Specific amino acid residues in both the PstB and PstC proteins are required for phosphate transport by the Escherichia coli Pst system.

Three mutant alleles of the pstC gene and one mutant allele of the pstB gene were produced by site-directed mutagenesis. The pstC gene encodes an integral membrane protein of the phosphate-specific transport (Pst) system of Escherichia coli. The amino acid substitutions resulting from the pstC gene mutations, Arg-237----Gln, Glu-240----Gln, or a combination of both, caused the loss of phosphate transport through the Pst system, but the alkaline phosphatase activity remained repressed. The pstB gene encodes a peripheral membrane protein of the Pst system which carries a putative nucleotide-binding site. The amino acid substitutions Gly-48----Ile and Lys-49----Gln, resulting from the pstB mutations, caused the loss of phosphate transport through the Pst system and the derepression of alkaline phosphatase activity. The residues Gly-48 and Lys-49 are key residues in the putative nucleotide-binding site.     

An additional acidic residue in the membrane portion of the b-subunit of the energy-transducing adenosine triphosphatase of Escherichia coli affects both assembly and function.

Glycine at position 9 is replaced by aspartic acid in the mutant b-subunit of Escherichia coli F1F0-ATPase coded for by the uncF476 allele. The mutant b-subunit is not assembled into the membrane in haploid strains carrying the uncF476 allele, but, if the mutant allele is incorporated into a multicopy plasmid, then some assembly of the mutant b-subunit occurs. Two revertant strains were characterized, one of which (AN2030) was a full revertant, the other (AN1953) a partial revertant. DNA sequencing indicated that in strain AN2030 the uncF476 mutation had reverted to give the sequence found in the normal uncF gene. The partial-revertant strain AN1953, however, retained the DNA sequence of the uncF476 allele, and complementation analysis indicated that the second mutation may be in the uncA gene. Membranes prepared from the partial-revertant strain carried out oxidative phosphorylation, although the membranes appeared to be impermeable to protons, and the ATPase activity was sensitive to the inhibitor dicyclohexylcarbodi-imide.     

Tam3 produces a suppressible allele of the DAG locus of Antirrhinum majus similar to Mu-suppressible alleles of maize

Tam3 from Antirrhinum majus belongs to the Ac/Ds family of transposable elements. An allele of the DAG locus of Antirrhinum ( dag ::Tam3), which is required for chloroplast development and leaf palisade differentiation, has been generated by Tam3 insertion into the untranslated leader sequence of the gene. This allele gives rise to a cold-sensitive phenotype, where mutant tissue containing wild-type revertant somatic sectors is observed in the leaves of plants grown at 15°C, while leaves of plants grown at 25°C appear near wild-type. The temperature sensitivity of dag ::Tam3 results from expression of the DAG locus responding to the activity of the transposable element, the transposition of which is very sensitive to growing temperature. Genetic suppression of Tam3 transposition, using the STABILISER locus, also results in suppression of the dag mutant phenotype. dag ::Tam3 represents a Tam3-suppressible allele similar to those described for Mu transposons in maize. Suppression of the dag mutant phenotype in response to element inactivation appears to result from use of an alternative promoter at the 3' end of the Tam3 element. The production of suppressible alleles by an Ac-like element is discussed in relation to the mutagenic potential of plant transposons in producing complex genetic diversity.     

Maltose-binding protein does not modulate the activity of maltoporin as a general porin in Escherichia coli.

Maltoporin (lambda receptor) is part of the maltose transport system in Escherichia coli and is necessary for the facilitated diffusion of maltose and maltodextrins across the outer membrane. Maltoporin also allows the diffusion of nonmaltodextrin substrates, albeit with less efficiency. The preference of maltoporin for maltodextrins in vivo is thought to be the result of an interaction of maltoporin with the maltose-binding protein, the malE gene product. In a recent report Heuzenroeder and Reeves (J. Bacteriol. 144:431-435, 1980) suggested that this interaction establishes a gating mechanism which inhibits the diffusion of nonmaltodextrin substrates, such as lactose. To reinvestigate this important conclusion, we constructed ompR malTc strains carrying either the malE+ gene, the nonpolar malE444 deletion, or the malE254 allele, which specifies an interaction-deficient maltose-binding protein. Lactose uptake was measured at different concentrations below the Km of this transport system and under conditions where transport was limited by the diffusion through maltoporin. We found no difference in the kinetics of lactose uptake irrespective of the malE allele. We conclude that the maltose-binding protein does not modulate the activity of maltoporin as a general outer membrane porin.     

Physiological and genetic regulation of the aldohexuronate transport system in Escherichia coli.

