Reconstitution of maltose transport in malB mutants of Escherichia coli through calcium-induced disruptions of the outer membrane

In Escherichia coli, the periplasmic maltose-binding protein (MBP), the product of the malE gene, is the primary recognition component of the transport system for maltose and maltodextrins. It is also the maltose chemoreceptor, in which capacity it interacts with the signal transducer Tar (taxis to aspartate and some repellents). In studies of the maltose system in other members of the family Enterobacteriaceae, we found that MBP is produced by Salmonella typhimurium, Klebsiella pneumoniae, Enterobacter aerogenes, and Serratia marcescens. MBP from all of these species cross-reacted with antibody against the E. coli protein and had a similar molecular weight (about 40,000). The Shigella flexneri and Proteus mirabilis strains we examined did not synthesize MBP. The isoelectric points of MBP from different species varied from the acid extreme of E. coli (4.8) to the basic extreme of E. aerogenes (8.9). All species with MBP transported maltose with high affinity, although the Vmax for K. pneumoniae was severalfold lower than that for the other species. Maltose chemotaxis was observed only in E. coli and E. aerogenes. In S. typhimurium LT2, Tar was completely inactive in maltose taxis, although it signaled normally in response to aspartate. MBP isolated from all five species could be used to reconstitute maltose transport and taxis in a delta malE strain of E. coli after permeabilization of the outer membrane with calcium.

Section: Discussionmentioning

confidence: 54%

Interspecific reconstitution of maltose transport and chemotaxis in Escherichia coli with maltose-binding protein from various enteric bacteria

Dahl¹,

Manson²

1985

“…In the present paper we investigated whether the Ca21induced uptake of protein into the periplasm (4,5) is mechanistically related to the CaU-induced uptake of DNA during transformation (23). Using an E. coli strain containing a nonpolar deletion in malE, we compared the conditions required for transformation by plasmid DNA with the conditions necessary for reconstitution of maltose transport by exogenous MBP (4,5).…”

Section: Discussionmentioning

confidence: 99%

“…We have recently shown that Ca2+ treatment of E. coli and Salmoniell(l tvyphimurium at a low temperature induces a reversible permeabilization of the outer membrane of these organisms. This phenomenon allows efficient reconstitution of transport and chemotaxis in binding-protein-deficient mutants after import of exogenous binding protein into the periplasm (4)(5)(6). Under optimal conditions, about 20% of the cells were fully competent for uptake of proteins into the periplasm.…”

mentioning

confidence: 99%

Ca2+-induced permeabilization of the Escherichia coli outer membrane: comparison of transformation and reconstitution of binding-protein-dependent transport

Bukau¹,

Brass²,

Boos³

1985

Self Cite

Ca2+ treatment renders the outer membrane of Escherichia coli reversibly permeable for macromolecules. We investigated whether Ca2+-induced uptake of exogenous protein into the periplasm occurs by mechanisms similar to Ca2+-induced uptake of DNA into the cytoplasm during transformation. Protein import through the outer membrane was monitored by measuring reconstitution of maltose transport after the addition of shock fluid containing maltose-binding protein. DNA import through the outer and inner membrane was measured by determining the efficiency of transformation with plasmid DNA. Both processes were stimulated by Preparation of shock fluid. Shock fluid of maltose-induced cells of strain poplO80 [HfrG6 lamB102(Am) metA trpE(Am) galE galY] (18), containing between 30 and 50% of total protein as MBP, was prepared by osmotic shock by the 61 on August 5, 2020 by guest

“…We show here that maltose chemotaxis in E. coli can be reconstituted by the addition of exogenous MBP to strains lacking this protein. The procedure, based on Ca2+-induced permeabilization of the outer membrane of gram-negative bacteria (10), was used previously to reconstitute transport in mutants lacking binding proteins (11). Hazelbauer and Adler (22) reported that galactose chemotaxis could be restored to osmotically shocked cells of wild-type strains by adding shock fluid containing GBP to the cells.…”

Section: Discussionmentioning

confidence: 99%

“…We have recently developed a technique whereby soluble substrate-binding proteins can be introduced into the periplasmic space after Ca2+induced permeabilization of the outer membrane of gramnegative bacteria. Import and function of these proteins can be demonstrated by the reconstitution of transport in mutants lacking binding protein (10,11).…”

mentioning

confidence: 99%

Reconstitution of maltose chemotaxis in Escherichia coli by addition of maltose-binding protein to calcium-treated cells of maltose regulon mutants

Brass¹,

Manson²

1984

Self Cite

Maltose chemotaxis was reconstituted in AmalE cells lacking maltose-binding protein (MBP). Purified MBP was introduced into intact cells during incubation with 250 mM CaCl2 in Tris-hydrochloride buffer at 0C. After removal of extracellular CaCl2 and MBP, chemotaxis was measured with tethered bacteria in a flow chamber or with free-swimming cells in a capillary assay. About 20% of tethered cells responded to 10-4 M maltose; the mean response times were about half those of CaCl2-treated wild-type cells (100 s as opposed to 190 s). In capillary tests, the maltose response of reconstituted cells was between 15 and 40% of the aspartate response, about the same percentage as in wild-type cells. The best reconstitution was seen with 0.5 to 1 mM MBP in the reconstitution mixture, which is similar to the periplasmic MBP concentration estimated for maltose-induced wild-type cells. Strains containing large deletions of the malB region and malT mutants lacking the positive regulator gene of the mal regulon also could be reconstituted for maltose chemotaxis, showing that no product of the mal regulon other than MBP is essential for maltose chemotaxis.1. Adler, J. 1973. A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli.