Human placental Na+-dependent multivitamin transporter. Cloning, functional expression, gene structure, and chromosomal localization.

We have cloned the human Na+-dependent multivitamin transporter (SMVT), which transports the water-soluble vitamins pantothenate, biotin, and lipoate, from a placental choriocarcinoma cell line (JAR). The cDNA codes for a protein of 635 amino acids with 12 transmembrane domains and 4 putative sites for N-linked glycosylation. The human SMVT exhibits a high degree of homology (84% identity and 89% similarity) to the rat counterpart. When expressed in HRPE cells, the cDNA-induced transport process is obligatorily dependent on Na+ and accepts pantothenate, biotin, and lipoate as substrates. The relationship between the cDNA-specific uptake rate of pantothenate or biotin and Na+ concentration is sigmoidal with a Na+:vitamin stoichiometry of 2:1. The human SMVT, when expressed in Xenopus laevis oocytes, induces inward currents in the presence of pantothenate, biotin, and lipoate in a Na+-, concentration-, and potential-dependent manner. We also report here on the structural organization and chromosomal localization of the human SMVT gene. The SMVT gene is approximately 14 kilobase pairs in length and consists of 17 exons. The SMVT gene is located on chromosome 2p23 as evidenced by somatic cell hybrid analysis and fluorescence in situ hybridization.     

Overproduction and rapid purification of the biotin operon repressor from Escherichia coli

The Escherichia coli biotin operon repressor protein (BirA) has been overexpressed at the level of 0.5-1% of the total cellular protein from the plasmid pMBR10. Four lines of evidence demonstrated that authentic BirA protein was produced. First, birA plasmids complemented birA mutants for both the repressor and biotin holoenzyme synthetase activities of BirA. Second, biotin holoenzyme synthase activity was increased in strains containing the overproducing plasmids. Third, deletion of sequences flanking the birA gene did not alter production of the 35-kDa BirA protein, but insertion of oligonucleotide linkers within the birA coding region abolished it. Fourth, the 35-kDa protein copurified with the biotin binding activity normally associated with BirA. The birA protein has been purified to homogeneity in a three-step process involving chromatography on phosphocellulose and hydroxyapatite columns.     

Nucleotide sequence of the promoter-distal region of the tra operon of plasmid R100, including traI (DNA helicase I) and traD genes

The nucleotide sequence of the promoter-distal region of the tra operon of R100 was determined. There are five open reading frames in the region between traT and finO, and their protein products were identified. Nucleotide sequences of plasmid F corresponding to the junction regions among the open reading frames seen in R100 were also determined. Comparison of these nucleotide sequences revealed strong homology in the regions containing traD, traI and an open reading frame (named orfD). The TraD protein (83,899 Da) contains three hydrophobic regions, of which two are located near the amino-terminal region. This protein also contains a possible ATP-binding consensus sequence at the amino-terminal region and a characteristic repeated peptide sequence (Gln-Gln-Pro)10 at the carboxy-terminal region. The TraI protein (191,679 Da) contains the sequence motif conserved in an ATP-dependent DNA helicase superfamily in its carboxy-terminal region. The protein product of orfD, which is probably a new tra gene (named traX), contains 65% hydrophobic amino acids, especially rich in alanine and leucine. There exist non-homologous regions between R100 and F that could be represented as four I-D (insertion or deletion) loops in heteroduplex molecules. Assignment of each loop to the strand of R100 or F was , however, found to be the reverse from that previously assumed. The three I-D loops that were located between traT and traD, between traD and traI, and between traI and finO had no terminal inverted repeat sequences nor had they any homology with known insertion sequences, while the fourth was IS3, located within the finO gene of F. The sequences in the I-D loops, except IS3, may also code for proteins that are, however, likely to be nonessential for transfer of plasmids.     

Spectroscopic changes during a single turnover of biotin synthase: destruction of a [2Fe-2S] cluster accompanies sulfur insertion.

Biotin synthase catalyzes the insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Catalysis requires AdoMet and flavodoxin and generates 5'-deoxyadenosine and methionine, suggesting that biotin synthase is an AdoMet-dependent radical enzyme. Biotin synthase (BioB) is aerobically purified as a dimer of 38.4 kDa monomers that contains 1-1.5 [2Fe-2S](2+) clusters per monomer and can be reconstituted with exogenous iron, sulfide, and reductants to contain up to two [4Fe-4S] clusters per monomer. The iron-sulfur clusters may play a dual role in biotin synthase: a reduced iron-sulfur cluster is probably involved in radical generation by mediating the reductive cleavage of AdoMet, while recent in vitro labeling studies suggest that an iron-sulfur cluster also serves as the immediate source of sulfur for the biotin thioether ring. Consistent with this dual role for iron-sulfur clusters in biotin synthase, we have found that the protein is stable, containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. In the present study, we demonstrate that this mixed cluster state is essential for optimal activity. We follow changes in the Fe and S content and UV/visible and EPR spectra of the enzyme during a single turnover and conclude that during catalysis the [4Fe-4S](2+) cluster is preserved while the [2Fe-2S](2+) cluster is destroyed. We propose a mechanism for incorporation of sulfur into dethiobiotin in which a sulfur atom is oxidatively extracted from the [2Fe-2S](2+) cluster.     

