Microscopic methods for distinguishing among three cell types in TOL plasmid-carrying Pseudomonas putida cultures

Microscopic methods were developed that enable the sensitive quantification of different cell types that are generated by plasmid instability processes when Pseudomonas putida PaW164 (X+), which carries a TOL plasmid (pWW0-164), is grown in chemostat culture. Cells that have lost the structural TOL genes (X-) or the entire TOL plasmid (X0) can be quantified in a background of 6000 X+ cells using catechol agarose miniplates. X0 cells can be quantified in a background of 3500 X+ or X- cells using carbenicillin agarose miniplates. These methods represent significant improvements in sensitivity over conventional plating methods.     

Kinetics of benzoate-induced loss of the TOL plasmid from Pseudomonas putida MT15 during growth in chemostat culture

Potassium-limited chemostat cultures of Pseudomonss putida MT15, grown on excess glucose, displayed approximately 100% plasmid loss after 60 generations of growth in the presence of 5 mM benzoate. The kinetics of plasmid loss indicated that plasmid-free cells displayed a growth rate advantage, which we attribute to selective inhibition of the growth of plasmid-containing cells by benzoate. However, stable, mixed populations of plasmid-free cells, deletants and plasmid-containing cells were selected during growth under glucose limitation in the presence of benzoate. This behaviour indicated that the plasmid-free cells in these cultures displayed a growth rate disadvantage and that their appearance was due entirely to benzoate-induced segregational instability of the plasmid.     

Leaching of Pseudomonas aeruginosa, and transconjugants containing pR68.45 through unsaturated, intact soil columns

Abstract Leaching of genetically engineered microbes (GEMs) through soil is a significant concern related to groundwater quality. The objective of this study was to examine the leaching, survival and gene transfer of a genetically engineered microbe and indigenous recipients of pR68.45 in nonsterile, undisturbed soil columns. Pseudomonas aeruginosa PAO25, containing the plasmid R68.45, was added to the surface of undisturbed soil columns (10 cm diameter × 80 cm length). Unsaturated flow conditions were maintained by 100 ml daily additions of 2 mM CaCl₂ for a period of 70 days. The population of the GEM exhibited a significant ( P = 0.05) linear decline with time. The GEM leached only to a depth of 30–40 cm in 70 days. Transfer of pR68.45 was shown to occur from P. aeruginosa into the indigenous bacterial population although relatively low numbers of transconjugants were observed (log 2 cfu g⁻¹ dry soil). The number of transconjugants also decreased with depth and time. Leaching of transconjugants, however, occured more readily than that of the GEM, probably as a result of plasmid transfer into smaller, more mobile bacteria. At 70 days incubation, no GEMs were detected in the columns, while transconjugants were observed at several depths. These results demonstrate the importance of examining both the survival and movement of GEMs and transconjugants in soil.     

Involvement of the plasmid pPGH1 in the phenol degradation of Pseudomonas putida strain H

Pseudomonas putida strain H (wildtype) was shown to harbour two plasmids with a molecular mass of about 50 kb and 200–220 kb, respectively. Evidence is presented that the larger one, pPGH1, is involved in the phenol degradation via the meta-cleavage pathway.     

Antimicrobial and anti-biofilm effect of a novel BODIPY photosensitizer against Pseudomonas aeruginosa PAO1

Photodynamic therapy (PDT) combines the use of organic dyes (photosensitizers, PSs) and visible light in order to elicit a photo-oxidative stress which causes bacterial death. GD11, a recently synthesized PS belonging to the boron-dipyrromethene (BODIPY) class, was demonstrated to be efficient against planktonic cultures of Pseudomonas aeruginosa, causing a 7 log unit reduction of viable cells when administered at 2.5?μM. The effectiveness of GD11 against P. aeruginosa biofilms grown in flow-cells and microtiter trays was also demonstrated. Confocal laser scanning microscopy of flow-cell-grown biofilms suggests that the treatment has a biocidal effect against bacterial biofilm cells.     

Characteristics of a newly created bioluminescent Pseudomonas putida harboring TOL plasmid for use in analysis of a bioaugmentation system

Insertion of a bacterial lux operon into the chromosome of Pseudomonas putida mt-2 holding TOL plasmid, yielded a new bioluminescent strain of P. putida BLU. Both in the cultures containing toluene and m-toluic acid as the sole carbon sources, P. putida BLU showed the same specific growth rate and cell yield as those of the wild strain. The bioluminescence output in the cell growth phases correlated with the cell concentration, indicating that the bioluminescent P. putida BLU can be monitored and quantified in a mixed culture in real time by the luminescence detection.     

Production of pyoverdine, the fluorescent pigment of Pseudomonas aeruginosa PAO1

Abstract The nutritional requirements for the production of pyoverdine were studied using Pseudomonas aeruginosa PAO₁ in a chemically defined medium, with shaking. Succinic acid and ammonium sulphate were found to be the best sources of carbon and nitrogen for pyoverdine production. The optimum carbon-to-nitrogen ratio was found to be 4:1. Elevated concentrations of phosphate inhibited pyoverdine production.     

