title: Single cell super-resolution imaging of E. coli OmpR during environmental stress.
abstract: Two-component signaling systems are a major strategy employed by bacteria, and to some extent, yeast and plants, to respond to environmental stress. The EnvZ/OmpR system in E. coli responds to osmotic and acid stress and is responsible for regulating the protein composition of the outer membrane. EnvZ is a histidine kinase located in the inner membrane. Upon activation, it is autophosphorylated by ATP and subsequently, it activates OmpR. Phosphorylated OmpR binds with high affinity to the regulatory regions of the ompF and ompC porin genes to regulate their transcription. We set out to visualize these two-components in single bacterial cells during different environmental stress conditions and to examine the subsequent modifications to the bacterial nucleoid as a result. We created a chromosomally-encoded, active, fluorescent OmpR-PAmCherry fusion protein and compared its expression levels with RNA polymerase. Quantitative western blotting had indicated that these two proteins were expressed at similar levels. From our images, it is evident that OmpR is significantly less abundant compared to RNA polymerase. In cross-sectional axial images, we observed OmpR molecules closely juxtaposed near the inner membrane during acidic and hyposomotic growth. In acidic conditions, the chromosome was compacted. Surprisingly, under acidic conditions, we also observed evidence of a spatial correlation between the DNA and the inner membrane, suggesting a mechanical link through an active DNA-OmpR-EnvZ complex. This work represents the first direct visualization of a response regulator with respect to the bacterial chromosome.
authors.affiliation: Mechanobiology Institute, T-Lab, 5A Engineering Drive 1, National University of Singapore, Singapore 117411.
authors.forename: Yong Hwee
authors.initials: YH
authors.lastname: Foo
authors.forename: Christoph
authors.initials: C
authors.lastname: Spahn
authors.forename: Hongfang
authors.initials: H
authors.lastname: Zhang
authors.forename: Mike
authors.initials: M
authors.lastname: Heilemann
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Kenney
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001696:Biomechanical Phenomena; D002876:Chromosomes, Bacterial / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004777:Environment; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D009997:Osmotic Pressure; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D059010:Single-Cell Analysis; D013050:Spectrometry, Fluorescence; D013312:Stress, Physiological; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; C057843:envZ protein, E coli