Metabolic pathway for the utilization of L-arginine, L-ornithine, agmatine, and putrescine as nitrogen sources in Escherichia coli K-12

The structural genes (melB) for the melibiose carrier of five mutants of Escherichia coli showing altered cation specificity for melibiose transport were cloned. The mutations were mapped in a 248-base-pair DNA fragment by a recombinational assay by using the mutants transformed with hybrid plasmids carrying various portions of the wild-type melB gene. The nucleotide sequences of the corresponding DNA fragments derived from mutated melB genes were determined, and the amino acid sequences of the carrier were deduced. Proline 122 was replaced with serine in the melibiose carrier of all five mutants (which were isolated independently). We conclude that this amino acid replacement caused the alteration in cation specificity (loss of coupling to H+) of the melibiose carrier.     

Melibiose permease and alpha-galactosidase of Escherichia coli: identification by selective labeling using a T7 RNA polymerase/promoter expression system

Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074]. Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiation codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested [Hanatani et al. (1984) J. Biol. Chem. 259, 1807].     

Nucleotide sequence of the melB gene and characteristics of deduced amino acid sequence of the melibiose carrier in Escherichia coli

The nucleotide sequence of the melB gene coding for the melibiose carrier in Escherichia coli has been determined. The melibiose carrier is predicted to consist of 469 amino acid residues, resulting in a protein with a molecular weight of 52,029. The predicted carrier protein is highly hydrophobic (70% nonpolar amino acid residues). The hydropathic profile suggests that there are 10 long hydrophobic segments in the primary structure of the carrier protein. Most of them seem to traverse the membrane. Although the hydropathic profile of the melibiose carrier is similar to that of the lactose carrier as a whole, homology in the primary structure between the two carriers is very low. Furthermore, no homology in the nucleotide sequence is found in the structural genes for the two carriers. However, the nucleotide sequences of the intergenic regions are very similar between the melibiose operon and the lactose operon. There is a typical intercistronic regulatory sequence in the 3'-flanking region of the melB as well as in that of the lacY, which suggests the presence of another gene downstream of the melB.     

Cloning and mapping of the genetic determinants for microcin C7 production and immunity.

Microcin C7, a peptide antibiotic inhibitor of protein synthesis, is produced by Escherichia coli K-12 strains that carry the 43-kilobase low-copy-number plasmid pMccC7. Microcin C7 production and immunity determinants of this plasmid have been cloned into the vectors pBR322 and pACYC184. The resulting plasmids overproduce microcin C7 and express immunity against the microcin. Mcc- and Mcc- Imm- mutants have been isolated on recombinant plasmids by inserting transposable elements. Physical and phenotypic characterization of these mutants shows that a DNA region of 5 kilobases is required to produce microcin C7, and that two small regions located inside the producing region are also required to express immunity. Analysis of plasmids carrying mcc-lacZ gene fusions indicates that all microcin DNA is transcribed in the same direction. The results suggest that a structure like a polycistronic operon is responsible for microcin C7 production and immunity.     

Clonal analysis of the lac operons from Klebsiella M5al and the Lac plasmid (pRE2) from Klebsiella V9A

The chromosomal lac region of the coliform bacterium Klebsiella M5al was cloned into the multicopy plasmid pBR322 to give pHE7 and pHE8. pHE8 contains 12.6 kb of M5al DNA, including its complete lac operon, and pHE7 contains 2.5 kb of M5al DNA and includes the complete lac Y gene and a small segment of lacZ. The M5al operon has the same gene order as the Escherichia coli lac operon. The lac genes of the Lac plasmid of Klebsiella V9A were cloned into pBR322 to give pHE1 and pHE2, of approximately 39 and 43 kb. Both plasmids were unstable in an E. coli RecA-strain, in contrast to the stability of pHE8. Polyacrylamide gel electrophoresis tests suggested that the M5al beta-galactosidase monomer is about 5% longer, i.e. has about 50 more amino acids, than that of the E. coli Z gene. Tests made on the enzymes coded by the lac operons of M5al, another Klebsiella strain (V9A) and its resident Lac plasmid, and several Lac+ Enterobacteria, led to the conclusion that only Escherichia coli among the Enterobacteria contains an active lacA gene.     

Galactoside-dependent proton transport by mutants of the Escherichia coli lactose carrier: substitution of tyrosine for histidine-322 and of leucine for serine-306

The lac Y genes from two Escherichia coli mutants, MAB20 and AA22, have been cloned in a multicopy plasmid by a novel 'sucrose marker exchange' method. Characterization showed that the plasmids express a lactose carrier with poor affinity for lactose. Neither mutant carried out concentrative uptake with methyl beta-D-galactopyranoside, lactose, or melibiose as the substrate. Nor did the mutants catalyze counterflow or exchange with methyl beta-D-galactopyranoside. Both mutants did, however, retain the capacity to carry out facilitated diffusion with lactose or melibiose. DNA sequencing revealed that MAB20 (histidine-322 to tyrosine) and AA22 (serine-306 to leucine) have amino acid substitutions within the putative 'charge-relay' domain thought to be responsible for proton transport. Galactoside-dependent H+ transport was readily measured in both mutants. We conclude, therefore, that the presence of a histidine residue at position 322 of the lactose carrier is not obligatory for H+ transport per se.     

