Charles F. Earhart

Characterization of Escherichia coli expressing an Lpp’OmpA(46-159)-PhoA fusion protein localized in the outer membrane

The Lpp′OmpA(46-159) hybrid protein can serve as an efficient targeting vehicle for localizing a variety of procaryotic and eucaryotic soluble proteins onto the E. coli surface, thus providing a system for several possible biotechnology applications. Here we show that fusions between Lpp′OmpA(46-159) and bacterial alkaline phosphatase (PhoA), a normally periplasmic dimeric enzyme, are also targeted to the outer membrane. However, protease accessibility experiments and immunoelectron microscopy revealed that, unlike other periplasmic proteins, the PhoA domain of these fusions is not exposed on the cell surface in cells having an intact outer membrane. Conditions that affect the formation of disulfide bonds and the folding of the PhoA domain in the periplasm not only did not facilitate targeting to the cell surface but led to lethality when the fusion was expressed from a high-copy-number plasmid. Furthermore, E. coli expressing the Lpp′OmpA(46-159)-PhoA fusion exhibited strain- and temperature-dependent alterations in outer-membrane permeability. Our results are consistent with previous studies with other vehicles indicating that PhoA is not displayed on the surface when fused to cell-surface expression vectors. Presumably, the enzyme rapidly assumes a tightly folded dimeric conformation that cannot be transported across the outer membrane. The large size and quaternary structure of PhoA may define a limitation of the Lpp′OmpA(46-159) fusion system for the display of periplasmic proteins on the cell surface. Alkaline phosphatase is a unique protein among a group of five periplasmic proteins (β-lactamase, alkaline phosphatase, Cex cellulase, Cex cellulose-binding domain, and a single-chain Fv antibody fragment), which have been tested as passengers for the Lpp′OmpA(46-159) expression system to date, since it was the only protein not displayed on the surface.

Effect of iron on the relative abundance of two large polypeptides of the Escherichiacoli outer membrane

Abstract The relative abundance of two polypeptides of the Escherichia coli outer membrane is affected by the growth medium. The polypeptides have molecular weights of 85,000 and 95,000 and, in cells grown in medium containing low concentrations of iron, are dominant outer membrane proteins.

The entD Gene of the Escherichia coli K12 Enterobactin Gene Cluster

The Escherichia coli entD gene encodes a product necessary for the synthesis of the iron-chelating and transport molecule enterobactin (Ent); cells harbouring entD mutations fail to grow in iron-deficient environments. For unknown reasons, it has not been possible to identify the entD product. The nucleotide sequence of the entD region has now been determined. An open reading frame extending in the same direction as the adjacent fepA gene and capable of encoding an approximately 24 kDa polypeptide was found; it contained a high percentage of rare codons and two possible translational start sites. Complementation data suggested that EntD proteins truncated at the carboxy terminus retain some activity. Two REP sequences were present upstream of entD and an IS186 sequence was observed downstream. RNA dot-blot hybridizations demonstrated that entD is transcribed from the strand predicted by the sequencing results. An entD-lacZ recombinant plasmid was constructed and shown to express low amounts of a fusion protein of the anticipated size (approximately 125 kDa). The evidence suggests a number of possible explanations for difficulties in detecting the entD product. Sequence data indicate that if entD has its own promoter, it is weak; the REP sequences suggest that entD mRNA may be destabilized; and translation may be slow because of the frequency of rare codons and a possible unusual start codon (UUG). The data are also consistent with previous evidence that the entD product is unstable.

The association of host and phage DNA with the membrane of Escherichia coli

Abstract The attachment of DNA to membrane in T4-infected cells was investigated by means of the Mg2+-sarkosyl crystals technique. Both parental and progeny phage DNA are associated with cell membrane throughout the eclipse period and are subsequently detached during the synthesis of mature phage. The initial attachment process does not require protein synthesis. Detachment appears to be due to a “late function” since it does not occur following infection with a DO mutant (amN82) or after treatment with chloramphenicol or tetracycline added 9 min after infection. Host DNA remains attached to membrane during the initial stages of infection and neither superinfection nor the addition of chloramphenicol alters this association.

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

The cytosolic proteins EntE, EntF, and EntB/G, which are Escherichia coli enzymes necessary for the final stage of enterobactin synthesis, are released by osmotic shock. Here, consistent with the idea that cytoplasmic proteins found in shockates have an affinity for membranes, a small fraction of each was found in membrane preparations. Two procedures demonstrated that the enzymes were enriched in a minor membrane fraction of buoyant density intermediate between that of cytoplasmic and outer membranes, providing indirect support for the notion that these proteins have a role in enterobactin excretion as well as synthesis.

Enterobactin synthase polypeptides of Escherichia coli are present in an osmotic-shock-sensitive cytoplasmic locality.

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 Ent 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.

Identification of hydrophobic proteins FepD and FepG of the Escherichia coli ferrienterobactin permease

In Escherichia coli, iron assimilation by means of the siderophore enterobactin requires two hydrophobic cytoplasmic membrane proteins, FepD and FepG, which are essential components of a binding-protein-dependent transport system. Such components are typically difficult to detect. Here we report observation of the fepD and fepG gene products in polyacrylamide gels; they appeared as diffuse bands at positions consistent with smaller sizes than those predicted by sequence analysis. Translational coupling was suggested by the lack of a detectable product from the fepG message in the absence of translation of the upstream fepD message. The orientation of FepD/FepG in the membrane was predicted based on their similarities in sequence and hydrophobicity to FhuB.

Suppression of iron uptake deficiency in Escherichia,coli K-12 by loss of two major outer membrane proteins

Abstract A novel iron uptake system was observed in pseudorevertants of Escherichia , coli strains defective in ferrienterochelin transport. The new system is unique in that it is an active transport system that does not utilize any known siderophore. Acquisition of the new uptake system occurs concomitantly with the loss of two major outer membrane proteins (b and c) believed to function as structural components of transmembrane pores.

Alterations in the cell envelope of Escherichia coli late in bacteriophage T4 infection.

The cell envelope of Escherichia coli was examined for changes during late stages of bacteriophage T4 infection. Late events in T4 infection are shown to result in (i) a reduction in the effectiveness of membrane separation procedures employing either isopycnic sucrose gradient centrifugation or selective solubilization of inner membrane by detergent (Sarkosyl or Triton X-100), (ii) the appearance of a 54 000 dalton host protein in membrane preparations, (iii) the adventitious presence of detergent-resistant phage morphogenetic structures in membrane preparation, and (iv) a decrease in the activity of NADH oxidase and an apparent alteration in its association with inner membrane. These modifications occur regardless of the state of the e and t genes of T4.

Iron uptake in pseudorevertants of Escherichia coli K-12 mutants with multiple defects in the enterochelin system

Iron uptake in pseudorevertants of Escherichia coli K-12 strains which lack the ability to synthesize enterochelin, 2,3-dihydroxybenzoate, and the ferrienterochelin receptor protein was characterized. In four independent pseudorevertants, the suppressor mutations which permitted growth in iron-poor environments appeared to be located in ompB, the regulatory locus for the porin proteins. Unlike wild-type cells, the pseudorevertants were unable to utilize ferrienterochelin and could acquire iron from citrate without induction by prior growth in citrate. The energy requirements of the pseudorevertant system appeared to be identical to those of the enterochelin system. Evidence that loss of the porin proteins results in the secretion by the pseudorevertants of a molecule with siderophore activity is presented; this siderophore is able to remove iron from the non-biological iron chelators nitrilotriacetic acid and α, α′-dipyridyl but not from the siderophores ferrichrome and enterochelin.