Selection for High Mutation Rates in Chemostats

Complementation and polarity suppression data are interpreted in terms of the genetic structure of the maltose B region. It is proposed that this region comprises two divergent operons. One operon includes malK, a cistron involved in maltose permeation, and lamB the only known cistron specifically involved in lambda receptor synthesis. The other operon includes malJ(1) and malJ(2) which are most probably two different cistrons, both involved in maltose permeation*. It is further assumed that expression of the two operons is controlled by malT, the positive regulatory gene of the maltose system, located in the malA region. The target(s) for the action of the malT product is (are) most likely to be located between malJ(1) and malK. There is an indication that the two operons might overlap in the region of their promoters. The structure of such an overlap as well as the possible function of the products of the different cistrons in malB are briefly discussed.     

On Some Genetic Aspects of Phage λ 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.     

Transposon Tn10-dependent expression of the lamB gene in Escherichia coli.

Among Tn10 insertions isolated in or near the malB region of Escherichia coli, one (zjb-729::Tn10) mapped between malK and lamB or late in malK and allowed MalT-independent expression of lamB. Tn10-dependent expression of a lamB-lacZ protein fusion was 25% of the expression of the fusion from the malK-lamB operon promoter in malTc constitutive strains. The maltoporin content of a strain carrying this Tn10 was about 20% that of a malTc 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 malTc 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.     

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.     

Temperature Sensitivity of Maltose Utilization and Lambda Resistance in Escherichia coli B

Escherichia coli B strains that have acquired the malB region from E. coli K-12 are able to utilize maltose and to adsorb phage lambda when grown at 30 C, but when grown at 40 C they do not absorb phage lambda and are devoid of amylomaltase activity. These Mal(ts) Lam(ts) cells can be mutated or transduced to become able to grow on maltose at 40 C, but they still have no detectable amylomaltase activity nor functional lambda receptors at that temperature. This Mal(40) phenotype is governed by a gene located near or at malA. It is suggested that the temperature sensitivity of both characters results from a defect in malT. However, transduction of malA from E. coli B to E. coli K-12 results in a wild-type phenotype, whereas E. coli B cells that have acquired malA from E. coli K-12 donors are still temperature sensitive for both amylomaltase and lambda-receptor production.     

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.     

Nucleotide sequence of the regulatory region of malB operons in E. coli

The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the promoter regions of the malB divergent operons was determined. The region of the proximal gene, malE of the malEFG operon, was identified on the basis of the known amino acid sequence of the precursor molecule of maltose-binding protein. The region of malK, the proximal gene of the malKlamB operon, was deduced from the observation that a cloned segment contains an amino-terminal portion of the malK gene. The non-coding region between malE and malK is 299 base pairs long and contains two long GC clusters. Another feature of this region that may be related to the regulation of gene expression is the presence of two palindromic structures between the GC clusters. The DNA regions binding to cyclic AMP binding protein were determined by a method using polyacrylamide gel electrophoresis. The sites are thought to be located close to GC clusters.     

Identification of the malK gene product. A peripheral membrane component of the Escherichia coli maltose transport system

The malK gene product of Escherichia coli has been identified through the use of a previously described technique that employs gene fusions (Shuman, H. A., Silhavy, T. J., and Beckwith, J. R. (1980) J. Biol. Chem. 255, 168-174). This protein, along with the four other products of the malB locus, comprise the complete maltose transport system. The malK protein has a molecular weight of approximately 40,000 and is located in the cell envelope. In mutant strains which lack another component of the transport system, the malG protein, the malK protein is located in the cytoplasm. This alteration in location suggests that the malK protein is associated with the inner surface of the cytoplasmic membrane via an interaction with the malG protein.     

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.     

Structure of the malB region in Escherichia coli K12. III. Correlation of the genetic map with the restriction map.

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.     

Induction of the lambda receptor is essential for effective uptake of trehalose in Escherichia coli.

Trehalose transport in Escherichia coli after growth at low osmolarity is mediated by enzyme IITre of the phosphotransferase system (W. Boos, U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma, J. Bacteriol. 172:3450-3461, 1990). The apparent Km (16 microM) of trehalose uptake is low. Since trehalose is a good source of carbon and the apparent affinity of the uptake system is high, it was surprising that the disaccharide trehalose [O-alpha-D-glucosyl(1-1)-alpha-D-glucoside] has no problems diffusing through the outer membrane at high enough rates to allow full growth, particularly at low substrate concentrations. Here we show that induction of the maltose regulon is required for efficient utilization of trehalose. malT mutants that lack expression of all maltose genes, as well as lamB mutants that lack only the lambda receptor (maltoporin), still grow on trehalose at the usual high (10 mM) trehalose concentrations in agar plates, but they exhibit the half-maximal rate of trehalose uptake at concentrations that are 50-fold higher than in the wild-type (malT+) strain. The maltose system is induced by trehalose to about 30% of the fully induced level reached when grown in the presence of maltose in a malT+ strain or when grown on glycerol in a maltose-constitutive strain [malT(Con)]. The 30% level of maximal expression is sufficient for maximal trehalose utilization, since there is no difference in the concentration of trehalose required for the half-maximal rate of uptake in trehalose-grown strains with the wild-type gene (malT+) or with strains constitutive for the maltose system [malT(Con)]. In contrast, when the expression of the lambda receptor is reduced to less than 20% of the maximal level, trehalose uptake becomes less efficient. Induction of the maltose system by trehalose requires metabolism of trehalose. Mutants lacking amylotrehalase, the key enzyme in trehalose utilization, accumulate trehalose but do not induce the maltose system.     

