Enterobactin Synthase Polypeptides of Escherichia Coli are Present in an Osmotic-Shock-Sensitive Cytoplasmic Locality

Abstract

The terminal reactions in the synthesis of the siderophore enterobactin (Ent) by Escherichia coli require the EntD, E, F and B/G polypeptides. The idea that these molecules form a complex (Ent synthase) that is membrane-associated was re-evaluated. In vitro results provided no evidence in support of the proposal : (i) Ent synthase activity occurred normally under conditions where membrane was either absent or disrupted by high concentrations of neutral detergents, and (ii) immunoprecipitation experiments conducted on extracts engaged in E n t synthesis failed to detect any association among the Ent polypeptides. However, Western blot analyses showed that EntE, F and B/G were released from cells by osmotic shock and freeze/thaw treatment but not by conversion of cells to spheroplasts. These results demonstrated that EntE, F and B/G belong to the Beacham group D class of proteins. The shockability of a given group D Ent protein was unaffected by the absence of either EntB/G or EntD and, for EntB/G, the N-terminus was sufficient for release by osmotic shock. The behaviour of group D proteins is generally attributed to their association (partial, loose or transient) with cytoplasmic membrane; therefore, the results are indirect evidence that Ent synthase interacts with membrane in vivo. At the very least, the data indicate that EntE, F and B/G are compartmentalized in E. coli and, because other biosynthetic enzymes for siderophores and surfactants are related to these Ent proteins, suggest that this entire protein class may be sequestered in vivo.

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

“…However, this protein might be prevented from exiting cells by binding the plasma membrane (2,3). The other apparent contradiction is presented by a large EntF polypeptide (142 kDa) that was shown to be partially released upon osmotic shock (9). It is easy to imagine, however, that a multidomain EntF may have an elongated or even flexible shape that would allow it to pass through a molecular sieve with a nominal 100-kDa cutoff.…”

“…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).…”

Siderophore-Based Iron Acquisition and Pathogen Control

“…Such a coupling would be reasonable in order to avoid the accumulation of nonloaded siderophore molecules in the cell and to prevent the degradation of newly synthesized siderophores, e.g., by esterases that are involved in iron release. In this context, indirect evidence for a membrane association of the enterobactin EntEBF synthetase was provided by the finding that enterobactin synthesis proteins are released from cells by osmotic shock but not by spheroblasting, thus behaving like Beacham group D class proteins that are released from cells in a similar way due to their association (partial, loose, or transient) with the CM (128). EntE, EntB, and EntF polypeptides were also detected in total membrane preparations from cells grown in iron-deficient medium (129).…”

Membrane Association of the Escherichia coli Enterobactin Synthase Proteins EntB/G, EntE, and EntF

“…An additional consideration is that certain exported proteins synthesized without their signal sequence are also released by osmotic shock (3,7,30). This led us to suggest that proteins released by osmotic shock but not by spheroplasting have temporary or fragile association with membrane sites at which transmembrane passage occurs (17).…”

“…EntE and EntF as well as EntB/G were detected in total membrane preparations from cells grown in iron-deficient medium (TMM) (17) (Fig. 2), but the extent of their membrane association varied significantly.…”