Biomedical subjects

Eric Batchelor

Publications and source records attributed to Eric Batchelor.

4 recordsLinked to original sources

PubMed2006

Imaging OmpR localization in Escherichia coli.

We have used a fusion of GFP to the response regulator OmpR to image the spatial distribution of OmpR in live cells of Escherichia coli. We observed foci of increased OmpR-GFP fluorescence that appear to be due to interactions with the histidine kinase EnvZ. We also observed colocalization of OmpR-GFP with clusters of plasmids carrying OmpR binding sites, which enabled us to develop a simple method for imaging the binding of OmpR to DNA in live cells. We used the peak fluorescence intensity within cells to quantify the extent of OmpR-GFP localization either due to interactions with EnvZ or due to binding DNA. With these assays we compared the effects of osmolarity and procaine, both of which are believed to modulate EnvZ activity. Our results suggest that, at least under our growth conditions, procaine activates EnvZ-OmpR signalling whereas osmolarity has, at best, a weak effect on the EnvZ-OmpR system.

Bacterial Outer Membrane Proteins↗

PubMed2005

The Escherichia coli CpxA-CpxR envelope stress response system regulates expression of the porins ompF and ompC.

We performed transposon mutagenesis of a two-color fluorescent reporter strain to identify new regulators of the porin genes ompF and ompC in Escherichia coli. Screening of colonies by fluorescence microscopy revealed numerous mutants that exhibited interesting patterns of porin expression. One mutant harbored an insertion in the gene encoding the histidine kinase CpxA, the sensor for a two-component signaling system that responds to envelope stress. The cpxA mutant exhibited increased transcription of ompC and a very strong decrease in transcription of ompF under conditions in which acetyl phosphate levels were high. Subsequent genetic analysis revealed that this phenotype is dependent on phosphorylation of the response regulator CpxR and that activation of CpxA in wild-type cells results in similar regulation of porin expression. Using DNase I footprinting, we demonstrated that CpxR binds upstream of both the ompF and ompC promoters. It thus appears that two distinct two-component systems, CpxA-CpxR and EnvZ-OmpR, converge at the porin promoters. Within the context of envelope stress, outer membrane beta-barrel proteins have generally been associated with the sigma E pathway. However, at least for the classical porins OmpF and OmpC, our results show that the Cpx envelope stress response system plays a role in regulating their expression.

Bacterial Outer Membrane Proteins↗

PubMed2004

Continuous control in bacterial regulatory circuits.

We show that for two well-characterized regulatory circuits in Escherichia coli, Tn10 tetracycline resistance and porin osmoregulation, the transcriptional outputs in individual cells are graded functions of the applied stimuli. These systems are therefore examples of naturally occurring regulatory circuits that exhibit continuous control of transcription. Surprisingly, however, we find that porin osmoregulation is open loop; i.e., the porin expression level does not feed back into the regulatory circuit. This mode of control is particularly interesting for an organism such as E. coli, which proliferates in diverse environments, and raises important questions regarding the biologically relevant inputs and outputs for this system.

Culture Media↗

PubMed2003

Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system.

The EnvZ/OmpR system in Escherichia coli, which regulates the expression of the porins OmpF and OmpC, is one of the simplest and best-characterized examples of two-component signaling. Like many other histidine kinases, EnvZ is bifunctional; it phosphorylates and dephosphorylates the response regulator OmpR. We have analyzed a mathematical model of the EnvZ-mediated cycle of OmpR phosphorylation and dephosphorylation. The model predicts that when EnvZ is much less abundant than OmpR, as is the case in E. coli, the steady-state level of phosphorylated OmpR (OmpR-P) is insensitive to variations in the concentration of EnvZ. The model also predicts that the level of OmpR-P is insensitive to variations in the concentration of OmpR when the OmpR concentration is sufficiently high. To test these predictions, we have perturbed the porin regulatory circuit in E. coli by varying the expression levels of EnvZ and OmpR. We have constructed two-color fluorescent reporter strains in which ompF and ompC transcription can be easily measured in the same culture. Using these strains we have shown that, consistent with the predictions of our model, the transcription of ompC and ompF is indeed robust or insensitive to a wide range of expression levels of both EnvZ and OmpR.

Bacterial Outer Membrane Proteins↗