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.

Fig. 1

Comparison of OmpR–PAmCherry with OmpR-mEos and wildtype OmpR. MH513 and MH225 are bacterial strains containing a chromosomal ompF-lacZ (A) or ompC-lacZ (B) transcriptional fusion, respectively. Wildtype OmpR (column 1) represented 100% and the other backgrounds were normalized to it. In the ompR null strain (ompR101), there was no activation of ompF or ompC. The PAmCherry fusion was 93% as active as the wildtype, when a 16 amino acid linker, GGSGx4, was placed between the 3ʹ end of ompR and the beginning of PAmCherry. Its activity was higher than the mEos2 fusion that contained the same linker length (striped column 4, 37%), but a longer linker (40 amino acids, GGSGx10) improved activity to 79% of the wildtype (column 5). Similarly, at ompC, the PAmCherry fusion was 71% of the wildtype activity and better than the mEos2 fusion (17% with a 16 amino acid linker and 60% with a 40 amino acid linker). The photoactivatable fusion affected ompC activity more than ompF as we have observed with other ompR mutants as well as with envZ photoactivatable fusions.

Fig. 2

Sequential super-resolution imaging of OmpR–PAmCherry (red hot), bacterial cell membrane (green) and DNA (cyan) in various growth media. LB (A), 100 mM Tris pH 7.2 (B), 100 mM MES pH 5.6 (C) and 0.5× M9 buffer (D). Evidence for DNA compaction is noticeable in the third row panels, where the green outlines the nucleoid edges. OmpR was distributed around the nucleoid edges (outlined in green) in LB, whereas it was more uniformly distributed in Tris buffer. In acidic (MES, pH 5.6) and hypotonic (0.5× M9) conditions, OmpR was recruited to the plasma membrane (scale bar 1 μm). Images in the bottom row show the averaged distribution of the bacterial membrane (green) and OmpR (red hot). These average images indicate a localization of OmpR near the membrane in acid and low osmolality. The recruitment of OmpR to DNA in LB is averaged out because of the nucleoid heterogeneity and asymmetric segregation. Cells grown in Tris and 0.5× M9 medium exhibited a smaller cell diameter (~0.8 μm), resulting in projection effects for the membrane average image. The number of images used for averaging was: 19, cell length 3.75–4.25 μm (A), 20 cell length 2.0–2.5 μm (B), 15 cell length 1.75–2.25 μm (C) and 13 cell length 1.5–2.0 μm (D).

Fig. 3

Cross-section of bacteria in 0.5× M9 (A) and MES pH 5.6 (B). Covering the bacterial cells with a hydrophilic gel matrix enabled some bacteria to orient axially, these provide cross-sectional images of the cells under various growth conditions. (i), (ii) and (iii) show the PALM, dSTORM and PAINT images of OmpR (red hot), DNA (cyan) and membrane (green), respectively. OmpR was distributed around the chromosome (iv) and along the plasma membrane (v), likely enabling recruitment to membrane-embedded EnvZ molecules during acidic and hypotonic shock. Interestingly, the chromosome seems to form a tube in the middle of the cell cylinder (vi) under acidic conditions, which exhibits small DNA fibers stretching from the tube towards the membrane (yellow arrows). (vii) Represents the composite image of all three SMLM channels (scale bar 0.5 μm).

Fig. 4

Representative super-resolution fluorescence images of OmpR (right panel, red hot) and RNAP (left panel, red hot) in E. coli cells grown in LB medium. The cell membrane was labeled with Nile red and visualized via PAINT imaging (green color). OmpR is clearly less abundant compared to RNAP (scale bar 1 μm).

Fig. 5

(A) FCCS measurement of EnvZc-A568 + OmpR-A488. Inset shows the close up of the relative amplitude of the CCF to the ACF of EnvZc-A568 (cross-correlation ratio). The fraction of OmpR-A488 bound to EnvZc-A568 is 0.14 ± 0.02. (B) FCCS measurement of EnvZc^M-A568 + OmpR-A488. The fraction of OmpR-A488 bound to EnvZc^M-A568 is 0.42 ± 0.08. (C) FCCS measurement of EnvZc^M-A568 + OmpR-A488 in the presence of 20% sucrose. The fraction of OmpR-A488 bound to EnvZc-A568 is 0.37 ± 0.03.

Fig. 6

Titration of EnvZc^M with 280 nM OmpR-A488. (A) The average number of OmpR-A488 (N). The dotted line indicates the value of N for the sample with only OmpR-A488. (B) Count rate per molecule (cpm) values were obtained by normalizing the fluorescence intensity by N. The lower dotted line indicates the cpm for the sample with only OmpR-A488. The upper dotted line indicates the expected cpm value (i.e., twice that of a monomer) of an OmpR-A488 dimer.