Cultivation at high osmotic pressure confers ubiquinone 8-independent protection of respiration on Escherichia coli.

Ubiquinone 8 (coenzyme Q8 or Q8) mediates electron transfer within the aerobic respiratory chain, mitigates oxidative stress, and contributes to gene expression in Escherichia coli In addition, Q8 was proposed to confer bacterial osmotolerance by accumulating during growth at high osmotic pressure and altering membrane stability. The osmolyte trehalose and membrane lipid cardiolipin accumulate in E. coli cells cultivated at high osmotic pressure. Here, Q8 deficiency impaired E. coli growth at low osmotic pressure and rendered growth osmotically sensitive. The Q8 deficiency impeded cellular O2 uptake and also inhibited the activities of two proton symporters, the osmosensing transporter ProP and the lactose transporter LacY. Q8 supplementation decreased membrane fluidity in liposomes, but did not affect ProP activity in proteoliposomes, which is respiration-independent. Liposomes and proteoliposomes prepared with E. coli lipids were used for these experiments. Similar oxygen uptake rates were observed for bacteria cultivated at low and high osmotic pressures. In contrast, respiration was dramatically inhibited when bacteria grown at the same low osmotic pressure were shifted to high osmotic pressure. Thus, respiration was restored during prolonged growth of E. coli at high osmotic pressure. Of note, bacteria cultivated at low and high osmotic pressures had similar Q8 concentrations. The protection of respiration was neither diminished by cardiolipin deficiency nor conferred by trehalose overproduction during growth at low osmotic pressure, but rather might be achieved by Q8-independent respiratory chain remodeling. We conclude that osmotolerance is conferred through Q8-independent protection of respiration, not by altering physical properties of the membrane.

Impacts of the osmolality and the lumenal ionic strength on osmosensory transporter ProP in proteoliposomes.

H(+) symporter ProP serves as a paradigm for the study of osmosensing. ProP attains the same activity at the same osmolality when the medium outside cells or proteoliposomes is supplemented with diverse, membrane-impermeant solutes. The osmosensory mechanism of ProP has been probed by varying the solvent within membrane vesicles and proteoliposomes. ProP activation was not ion specific, did not require K(+), and could be elicited by large, uncharged solutes polyethylene glycols (PEGS). We hypothesized that ProP is an ionic strength sensor and lumenal macromolecules activate ProP by altering ion activities. The attainable range of lumenal ionic strength was expanded by lowering the phosphate concentration within proteoliposomes. ProP activity at high osmolality, but not the osmolality, yielding half-maximal activity (Π(1/2)/RT), decreased with the lumenal phosphate concentration. This was attributed to acidification of the proteoliposome lumen due to H(+)-proline symport. The ionic strength yielding half-maximal ProP activity was more anion-dependent than Π(1/2)/RT for proteoliposomes loaded with citrate, sulfate, phosphate, chloride, or iodide. The anion effects followed the Hofmeister series. Lumenal bovine serum albumin (BSA) lowered the lumenal ionic strength at which ProP became active. Osmolality measurements documented the non-idealities of solutions including potassium phosphate and other solutes. The impacts of PEGS and BSA on ion activities did not account for their impacts on ProP activity. The effects of the tested solutes on ProP appear to be non-coulombic in nature. They may arise from effects of preferential interactions and macromolecular crowding on the membrane or on ProP.

Transcriptional regulation and posttranslational activity of the betaine transporter BetL in Listeria monocytogenes are controlled by environmental salinity.

While the genetic elements contributing to the salinity tolerance of Listeria monocytogenes have been well characterized, the regulatory signals and responses (genetic and/or biochemical) that govern these mechanisms have yet to be elucidated. Encoded by betL, the first genetic element to be linked to listerial osmotolerance, the secondary betaine uptake system BetL is a member of the betaine-carnitine-choline transporter family. Preceded by consensus sigma(A)- and sigma(B)-dependent promoter sites, betL is constitutively expressed and transcriptionally up-regulated in response to salt stress. The nisin-controlled expression system was used to achieve salinity-independent, controlled betL expression in Listeria. In the absence of NaCl-activated transcriptional control, BetL activity was found to be a function of environmental salinity, showing optimal activity in buffer supplemented with 1 to 2% NaCl (osmolality, 417 to 719 mosmol/kg). In addition, BetL was activated rapidly (half-life, 2 min) in response to an osmotic upshift imposed by adding 2% NaCl to 50 mM potassium phosphate buffer.