Resistance of the melibiose carrier to inhibition by the phosphotransferase system due to substitutions of amino acid residues in the carrier of Salmonella typhimurium.

The melibiose carrier of Salmonella typhimurium is under the control of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). We isolated mutants of the melibiose carrier that showed resistance to inhibition via the PTS. Growth of the mutants on melibiose was not inhibited by 2-deoxyglucose, a non-metabolizable substrate of the PTS, although growth of the parent strain was inhibited. Transport activity of the melibiose carrier in the mutants was fairly resistant to inhibition by 2-deoxyglucose, although the activity in the parent was sensitive to inhibition. We cloned the mutated melB gene that encodes the melibiose carrier, determined the nucleotide sequences, and identified replaced nucleotides. The mutations resulted in substitutions of Asp-438 with Tyr, Arg-441 with Ser, or Ile-445 with Asn. All of these residues are in the COOH-terminal region of the carrier. The secondary structure of this region is predicted to be an alpha-helix, and the mutated residues were on the same side of the helix. This region showed sequence similarity to a region of the MalK protein, in which substitution of amino acid residues also resulted in PTS-resistant mutants. Thus the COOH-terminal portion of the melibiose carrier is important for the interaction of dephosphorylated IIIGlc, which is an entity causing reversible inactivation of the carrier.     

In vitro and in vivo manipulations of bacteriophage Mu DNA: cloning of Mu ends and construction of mini-Mu's carrying selectable markers

Recombinant plasmids carrying one or both ends of the bacteriophage Mu genome were constructed by molecular cloning. Transposable mini-Mu's with selectable markers (ampicillin resistance, kanamycin resistance or the entire lac operon of Escherichia coli) inserted between the Mu ends were also constructed. As a source of lac operon DNA, a pBR322 derivative with a 27 kb insert containing the lac operon was constructed. The plasmids with both ends of Mu (mini-Mu's) conferred full Mu immunity upon the host cells. However, the same mini-Mu's containing kan or lac inserts were defective in immunity. A summary of the construction and physical characterization, including restriction endonuclease cleavage maps and some of the biological properties of the plasmids, is presented.     

Plasmid vectors for positive galactose-resistance selection of cloned DNA in Escherichia coli

Insertion of a HindIII-EcoRI fragment carrying part of the gal operon from lambda gal+ into pBR322 yields a plasmid (pAA3) which confers strong galactose sensitivity on E. coli strains deleted for the gal operon. Sensitivity to galactose is caused by the expression of kinase and transferase (but not epimerase) genes from a promoter located in the tet gene of pBR322. Insertion of a DNA fragment carrying Tn9 at the HindIII junction blocks gal expression and produces a galactose-resistant phenotype. Hence, galactose resistance can be used to select DNA fragments cloned at the HindIII site. The system was used efficiently for cloning lambda, yeast, and human DNA. The cloned fragments can be screened directly for the presence of promoters by testing for tetracycline resistance. Alternatively, these plasmids can be used as cosmids for cloning large fragments of DNA at a number of sites. Construction of several related vectors is described.     

Amino acid substitutions and alteration in cation specificity in the melibiose carrier of Escherichia coli

We isolated mutants of Escherichia coli which showed Li+-resistant growth on melibiose. The melibiose carrier of the mutants lost the ability to couple to H+, whereas it retained the ability to couple to Na+. The mutated gene, melB, of the mutants was cloned, and the nucleotide sequence was determined. The nucleotide replacements caused the following substitutions of amino acid residues in the melibiose carrier: Pro-142 with Ser, Leu-232 with Phe, or Ala-236 with Thr or Val. These amino acid residues are located in slightly hydrophobic regions of the melibiose carrier. The results provide strong support for the idea that such regions or their vicinities which contain those amino acid residues play an important role in H+ (or Li+) recognition or H+ (or Li+) transport by the melibiose carrier.     

Cloning of the Bacillus subtilis DNA fragment containing the genes for lysine and riboflavin biosynthesis

The SalI fragment of chromosomal DNA of Bacillus subtilis carrying the gene for lysine biosynthesis and the regulatory operator region (ribO) from the riboflavin gene was cloned into Escherichia coli cells. This fragment was shown to contain the gene coding for lysine synthesizing enzyme. Localization of this gene in Bac. subtili was determined. New plasmids pLRS33 and pLRB4 were constructed using pBR322; they carry a fragment homologous to pLP102 plasmid containing the operon for riboflavin biosynthesis.     

