Transposon-insertion mutants of Escherichia coli K12 defective in a component common to galactose and ribose chemotaxis.

Summary From a collection of 8,000 transposon-insertion mutants of Escherichia coli K12 we identified two mutations, trg-1::Tn5 and trg-2::Tn10, that simultaneously eliminate chemotactic response to ribose and galactose, two attractants recognized by independent receptors. We show that these transposon-insertions confer a Trg phenotype, indicating that this specific pattern of tactic defects is a null phenotype. The two mutation sites are cotransductionally linked to an extend consistent with placement in the same gene. The Trg phenotype of a family of deletion mutants produced by curing trg-2::Tn10 implies that trg is a single gene. Experiments with appropriate F-primes and Hfr's locate the trg locus at approximately 31 min on the linkage map, with a marker order: pyrF-rac-(P.O. 43)-trg-man.We also found one trg mutant whose Trg phenotype was not linked to a transposon-insertion but is probably the result of a mutator activity in the parent strain. Selection of transposon-insertions near, but not in trg allowed demonstration of a very close linkage between the spontaneous trg-3 and the transposon-generated trg's, indicating all three mutations are probably in the same gene. In our manipulations of transposon-insertions we found that Tn5 had a tendency to translocate from its initial site of insertion while Tn10 was relatively stable.The trg-product is probably a chemotactic signal transducer, which interacts directly with two independent receptor proteins and transmits information to the central chemotactic machinery.     

Nucleotide sequence and analysis of the mgl operon of Escherichia coli K12

Summary The nucleotide sequence of the Escherichia coli K12 -methylgalactoside transport operon, mgl, was determined. Primer extension analysis indicated that the synthesis of mRNA initiates at guanine residue 145 of the determined sequence. The operon contains three open reading frames (ORF). The operator proximal ORF, mglB, encodes the galactose binding protein, a periplasmic protein of 332 amino acids including the 23 residue amino-terminal signal peptide. Following a 62 nucleotide spacer, the second ORF, mglA, is capable of encoding a protein of 506 amino acids. The amino-terminal and carboxyl-terminal halves of this protein are homologous to each other and each half contains a putative nucleotide binding site. The third ORF, mglC, is capable of encoding a hydrophobic protein of 336 amino acids which is thought to generate the transmembrane pore. The overall organization of the mglBAC operon and its potential to encode three proteins is similar to that of the ara FGH high affinity transport operon, located approximately 1 min away on the E. coli K12 chromosome.     

The mglB sequence of Salmonella typhimurium LT2; promoter analysis by gene fusions and evidence for a divergently oriented gene coding for the mgl repressor

Summary The mglB gene of Salmonella typhimurium LT2 coding for the galactose-binding protein (GBP) was sequenced. We compared the deduced amino acid sequence with the GBP sequence of Escherichia coli K12. The mature proteins differ in only 19 of 309 amino acid residues, corresponding to 94% homology. Analysis of the mglB control region by promoter-probe vectors revealed that two promoters, P1 and P2, constitute the mgl control region (P _mgl ). P1 and P2 function in a synergistic way. P1 is the main promoter of the operon; its activity is 20 times the activity of P2. Both promoters are activated by the cyclic adenosine monophosphate catabolite activator protein (cAMP/CAP) complex. While P1 is inactive in the absence of the cAMP/CAP complex, there is residual activity of P2 under these conditions. Studies on the inducibility of the mglBAEC operon using multicopy plasmid promoter-probe vectors were hampered by the titration of the mgl repressor resulting in a partially constitutive expression of the mgl operon. The results indicate that only P1 is responding to induction by D-fucose. A weak promoter, P _D , within the P1 region but divergent to it was found. P _D is neither stimulated by the cAMP/CAP complex nor by D-fucose. We cloned the gene located downstream to P _D and found it to strongly repress the expression of the mgl operon. We termed this gene mglD. The presence of D-fucose abolished the repression caused by the plasmid-encoded mglD gene product.Abbreviations IPTG isopropyl-1-thic--D-galatopyranoside - ONPG 2-nitrophenyl--D-galatopyranoside - XG 5-bromo-4-chloro-3-indolyl--D-galatopyranoside - Kan^r Kanamycin resistance     

Mutations specifically affecting ligand interaction of the Trg chemosensory transducer.

