• Journal of Microbiology and Biotechnology
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• v.4 no.1
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• pp.13-19
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• 1994

A model equation to describe the plasmid instability in recombinant Escherichia coli fermentation is proposed. The equation allows one to estimate easily the two model parameters; (1) the difference in the specific growth rates between plasmid-free cells and plasmid-harboring cells ($\delta$), and (2) the probability of plasmid loss by plasmid-harboring cells ($\rho$). The estimated values of $\delta and \rho$ were in the range of 0.02-0.07 and $10^{-3}-10^{-5}$, respectively, and were strongly dependent on the dilution rate. As another parameter, the ratio of specific growth rates of plasmid-free cells and plasmid-harboring cells ($\alha$) was calculated and the result showed the highest value of 1.28 at the lowest dilution rate of 0.075 $hr^{-l}$, examined in this work. By the sensitivity analyses on the estimates of $\delta and \rho$, it was found that the growth rate difference ($\delta$) affected the plasmid instability more seriously than the probability of plasmid loss ($\rho$). Furthermore, the profound instability of plasmid at low dilution rate could be explained by the high values of $\alpha and \rho$.

• Journal of Microbiology and Biotechnology
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• v.2 no.2
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• pp.129-134
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• 1992

Segregational instability of a recombinant plasmid, pDML6, encoding extracellular $\beta$-lactamase in Streptomyces lividans PD6 was characterized by growth kinetic analysis. The quantitative determination of the plasmid harbored in the mycelia was evaluated with mycelia fragmented mechanically, and also with colonies regenerated from protoplasts. Conditions for the formation of protoplasts and regeneration of protoplasts were established. The maximal specific growth rates of the host strain and the plasmid-harboring strain in a chemically defined medium without selection pressure were the same. The probability of plasmid loss from the harbouring cells was higher at higher growth rates. Mathematical models for the prediction of cell growth, substrate uptake, and accumulation of the cloned gene product were developed.

• Journal of Microbiology
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• v.44 no.3
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• pp.255-262
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• 2006

Titanium and its alloys are technically superior and cost-effective materials, with a wide variety of aerospace, industrial, marine, and commercial applications. In this study, the effects of titanium ions on bacterial growth were evaluated. Six strains of bacteria known to be resistant to both metal ions and antibiotics were used in this study. Two strains, Escherichia coli ATCC 15489, and Pseudomonas aeruginosa ATCC 10145, proved to be resistant to titanium ions. Plasmid-cured p. aeruginosa resulted in the loss of one or move resistance markers, indicating plasmid-encoded resistance. The plasmid profile of p. aeruginosa revealed the presence of a 23-kb plasmid. The plasmid was isolated and transformed into $DH5{\alpha}$. Interestingly, the untransformed $DH5{\alpha}$ did not grow in 300 mg/l titanium ions, but the transformed $DH5{\alpha}$ grew quite well under such conditions. The survival rate of the transformed $DH5{\alpha}$ also increased more than 3-fold compared to that of untransformed $DH5{\alpha}$.

• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1621-1628
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• 2015

Chlamydia possesses a conserved 7.5 kb plasmid that is known to play an important role in chlamydial pathogenesis, since some chlamydial organisms lacking the plasmid are attenuated. The chlamydial transformation system developed recently required the use of plasmid-free organisms. Thus, the generation and identification of plasmid-free organisms represent a key step in understanding chlamydial pathogenic mechanisms. A tricolor immunofluorescence assay for simultaneously detecting the plasmid-encoded Pgp3 and whole organisms plus DNA staining was used to screen C. muridarum organisms selected with novobiocin. PCR was used to detect the plasmid genes. Next-generation sequencing was then used to sequence the genomes of plasmid-free C. muridarum candidates and the parental C. muridarum Nigg strain. We generated five independent clones of plasmid-free C. muridarum organisms by using a combination of novobiocin treatment and screening plaque-purified clones with anti-Pgp3 antibody. The clones were confirmed to lack plasmid genes by PCR analysis. No GlgA protein or glycogen accumulation was detected in cells infected with the plasmid-free clones. More importantly, whole-genome sequencing characterization of the plasmid-free C. muridarum organism and the parental C. muridarum Nigg strain revealed no additional mutations other than loss of the plasmid in the plasmid-free C. muridarum organism. Thus, the Pgp3-based immunofluorescence assay has allowed us to identify authentic plasmid-free organisms that are useful for further investigating chlamydial pathogenic mechanisms.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.79-84
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• 2001

An aniline-utilizing microorganism identified as a species of Pseudomonas was isolated from soil contaminated highly with aniline and urea-herbicide. This strain was able to utilize aniline as the sole source of carbon and energy, and was shown to harbor a single large plasmid mediating the aniline assimilation. Subsequent plasmid-curing of this bacterium resulted in the abolishment of the aniline utilizing phenotype and the loss of catechol-C2,3O-oxygenase. The reestablishment of the plasmid, denoted pB10, in cured Pseudomonas sp. via filter surface mating, resulted in restoration of the aniline assimilation abilities and enzyme activity.

