Biomedical subjects

Kyoung-Hee Choi

Publications and source records attributed to Kyoung-Hee Choi.

5 recordsLinked to original sources

PubMed2006

Two aerobic pathways for the formation of unsaturated fatty acids in Pseudomonas aeruginosa.

The double bond in anaerobic unsaturated fatty acid (UFA) biosynthesis is introduced by the FabA dehydratase/isomerase of the bacterial type II fatty acid biosynthetic pathway. A DeltafabA mutant of Pseudomonas aeruginosa grew aerobically, but required a UFA supplement for anaerobic growth. Wild-type cells produced 18:1Delta11 as the principal UFA, whereas the DeltafabA strain produced only 16:1Delta9. The double bond in the 16:1Delta9 was introduced after phospholipid formation and was localized in the sn-2 position. Two predicted membrane proteins, DesA and DesB, possessed the conserved histidine clusters characteristic of fatty acid desaturases. The DeltafabADeltadesA double mutant required exogenous fatty acids for growth but the DeltafabAdesB double mutant did not. Exogenous stearate was converted to 18:1Delta9 and supported the growth of DeltafabADeltadesA double mutant. A DeltafabADeltadesAdesB triple mutant was unable to desaturate exogenous stearate and was an UFA auxotroph. We detected a 2.5-fold increase in desA expression in DeltafabA mutants, whereas desB expression was derepressed by the deletion of the gene encoding a transcriptional repressor DesT. These data add two aerobic desaturases to the enzymes used for fatty acid metabolism in proteobacteria: DesA, a 2-position phospholipid Delta9-desaturase that supplements the anaerobic FabA pathway, and DesB, an inducible acyl-CoA Delta9-desaturase whose expression is repressed by DesT.

Aerobiosis↗

PubMed2005

A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation.

A rapid microcentrifuge-based method is described for preparation of Pseudomonas aeruginosa electrocompetent cells with up to 10,000-fold increased transformation efficiencies over existing procedures. This increased efficiency now enables the use of transformation for all applications requiring DNA transfer. These include transfer of chromosomal mutations marked with antibiotic resistance genes between P. aeruginosa strains, which solves the riddle of not having an efficient and reliable transduction procedure for this bacterium. Not surprisingly, the method also allows for very efficient transformation with replicative plasmids, with transformation efficiencies ranging from 10(7) to >10(11) transformants per microgram of DNA. Lastly, with efficiencies of up to >10(3) transformants per microgram of DNA the method replaces in most instances conjugation for the transfer of non-replicative plasmids used in gene replacement, site-specific gene integration and transposon mutagenesis experiments.

Chromosomes, Bacterial↗

PubMed2005

An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants.

BACKGROUND: Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process. RESULTS: We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family. CONCLUSION: While maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria.

Bacterial Proteins↗

PubMed2005

A Tn7-based broad-range bacterial cloning and expression system.

For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.

Chromosomes, Bacterial↗

PubMed2005

Molecular basis of azithromycin-resistant Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa biofilms are extremely recalcitrant to antibiotic treatment. Treatment of cystic fibrosis patients with azithromycin (AZM) has shown promise. We used DNA microarrays to identify differentially expressed transcripts in developing P. aeruginosa biofilms exposed to 2 mug/ml AZM. We report that transcripts for multiple restriction-nodulation-cell division (RND) efflux pumps, known to be involved in planktonic antibiotic resistance, and transcripts involved in type III secretion were upregulated in the resistant biofilms that developed in the presence of AZM. Interestingly, the MexAB-OprM and MexCD-OprJ efflux pumps, but not type III secretion, appear to be integral to biofilm formation in the presence of AZM, as evidenced by the fact that a mutant deleted in both mexAB-oprM and mexCD-oprJ was unable to form a biofilm in the presence of AZM. A mutant deleted in type III secretion was still able to form biofilms in the presence of drug. Furthermore, single mexAB-oprM- and mexCD-oprJ-null mutants were able to form a biofilm in the presence of drug, indicating that either of the pumps can confer resistance to AZM during biofilm development. In contrast to planktonically grown cells, where no mexC expression was detectable regardless of the presence of AZM, biofilms exhibited induction of mexC expression from the outset of their formation, but only in the presence of AZM. mexA, which is constitutively expressed in planktonic cells, was uniformly expressed in biofilms regardless of the presence of AZM. These data indicate that the MexCD-OprJ pump acts as a biofilm-specific mechanism for AZM resistance.

Anti-Bacterial Agents↗