The Trg transducer mediates chemotactic response to galactose and ribose by interacting, respectively, with sugar-occupied galactose- and ribose-binding proteins. Adaptation is linked to methylation of specific glutamyl residues of the Trg protein. This study characterized two trg mutations that affect interaction with binding protein ligands but do not affect methylation or adaptation. The mutant phenotypes indicated that the steady-state activity of methyl-accepting sites is independent of ligand-binding activity. The mutation trg-8 changed arginine 85 to histidine, and trg-19 changed glycine 151 to aspartate. The locations of the mutational changes provided direct evidence for functioning of the amino-terminal domain of Trg in ligand recognition. Cross-inhibition of tactic sensitivity by the two Trg-linked attractants implies competition for a common site on Trg. However, the single amino acid substitution caused by trg-19 greatly reduced the response to galactose but left unperturbed the response to ribose. Thus Trg must recognize the two sugar-binding proteins at nonidentical sites, and the complementary sites on the respective binding proteins should differ. trg-8 mutants were substantially defective in the response to both galactose and ribose. An increase in cellular content of Trg-8 protein improved the response to galactose but not to ribose. It appears that Trg-8 protein is defective in the generation of the putative conformational change induced by ligand interaction. The asymmetry of the mutational defect implies that functional separation of interaction sites could persist beyond the initial stage of ligand binding.     

Regulation of methyl beta-galactoside permease activity in pts and crr mutants of Salmonella typhimurium

Summary We have studied the regulation of the synthesis and activity of a major galactose transport system, that of methyl -galactoside (MglP), in mutants of Salmonella typhimurium. Two classes of mutation that result in a (partially) defective phosphoenolpyruvate: sugar phosphotransferase system (PTS) interfere with MglP synthesis. pts mutations, which eliminate the general proteins of the PTS Enzyme I and/or HPr and crr mutations, which result in a defective glucose-specific factor III^Gle of the PTS, lead to a low MglP activity, as measured by methyl -galactoside transport. In both ptsH,I, and crr mutants the amount of galactose binding protein, one of the components of MglP, is only 5%–20% of that in wild-type cells, as measured with a specific antibody. We conclude that synthesis of MglP is inhibited in pts and crr mutants. Once the transport system is synthesized, its transport activity is not sensitive to PTS sugars (i.e., no inducer exclusion occurs). The defect in pts and crr mutants with respect to MglP synthesis can be relieved in two ways: by externally added cyclic adenosine 3, 5-monophosphate (cAMP) or by a mutation in the cAMP binding protein. The conclusion that MglP synthesis is dependent on cAMP is supported by the finding that its synthesis is also defective in mutants that lack adenylate cyclase. pts and crr mutations do not affect growth of S. typhimurium on galactose, however, since the synthesis and activity of the other major galactose transport system, the galactose permease (GalP), is not sensitive to these mutations. If the galactose permease is eliminated by mutation, growth of pts and crr mutants on low concentrations of galactose becomes very slow due to inhibited MglP synthesis. Residual growth observed at high galactose concentrations is the result of yet another transport system with low affinity for galactose.     

Genetic structure and domains of DNA polymerase III of Bacillus subtilis

Summary We have determined the nucleotide sequence of the polC gene of Bacillus subtilis which codes for DNA polymerase III. Our recent analysis has revealed that the gene comprises 4311 nucleotides, from the start to the stop codon, 306 nucleotides more than we reported earlier. The plasmid reported by us and by N.C. Brown's laboratory contained a sequence at the end of the gene which is not related to the polC region of B. subtilis. We have isolated the rest of the gene, the sequence of which is presented in this paper. The new stop codon is followed by a hyphenated palindromic sequence of 13 nucleotides. The C-terminus' of the coding region contains the novel mutation, dnaF, which results in a defect in the initiation of replication due to a change in the codon TCC to TTC (serine to phenylalanine). The hypermutator mutation mut-1 is due to two point mutations in the 3 to 5 exonuclease domain, the proof reading function. The codon changes are GGA to GAA (glycine to glutamic acid) and AGC to AAC (serine to asparagine). The elongation defective mutation, polC26, affecting the catalytic site that adds nucleotides to the growing chain, is due to a change in the codon GTC to GAC (valine to aspartic acid). It is separated from the mutation reported earlier, azp-12, by 306 nucleotides. Knowing the locations of the mutational sites allowed us to deduce the domains of the gene and the enzyme it encodes, and permitted us to present a precise map of the gene at the molecular level.Abbreviations HPUra p-hydroxyphenyl azouracil - nt nucleotide - PCR polymerase chain reaction     