In Escherichia coli K-12, the specificity of the aldohexuronate transport system (THU) is restricted to glucuronate and galacturonate. There is a relatively high basal-level activity in uninduced wild-type or isomeraseless strains. Supplementary activity is obtained with the inducers mannonic amide (five-fold), galacturonate (fourfold), fructuronate (fivefold), and tagaturonate (sevenfold). Specific THU- mutants were selected as strains unable to grow on either aldohexuronate but able to grow on fructuronate or tagaturonate. The remaining transport activity in uninduced and induced THU- starins represents less than 20% of that found in the wild type. Conjugation and transduction experiments indicate that all of the THU- mutations are located in a unique locus, exuT, half-way between the tolC (59 min) and argG (61 min) markers. exuT is closely linked to the uxaC-uxaA operon (60 min) and to the regulatory gene exuR (60 min), which controls the above-mentioned operon and the uxaB operon (45 min). Growth on either aldohexuronate and transport activity are fully recovered when exuT mutants are allowed to revert to exuT+ on galacturonate or glucuronate. Reversion on glucuronate alone may lead to the mutational derepression of the 2-keto-3-deoxygluconate transport system, which is uninducible in the wild type, which also takes up glucuronate, and whose structural gene belongs to the kdg regulon. Such strains, which remain unable to grow on galacturonate, are exuT and kdgR (constitutive allele of the regulatory gene kdgR of the kdg regulon). THU activity is superrepressed in an exuR mutant in which the uxaC-uxaA operon and the uxaB operon are superrepressed; exuR+/exuR merodiploids are also superrepressed. In a thermosensitive exuR mutant in which the above-mentioned operons are constitutive at 42 degrees C, the THU activity is fully derepressed at this temperature. On the basis of these and other results, it is concluded that THU is coded for by the structural gene exuT, which is negatively controlled by the exuR gene product and which probably belongs to an operon distinct from the uxaA-uxaC operon.     

Arg-220 of the PstA protein is required for phosphate transport through the phosphate-specific transport system in Escherichia coli but not for alkaline phosphatase repression.

The pstA gene encodes an integral membrane protein of the phosphate-specific transport system of Escherichia coli. The nucleotide change in the previously described pstA2 allele was found to be a G----A substitution at position 276 of the nucleotide sequence, resulting in the premature termination of translation. Three mutations in the pstA gene were produced by site-directed mutagenesis. The amino acid substitutions resulting from the three site-directed mutations were Arg-170----Gln, Glu-173----Gln, and Arg-220----Gln. These amino acid residues were selected because a previous PstA protein structure prediction placed them within the membrane. The Arg-220----Gln mutation resulted in the loss of phosphate transport through the phosphate-specific transport system, but the alkaline phosphatase activity remained repressed. Neither the Arg-170----Gln nor the Glu-173----Gln mutation affected phosphate transport. The results are discussed in relation to a proposed structure of the PstA protein.     

Identification and sequence of a Na+-linked gene from the marine bacterium Alteromonas haloplanktis which functionally complements the dagA gene of Escherichia coli

A 4.0 kb fragment from a plasmid genomic DNA library of the marine bacterium Alteromonas haloplanktis ATCC 19855 was found in the presence of Na+ to complement the dagA gene of Escherichia coli. We have completely sequenced this fragment and the position of the Na(+)-linked D-alanine glycine permease gene (dagA) on the fragment has been determined by complementation. The predicted carrier protein consists of 542 amino acid residues (M(r) 58,955). Its hydropathy profile suggests it is composed of eight transmembrane segments with a long hydrophilic region between segments six and seven. Significant similarity has been found between this Na(+)-linked permease and the Na+/proline permeases of E. coli and Salmonella typhimurium and the human and rabbit intestinal Na+/glucose cotransporters.     

Transport of long-chain fatty acids in Escherichia coli. Evidence for role of fadL gene product as long-chain fatty acid receptor

Transport of long-chain fatty acids (LCFA) across the cytoplasmic membrane of Escherichia coli requires functional fadL and fadD genes. The fadD gene codes for an acyl-CoA synthetase (fatty acid: CoA ligase (AMP forming] which has broad chain length specificity and is loosely bound to the cytoplasmic membrane. The fadL gene codes for a 43,000-dalton cytoplasmic membrane protein which, acting by an unknown mechanism, is needed specifically for LCFA transport. As a first step to define the role of the fadL gene product, studies were performed to determine if it functions as a LCFA receptor. The LCFA-binding activity was quantitated in intact cells in the absence of LCFA transport by comparing the binding of LCFA in fadD fadL and fadD fadL+ strains. These studies revealed that (i) fadD fadL+ strains bind 6-fold more LCFA than fadD fadL strains; (ii) fadD fadL strains harboring a plasmid containing the fadL gene bind 16-fold more LCFA than fadD fadL strains harboring only the plasmid vector; and (iii) the fadL-specific LCFA-binding activity is regulated by the fadR gene and catabolite repression. Studies with fadL strains harboring fadL plasmids containing in vitro constructed deletions indicate that mutations which alter the physical properties of the 43,000-dalton fadL gene product also affect fadL gene product-specific LCFA-binding activity. Overall, these studies suggest that one role of the fadL gene product in the LCFA transport process is to sequester LCFA at sites in the cell membrane for transport.     