Cloning and characterization of gdhA, the structural gene for glutamate dehydrogenase of Salmonella typhimurium.

Glutamic acid is synthesized in enteric bacteria by either glutamate dehydrogenase or by the coupled activities of glutamate synthase and glutamine synthetase. A hybrid plasmid containing a fragment of the Salmonella typhimurium chromosome cloned into pBR328 restores growth of glutamate auxotrophs of S. typhimurium and Escherichia coli strains which have mutations in the genes for glutamate dehydrogenase and glutamate synthase. A 2.2-kilobase pair region was shown by complementation analysis, enzyme activity measurements, and the maxicell protein synthesizing system to carry the entire glutamate dehydrogenase structural gene, gdhA. Glutamate dehydrogenase encoded by gdhA carried on recombinant plasmids was elevated 5- to over 100-fold in S. typhimurium or E. coli cells and was regulated in both organisms. The gdhA promoter was located by recombination studies and by the in vitro fusion to, and activation of, a promoter-deficient galK gene. Additionally, S. typhimurium gdhA DNA was shown to hybridize to single restriction fragments of chromosomes from other enteric bacteria and from Saccharomyces cerevisiae.     

Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751.

The primase genes of RP4 are part of the primase operon located within the Tra1 region of this conjugative plasmid. The operon contains a total of seven transfer genes four of which (traA, B, C, D) are described here. Determination of the nucleotide sequence of the primase region confirmed the existence of an overlapping gene arrangement at the DNA primase locus (traC) with in-phase translational initiation signals. The traC gene encodes two acidic and hydrophilic polypeptide chains of 1061 (TraC1) and 746 (TraC2) amino acids corresponding to molecular masses of 116,721 and 81,647 Da. In contrast to RP4 the IncP beta plasmid R751 specifies four large primase gene products (192, 152, 135 and 83 kDa) crossreacting with anti-RP4 DNA primase serum. As shown by deletion analysis at least the 135 and 83 kDa polypeptides are two separate translational products that by analogy with the RP4 primases, arise from in-phase translational initiation sites. Even the smallest primase gene products TraC2 (RP4) and TraC4 (R751) exhibit primase activity. Nucleotide sequencing of the R751 primase region revealed the existence of three in-phase traC translational initiation signals leading to the expression of gene products with molecular masses of 158,950 Da, 134,476 Da, and 80,759 Da. The 192 kDa primase polypeptide is suggested to be a fusion protein resulting from an in frame translational readthrough of the traD UGA stopcodon. Distinct sequence similarities can be detected between the TraC proteins of RP4 and R751 gene products TraC3 and TraC4 and in addition between the TraD proteins of both plasmids. The R751 traC3 gene contains a stretch of 507 bp which is unrelated to RP4 traC or any other RP4 Tra1 gene.     

Biotin synthase is a pyridoxal phosphate-dependent cysteine desulfurase

Biotin synthase (BioB) is an iron-sulfur dimeric enzyme which catalyzes the last step in biotin synthesis. The reaction consists of the introduction of a sulfur atom into dethiobiotin. It is shown here that BioB displays a significant cysteine desulfurase activity, providing it with the ability to mobilize sulfur from free cysteine. This activity is dependent on pyridoxal 5'-phosphate (PLP) and dithiothreitol and proceeds through a protein-bound persulfide. Like other cysteine desulfurases, BioB binds 1 equiv of PLP. By site-directed mutagenesis, two conserved cysteines, Cys97 and Cys128, are shown to be critical for cysteine desulfuration and are good candidates as the site for a persulfide. Since biotin synthase activity is greatly increased by PLP and cysteine, even though it does not exceed 1 nmol of biotin/nmol of monomer, it is proposed that cysteine desulfuration is intimately linked to biotin synthesis. New scenarios for sulfur insertion into dethiobiotin, in which cysteine persulfides play a key role, are discussed.     

The lipoate synthase from Escherichia coli is an iron-sulfur protein.

Lipoate synthase catalyzes the last step of the biosynthesis of lipoic acid in microorganisms and plants. The protein isolated from an overexpressing Escherichia coli strain was purified from inclusion bodies. Spectroscopic (UV-visible and electron paramagnetic resonance) properties of the reconstituted protein demonstrate the presence of a (2Fe-2S) center per protein. As observed in biotin synthase, these clusters are converted to (4Fe-4S) centers during reduction under anaerobic conditions. The possible involvement of the cluster in the insertion of sulfur atoms into the octanoic acid backbone is discussed.     

Bacteriophage SPO2-mediated plasmid transduction in transpositional mutagenesis within the genus Bacillus.