Cloning of mglB, the structural gene for the galactose-binding protein of Salmonella typhimurium and Escherichia coli

Summary From libraries of EcoRI fragments of Salmonella thyphimurium and Escherichia coli DNA in gt7, phages could be isolated that carry mglB, the structural gene of the galactose-binding protein as well as other mgl genes. Lysogenization of an E. coli mutant carrying a defective galactose-binding protein with gt7 mglB (Salmonella) restores full galactose transport and galactose chemotaxis. Both the E. coli mutant protein as well as the wild-type Salmonella galactose-binding protein are synthesized in this strain. The EcoR1 fragments of both organisms carrying the mgl genes were 6 Kb long. They were subcloned into the multicopy plasmid pACUC184. The hybrid plasmid containing the Salmonella mgl DNA gives rise to the synthesis of large amounts of galactose-binding protein in the periplasm of E. coli. The protein can be precipitated by antibodies against the E. coli binding protein and is identical to the fully processed protein isolated from Salmonella typhimurium LT2. In vitro protein synthesis (Zubay-system) with either gt7 mgl phages as well as the hybrid plasmid as DNA matrix produces the galactose-binding protein mainly in precursor form that is precipitable by specific antibodies.     

A mutation affecting amdS expression in Aspergillus nidulans contains a triplication of a cis-acting regulatory sequence

Summary In Aspergillus nidulans expression of the acetamidase structural gene, amdS, is under the control of at least four regulatory genes including the trans-acting amdA regulatory gene. A cis-acting mutation (amdI66) consisting of an 18 by duplication in the 5 region of the amdS gene results in very high levels of acetamidase activity but only in strains carrying semi-dominant mutations in the amdA gene. In selecting for increased amdS expression in an amdI66 amdA ^– strain, an A. nidulans strain with a mutation in the 5 region of the amdS gene was isolated. The nucleotide sequence was determined of the region containing the mutation, designated amdI666. The mutant strain carries three tandem copies of the 18 by sequence that is duplicated in the amdI66 mutation. Thus, from a strain carrying a duplication of an apparent regulatory protein binding site with little effect on gene expression, a strain has been derived that carries a triplication of the site with consequent major effects on regulation. The multiple copies of regulatory sites present in many genes may have been generated by a similar mechanism.     

Unique active site formation in a novel galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum

A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.     

Somatic cell variants of H-2k: b: A point mutation in the first extracellular domain results in altered immune recognition

A cell-surface-associated variant H-2K product was expressed by an Abelson virus-induced pre-B-cell line after chemical mutagenesis with ethyl methane sulfonate. The variant cell line (R8.313) was previously demonstrated to have altered allodeterminants in K^b as demonstrated by both K^b-specific monoclonal antibody binding and alloreactive cytotoxic T lymphocyte (CTL) cytolysis. The mutant H-2K ^b gene from R8.313 was cloned and characterized in detail. DNA sequence analysis of the region of the gene corresponding to the three extracellular domains identified a single point mutation resulting in a leucine-to-phenylalanine substitution at amino acid residue 82. The site of mutation within the 1 domain was confirmed by oligonucleotide hybridization analysis. Mouse L-cell fibroblasts transfected with the mutant gene were recognized with the same monoclonal antibody binding and CTL lytic pattern as the R8.313 cell line, confirming that the altered phenotype of the mutant cell line was due to a point mutation in the H-2K ^b gene. These data further extend the hypothesis that the region of amino acid residues 70–90 in the 1 domain is important in the formation of both antibody and CTL-defined recognition structures on major histocompatibility complex class I molecules.     

