functional bacteriophage that we have designated Pspu28. Integrated and rarely excised forms of Pspu28 coexist in KT2440 populations. Pspu28 is self-transmissible, and an excisionase is essential for its removal from the bacterial chromosome. The excised Pspu28 forms a circular element that can integrate into the chromosome at a specific location, att sites containing a 17-bp direct repeat sequence. Excision/insertion of Pspu28 alters the promoter sequence and changes the expression level of PP_1531, which encodes a predicted arsenate reductase. Finally, we show that the presence of Pspu28 in the lysogenic state has a negative effect on bacterial fitness in the rhizosphere under conditions of intraspecific competition, thus explaining why clones having lost this mobile element are recovered from that environment.     

Novel system for recognizing and eliminating foreign DNA in Pseudomonas putida.

Derivatives of pQSR49 (R1162::Tn1) containing cloned fragments of Escherichia coli chromosomal DNA are stable in E. coli but unstable in Pseudomonas putida. Similar derivatives containing P. putida chromosomal DNA are stable in both species. Instability is a consequence of plasmid loss during growth and is not due to death or inhibition of growth of plasmid-containing cells. Average copy numbers per cell of the unstable hybrid plasmids are similar to that of pQSR49, indicating that instability is not the result of reduced replication of plasmid DNA. A plasmid unrelated to pQSR49, RK2, becomes unstable in the presence of the unstable hybrids but only when it also contains foreign DNA. The data suggest an inducible mechanism in P. putida active in the elimination of plasmid-borne foreign DNA from the cell.     

Analysis of Dibenzothiophene Desulfurization in a Recombinant Pseudomonas putida Strain

Biodesulfurization was monitored in a recombinant Pseudomonas putida CECT5279 strain. DszB desulfinase activity reached a sharp maximum at the early exponential phase, but it rapidly decreased at later growth phases. A model two-step resting-cell process combining sequentially P. putida cells from the late and early exponential growth phases was designed to significantly increase biodesulfurization.     

Identification of Pseudomonas alcaligenes chromosomal DNA in the plasmid DNA of the chlorobenzene-degrading recombinant Pseudomonas putida strain CB1-9

The recombinant Pseudomonas putida strain CB1-9, which acquired the ability to grow on chlorobenzenes, contains a 33-kilobase (kb) plasmid (pKFL3) which lacked homology to an indigenous 15-kb plasmid (pKFL1) in Pseudomonas alcaligenes C-0 parent but was homologous to a 55-kb plasmid (pKFL2) from the P. putida R5-3 parent. Chromosomal DNA of P. alcaligenes C-0 hybridized to probes prepared from pKFL3 but not to probes prepared from pKFL2. A single clone from a genomic library of P. alcaligenes C-0 hybridized to EcoRI-digested pKFL3. Southern blot hybridization with the insert DNA from that clone identified homology with specific restriction enzyme fragments in pKFL3. The ability of the recombinant to utilize 3-chlorobenzoate, chlorobenzene, and 1,4-dichlorobenzene as well as its loss of utilization of xylenes and methylbenzoates appears to be associated with the transfer and integration of chromosomal DNA from P. alcaligenes into a Tol-like plasmid of P. putida R5-3.