The reconstitution of active transport by the Ca2+-induced import of exogenous binding protein was studied in detail in whole cells of a malE deletion mutant lacking the periplasmic maltose-binding protein. A linear increase in reconstitution efficiency was observed by increasing the Ca2+concentration in the reconstitution mixture up to 400 mM. A sharp pH optimum around pH 7.5 was measured for reconstitution. Reconstitution efficiency was highest at 0°C and decreased sharply with increasing temperature. The time necessary for optimal reconstitution at 0°C and 250 mM Ca was about 1 min. The competence for reconstitution was highest in exponentially growing cultures with cell densities up to 1 x 109/ml and declined when the cells entered the stationary-growth phase. The apparent Km for maltose uptake was the same as that of wild-type cells (1 to 2 puM). Vmax at saturating maltose-binding protein concentration was 125 pmol per min per 7.5 x 107 cells (30% of the wild-type activity). The concentration of maltose-binding protein required for half-maximal reconstitution was about 1 mM. The reconstitution procedure appears to be generally applicable. Thus, galactose transport in Escherichia coli could also be reconstituted by its respective binding protein. Maltose transport in E. coli was restored by maltose-binding protein isolated from Salmonella typhimurium. Finally, in S. typhimurium, histidine transport was reconstituted by the addition of shock fluid containing histidinebinding protein to a hisJ deletion mutant lacking histidine-binding protein. The method is fast and general enough to be used as a screening procedure to distinguish between transport mutants in which only the binding protein is affected and those in which additional transport components are affected.

The barrier function of the Escherichia coli outer membrane against low concentrations of maltose in strains missing the lambda receptor was partially overcome by treating the cells for 3 h with 25 mM Ca2+. Kinetic analysis of maltose-transport revealed a Ca2+-induced shift of the apparent Km of the system from about 100 microM in cells pretreated with Tris to about 15 microM in cells pretreated with Tris plus Ca2+. In contrast to maltose transport in untreated cells, that of Ca2+-treated lamB cells was inhibited by molecules with a high molecular weight, such as amylopectin (molecular weight, 20,000), and anti-maltose-binding protein antibodies. In addition, lysozyme was shown to attack Ca2+-treated cells in contrast to untreated cells. The Ca2+-induced permeability increase of the outer membrane allowed reconstitution of maltose transport in a mutant missing the maltose-binding protein with osmotic shock fluid containing the maltose-binding protein. Even though Ca2+-treatment allowed the entry of large molecules, the release of the periplasmic maltose-binding protein or alkaline phosphatase was negligible.

Among TnlO insertions isolated in or near the malB region of Escherichia coli, one (zjb-729::TnlO) mapped between malK and lamB or late in malK and allowed MalT-independent expression of lamB. TnlO-dependent expression of a lamB-lacZ protein fusion was 25% of the expression of the fusion from the malK-lamB operon promoter in maiTh constitutive strains. The maltoporin content of a strain carrying this TnlO was about 20% that of a maiTh malB+ strain. Transport of maltose at concentrations of below 10-6 M was reduced about threefold. When maltoporin was present at about 50% of the level of mal7 malB+ strains, maltose transport was largely restored. We conclude that maltoporin is not rate limiting for maltose transport in wild-type cells but becomes rate limiting when the ratio of maltoporin to other maltose transport components is reduced more than twofold.

The E. coli biotin (bio) operon was modified to improve biotin production by host cells: (a) the divergently transcribed wild-type bio operon was re-organized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ribosomal binding site (RBS) was replaced with an artificial RBS that resulted in improved bioB expression. The effects of the modifications on the bio operon were studied in E. coli by measuring biotin and dethiobiotin production, and bio gene expression with mini-cells and two-dimensional polyacrylamide gel electrophoresis. The modified E. coli bio operon was introduced into a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material. Biotin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth conditions.

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.