Identification of the Klebsiella pneumoniae glnB gene: nucleotide sequence of wild-type and mutant alleles

Summary The glnB gene of Klebsiella pneumoniae, which encodes the nitrogen regulation protein P_II has been cloned and sequenced. The gene encodes a 12429 dalton polypeptide and is highly homologous to the Escherichia coli glnB gene. The sequences of a glnB mutation which causes glutamine auxotrophy and of a Tn5 induced Gln⁺ suppressor of this mutation were also determined. The glutamine auxotrophy was deduced to be the result of a modification of the uridylylation site of P_II and the suppression was shown to be caused by Tn5 insertion in glnB. The 3 end of an open reading frame of unknown function was identified upstream of glnB and may be part of an operon containing glnB. Potential homologues of glnB encoding polypeptides extremely similar in sequence to P_II were identified upstream of published sequences of the glutamine synthetase structural gene (glnA) in Rhizobium leguminosarum, Bradyrhizobium japonicum and Azospirillum brasilense.     

An isolated reaction center complex from the green sulfur bacterium Chlorobium vibrioforme can photoreduce ferredoxin at high rates

Chlorosome-depleted membranes and a reaction center complex with well-defined subunit composition were prepared from the green sulfur bacterium Chlorobium vibrioforme under anaerobic conditions. The reaction center complex contains a 15-kDa polypeptide with the N-terminal amino acid sequence MEPQLSRPETASNQVR/. This sequence is nearly identical to the N-terminus of the pscD gene product from Chlorobium limicola (Hager-Braun et al. (1995) Biochemistry 34: 9617–9624). In the presence of ferredoxin and ferredoxin:NADP⁺ oxidoreductase, the membranes and the isolated reaction center complex photoreduced NADP⁺ at rates of 333 and 110 mol (mg bacteriochlorophyll a)^–1 h^–1, respectively. This shows that the isolated reaction center complex contains all the components essential for steady state electron transport. Midpoint potentials at pH 7.0 of 160 mV for cytochrome c ₅₅₁ and of 245 mV for P840 were determined by redox titration. Antibodies against cytochrome c ₅₅₁ inhibit NADP⁺ reduction while antibodies against the bacteriochlorophyll a-binding Fenna-Matthews-Olson protein do not.Abbreviations FMO protein Fenna-Matthews-Olson protein - TMBZ 3,3,5,5-tetramethylbenzidine     

Mitochondrial DNA restriction map for the Caribbean fruit fly,Anastrepha suspensa,and occurrence of mitochondrial DNA diversity within highly inbred colonies

A restriction map has been constructed for Anastrepha suspensa mitochondrial DNA. One HaeIII site was found to be polymorphic among individuals in highly inbred colonies and a feral population. Based on mapping information, the polymorphic site was determined to be in the ATPase 6 gene. Primers TK-J-3804 and C3-N-5460 amplified this region. The amplicon was cut by HaeIII in flies of one haplotype and not cut in flies of the other haplotype. From 30 to 43% of the individual flies studied had this additional HaeIII site. After cloning of the 5200 bp XbaI fragment, the two mitotypes were identified. A 988 base fragment, coding for the entire tRNA-Lys(AAG), tRNA-Asp(GAC), and ATPase 8genes, and a partial ATPase 6gene was sequenced Four silent mutations, including the one at the informative site were located. The HaeIII polymorphism and other sequence differences may prove useful as a diagnostic for identification of the origin of introduced fruitflies.     

Organization of specific DNA sequence elements in the region of the replication origin and matrix attachment site in the chicken alpha-globin gene domain.

Summary The distribution of specific DNA sequence elements in a 2.9 kb HindIII fragment of chicken DNA containing the replication origin and the upstream matrix attachment site (MAR) of the -globin gene domain was investigated. The fragment was shown to contain a CR1-type repetitive element and two stably bent DNA sequences. One of them colocalizes with the previously described MAR element and with the recognition site for a proliferating-cell-specific, DNA-binding protein. The melting pattern of a set of subfragments of the region proved to be non random. No correlation between the distribution of readily melting sequences and bent DNA was found. The possible importance of curved, low-melting and repetitive DNA sequences for the organization of the upstream boundary of the -globin gene domain and the function of the replication origin is discussed.     

