Journal of Microbiology

Research Support, Non-U.S. Gov't

Overexpression of Outer Membrane Protein OprT and Increase of Membrane Permeability in phoU Mutant of Toluene-Tolerant Bacterium Pseudomonas putida GM730: Kyunghee Lee , Juna Jung , Kwang Kim , Dongwon Bae , Dongbin Lim; J. Microbiol. 2009;47(5):557-562. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0105-y

Abstract: Eight toluene-sensitive mutants were previously isolated from the toluene-tolerant bacterium Pseudomonas putida GM730. One of these mutants was TOS6, in which Tn5 had been inserted into phoU. Susceptibility to multiple antibiotics, as well as toluene sensitivity, was increased in the phoU mutant of P. putida GM730. We compared the outer membrane proteins from the phoU mutant and wild-type via two-dimensional gel electrophoresis. A 45 kDa protein was dramatically overexpressed as the result of phoU inactivation, and this protein was identified by peptide mass fingerprinting and microsequencing as a conserved hypothetical protein consisting of 414 amino acids. The protein, designated as OprT, harbors a signal sequence and extended β-sheets, both of which are features common to the bacterial porins. The rate of ethidium bromide accumulation in TOS6 was higher than in GM730, which indicates that the TOS6 membranes may be more permeable to ethidium bromide than are the membranes of GM730. We propose that the toluene sensitivity and increased antibiotic susceptibility observed in the phoU mutant may be attributable to increased membrane permeability.

Transcriptional Induction of a Carbon Starvation Gene during Other Starvation and Stress Challenges in Pseudomonas putida MK1: A Role of a Carbon Starvation Gene in General Starvation and Stress Responses: Chitra, Subramanian+ , Lee, Ho Sa , Kim, Young Jun; J. Microbiol. 1999;37(3):141-147.

Abstract: Thirteen transcriptionally-fused carbon starvation mutants, derived from Pseudomonas putida ATCC 12633, were analyzed for their survivability and transcriptional induction profiles upon carbon starvation. One of these mutants, MK114, which exhibited the lowest survivability and the highest induction rate, was selected and further examined under different starvation (nitrogen and phosphate) and stress (osmolarity, H₂O₂, salts, alcohol, and heat) conditions. Under all tested conditions MK114 induced β-galactosidase activity, implying that the interrupted gene (cst114) is a general starvation and stress response gene. The rate of induction ranged from 2.6-fold for phosphate starvation to 3.7-fold for osmotic shock. The mini-Tn5 flanking DNA was cloned from the chromosome of MK114. The cloned DNA fragment exhibited carbon starvation activity, indicating that this fragment contains a carbon starvation-related promoter region. This region was partially sequenced. Possible physiological roles of Cst114 in a carbon sensing mechanism and in other stress responses are also discussed.

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