Bacterial protein secretion is important in the life cycles of most bacteria, in which it contributes to the formation of pili and flagella and makes available extracellular enzymes to digest polymers for nutritional purposes and toxins to kill host cells in infections of humans, animals and plants. It is generally accepted that nonpathogenic laboratory strains of Escherichia coli, particularly K12 strains, do not secrete proteins into the extracellular medium under routine growth conditions. In this study, we report that commonly used laboratory strains secrete YebF, a small (10.8 kDa in the native form), soluble endogenous protein into the medium, challenging the status quo view that laboratory strains do not secrete proteins to the medium. We further show that 'passenger' proteins linked to the carboxyl end of YebF are efficiently secreted. The function of YebF is unknown, but its use as a carrier for transgenic proteins provides a tool to circumvent toxicity and other contamination issues associated with protein production in E. coli.

We report the biochemical and biophysical characterization of YedYZ, a sulfite oxidase homologue from Escherichia coli. YedY is a soluble catalytic subunit with a twin arginine leader sequence for export to the periplasm by the Tat translocation system. YedY is the only molybdoenzyme so far isolated from E. coli with the Mo-MPT form of the molybdenum cofactor. The electron paramagnetic resonance (EPR) signal of the YedY molybdenum is similar to that of known Mo-MPT containing enzymes, with the exception that only the Mo(IV) --> Mo(V) transition is observed, with a midpoint potential of 132 mV. YedZ is a membrane-intrinsic cytochrome b with six putative transmembrane helices. The single heme b of YedZ has a midpoint potential of -8 mV, determined by EPR spectroscopy of YedZ-enriched membrane preparations. YedY does not associate strongly with YedZ on the cytoplasmic membrane. However, mutation of the YedY active site Cys102 to Ser results in very efficient targeting of YedY to YedZ in the membrane, demonstrating a clear role for YedZ as the membrane anchor for YedY. Together, YedYZ comprise a well-conserved bacterial heme-molybdoenzyme found in a variety of bacteria that can be assigned to the sulfite oxidase class of enzyme.

The blood-testis barrier and blood-brain barrier are responsible for protecting the male genital tract and central nervous system from xenobiotic exposure. In HIV-infected patients, low concentrations of antiretroviral drugs in cerebrospinal fluid and seminal fluid have been reported. One mechanism that may contribute to reduced concentrations is the expression of ATP-binding cassette drug efflux transporters, such as P-glycoprotein (P-gp). The objective of this study was to investigate in vivo the tissue distribution of the HIV protease inhibitor atazanavir in wild-type (WT) mice, P-gp/breast cancer resistance protein (Bcrp)-knockout (Mdr1a ؊/؊ , Mdr1b ؊/؊ , and Abcg2 ؊/؊ triple-knockout [TKO]) mice, and Cyp3a ؊/؊ (Cyp) mice. WT mice and Cyp mice were pretreated with a P-gp/Bcrp inhibitor, elacridar (5 mg/kg of body weight), and the HIV protease inhibitor and boosting agent ritonavir (2 mg/kg intravenously [i.v.]), respectively. Atazanavir (10 mg/kg) was administered i.v. Atazanavir concentrations in plasma (C plasma ), brain (C brain ), and testes (C testes ) were quantified at various times by liquid chromatography-tandem mass spectrometry. In TKO mice, we demonstrated a significant increase in atazanavir C brain /C plasma (5.4-fold) and C testes /C plasma (4.6-fold) ratios compared to those in WT mice (P < 0.05). Elacridar-treated WT mice showed a significant increase in atazanavir C brain /C plasma (12.3-fold) and C testes /C plasma (13.5-fold) ratios compared to those in vehicle-treated WT mice. In Cyp mice pretreated with ritonavir, significant (P < 0.05) increases in atazanavir C brain /C plasma (1.8-fold) and C testes /C plasma (9.5-fold) ratios compared to those in vehicle-treated WT mice were observed. These data suggest that drug efflux transporters, i.e., P-gp, are involved in limiting the ability of atazanavir to permeate the rodent brain and genital tract. Since these transporters are known to be expressed in humans, they could contribute to the low cerebrospinal and seminal fluid antiretroviral concentrations reported in the clinic.

Escherichia coli export the protein YebF into the extracellular medium by a two-step process. However, as no general outer membrane protein secretion system common to all E. coli strains has been reported, the mechanism of export has remained unclear. Herein, we identify the outer membrane proteins OmpF, OmpC, and OmpX as central to the YebF export mechanism using both genetic and planar lipid bilayer experiments. The nuclear magnetic resonance structural ensemble of YebF reveals a cystatin-like fold consisting of a structured core and an extended dynamic surface in a state of conformational exchange. This surface, conserved throughout YebF orthologs of Enterobacteriaceae, may facilitate the porin-mediated transport of YebF as amino acid substitutions of dynamic residues reduced secretion to the extracellular medium. Our results demonstrate that OmpF and OmpC not only operate to import ions and protein toxins but may also contribute to the export of the YebF protein family.

Dimethyl sulfoxide reductase is a heterotrimeric enzyme (DmsABC) localized to the cytoplasmic surface of the inner membrane. Targeting of the DmsA and DmsB catalytic subunits to the membrane requires the membrane targeting and translocation (Mtt) system. The DmsAB dimer is a member of a family of extrinsic, cytoplasmic facing membrane subunits that require Mtt in order to assemble on the membrane. We show that the MttA 2 , MttB, and presumably MttA 1 but not the MttC proteins are required for targeting DmsAB to the membrane. Unlike other Mtt substrates such as trimethylamine N-oxide reductase, the soluble cytoplasmic DmsAB dimer that accumulates in the mtt deletions is very labile. Deletion of the mttA 2 or mttB genes also prevents anaerobic growth on fumarate even though fumarate reductase does not require Mtt for assembly. This was due to the lethality of membrane insertion of DmsC in the absence of the DmsAB subunits. In the absence of DmsC, DmsAB accumulates in the cytoplasm. A 45-amino acid leader on DmsA is removed during assembly. Processing does not require DmsC but does require Mtt. Translocation of DmsAB to the periplasm is not required for processing. The leader may be cleaved by a novel leader peptidase, or the long DmsA leader may traverse the membrane through the Mtt system resulting in cleavage by the periplasmic leader peptidase I followed by release of DmsA into the cytoplasm.