Carbohydrate uptake genes inEscherichia coli are induced by carbon starvation

Escherichia coli produces several membrane-associated and periplasmic proteins in response to deprivation for a carbon source. The carbon starvation response involves a two- to fourfold, cAMP-dependent induction of operons involved in carbohydrate uptake and utilization, including thelac operon. Threecarbon-starvation-inducible (sci) gene fusions to aphoA reporter sequence were characterized. ThephoA-fusions werecya ⁺/crp ⁺-dependent and located in three previously characterized genes involved in high-affinity uptake of alternative carbon sources:mglB, encoding the periplasmic galactose binding protein;rbsB, encoding the periplasmic ribose binding protein; andlamB, encoding the maltodextrin-specific outer membrane porin.     

Acute intermittent porphyria caused by a C→T mutation that produces a stop codon in the porphobilinogen deaminase gene

Summary A mutation of the porphobilinogen (PBG) deaminase gene that produces the cross-reacting immunological material (CRIM)-negative type of acute intermittent porphyria (AIP) has been identified in one of 43 unrelated patients with this form of the disorder. The mutation is a CT transition that abolishes a PstI recognition site in exon 9 of the gene and converts a codon for glutamine to a stop codon.     

Genomic organization of the canrep repetitive DNA in Brassica juncea

Canrep is a heterogeneous, tandemly repeated, 176 bp nucleotide sequence that contains a single Hind III site and is present in high copy numbers in the genomes of many Brassica species. Complete clusters of repeats of this DNA were cloned from the nuclear DNA of Brassica juncea. Restriction-fragment dimers and higher multimers of the 176 bp sequence have arisen by mutations within the Hind III recognition sequence. Adjacent repeats from within the same cluster usually have different nucleotide sequences with features indicating that diversity is generated by a mechanism that causes site-specific base substitutions. While most of the units of canrep DNA are clustered in long arrays of tandem repeats, some are dispersed throughout the genome as isolated copies or in small clusters. Regardless of the size of the arrays, each cluster begins and ends with a variable-length, truncated repeat and is flanked by inverted copies of the sequence 5-ATCTCAT3-,which is not part of the basic sequence of the canrep family of DNAs. Furthermore, some clusters are located close to nucleotide sequences related to those of known plant transposons. Thus, canrep elements may be dispersed by transposition. There are two distinct subfamilies of canrep sequences in B. juncea, and one of these is closely related to one of the two subfamilies of this type of DNA from B. napus, indicating that it originated from B. campestris, the common diploid ancestor of both amphidiploid species. Neither the repetitive DNA nor nucleotide sequences flanking canrep clusters are transcribed in seedlings, suggesting that even small arrays of repeats are located in heterochromatic regions and might be involved in chromatin condensation and/or chromosome segregation.     

The genomic structure of the proto-oncogene c-kit encoded at the murineWhite spotting locus

The proto-oncogene c-kit encodes a transmembrane receptor with tyrosine kinase activity, which transduces signal fromkit ligand (KL), and is responsible for hematogenesis, melanogenesis and gametogenesis during fetal development and adult life. Partial or complete loss of c-kit function due to mutation of the c-kit or KL gene accounts for the phenotypes of the murineWhite-spotting andSteel mutations, respectively. The c-kit protein has the structural features of extracellular immunoglobulin-like domains and intracellular kinase domain with a hydrophilic insert. These features have categorized c-kit along with platelet-derived growth factor receptors, colony-stimulating factor 1 receptor (c-fms) and others to subclass III of the receptor tyrosine kinases. We report the structure of the murine c-kit gene. The c-kit gene consists of 21 exons and spans at least 70 kb. The 5 and 3 flanking exons encode the untranslated sequences as well as part of the coding sequence. The internal exons are typically small with each of them encoding a structurally important subunit of the protein. Comparison of gene structures of members of the subclass III receptor tyrosine kinases has improved our understanding of the structure-functional relationship of the c-kit protein.