• Journal of Microbiology
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• v.44 no.5
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• pp.473-479
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• 2006

A well characterized naphthalene-degrading strain, Pseudomonas putida PpG7 was observed to utilize limonin, a highly-oxygenated triterpenoid compound as a sole source of carbon and energy. Limonin concentrations evidenced a 64% reduction over 48 h of growth in batch cultures. Attempts were made to acquire a plasmid-less derivative via various methods (viz. Ethidium Bromide, SDS, elevated temperature & mitomycin C), among which the method involving mitomycin C ($20{\mu}g/ml$) proved successful. Concomitant with the loss of plasmid in P. putida PpG7 strain, the cured derivative was identified as a $lim^-$ phenotype. The $lim^+$ phenotype could be conjugally transferred to the cured derivative. Based on the results of curing with mitomycin C, conjugation studies and presence of ndo gene encoding naphthalene 1,2 dioxygenase, it was demonstrated that genes for the limonin utilization were encoded on an 83 kb indigenous transmissible Inc. P9 NAH plasmid in Pseudomonas putida PpG7 strain.

• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1702-1708
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• 2015

We have developed a new shuttle plasmid, designated as pLK1-MCS that can replicate in both Clostridium acetobutylicum and Escherichia coli, by combining the pUB110 and pUC19 plasmids. Plasmid pLK1-MCS replicated more stably than previously reported plasmids containing either the pIM13 or the pAMβ1 replicon in the absence of antibiotic selective pressure. The transfer frequency of pLK1-MCS into C. acetobutylicum was similar to the transfer frequency of other shuttle plasmids. We complemented C. acetobutylicum ML1 (that does not produce solvents such as acetone, butanol, and ethanol owing to loss of the megaplasmid pSOL1 harboring the adhE1-ctfAB-adc operon) by introducing pLK1-MCS carrying the adhE1-ctfAB-adc operon into C. acetobutylicum ML1. The transformed cells were able to resume anaerobic solvent production, indicating that the new shuttle plasmid has the potential for practical use in microbial biotechnology.

• Journal of Microbiology
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• v.43 no.2
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• pp.196-203
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• 2005

Here, we attempted to evaluate the activity of artificially transconjugated multiple plasmids in 'designer biocatalysts' for the bioremediation of cocontaminated sites under nonselective conditions. We observed profound losses in the percent survivals of artificially transconjugated plasmid activity ($66 - 78\%$ loss immediately after freeze-drying, $99.45 - 99.88\%$ loss by the end of 6 months storage) in reconstituted Pseudomonas sp. KM12TC. Such unpredictable high losses of this particular plasmid appeared to clearly be a deleterious effect. However, even after 6 months of storage, the cells remained able to degrade $95\%$ of phenol within 9 days, and the full effiux of$^%${73} As, as compared to that of the non-freeze-dried cells, was successfully achieved 4 to 9 days later. These results indicate that 'stable designer biocatalysts' can remain viable, even after freeze-drying and 6 months of storage.

• Microbiology and Biotechnology Letters
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• v.18 no.1
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• pp.89-93
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• 1990

To improve the stability of recombinant pBR322-trip$^+$ plasmid (pLTW24) in E. coli culture, a positive selection system was devised. A DNA fragment containing pheW$^+$ gene (a structural gene for tRNA$^{phe}$ was isolated and inserted into the pBR322-trip$^+$ plasmid(pLTP24). A temperature sensitive host strain. LC901-pheS$^{-ts}$, was constructed for this plasmid by transducing pheS$^{-ts}$ allele (phenylalanyl-tRNA synthetase) to E. coli LC901 using P1kc bacteriophage. The LC901-pheS$^{-ts}$ cells were unable to grow at a restrictive temperature when they had lost the pBR322 :: pheW$^+$ (pLTP24) plasmid. The effects of pheW$^+$ gene on the plasmid stability and the expression level of trip$^+$ gene in LC901-pheS$^{-ts}$ strain were investigated. The proportion of Trip$^+$ colonies among LC901-pheS$^{-ts}$ strain carrying plasmid pLTP24 was 99%, whereas that of LC901 strain carrying plasmid pLTW24 was 7% at the end of 20 generations. After 100 generations of growth, the strain LC901-pheS$^{-ts}$ carrying plasmid pLTP24 showed little loss of plasmids. While the majority of plasmid pLTW24 in LC901 strain were lost in the same period. The activities of tryptophan synthetase (T. Sase) and anthranilate synthetase (A. Sase) in LC901 strain carrying pLTW24 were about 1.2 times and 1.8 times respectively of those in LC901-pheS$^{-ts}$ strain carrying pLTP24.

• Journal of Microbiology and Biotechnology
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• v.6 no.5
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• pp.321-325
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• 1996

Mutants having altered levels and/or types of EPS in exopolysaccharide biosynthesis were isolated after NTG mutagenesis of Zoogloea ramigera 115SLR. Mutant candidates were classfied with five groups based on the observed characteristics for EPS biosynthesis pattern. The recombinant plasmids pLEX3BS and pLEX3BM were constructed from pEX3B which was previously isolated from genomic DNA of Z. ramigera 115SLR. Plasmid pLEX3BM with a 7.8 kb insert DNA contains an additional 1.8kb DNA fragment which is not present in pLEX3BS containing 13 kb insert DNA. Plasmid pLEX3BM was able to complement the mutation responsible for the changes in morphology of Z. ramigera 115SLR. However, the complementation of EPS negative mutant strains was not successful with pLEX3BM. Plasmid pLEX3BS on the other hand complemented the mutation responsible for the loss of EPS biosynthesis, resulting in the restoration of Z. ramigera 115SLR phenotype. But this plasmid was not able to complement the morphological mutant strain, Z. ramigera 115SLR.