malB Region Research Articles

Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein.

Mal+ lacZ operon fusions, inducible by maltose, were isolated in Escherichia coli, strain MC4100. One fusion strain, SF1707, was analyzed in detail. This fusion did not map in any of the known genes of the malA or malB region, but its expression was under control of malT, the positive regulator gene of the maltose regulon. The gene in which the fusion occurred mapped between xyl and mtl at 80 min on the linkage map and was transcribed clockwise. We define this gene as malS. The malS-lacZ fusion was transferred onto a phage lambda vector and the 5' portion of malS was subcloned into pBR322. The resulting plasmid was used as a probe to identify the intact malS gene in a lambda library of E. coli chromosomal HindIII fragments. The phage that hybridized with the probe contained a 12-kilobase insert. The malS containing portion was subcloned into pBR322 as a 4-kilobase ClaI-HindIII fragment. This plasmid directed the malT and maltose-dependent synthesis of a periplasmic protein of 66,000 apparent molecular weight. The purified enzyme hydrolyzed maltodextrins longer than maltose including cyclic dextrins. The primary products of hydrolysis were glucose, maltose, and maltotriose, even when maltotetraose was used as a substrate. These properties differentiate this periplasmic enzyme from the cytoplasmic amylomaltase and define it as an alpha-amylase.

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

Maltose chemotaxis was reconstituted in delta malE cells lacking maltose-binding protein (MBP). Purified MBP was introduced into intact cells during incubation with 250 mM CaCl2 in Tris-hydrochloride buffer at 0 degrees C. 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.

Mutations in the promoter regions of the malEFG and malK-lamB operons of Escherichia coli K12

The malB region of Escherichia coli is composed of two operons, malEFG and malK-lamB, transcribed divergently from a control region located between the malE and malK genes. Expression of the malB operons is under the positive control of the malT gene product (MalT) and maltose and of the crp gene product (CRP) and cyclic AMP. Strains in which the lac genes have been fused to malE or malK are unable to use lactose as carbon source if they have been deleted for malT or crp. Mutations in the malB region allowing such fusion strains to grow on lactose have been isolated. These and previously isolated mutations were genetically characterized. As regards the malEp promoter mutations, malEp9, malEp1 and malEp6 create new promoters that are MalT and CRP independent. malEp9 and malEp1 change residues -1 and -2, respectively, of malEp without altering its activity. malEp6 duplicates six base-pairs between residues -22 and -23. malEp3 improves the -10 region hexamer. malEp5 deletes residues -29 to -62. It creates a new promoter that is MalT independent, CRP dependent, likely by fusing together functional regions of malEp that are normally apart. malEp5 also reduces the expression of malK-lamB, suggesting the existence of a link between the malEp and malKp promoters. As regards the malKp mutations, malKp6 changes residue -81 of malKp without altering its activity. It creates a new promoter, which is MalT independent, CRP dependent, likely by using a pre-existing cyclic AMP/CRP binding site. malKp102 changes residue -36, two bases upstream of the -35 region hexamer. It decreases the activity of malKp by at least four orders of magnitude and likely alters the MalT binding site. These results are discussed in terms of regulatory interactions within the malB control region.

Promoters of the malEFG and malK-lamB operons in Escherichia coli K12

The malB region is composed of two operons, malEFG and malK-lamB, transcribed divergently from a control region located between malE and malK. We determined the precise sites at which transcription of the malB operons starts in vivo. These initiation sites define the promoter signals for malEFG and malK-lamB. Comparison of the malE and malK promoter sequences reveals homologies that are likely to correspond to the sites at which the malT product and the cyclic-AMP receptor protein activate expression of the malB operons.

A novel intercistronic regulatory element of prokaryotic operons.

The nucleotide sequences of the intercistronic regions from several multicistronic operons have recently been determined. In many cases such intercistronic regions consist of only a few base pairs (bp); in certain extreme cases, where the initiation codon of one gene overlaps the termination codon of the previous gene, intercistronic regions are essentially absent1,2. However, in other cases intercistronic regions can be up to several hundred base pairs long; it seems reasonable to suppose that, at least in these cases, such regions have a role in regulating expression of the operon. Indeed, a regulatory function for the large intercistronic region of the rplJL–rpoBC operon has been demonstrated3. We have now identified a genetic element, common to intercistronic regions from three independent bacterial operons (the histidine transport and the histidine biosynthetic operons of Salmonella typhimurium and the malK–lamB operon of the malB region of Escherichia coli), which we believe to be important in regulating the differential expression of individual genes in these multicistronic operons. A similar element is also found immediately following the trpR gene of E. coli although in this case it is not known whether or not a distal gene exists. Each of these elements consists of a long dyad symmetry which, once transcribed, could form an exceptionally stable stem–loop structure with a stem of up to 35 bp. In each case smaller palindromic units are also found, some of which overlap the main symmetry. The stem–loop structures show remarkable homology (---90%) and probably have a common evolutionary origin, possibly an insertion element. It seems that the function of these structures is to effect a decrease in expression of distal genes in an operon.

A DNA sequence containing the control regions of the malEFG and malK-lamB operons in Escherichia coli K12.

The malB region in E. coli is composed of two operons, malEFG and malK-lamB, transcribed divergently from a control region located between the malE and malK genes. We have established the DNA sequence of a 600 base pair segment which contains the sites necessary for initiation of transcription of the malB operons and the translation start sites for malE and malK. We have also determined the position of a 147 base pair deletion, malB delta 100, in this sequence. The control region contains two short segments (about 35 base pairs) showing unusual distributions of bases: the GC content (85%) is higher than average and most of the purines (85%) are on one strand. We discuss the possible relevance of this and other salient features of the DNA sequence to the mechanisms by which the expression of the malB region is positively regulated.