A single copy of the Streptococcus faecalis transposon Tn917, located in the Bacillus subtilis chromosome, was able to transpose onto the SPO2 cos plasmid pPL1017, which codes for chloramphenicol resistance and contains the bacteriophage phi 105 immunity region. Selection for pPL1017::Tn917 chimeras was performed by SPO2-mediated plasmid transduction of transposon-borne resistance to macrolide-lincosamide-streptogramin B antibiotics (MLSr). The transposition of Tn917 onto plasmid pPL1017 occurred with a frequency of 10(-5) and was dependent on the presence of a subinhibitory dose of erythromycin. Twelve chimeras were subjected to genetic and physical analyses. Two Cams transductants harbored plasmids whose chloramphenicol acetyltransferase genes had been insertionally inactivated by Tn917. Several transpositions in the vicinity of the phi 105 immunity region were detected. However, all of the 300 MLSr, Camr transductants screened were immune to phi 105 infectious activity. One pPL1017::Tn917 chimera, pLK200, was transferred by SPO2 plasmid transduction into the Bacillus amyloliquefaciens prototrophic strain DSM7. Plasmid pLK200 was effective in the mutagenesis of the DSM7 chromosome and yielded auxotrophs at a frequency of 0.5 to 5.3%. Generation of auxotrophs was also dependent on the presence of a subinhibitory dose of erythromycin. Forty-four auxotrophs representing at least nine amino acid requirements were recovered.     

Expression and purification of the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase subunits of the Escherichia coli pyruvate dehydrogenase multienzyme complex: a mass spectrometric assay for reductive acetylation of dihydrolipoamide acetyltransferase

Plasmids were constructed for overexpression of the Escherichia coli dihydrolipoamide acetyltransferase (1-lip E2, with a single hybrid lipoyl domain per subunit) and dihydrolipoamide dehydrogenase (E3). A purification protocol is presented that yields homogeneous recombinant 1-lip E2 and E3 proteins. The hybrid lipoyl domain was also expressed independently. Masses of 45,953+/-73Da (1-lip E2), 50,528+/-5.5Da (apo-E3), 51,266+/-48Da (E3 including FAD), and 8982+/-4.0 (lipoyl domain) were determined by MALDI-TOF mass spectrometry. The purified 1-lip E2 and E3 proteins were functionally active according to the overall PDHc activity measurement. The lipoyl domain was fully acetylated after just 30 s of incubation with E1 and pyruvate. The mass of the acetylated lipoyl domain is 9019+/-2Da according to MALDI-TOF mass spectrometry. Treatment of the 1-lip E2 subunit with trypsin resulted in the appearance of the lipoyl domain with a mass of 10,112+/-3Da. When preincubated with E1 and pyruvate, this tryptic fragment was acetylated according to the mass increase. MALDI-TOF mass spectrometry was thus demonstrated to be a fast and precise method for studying the reductive acetylation of the recombinant 1-lip E2 subunit by E1 and pyruvate.     

Characterization of the cit-1 gene from Neurospora crassa encoding the mitochondrial form of citrate synthase

We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.     

Characterization of the cit-1 gene from Neurospora crassa encoding the mitochondrial form of citrate synthase

We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.     

Genome structure of Halobacterium halobium: plasmid dynamics in gas vacuole deficient mutants

Halobacterium halobium contains two gas vacuole protein genes that are located in plasmid pHH1 (p-vac) and in the chromosomal DNA (c-vac). The mutation frequency for these genes is different: the constitutively expressed p-vac gene is mutated with a frequency of 10(-2), while the chromosomal gene expressed in the stationary phase of growth is mutated with a frequency of 10(-5). The difference in the mutation susceptibility is due to the dynamics of plasmid pHH1. p-vac gene mutations are caused (i) by the integration of an insertion element or (ii) by a deletion event encompassing the p-vac gene region. In contrast, c-vac mutants analyzed to date incurred neither insertion elements nor deletions. Deletion events within pHH1 occur at high frequencies during the development of a H. halobium culture. The investigation of the fusion regions resulting from deletion events indicates that insertion elements are involved. The analysis of pHH1 deletion variants led to a 4 kilobase pair DNA region containing the origin of replication of the pHH1 plasmid.     

The enzymology of sulfur activation during thiamin and biotin biosynthesis.

The thiamin and biotin biosynthetic pathways utilize elaborate strategies for the transfer of sulfur from cysteine to cofactor precursors. For thiamin, the sulfur atom of cysteine is transferred to a 66-amino-acid peptide (ThiS) to form a carboxy-terminal thiocarboxylate group. This sulfur transfer requires three enzymes and proceeds via a ThiS-acyladenylate intermediate. The biotin synthase Fe-S cluster functions as the immediate sulfur donor for biotin formation. C-S bond formation proceeds via radical intermediates that are generated by hydrogen atom transfer from dethiobiotin to the adenosyl radical. This radical is formed by the reductive cleavage of S-adenosylmethionine by the reduced Fe-S cluster of biotin synthase.