铜绿假单胞菌lasI,rhlI基因功能缺陷株的构建和生物学功能验证

构建铜绿假单胞菌lasI,rhlI基因功能缺陷株,为进一步阐明氦氧饱和高气压暴露条件诱导lasI,rhlI基因介导铜绿假单胞菌毒力调节的分子机制研究奠定基础。用双亲株接合转移法删除lasI,rhlI基因ORF编码区,通过RT-PCR方法验证目标基因编码序列mRNA的缺失;通过对细菌生长增殖能力、弹性蛋白酶代谢活性和细菌绿脓菌素分泌能力等表型的测定,验证目标基因编码序列缺失后的基因调节功能的缺陷。结果表明成功构建铜绿假单胞菌lasI,rhlI基因功能缺陷株,可作为进一步研究的基因工程菌。     

Effect of carbon source addition on toluene biodegradation by an Escherichia coli DH5α transconjugant harboring the TOL plasmid

Horizontal gene transfer (HGT) of plasmids is a naturally occurring phenomenon which could be manipulated for bioremediation applications. Specifically, HGT may prove useful to enhance bioremediation through genetic bioaugmentation. However, because the transfer of a plasmid between donor and recipient cells does not always result in useful functional phenotypes, the conditions under which HGT events result in enhanced degradative capabilities must first be elucidated. The objective of this study was to determine if the addition of alternate carbon substrates could improve toluene degradation in Escherichia coli DH5α transconjugants. The addition of glucose (0.5–5 g/L) and Luria–Bertani (LB) broth (10–100%) resulted in enhanced toluene degradation. On average, the toluene degradation rate increased 14.1 (±2.1)‐fold in the presence of glucose while the maximum increase was 18.4 (±1.7)‐fold in the presence of 25% LB broth. Gene expression of xyl genes was upregulated in the presence of glucose but not LB broth, which implies different inducing mechanisms by the two types of alternate carbon source. The increased toluene degradation by the addition of glucose or LB broth was persistent over the short‐term, suggesting the pulse amendment of an alternative carbon source may be helpful in bioremediation. While the effects of recipient genome GC content and other conditions must still be examined, our results suggest that changes in environmental conditions such as alternate substrate availability may significantly improve the functionality of the transferred phenotypes in HGT and therefore may be an important parameter for genetic bioaugmentation optimization. Biotechnol. Bioeng. 2010;107: 269–277. © 2010 Wiley Periodicals, Inc.     

High frequency conjugal transfer of tylosin genes and amplifiable DNA in Streptomyces fradiae

Summary Genes encoding enzymes for tylosin biosynthesis, genes involved in the expression of resistance to tylosin (Tyl), hygromycin B (Hm), chloramphenicol (Cm), and mitomycin C (MC), and a single copy of an amplifiable unit of DNA (AUD) were jointly transferred at very high frequencies by conjugation from several different Streptomyces fradiae strains to S. fradiae JS85, a mutant defective in many or possibly all tylosin biosynthetic reactions and containing a multiple tandem reiteration of the AUD. No recombination was observed between nar, rif and spc genes in conjugal matings, but recombination was observed between these genes after protoplast fusion. Tylosin biosynthetic genes were transferred at a much lower frequency to S. fradiae JS87, another mutant defective in many or all tylosin biosynthetic reactions, but deleted for the AUD and other DNA sequences. These findings suggest that tylosin structural genes, several genes encoding antibiotic resistance determinants, and amplifiable DNA are present on a self-transmissible element that does not mobilize chromosomal genes, and that JS85 and JS87 contain deletions, and JS85 an amplification, of overlapping portions of this element.     

TOL plasmid in Pseudomonas aeruginosa PAO: thermosensitivity of self-maintenance and inhibition of host cell growth.

The TOL plasmid originally isolated in Pseudomonas putida (arvilla) mt-2 was transmissible to strains of the fluorescens group of Pseudomonas, i.e., P. putida, P. fluorescens, and P. aeruginosa, except for a strain of P. aeruginosa, strain PAO. The same strain, however, could accept the plasmid when its restriction and modification abilities were lost by mutations or by growing at high temperature. In addition, the transmissibility of the TOL plasmid from strain PAO to P. putida was low when the plasmid was modified by the donor. By using P. aeruginosa PAO carrying the TOL plasmid, the stability and genetic expression of the plasmid as well as its effect on the host cell growth were examined. Thus the self-maintenance of the plasmid was found to be thermosensitive. Furthermore, the TOL plasmid inhibited the growth of strain PAO at high temperature, accompanied by the formation of some filamentous cells. These thermosensitive properties of the TOL plasmid were host dependent and not exhibited in another strain of P. aeruginosa.     

Chromosomal insertion of TOL transposons in Pseudomonas aeruginosa PAO.

Insertions of the TOL plasmid transposons Tn4651 and Tn4653 into the Pseudomonas aeruginosa PAO chromosome were isolated by a temperature selection technique. The locations and orientations of 16 insertions were determined by pulsed field gel electrophoresis and Southern hybridization with genomic and TOL DNA probes. All insertions occurred within a 334 kb region of the chromosome (representing less than 6% of the genome) with nine of the inserts clustered within a 10 kb area. Each transposon was able to insert in either orientation. An internal duplication of the 39 kb excisable region of pWW0 was seen in two independent insertions.