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Pseudomonas putida encodes 20 extracytoplasmic sigma factors (ECFs). In this study, we show that one of
these ECFs, known as ECF-Pp12 (PP3006), plays a role in tolerance of toluene and other organic solvents.
Based on this finding, we have called the gene that encodes this new ECF rpoT. The rpoT gene forms an operon
with the preceding gene and with the gene located downstream. The translated gene product of the open
reading frame PP3005 is an inner membrane protein, whereas the PP3007 protein is periplasmic. A nonpolar
rpoT mutant was generated by homologous recombination, and survival of the mutant was tested under
various stress conditions. The mutant strain was hypersensitive to toluene and other solvents but just as
tolerant as the wild type of stress imposed by heat, antibiotics, NaCl, paraquat, sodium dodecyl sulfate, H2O2,
and benzoate. In the rpoT mutant background, expression of around 50 transcriptional units was affected: 31
cistrons were upregulated, and 23 cistrons were downregulated. This indicates that about 1% of all P. putida
genes are under the direct or indirect influence of RpoT. The rpoT gene controls the expression of a number
of membrane proteins, including components of the respiratory chains, porins, transporters, and multidrug
efflux pumps. Hypersensitivity of the P. putida RpoT-deficient mutant to organic solvents can be attributed to
the fact that in the rpoT strain, expression of the toluene efflux pump ttgGHI genes is severalfold lower than
in the parental strain. |
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