Eliminating a set of four penicillin binding proteins triggers the Rcs phosphorelay and Cpx stress responses in Escherichia coli.

Penicillin binding proteins (PBPs) are responsible for synthesizing and modifying the bacterial cell wall, and in Escherichia coli the loss of several nonessential low-molecular-weight PBPs gives rise to abnormalities in cell shape and division. To determine whether these proteins help connect the flagellar basal body to the peptidoglycan wall, we surveyed a set of PBP mutants and found that motility in an agar migration assay was compromised by the simultaneous absence of four enzymes: PBP4, PBP5, PBP7, and AmpH. A wild-type copy of any one of these restored migration, and complementation depended on the integrity of the PBP active-site serine. However, the migration defect was caused by the absence of flagella instead of improper flagellar assembly. Migration was restored if the flhDC genes were overexpressed or if the rcsB or cpxR genes were deleted. Thus, migration was inhibited because the Rcs and Cpx stress response systems were induced in the absence of these four specific PBPs. Furthermore, in this situation Rcs induction depended on the presence of CpxR. The results imply that small changes in peptidoglycan structure are sufficient to activate these stress responses, suggesting that a specific cell wall fragment may be the signal being sensed. The fact that four PBPs must be inactivated may explain why large perturbations to the envelope are required to induce stress responses.

Profiles and some initial identifications of (anti)androgenic compounds in fish exposed to wastewater treatment works effluents.

Exposure of fish to wastewater treatment works (WwTWs) effluents can result in reproductive anomalies consistent with exposure to estrogenic compounds. However, UK WwTWs effluents also contain compounds with androgen receptor activities which may contribute to reproductive dysfunction in fish. A toxicity identification and evaluation (TIE) approach was used to profile (anti)androgenic compounds in bile of fish exposed to two WwTWs effluents. Extracts of bile from exposed fish and effluent were fractionated by liquid chromatography and tested for (anti)androgenic activity using a yeast androgen receptor transcription screen (YAS). A number of bile fractions contained (anti)androgenic activity unique to the effluent-exposed fish. Some of these fractions contained di(chloromethyl)anthracene or dichlorophene, and these contaminants showed antagonistic activity in the YAS when tested as pure compounds. No androgenic activity was detected in the effluents, but TIE analysis of bile revealed a number of androgenic fractions which contained testosterone metabolites that were unique to effluent-exposed fish. This is the first work reported on the nature of some of the (anti)androgenic compounds that bioaccumulate in fish from WwTWs effluents and indicates that other contaminants, besides estrogenic substances, need to be considered for their potential to contribute to the disruption of reproductive system of fish in UK waters.

CYP2E1 overexpression inhibits microsomal Ca2+-ATPase activity in HepG2 cells.

Cytochrome P450 2E1 (CYP2E1) is a microsomal enzyme that generates reactive oxygen species during its catalytic cycle. We previously found an important role for calcium in CYP2E1-potentiated injury in HepG2 cells. The possibility that CYP2E1 may oxidatively damage and inactivate the microsomal Ca2+-ATPase in intact liver cells was evaluated, in order to explain why calcium is elevated during CYP2E1 toxicity. Microsomes were isolated by differential centrifugation from two liver cell line: E47 cells (HepG2 cells transfected with the pCI neo expression vector containing the human CYP2E1 cDNA, which overexpress active microsomal CYP2E1), and control C34 cells (HepG2 cells transfected with the pCI neo expression vector alone, which do not express significantly any cytochrome P450). The Ca2+-dependent ATPase activity was determined by measuring the accumulation of inorganic phosphate from ATP hydrolysis. CYP2E1 overexpression produced a 45% decrease in Ca2+-dependent ATPase activity (8.6 nmol Pi/min/mg protein in C34 microsomes versus 4.7 nmol Pi/min/mg protein in microsomes). Saturation curves with Ca2+ or ATP showed that CYP2E1 overexpression produced a decrease in Vmax but did not affect the Km for either Ca2+ or ATP. The decrease in activity was not associated with a decrease in SERCA protein levels. The ATP-dependent microsomal calcium uptake was evaluated by fluorimetry using fluo-3 as the fluorogenic probe. Calcium uptake rate in E47 microsomes was 28% lower than in C34 microsomes. Treatment of E47 cells with 2mM N-acetylcysteine prevented the decrease in microsomal Ca2+-ATPase found in E47 cells. These results suggest that CYP2E1 overexpression produces a decrease in microsomal Ca2+-ATPase activity in HepG2 cells mediated by reactive oxygen species. This may contribute to elevated cytosolic calcium and to CYP2E1-potentiated injury.