The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli

The stability of different derivatives of plasmid vectors pBR322 and pACYC184 carrying the tryptophan operon of Escherichia coli was monitored in various media. It was found that in the absence of any special selective pressure, all plasmids were lost from the culture. The stability varied depending both on the orientation of the inserted tryptophan fragment and the growth media used. The pBR322::trp+ plasmids were lost at an average frequency of 0.3 to 0.8% per cell generation, while the pACYC184::trp+ plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost, indicating a high stability of the plasmid::cloned DNA as such. To increase the stability of the cloning vectors, the partition locus of plasmid pSC101 was added to both the pBR322::trp+ and pACYC184::trp+ plasmids. The addition of this gene increased the replicon stability at least 3- to 10-fold, with the pBR322::trp+-par+ plasmids being the most stable. Also in this case, the stability was dependent on the plasmid type and on the growth medium. In no case was there a discoordinate loss of the antibiotic-resistance and tryptophan genes from the vectors.     

Directed modification of the Tcr gene region of the plasmid pBR322 using complementary single-stranded DNA fragments carrying alkylating groups

A method is suggested for chemical modification of preselected regions of plasmid DNA by complementary single-stranded restriction fragments of DNA (srf DNA), carrying alkylating reagents. The gene coding for tetracycline resistance of plasmid pBR322 was used as a target. Srf DNA was prepared by a partial digestion of a double-stranded EcoRI-BamHI restriction fragment (377 base pairs) from Tcr by E. coli exonuclease III. The residues of an alkylating reagent N,N,N'-tri(beta-chlorethyl)-N'-(p-formylphenyl) propylenediamine 1,3 (TFP) were attached covalently to 4-5% of sfr DNA bases. The alkylating derivative of the sfr DNA was hybridized with supercoiled pBR322 plasmid DNA. The hybridization conditions (37 degrees C, 40% formamide, 0,2 M NaCl, 0,1 M Tris-HCl pH 7,5, 0,001 M EDTA) under which the bases carrying TFP residues are not eliminated from the sfr DNA, and transforming activity of pBR322 DNA does not decrease were established. It was shown that about 20% of plasmid pBR322 molecules form D-loops with alkylating sfr DNA under these conditions. It was shown that sfr DNA, carrying TFP can alkylate the complementary region of plasmid DNA, forming cross-linked D-loops. A method for the site-directed mutagenesis of switching off the preselected genes or non-transcribed DNA functional regions (promotors, introns etc) integrated into plasmids of other vectors is suggested.     

Transformation of rat fibroblasts by cloned defective polyoma DNA

Defective polyoma DNA molecules isolated from mouse cells infected with high-multiplicity-passaged virus were cloned in pBR322, and the recombinant plasmids were tested for their capacity to transform Fischer rat 3T3 cells in culture. Recombinants carrying an intact proximal portion of the early region, i.e., the region coding for both small and middle T antigens, were able to induce the transformed phenotype. A recombinant plasmid containing a defective polyoma genome with a deletion of about 300 base pairs in the region coding for the C-terminal segment of middle T antigen failed to transform.     

The induction of auxotrophic mutations for riboflavin by the integration of plasmid pLRS33 into the chromosome of Bacillus subtilis

The transformation of Bacillus subtilis Lys- strains with plasmid pLRS33 containing pBR322 and the Bac. subtilis chromosomal fragment carrying the genes for lysin biosynthesis and the riboflavin operon regulatory operator region (ribO) leads to the appearance of Rib- mutants. It was shown that these mutants contained long deletions covering a great portion of the riboflavin operon.     

Effects of 3' end deletions from the Vibrio harveyi luxB gene on luciferase subunit folding and enzyme assembly: generation of temperature-sensitive polypeptide folding mutants

Ten recombinant plasmids have been constructed by deletion of specific regions from the plasmid pTB7 that carries the luxA and luxB genes, encoding the alpha and beta subunits of luciferase from Vibrio harveyi, such that luciferases with normal alpha subunits and variant beta subunits were produced in Escherichia coli cells carrying the recombinant plasmids. The original plasmid, which conferred bioluminescence (upon addition of exogenous aldehyde substrate) on E. coli carrying it, was constructed by insertion of a 4.0-kb HindIII fragment of V. harveyi DNA into the HindIII site of plasmid pBR322 [Baldwin, T.O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., & Ziegler, M. M. (1984) Biochemistry 23, 3663-3667]. Deletion mutants in the 3' region of luxB were divided into three groups: (A) those with deletions in the 3' untranslated region that left the coding sequences intact, (B) those that left the 3' untranslated sequences intact but deleted short stretches of the 3' coding region of the beta subunit, and (C) those for which the 3' deletions extended from the untranslated region into the coding sequences. Analysis of the expression of luciferase from these variant plasmids has demonstrated two points concerning the synthesis of luciferase subunits and the assembly of those subunits into active luciferase in E. coli. First, deletion of DNA sequences 3' to the translational open reading frame of the beta subunit that contain a potential stem and loop structure resulted in dramatic reduction in the level of accumulation of active luciferase in cells carrying the variant plasmids, even though the luxAB coding regions remained intact.