75] Maltose-binding protein from Escherichia coli

Publisher Summary This chapter describes the assay method, the purification procedure, and the properties of maltose-binding protein from Escherichia coli . The maltose-binding protein is a periplasmic protein of Escherichia coli that is involved in the transport of maltose and maltodextrins across the bacterial envelope and in the chemotaxis toward these sugars. Maltose binding is measured by equilibrium dialysis using [ 14 C]maltose. The chapter presents two purification procedures for maltose-binding protein—growth of cell and osmotic shock treatment, and QAE-Sephadex chromatography. The alternative purification procedure is affinity chromatography. Both methods yield a protein that is more than 90% pure as estimated by electrophoresis on polyacrylamide gels in different conditions. A molecular weight of 40,000 to 44,000 of the protein is determined by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate. The maltose binding protein is coded by gene malE , one of the five cistrons of the malB region involved in the transport of maltose and maltodextrins across the envelope of E.coli.

Rho Mutations restore lamB expression in E. coli K12 strains with an inactive malB region.

lamB, the structural gene for lambda receptor, is the second gene of the malK-lamB operon in the malB region of the Escherichia coli K12 chromosome. lamB is essentially not expressed in the absence of an active malT gene product, the activator of the maltose regulon. A malT strain is resistant to phage lambda. We show that: (i) Introduction of rho mutations in malT mutants restores lamB expression to a level sufficient to render the strain sensitive to phage lambda; (ii) This restoration is not dependent on the main promoter of the malK lamB operon. It depends on the distal part of the malK gene. We propose that rho inactivation unmasks the activity of a promoter located near the distal end of malK. Experiments with Mu insertions in gene malK suggest that in the (-) orientation a Mu promoter is also able to allow lamB expression in a rho background.

Cosmid cloning and transposon mutagenesis in Salmonella typhimurium using phage lambda vehicles.

We have constructed a strain of Salmonella typhimurium which contains the malB region from Escherichia coli and carries the bacteriophage lambda receptor protein in its outer membrane. Phage lambda adsorbs to this strain but cannot grow, thus providing a very useful system for transposon mutagenesis of S. typhimurium using lambda vehicles carrying transposons. This system can also be used for cosmid cloning.

Structure of the malB region in Escherichia coli K12

Starting with a strain containing a malK-lacZ fusion, a series of lambda plaque-forming phages which carry varying amounts of the malE,F operon have been isolated. We have used these phages to construct a deletion map of the malE,F operon. The construction of this deletion map has led to the identification of a new gene, malG. The malG gene is located distal to malF. The malG gene product is a protein required for the active transport of maltose and maltodextrins.

Structure of the malB region in Escherichia coli K12

Structure of the malB region in Escherichia coli K12

Structure of the malB region in Escherichia coli K12

A correlation between the genetic and physical maps of the malB region was obtained by performing a restriction cleavage analysis of DNA's carrying various genetically characterized malB deletions. This also allowed to localize the boundaries between malF and malE, malE and malK, mal K and lamB on the restriction map. The genetic map is not grossly distorted with respect to the physical map.

MalB region in Escherichia coli K-12: specialized transducing bacteriophages and first restriction map

By starting from an Escherichia coli K-12 strain with a lambda phage integrated in the malB region, series of transducing phages carrying part or all of the malB region have been isolated. Genetic mapping of the transduced malB fragments was accomplished by complementation and recombination with known mutations in the region. By using the DNA of these phages, it was found that the malB region is cleaved by the restriction enzymes BglII, EcoRI, HaeII, HincII, SalI, and SstI, but not BamHI, HindIII, KpnI, PstI, XbaI, or XhoI. A physical map was constructed and tentatively correlated with the genetic map.

Active transport of maltose in Escherichia coli K12. Involvement of a "periplasmic" maltose binding protein.

A “periplasmic” maltose binding protein was purified from Escherichia coli. This protein is shown to be under the same positive control as the whole maltose system, and its synthesis is inducible by maltose. This binding protein is coded by malE, one of the three cistrons of the malB region involved in the transport of maltose.The binding characteristics of this protein, as well as the kinetics of release of bound maltose, suggest that the binding protein can exist in two states differing by their affinity towards maltose. The significance of this result is discussed in view of the role that the protein is believed to play in maltose transport.

On some genetic aspects of phage lambda resistance in E. coli K12.

Most mutations rendering E. coli K12 resistant to phage lambda, map in two genetic regions malA and malB.-The malB region contains a gene lamB specifically involved in the lambda receptor synthesis. Twenty-one independent lamB mutations studied by complementation belonged to a single cistron. This makes it very likely that lamB is monocistronic. Among the lamB mutants some are still sensitive to a host range mutant of phage lambda. Mutations mapping in a proximal gene essential for maltose metabolism inactivate gene lamB by polarity confirming that both genes are part of the same operon. Because cases of intracistronic complementation have been found, the active lamB product may be an oligomeric protein.-Previously all lambda resistant mutations in the malA region have been shown to map in the malT cistron. malT is believed to be a positive regulatory gene necessary for the induction of the "maltose operons" in the malA region and in the malB region of the E. coli K12 genetic map. No trans dominant malT mutation have been found. Therefore if they exist, they occur at a frequency of less than 10(-8), or strongly reduce the growth rate of the mutants.

Genes affecting sensitivity to ultraviolet light in the malB region of the chromosome of Escherichia coli.

Genes affecting sensitivity to ultraviolet light in the malB region of the chromosome of Escherichia coli.