Changes in Permeability of Gram Negative Bacteria:  Given our interest in biocides that react with cells with near-diffusion-controlled kinetics, we are naturally interested in how they inflict their damage.  For Gram negative bacteria, the biocide probably needs to get past the outer membrane (since it is not likely that mortal blows can be inflicted through damage to the outer membrane).  Indeed, the aforementioned results for MG1655-lux evidence that HOCl imposes damage to inner membrane functions and/or cytoplasmic functions synchronously with the reaction of the HOCl.  The question naturally arises as to whether inner membrane function (e.g., respiration) is destroyed or whether HOCl is capable of efficiently traversing the inner membrane.  We note the inner membrane is considerably less porous than the outer membrane.  Nonetheless, there is good evidence that respiratory function is lost well after fatal damage is inflicted by HOCl.  Accordingly, we have begun to investigate the possibility that HOCl enters the cytoplasm vis-à-vis a porin.  Our first investigation has involved aquaporin Z (AqpZ), a porin that is believed to traffic exclusively in water, although this point has not been proven.  AqpZ, which was identified a decade ago in wild-type E. coli on the basis of sequence homology to other aquaporins, was the first water channel to be recognized in prokaryotes. In contrast to earlier reports that expression of E. coli aqpZ peaks during mid-log phase, a recent study by Kustu and co-workers challenged the hypothesis that expression of the gene is an adaptive response by rapidly growing cells, and instead found that increased transcription is under control of the RpoS sigma factor. Although it has been previously demonstrated that purified E. coli AqpZ increases the permeability of water in liposomes, there is a notable absence of direct evidence that AqpZ mediates water permeability in vivo.  We have recently reported (Ashby, M. T.; Mallo, R. C., J. Bacteriol., JB01106-05, submitted) that the water permeability of wild-type E. coli during log growth is comparable to that of an aqpZ knockout mutant. In contrast, an increase in permeability is observed for the wild-type at the onset of the stationary stage with no significant corresponding change in the permeability of the mutant (Figure 3).  However, no difference in the minimum inhibitory concentration (MIC) of HOCl was observed for the wild-type or mutant in either log or stationary stage.  Accordingly, we are presently investigating other porins.  

Acknowledgement:  We are very grateful to the National Science Foundation (CHE-0503984) and the Department of Education (GAANN) for their financial support of this project.