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.     

Maltose chemoreceptor of Escherichia coli.

Strains carrying mutations in the maltose system of Escherichia coli were assayed for maltose taxis, maltose uptake at 1 and 10 muM maltose, and maltose-binding activity released by osmotic shock. An earlier conclusion that the metabolism of maltose is not necessary for chemoreception is extended to include the functioning of maltodextrin phosphorylase, the product of malP, and the genetic control of the maltose receptor by the product of malT is confirmed. Mutants in malF and malK are defective in maltose transport at low concentrations as well as high concentrations, as previously shown, but are essentially normal in maltose taxis. The product of malE has been previously shown to be the maltose-binding protein and was implicated in maltose transport. Most malE mutants are defective in maltose taxis, and all those tested are defective in maltose transport at low concentrations. Thus, as previously suggested, the maltose-binding protein probably serves as the recognition component of the maltose receptor, as well as a component of the transport system. tsome malE mutants release maltose-binding activity and are tactic toward maltose, although defective in maltose transport, implying that the binding protein has separate sites for interaction with the chemotaxis and transport systems. Some mutations in lamB, whose product is the receptor for the bacteriophage lamba, cause defects in maltose taxis, indicating some involvement of that product in maltose reception.     

Sequence of the malK gene in E.coli K12.

We present the sequence of gene malK which encodes a component of the system for maltose transport in E.coli K12. We also determined the position of deletion (S50) which fuses malK to the following gene lamB; the malK-lamB protein hybrid contains all of the malK protein. The mRNA corresponding to the last two thirds of gene malK could form stable stem and loop structures. The malK protein, as deduced from the gene sequence, would include 370 residues and correspond to a molecular weight of 40700. The sequence as well as sequence comparisons with the ndh protein of E.coli are discussed in terms of the location and function of the malK protein.     

Maltose transport in Escherichia coli K-12: involvement of the bacteriophage lambda receptor.

Mutants affected in lamB, the structural gene for phage lambda receptor, are unable to utilize maltose when it is present at low concentrations (less than or equal 10 muM). During growth in a chemostat at limiting maltose concentrations, the lamB mutants tested were selected against in the presence of the wild-type strain. Transport studies demonstrate that most lamB mutants have deficient maltose transport capacities at low maltose concentrations. When antibodies against purified phage lambda receptor are added to a wild-type strain, transport of maltose at low concentrations is significantly reduced. These results strongly suggest that the phage lambda receptor molecule is involved in maltose transport.     

Role of the receptor for bacteriophage lambda in the functioning of the maltose chemoreceptor of Escherichia coli.

Chemotaxis towards maltose is specifically defective in many strains of Escherichia coli carrying mutations affecting lamB, the gene coding for the outer membrane receptor for bacteriophage lambda. However, with one exception, the most extreme effect of lamB mutants on the maltose response as determined in the capillary assay is a shift to higher sugar concentrations and a reduction in the number of bacteria accumulated to about 25% of the wild-type level. The severity of the taxis defect is strongly correlated with reduced ability of the cells to take up the maltose present at 1 and 10 muM. Evidence presented here and in the accompanying paper indicates that the lambda receptor is involved in the transport of maltose at these concentrations. The effects of lamB mutations on maltose taxis can be explained by postulating that the high-affinity maltose transport system in which the lambda receptor participates transfers maltose from the surrounding medium across the outer membrane and into the periplasmic space. If the maltose chemoreceptor detects sugar present in the periplasmic space, and not molecules external to the outer membrane, then defective transport of low concentrations of maltose into the periplasm would result in the observed apparent reduction in the sensitivity of the maltose receptor. Thus, the lambda receptor protein would participate in maltose chemorecepton only indirectly through its role in maltose transport.     

Distribution of mannosamine and mannosaminuronic acid among cell walls of Bacillus species

Some Escherichia coli K-12 lamB mutants, those producing reduced amounts of LamB protein (one-tenth the wild type amount), grow normally on dextrins but transport maltose when present at a concentration of 1 microM at about one-tenth the normal rate. lamB Dex- mutants were found as derivatives of these strains. These Dex- mutants are considerably impaired in the transport of maltose at low concentrations (below 10 microM), and they have a structurally altered LamB protein which is impaired in its interaction with phages lambda and K10 but still interacts with a lambda host range mutant lambda hh*. The Dex- mutants are double lamB mutants carrying one mutation, already present in the parental strains, that reduces LamB synthesis and a second that alters LamB structure. The secondary mutations, present in different independent Dex- mutants, are clustered in the same region of the lamB gene. Dex+ revertants were isolated and analyzed: when the altered LamB protein is made in wild-type amount, due to a reversion of the first mutation, the phenotype reverts to Dex+. However, these Dex+ revertants are still very significantly impaired in maltose transport at low concentrations (below 10 microM).