Molecular Sieve Mechanism of Selective Release of Cytoplasmic Proteins by Osmotically Shocked Escherichia coli

Abstract

Escherichia coli cells, the outer membrane of which is permeabilized with EDTA, release a specific subset of cytoplasmic proteins upon a sudden drop in osmolarity in the surrounding medium. This subset includes EF-Tu, thioredoxin, and DnaK among other proteins, and comprises ϳ10% of the total bacterial protein content. As we demonstrate here, the same proteins are released from electroporated E. coli cells pretreated with EDTA. Although known for several decades, the phenomenon of selective release of proteins has received no satisfactory explanation. Here we show that the subset of released proteins is almost identical to the subset of proteins that are able to pass through a 100-kDa-cutoff cellulose membrane upon molecular filtration of an E. coli homogenate. This finding indicates that in osmotically shocked or electroporated bacteria, proteins are strained through a molecular sieve formed by the transiently damaged bacterial envelope. As a result, proteins of small native sizes are selectively released, whereas large proteins and large protein complexes are retained by bacterial cells.The procedure of osmotic shock was originally introduced for the purpose of extracting periplasmic enzymes of Escherichia coli (20). In this procedure, bacterial cells are preincubated in a hyperosmotic sucrose solution supplemented with EDTA to permeabilize their outer membrane and then transferred into a solution of low osmolarity. The resulting osmotic pressure within shocked cells was believed to cause extrusion of the contents of the periplasm. Later experiments have shown, however, that osmotically shocked bacteria, while remaining viable, also release a number of cytoplasmic molecules, including ions, metabolites (22), and certain proteins. In fact, the major protein released from the shocked cells is a cytoplasmic constituent, translation elongation factor EF-Tu, at least half of which is extracted by the osmotic shock procedure (12, 13). The subset of released cytoplasmic proteins also includes thioredoxin (1, 17), molecular chaperone DnaK (6,8), and proteins involved in the biosynthesis of enterobactin (9), among others (5). Some foreign proteins expressed in the cytoplasm of E. coli have also been successfully extracted by the osmotic shock procedure (16, 23).It has been unclear what determines the selectivity of protein release from osmotically shocked bacteria, since the released cytoplasmic proteins share no apparent common characteristics distinguishing them from the majority of proteins, which are retained by the cells. How these proteins leave the cytoplasm in the absence of cell lysis has also remained an enigma. The most frequently entertained hypothesis postulates that these proteins normally concentrate in a hypothetical "osmotically sensitive compartment" of the bacterial cytoplasm, which is presumably adjacent to the zones of adhesion between the plasma membrane and the outer membrane and, therefore, is selectively extruded from the shocked cells (4, 8-10, 18, 23).This hypothesis leaves open, however, a s...

Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B

“…5 shows LpoB). The globular domain of LpoB and the rigid body of LpoA both have a diameter of ∼30 Å, allowing them to traverse the PG layer, which has ∼40-60-Å-wide pores depending on turgor (20,21). As interlayer distances have been measured only for the lateral part of the E. coli envelope, length constraints and membrane separation could be different in the leading edge of the inward growing septum, where PBP1B-LpoB predominantly localizes and acts.…”

The Architecture of the Murein (Peptidoglycan) in Gram-Negative Bacteria: Vertical Scaffold or Horizontal Layer(s)?

“…The latter molecule would have a reduced number of connected tesseras because the slightly lower level of cross-linkage is more than compensated for by the three-to fivefold longer coli has pores with a mean radius of 2.06 nm (17). In vivo, the stretched murein allows penetration of proteins with molecular masses of up to 100 kDa (17,74), indicating that larger pores with a radius of about 3 nm may exist (according to the formula given in reference 17, a globular protein with a molecular mass of 100 kDa has an estimated radius of 3.1 nm). The possible existence of larger holes indicates that the murein net is not perfect and is consistent with the data on the glycan length distribution and on the degree of cross-linkage.…”

“…Because in the living cell the murein is under tension, it was roughly estimated that in vivo the stretched murein may allow free diffusion of globular proteins with a maximum molecular mass of 50 kDa. In another study it was demonstrated that EDTA treatment of E. coli cells in combination with a hyperosmotic shock released a subset of cytoplasmic proteins that were almost identical to the proteins that are able to pass through a 100-kDa-cutoff filter (74). It was speculated that this limitation was caused by the molecular sieving property of the murein sacculus that was impermeable for proteins with molecular masses of more than 100 kDa under the osmotic shock conditions.…”

Cloning and Characterization of a Novel Carboxylesterase Gene from Cow Rumen Metagenomic Library

“…A genomic library was constructed in the fosmid vector pCC1FOS as previously described [35]. Total genomic DNA from cow rumen was sheared into approximately 40 kb fragments using a syringe needle, size-fractionated on a 5 to 40% linear sucrose gradient and then end-repaired to yield blunt, 5'-phosphorylated ends.…”