Translocation of Jellyfish Green Fluorescent Protein via the Tat System of Escherichia coli and Change of Its Periplasmic Localization in Response to Osmotic Up-shock

Abstract

The bacterial twin arginine translocation (Tat) pathway is capable of exporting cofactor-containing enzymes into the periplasm. To assess the capacity of the Tat pathway to export heterologous proteins and to gain information about the property of the periplasm, we fused the twin arginine signal peptide of the trimethylamine N-oxide reductase to the jellyfish green fluorescent protein (GFP). Unlike the Sec pathway, the Tat system successfully exported correctly folded GFP into the periplasm of Escherichia coli. Interestingly, GFP appeared as a halo in most cells and occasionally showed a polar localization in wild type strains. When subjected to a mild osmotic up-shock, GFP relocalized very quickly at the two poles of the cells. The conversion from the halo structure to a periplasmic gathering at particular locations was also observed with spherical cells of the ⌬rodA-pbpA mutant or of the wild type strain treated with lysozyme. Therefore, the periplasm is not a uniform compartment and the polarization of GFP is unlikely to be caused by simple invagination of the cytoplasmic membrane at the poles. Moreover, the polar gathering of GFP is reversible; the reversion was accelerated by glucose and inhibited by azide and carbonyl cyanide m-chlorophenylhydrazone, indicating an active adaptation of the bacteria to the osmolarity in the medium. These results strongly suggest a relocalization of periplasmic substances in response to environmental changes. The polar area might be the preferential zone where bacteria sense the change in the environment.The periplasmic space lies between the inner and the outer membranes of Gram-negative bacteria. A number of processes that are vital to the growth and viability of the cell occur within this compartment. Proteins residing in the periplasmic space fulfill important functions in the detection, processing, and uptake of essential nutrient substances. These proteins are exported into the periplasm mainly via two pathways: the unfolded proteins via the Sec system (1) and the folded enzymes containing redox cofactor via the Tat 1 (or Mtt) pathway (2-4).The periplasm might not be a uniformly homogenous compartment; fine structures known as Bayer patches/bridges (5) and periseptal and polar annuli (6 -9) have been described. The existence of these structures under physiological conditions is a subject of contention (10,11). Nevertheless, these structures were proposed to provide sites required for the export of outer membrane components, murein synthesis, secretion of bacteriophages, and cell divisions (12).On the other hand, polar bacterial organization was observed with a variety of bacterial species and concerns a disparate array of cellular functions (13). In addition to the well known examples of polar organelles such as flagella, pili, and stalklike appendages at the bacterial surface, accumulating evidence shows that periplasmic, inner membranous, and cytoplasmic proteins may also exhibit polar localization under certain condition. These proteins participate in various cellula...

Versatile selection technology for intracellular protein–protein interactions mediated by a unique bacterial hitchhiker transport mechanism

“…35). Interestingly, c-myc-HybC was notably absent from both the cytoplasmic and periplasmic fractions of ⌬tatC cells, consistent with reports that some nonexported Tat substrates are efficiently degraded by an unidentified ''housecleaning'' mechanism (30,36). Importantly, these results confirm that hitchhiker export can be functionally reconstituted using multicopy expression plasmids.…”

A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant

“…In a strain expressing TorAss-YFP, most cells showed bright, uniform fluorescence, but some cells (about 10%) exhibited the same fluorescent haloes as those described in previous studies ( Fig. 7A4) (Santini et al, 2001;Thomas et al, 2001). We attributed the uniform fluorescence to overproduction of the fusion protein and inefficient export by the Tat system.…”

Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway

“…In the FA113 tatC::spec strain, no F AB could be detected in the periplasmic fraction and a significant reduction in the amount of protein remaining in the cytoplasm was also observed. We and others have observed that depletion of the tat genes often results in inactivation or degradation of Tat substrate proteins in the cytoplasm (32,33). Similarly, no light-chain protein or F AB -binding activity could be detected in E. coli DHB4 (Fig.…”