Cloning and nucleotide sequence of braC, the structural gene for the leucine-, isoleucine-, and valine-binding protein of Pseudomonas aeruginosa PAO.

The gene for the leucine-, isoleucine-, and valine-binding protein (LIVAT-BP) in Pseudomonas aeruginosa PAO was isolated, and its nucleotide sequence was determined. The gene consisted of 1,119 nucleotides specifying a protein of 373 amino acid residues. Determination of the N-terminal amino acid sequence of the LIVAT-BP purified from P. aeruginosa shock fluid suggested that the N-terminal 26 residues of the gene product are cleaved off posttranslationally, showing the characteristic features of procaryotic signal peptides. The amino acid composition of the mature product predicted from the nucleotide sequence was in good agreement with that of the purified LIVAT-BP. The plasmid carrying the LIVAT-BP gene restored the activity of the high-affinity branched-chain amino acid transport system (the leucine, isoleucine, valine [LIV-I] transport system) in the braC310 mutant of P. aeruginosa, confirming that braC is the structural gene for LIVAT-BP. The mutant LIVAT-BP lacking a 16-amino-acid peptide in the middle was found to be functional in the LIV-I transport system. LIVAT-BP showed extensive homology (51% identical) to the LIV- and leucine-specific-binding proteins of Escherichia coli K-12, which are coded for by the livJ and livK genes, respectively, suggesting that the role of the proteins in the LIV-I transport systems is analogous in both organisms.     

Characterization of the mgl operon of Escherichia coli by transposon mutagenesis and molecular cloning

We used transposon insertion mutagenesis, molecular cloning, and a novel procedure for in vitro construction of polar and nonpolar insertion mutations to characterize the genetic organization and gene products of the beta-methylgalactoside (Mgl) transport system, which utilizes the galactose-binding protein. The data indicate that the mgl operon contained three genes, which were transcribed in the order mglB, mglA, and mglC. The first gene coded for the 31,000 Mr galactose-binding protein, which was synthesized as a 3,000-dalton-larger precursor form. The mglA product was a 50,000 Mr protein which was tightly associated with the membrane, and the mglC product was a 38,000 Mr protein which was apparently loosely associated with the membrane and was probably located on the internal face of the cytoplasmic membrane. Identification of gene products was facilitated by in vitro insertion of a fragment of Tn5 containing the gene conferring kanamycin resistance into a restriction site in the operon. The fragment proved to have a polar effect on the expression of promoter-distal genes only when inserted in one of the two possible orientations. The three identified gene products were necessary and apparently sufficient for transport activity, but only the binding protein was required for chemotaxis towards galactose. The transport system appeared to contain the minimum number of components for a binding protein-related system: a periplasmic recognition component, a transmembrane protein, and a peripheral membrane protein that may be involved in energy linkage.     

A Fifth Gene (uncE) in the Operon Concerned with Oxidative Phosphorylation in Escherichia coli

Three mutant unc alleles (unc-408, unc-410, and unc-429) affecting the coupling of electron transport to oxidative phosphorylation in Escherichia coli K-12 have been characterized. Genetic complementation analyses using previously defined mutant unc alleles indicated that the new mutant unc alleles affect a previously undescribed gene designated uncE. The phenotype of strains carrying the uncE408 or uncE429 allele is similar in that Mg(2+)-adenosine triphosphatase activity is only found in the cytoplasmic fraction, and membranes do not bind the F(1) portion of adenosine triphosphatase purified from a normal strain. In contrast, adenosine triphosphatase activity is present both in the cytoplasm and on the membranes from a strain carrying the unc-410 allele, and normal F(1) binds to F(1)-depleted membranes from this strain. The adenosine triphosphatase solubilized from membranes of a strain carrying the unc-410 allele reconstituted ATP-dependent membrane energization in F(1)-depleted membranes from a normal strain. Genetic complementation tests using various Mu-induced unc alleles in partial diploid strains show that the uncE gene is in the unc operon and that the order of genes is uncB E A D C. The unc-410 allele differs from the uncE408 and uncE429 alleles in that complementation tests with the Mu-induced unc alleles indicate that more than one gene is affected. It is concluded that this is due to a deletion which includes part of the uncE gene and another gene, or genes, between the uncE and uncA genes.     

