Osmoregulation of the maltose regulon in Escherichia coli.

The maltose regulon consists of four operons that direct the synthesis of proteins required for the transport and metabolism of maltose and maltodextrins. Expression of the mal genes is induced by maltose and maltodextrins and is dependent on a specific positive regulator, the MalT protein, as well as on the cyclic AMP-catabolite gene activator protein complex. In the absence of an exogenous inducer, expression of the mal regulon was greatly reduced when the osmolarity of the growth medium was high; maltose-induced expression was not affected, and malTc-dependent expression was only weakly affected. Mutants lacking MalK, a cytoplasmic membrane protein required for maltose transport, expressed the remaining mal genes at a high level, presumably because an internal inducer of the mal system accumulated; this expression was also strongly repressed at high osmolarity. The repression of mal regulon expression at high osmolarity was not caused by reduced expression of the malT, envZ, or crp gene or by changes in cellular cyclic AMP levels. In strains carrying mutations in genes encoding amylomaltase (malQ), maltodextrin phosphorylase (malP), amylase (malS), or glycogen (glg), malK mutations still led to elevated expression at low osmolarity. The repression at high osmolarity no longer occurred in malQ mutants, however, provided that glycogen was present.     

Characterization of the aes gene of Escherichia coli encoding an enzyme with esterase activity.

malQ mutants of Escherichia coli lacking amylomaltase cannot grow on maltose. They express the maltose system constitutively and are sensitive to maltose when grown on another carbon source. In an attempt to isolate a multicopy suppressor that would result in growth on maltose, we transformed a malQ mutant with a gene bank of E. coli DNA which had been digested with Sau3a and cloned in pBR322. We screened the transformants on MacConkey maltose plates. A colony was isolated that appeared to be resistant to maltose and was pink on these plates, but it was still unable to grow on minimal medium with maltose as the carbon source. The plasmid was isolated, and the gene causing this phenotype was characterized. The deduced amino acid sequence of the encoded protein shows homology to that of lipases and esterases. We termed the gene aes, for acetyl esterase. Extracts of cells harboring plasmid-encoded aes under its own promoter exhibit a fivefold higher capacity to hydrolyze p-nitrophenyl acetate than do extracts of cells of plasmid-free strains. Similarly, strains harboring plasmid-encoded aes are able to grow on triacetyl glycerol (triacetin) whereas the plasmid-free strains are not. The expression of plasmid-encoded aes resulted in strong repression of the maltose transport genes in malT+ strains (10-fold reduction), but not in a malT(Con) strain which is independent of the inducer. Also, overproduction of MalT counteracted the Aes-dependent repression, indicating a direct interaction between MalT and Aes.     

Role of the catabolite activator protein in the maltose regulon of Escherichia coli.

The maltose regulon consists of three operons controlled by a positive regulatory gene, malT. Deletions of the gene crp were introduced into strains which carried a malT-lacZ hybrid gene. From the observed reduction in beta-galactosidase activity it was concluded that the expression of malT-lacZ, and therefore of malT, is controlled by the catabolite activator protein (CAP), the product of the gene crp. Mutations were obtained which allowed a malT-lacZ hybrid gene to be expressed at a high level even in the absence of CAP. These mutations were shown to be located in or close to the promoter of the malT gene and were called malTp. The malTp mutations were transferred in the cis position to a wild-type malT gene. In the resulting strains, the expression of two of the maltose operons, malEFG and malK-lamB, still required the action of CAP, whereas that of the third operon, malPQ, was CAP independent. Therefore, in wild-type cells, CAP appears to control malPQ expression mainly, if not solely, by regulating the concentration of MalT protein in the cell. On the other hand, it controls the other two operons more stringently, both by regulating malT expression and by a more direct action, probably exerted in the promoters of these operons.     

The malX malY operon of Escherichia coli encodes a novel enzyme II of the phosphotransferase system recognizing glucose and maltose and an enzyme abolishing the endogenous induction of the maltose system.

Mutants lacking MalK, a subunit of the binding protein-dependent maltose-maltodextrin transport system, constitutively express the maltose genes. A second site mutation in malI abolishes the constitutive expression. The malI gene (at 36 min on the linkage map) codes for a typical repressor protein that is homologous to the Escherichia coli LacI, GalR, or CytR repressor (J. Reidl, K. R?misch, M. Ehrmann, and W. Boos, J. Bacteriol. 171:4888-4899, 1989). We now report that MalI regulates an adjacent and divergently oriented operon containing malX and malY. MalX encodes a protein with a molecular weight of 56,654, and the deduced amino acid sequence of MalX exhibits 34.9% identity to the enzyme II of the phosphototransferase system for glucose (ptsG) and 32.1% identity to the enzyme II for N-acetylglucosamine (nagE). When constitutively expressed, malX can complement a ptsG ptsM double mutant for growth on glucose. Also, a delta malE malT(Con) strain that is unable to grow on maltose due to its maltose transport defect becomes Mal+ after introduction of malI::Tn10 and the plasmid carrying malX. MalX-mediated transport of glucose and maltose is likely to occur by facilitated diffusion. We conclude that malX encodes a phosphotransferase system enzyme II that can recognize glucose and maltose as substrates even though these sugars may not represent the natural substrates of the system. The second gene in the operon, malY, encodes a protein of 43,500 daltons. Its deduced amino acid sequence exhibits weak homology to aminotransferase sequences. The presence of plasmid-encoded MalX alone was sufficient for complementing growth on glucose in a ptsM ptsG glk mutant, and the plasmid-encoded MalY alone was sufficient to abolish the constitutivity of the mal genes in a malK mutant. The overexpression of malY in a strain that is wild type with respect to the maltose genes strongly interferes with growth on maltose. This is not the case in a malT(Con) strain that expresses the mal genes constitutively. We conclude that malY encodes an enzyme that degrades the inducer of the maltose system or prevents its synthesis.     

Overproduction of MalK protein prevents expression of the Escherichia coli mal regulon.

The mal regulon of Escherichia coli comprises a large family of genes whose function is the metabolism of linear maltooligosaccharides. Five gene products are required for the active accumulation of maltodextrins as large as maltoheptaose. Two cytoplasmic gene products are necessary and sufficient for the intracellular catabolism of these sugars. Two newly discovered enzymes have the capacity to metabolize these sugars but are not essential for their catabolism in wild-type cells. A single regulatory protein, MalT, positively regulates the expression of all of these genes in response to intracellular inducers, one of which has been identified as maltotriose. In the course of studying the mechanism of the transport system, we have placed the structural gene for one of the transport proteins, MalK, under the control of the Ptrc promoter to produce large amounts of this protein. We found that although high-level expression of MalK was not detrimental to E. coli, the increased amount of MalK decreased the basal-level expression of the mal regulon and prevented induction of the mal system even in the presence of external maltooligosaccharides. Constitutive mutants in which MalT does not depend on the presence of the internal inducer(s) were unaffected by the increased levels of the MalK protein. These results are consistent with the idea that MalK protein somehow interferes with the activity of the MalT protein. Different models for the regulatory function of MalK are discussed.