Characterization of a LuxR repressor for 3,17ß-HSD in Comamonas testosteroni ATCC11996.

3,17ß-Hydroxysteroid dehydrogenase in Comamonas testosteroni (C. testosteroni) is a key enzyme involved in the degradation of steroid compounds. Recently, we found that LuxR is a negative regulator in the expression of the 3,17ß-HSD gene. In the present work, we cultured wild-type and LuxR knock-out mutants of C. testosteroni with inducers such as testosterone, estradiol, progesterone or estrone. HPLC analysis showed that the degradation activities towards testosterone, estradiol, progesterone, and estrone by C.T.-LuxR-KO1 were increased by 7.1%, 9.7%, 11.9% and 3.1%, respectively compared to the wild-type strain. Protein conformation of LuxR was predicted by Phyre 2 Server software, where the N-terminal 86(Ile), 116(Ile), 118(Met) and 149(Phe) residues form a testosterone binding hydrophobic pore, while the C-terminus forms the DNA binding site (HTH). Further, luxr point mutant plasmids were prepared by PCR and co-transformed with pUC3.2-4 into E. coli HB101. ELISA was used to determine 3,17ß-HSD expression after testosterone induction. Compared to wild-type luxr, 3,17ß-HSD expression in mutants of I86T, I116T, M118T and F149S were decreased. The result indicates that testosterone lost its capability to bind to LuxR after the four amino acid residues had been exchanged. No significant changes of 3,17ß-HSD expression were found in K354I and Y356 N mutants compared to wild-type luxr, which indicates that these two amino acid residues in LuxR might relate to DNA binding. Native LuxR protein was prepared from inclusion bodies using sodium lauroylsarcosinate. Molecular interaction experiments showed that LuxR protein binds to a nucleotide sequence which locates 87 bp upstream of the ßhsd promoter. Our results revealed that steroid induction of 3,17ß-HSD in C. testosteroni in fact appears to be a de-repression, where testosterone prevents the LuxR regulator protein binding to the 3,17ß-HSD promoter domain.

Nutrition effects on the biofilm immobilization and 3,5-DNBA degradation of Comamonas testosteroni A3 during bioaugmentation treatment.

The survival of inoculated microbes is critical for successful bioaugmentation in wastewater treatment. The influence of readily available nutrients (RANs) on the colonization of two functional bacteria, Pseudomonas putida M9, a strong biofilm-forming strain, and Comamonas testosteroni A3, a 3,5-dinitrobenzoic acid (3,5-DNBA)-degrading strain, in biofilms was studied with 3,5-dinitrobenoic acid synthetic wastewater (DCMM) complemented with various ratios of Luria-Bertani broth (LB). With the increase in LB rate, the biofilm biomass was increased, the percentage of gfp-labeled M9 measured in the mixed culture enhanced, and also M9 became dominant. In laboratory-scale sequencing batch biofilm reactors, with the increase in 3,5-DNBA concentration and extension of the running time, the 3,5-DNBA removal in DCMM wastewater complemented with RANs tended to be more efficient and its removal rates increased gradually over the experimental period. Our study demonstrated that supplementing RANs could be a useful strategy for enhancing colonization of degrading bacteria in wastewater treatment systems.

Impact of membrane fatty acid composition on the uncoupling sensitivity of the energy conservation of Comamonas testosteroni ATCC 17454.

The fatty acid composition of pyruvate-grown Comamonas testosteroni ATCC 17454 was analyzed after growth at 30 and 20 degrees C and after half-maximum growth inhibition caused by different membrane-active chemicals at 30 degrees C. Palmitic acid (16:0), palmitoleic acid (16:1 omega7c) and vaccenic acid (18:1 omega7c) were the dominant fatty acids. At 20 degrees C, the proportion of palmitic acid decreased and those of palmitoleic and vaccenic acid increased. Saturation degree was also lowered when half-maximum growth inhibition was caused by 4-chlorosalicylic acid, 2,4-dichlorophenoxyacetic acid and 2,4-dinitrophenol and, to a lesser extent, in the presence of 2,4-dichlorophenol, phenol and ethanol. It appeared that the dissociated forms of the former group of chemicals were preferentially incorporated near the head group region of the lipid bilayer, thereby somewhat extending the outer region of the membranes, and that the increased amount of bent, unsaturated fatty acids helped to maintain membrane integrity. Irrespective of how the decrease of the saturation degree was triggered, it caused electron transport phosphorylation (adenosine triphosphate synthesis driven by n-hexanol oxidation) to become more sensitive to uncoupling. Apparently, the viscosity and phase stability of the cytoplasmic membrane of C. testosteroni were maintained at the price of a reduced protection against energy toxicity.