Abstract
Analysis of suppressors that alleviate the acute envelope stress phenotype of a ΔbamBΔdegP strain of Escherichia coli identified a novel protein MzrA and pleiotropic envZ mutations. Genetic evidence shows that overexpression of MzrA – formerly known as YqjB and EcfM – modulates the activity of EnvZ/OmpR similarly to pleiotropic EnvZ mutants and alter porin expression. However, porin expression in strains devoid of MzrA or overexpressing it is still sensitive to medium osmolarity, pH and procaine, all of which modulate EnvZ/OmpR activities. Thus, MzrA appears to alter the output of the EnvZ/OmpR system but not its ability to receive and respond to various environmental signals. Localization and topology experiments indicate that MzrA is a type II membrane protein, with its N-terminus exposed in the cytoplasm and C-terminus in the periplasm. Bacterial two-hybrid experiments determined that MzrA specifically interacts with EnvZ but not with OmpR or the related membrane sensor kinase, CpxA. This and additional genetic and biochemical evidence suggest that the interaction of MzrA with EnvZ would either enhance EnvZ's kinase activity or reduce its phosphatase activity, thus elevating the steady state levels of OmpR∼P. Furthermore, our data show that MzrA links the two-component envelope stress response regulators, CpxA/CpxR and EnvZ/OmpR.
Highlights
The outer membrane of Gram-negative bacteria is the first defence barrier between the often hostile milieu in which the bacteria live and their intracellular contents
To identify potentially new genes involved in the response to envelope stress, we introduced a random plasmid gene library, prepared from the MC4100 chromosome, into the DbamB DdegP strain and sought transformants that improved growth at 37°C
We focused on the exuT-yqj clone because the yfgM-bamB clone most likely improved growth by complementing the DbamB mutation of the recipient strain
Summary
The outer membrane of Gram-negative bacteria is the first defence barrier between the often hostile milieu in which the bacteria live and their intracellular contents. The outer membrane is home to b-barrel-forming outer membrane proteins (OMPs) as well as lipoproteins (Nikaido and Vaara, 1985). The EnvZ/OmpRmediated regulation of OmpF and OmpC, the two major pore-forming OMPs, has been a paradigm for understanding gene regulation by two-component signal transduction systems (for reviews, see Hoch and Silhavy, 1995). EnvZ is a membrane-bound sensor kinase, the signal it senses is not known. OmpR is a cytosolic response regulator, which binds to the promoter region of the porin genes. Other factors, including local anaesthetics (Villarejo and Case, 1984), pH (Heyde and Portalier, 1987; Sato et al, 2000) and nutrition limitation (Liu and Ferenci, 1998), influence ompF and ompC transcription in an EnvZ/OmpR-dependent manner
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