Sequences and expression of alleles of polymorphic arylamine N-acetyltransferase of human liver.

Fifty human livers obtained at autopsy were analyzed for N-acetyltransferase and classified into six genotypes. Determination of N-acetyltransferase activity and proteins from supernatants of liver homogenates indicate that genotype I corresponds to rapid acetylator, genotypes II and III to intermediate acetylator, and genotypes IV, V, and VI to slow acetylator phenotypes. Northern blot analysis shows that levels of mRNA for N-acetyltransferase in the livers do not markedly differ among the six genotypes. Three alleles of the N-acetyltransferase gene were cloned and sequenced. mRNA is coded in two exons. Comparison of alleles 2 and 3, which correspond to low N-acetyltransferase activity, with allele 1, which corresponds to high N-acetyltransferase activity, revealed several polymorphisms. Two gene sequence differences occur in the coding exons of alleles 2 and 3, one of which would produce different amino acids in the proteins. Those sequence differences that lead to amino acid substitutions result in a loss of BamHI and TaqI sites for alleles 2 and 3, respectively. Expression studies of the alleles in Chinese hamster ovary cells show that allele 1 expresses high levels of N-acetyltransferase activity and enzyme protein, while alleles 2 and 3 express low levels of both protein and activity.     

Escherichia coli K-12 mutants that allow transport of maltose via the beta-galactoside transport system.

We have isolated mutants of Escherichia coli that have an altered beta-galactoside transport system. This altered transport system is able to transport a sugar, maltose, that the wild-type beta-galactoside transport system is unable to transport. The mutation that alters the specificity of the transport system is in the lacY gene, and we refer to the allele as lacYmal. The lacYmal allele was detected originally in strains in which the lac genes were fused to the malF gene. Thus, as a result of gene fusion and isolation of the lacYmal mutation, a new transport system was evolved with regulatory properties and specificity similar to those of the original maltose transport system. Maltose transport via the lacYmal gene product is independent of all of the normal maltose transport system components. The altered transport system shows a higher affinity than the wild-type transport system for two normal substrates of the beta-galactoside transport system, thiomethyl-beta-D-galactoside and o-nitrophenyl-beta-D-galactoside.     

Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominate type of mechanism, although both systems may have components in common.     

Temperature sensitivity caused by missense suppressor supH and amber suppressor supP in Escherichia coli.

The temperature-sensitive missense suppressor supH and amber suppressor supP in Escherichia coli are mutations of the serU and leuX genes, respectively. The supH tRNA, tRNA(SerCAA), is expected to recognize UUG codons, which are normally read by tRNA(LeuCAA) and tRNA(LeuUAA), coded for by the leuX gene and the leuZ gene, respectively. We show that supP and supH are incompatible and that strains carrying both supP and a restrictive rpsL allele are temperature sensitive. It is suggested that the temperature sensitivity of both supH and supP strains is caused by deficient reading of UUG codons by tRNA(LeuUAA).     

Signal strains that can detect certain DNA replication and membrane mutants of Escherichia coli: isolation of a new ssb allele, ssb-3.

Mutations in several dna genes of Escherichia coli, when introduced into a strain with a lac fusion in the SOS gene sulA, resulted in formation of blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal). Unexpectedly, several lines of evidence indicated that the blue colony color was not primarily due to induction of the SOS system but rather was due to a membrane defect, along with the replication defect, making the cell X-Gal extrasensitive (phenotypically Xgx), possibly because of enhanced permeability to X-Gal or leakage of beta-galactosidase. (i) In most cases, beta-galactosidase specific activity increased only two- to threefold. (ii) Mutations conferring tolerance to colicin E1 resulted in blue colony color with no increase in beta-galactosidase specific activity. (iii) Mutations in either the dnaA, dnaB, dnaC, dnaE, dnaG, or ssb gene, when introduced into a strain containing a bioA::lac fusion, produced a blue colony color without an increase in beta-galactosidase synthesis. These lac fusion strains can serve as signal strains to detect dna mutations as well as membrane mutations. By localized mutagenesis of the 92-min region of the chromosome of the sulA::lac signal strain and picking blue colonies, we isolated a novel ssb allele that confers the same extreme UV sensitivity as a delta recA allele, which is a considerably greater sensitivity than that conferred by the two well-studied ssb alleles, ssb-1 and ssb-113. The technique also yielded dnaB mutants; fortuitously, uvrA mutants were also found.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli.

Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.