Cells from diverse organisms can accumulate similar arrays of organic compounds, all known to be compatible with and/or to stabilize protein structure, when challenged by hypertonic environments (35
). The machinery of compatible solute accumulation has been described for some organisms (e.g., E. coli
]), but its regulation is not well understood. Although proteins Fis and CAP are involved, no trans
-acting transcriptional regulatory element specific to locus proP
has been implicated in the impressive modulation of its transcription by osmotic stress. Transporter ProP is activated, in the absence of protein synthesis, when whole bacteria (11
), cytoplasmic membrane vesicles (26
), or proteoliposomes incorporating purified ProP (30
) are subjected to an osmotic upshift with a membrane-impermeant osmolyte. Our research is designed to elucidate the mechanisms by which ProP senses osmolality changes and mounts its osmoregulatory response. Since ProP activity is impaired by insertion proQ220
), we are exploring the structure and function of proQ
as well as its relationship to ProP.
In this study we establish that the effects of the insertion on ProP are due to the altered expression of locus proQ and not to polar effects on downstream locus prc (see the text and Fig. and ). The previous conclusion that the Tn5 insertion in proQ does not influence proP transcription was confirmed. Database analysis identified two proteins with weak sequence similarities to ProQ. Within this group of homologues, the relationship that appeared most interesting was the weak similarity with translational regulator FinO. This raised the possibility that ProQ could be acting at a translational level to alter the levels of ProP protein. However, this study has shown that the level of ProP protein present in either whole cells (data not shown) or membrane vesicles is not altered by the Tn5 insertion in locus proQ (Fig. ).
This study has further shown that the rate and extent of ProP activation are significantly reduced in a proQ220::Tn5 strain of E. coli. These reports are significant in documenting the only trans-acting factor which is known to influence the osmotic activation of ProP. Gene proQ is predicted to encode a 232-amino-acid protein that is both basic and hydrophilic in nature. SDS-PAGE and Western blot analysis indicate that the overexpressed ProQ protein is soluble, as predicted (Fig. ). The subcellular location of the protein in wild-type bacteria remains to be determined, however.
ProP activity is observed in cytoplasmic membrane vesicles (26
) and proteoliposomes prepared with purified ProP (30
). There are some significant differences between the ProP activities of these vesicle systems and those of whole cells, however. The hyperosmotic shift which gives maximal ProP activity in cells (0.2 osM) is lower than that required in membrane vesicles (0.8 osM) (22
). As well, ProP is active in whole cells even without an osmotic shift, whereas ProP activities in proteoliposomes and membrane vesicles absolutely require a hyperosmotic shift (22
). Given these contrasting features of ProP activities in cells and vesicle systems, we now believe that ProQ is a structural element which influences the osmotic activation of ProP at a posttranslational level.