The sensitivity of the outer and cytoplasmic membranes of Escherichia coli to detergent was examined by isopycnic sucrose density gradient centrifugation. Sodium lauryl sarcosinate (Sarkosyl) was found to disrupt the cytoplasmic membrane selectively under conditions in which Triton X-100 and dodecyl sodium sulfate solubilized all membrane protein. These results were verified by gel electrophoresis; membrane proteins solubilized by Sarkosyl were identical to those of the cytoplasmic membrane. The presence of Mg2+ during treatment with Sarkosyl was found to afford partial protection of the cytoplasmic membrane from dissolution.

The outer membrane of Gram-negative bacteria presents an effective barrier that restricts the release of proteins from the cell. Virtually all extracellular proteins of Gram-negative bacteria are exported by specialized systems requiring the action of several gene products. We have constructed a tripartite fusion consisting of (i) the signal sequence and rust nine N-terminal amino acids of the mature major Escherichia coli lipoprotein, (it) amino acids 46-159 ofthe outer membrane protein OmpA, and (Mi) the complete mature ,-lactamase (EC 3.5.2.6) sequence. This protein had an enzymatically active ,-lactamase and was found predominantly in the outer membrane. Immunofluorescence microscopy, the accessibility of the fusion protein to externally added proteases, and the rates of hydrolysis of nitrocefin and penicillin G by whole cells demonstrated that a substantial fraction (20-30%) of the ,t-lactamase domain of the fusion protein was exposed on the external surface of E. coil. In cells grown at 240C the localization of 8-lactamase on the cell surface was almost quantitative (>80% of the enzymatically active protein was exposed to the extracellular fluid) as determined by nitrocefin and penicillin G hydrolysis and trypsin accessibility. These results demonstrated that a soluble protein, 13-lactamase, can be transported through-and become anchored on-the outer membrane by fusion to the proper targeting and localization signals.

Escherichia coli genes specifically required for transport of iron by the siderophore enterobactin are designatedfep. The studies reported here were initiated to identify and localize the fepB product. The plasmid pCP111, which consisted of an ll-kilobase E. coli DNA fragment containingfepB ligated to pACYC184, was constructed. The fepB gene was subcloned; in the process, complementation tests and TnS mutagenesis results provided evidence for the existence of a new fep gene, fepC. The order of the transport genes in the ent gene cluster is as follows: fepA fes entF fepC fepB entE. Minicell, maxicell, and in vitro DNA-directed protein synthesizing systems were used to identify thefepB andfepC products. ThefepC polypeptide was 30,500 daltons in standard sodium dodecyl sulfate-polyacrylamide gels. The fepB gene was responsible for the appearance of three or four bands (their apparent molecular weights ranged from 31,500 to 36,500) in sodium dodecyl sulfate-polyacrylamide gels, depending on the gel system employed. The largest of these was tentatively designated proFepB, since it apparintly had a leader sequence. Localization experiments showed that FepC was a membrane constituent and that mature FepB was present in the periplasm. An additional polypeptide (X) was also encoded by the bacterial DNA of pCPI11, but its relationship to iron transport is unknown. The results indicated that fetrienterobactin uptake is mediated by a periplasmic transport system and that genes coding for outer membrane (fepA), periplasmic (fepB), and cytoplasmic membrane (fepC) components have now been identified.The endogenous high-affinity system for iron transport of the gram-negative bacterium Escherichia coli utilizes the siderophore enterobactin (enterochelin) (for a review, see reference 39). Under conditions of iron deprivation, enterobactin is synthesized and released into the environment, where it binds iron; the resulting ferrienterobactin complexes are then actively transported back into the cell. The mechanism by which ferrienterobactin enters cells is not well understood and is the subject of this work.Four genes are known to produce products that influence the passage of ferrienterobactin through the cell envelope; two of the genes (tonB and exbB) have pleiotropic phenotypes, whereas the other two, fepA and fepB, seem to be specific for ferrienterobactin transport. A functional tonB gene is required for all high-affinity iron transport systems, for vitamin B12 uptake, and for sensitivity to many phages and colicins (22

Bacterial cell-surface exposure of foreign peptides and soluble proteins has been achieved recently by employing a fusion protein methodology. An Lpp'-OmpA(46-159)-Bla fusion protein has been shown previously to display the normally periplasmic enzyme beta-lactamase (Bla) on the cell surface of the Gram-negative bacterium Escherichia coli. Here, we have investigated the role of the OmpA domain of the tripartite fusion protein in the surface display of the passenger domain (Bla) and have characterized the effects of the fusion proteins on the integrity and permeability of the outer membrane. We show that in addition to OmpA(46-159), a second OmpA segment, consisting of amino acids 46-66, can also mediate the display of Bla on the cell surface. Other OmpA domains of various lengths (amino acids 46-84, 46-109, 46-128, 46-141 and 46-145) either anchored the Bla domain on the periplasmic face of the outer membrane or caused a major disruption of the outer membrane, allowing the penetration of antibodies into the cell. Detergent and antibiotic sensitivity and periplasmic leakage assays showed that changes in the permeability of the outer membrane are an unavoidable consequence of displaying a large periplasmic protein on the surface of E. coli. This is the first systematic report on the effects that cell surface engineering may have on the integrity and permeability properties of bacterial outer membranes.

Transposon mutagenesis and plasmid complementation studies have identified two genes, fepD and fepG, which are essential for ferrienterobactin transport in Escherichia coli. These genes mapped in the enterobactin gene cluster and genetic evidence indicated that they are transcribed as part of an operon (fepD, fepG, fepC). The nucleotide sequence of fepD was determine; it could encode a hydrophobic 33.8 kDa protein with sequence homologies to other iron and vitamin B12 transport proteins. Also identified, between fepD and fepB, was an open reading frame (ORF43) with no detectable function; its 43 kDa protein product (P43) was seen on polyacrylamide gels. The fepD-C operon and ORF43 were divergently transcribed from a 110bp region containing a binding site for the repressor protein Fur.