Biotin Biosynthetic Genes Research Articles

Control of biotin biosynthesis in mycobacteria by a pyruvate carboxylase dependent metabolic signal.

Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. To understand biotin biosynthesis in mycobacteria, we executed a genetic screen in Mycobacterium smegmatis for biotin auxotrophs and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. The biotin auxotrophy of the pyc::tn strain is due to failure to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of bioQ, the repressor of biotin biosynthesis, in the pyc::tn strain reverted biotin auxotrophy, as did reconstituting the last step of the pathway through heterologous expression of BioB and provision of its substrate DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Quantitation of the kinetics of depletion of biotinylated proteins after biotin withdrawal revealed that Pyc is the most rapidly depleted biotinylated protein and metabolomics revealed a broad metabolic shift in wild type cells upon biotin withdrawal which was blunted in cell lacking Pyc. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism.

Biotin Auxotrophy and Biotin Enhanced Germ Tube Formation in Candida albicans.

Due to the increased number of immunocompromised patients, infections with the pathogen Candida albicans have significantly increased in recent years. C. albicans transition from yeast to germ tubes is one of the essential factors for virulence. In this study we noted that Lee’s medium, commonly used to induce filamentation, contained 500-fold more biotin than needed for growth and 40-fold more biotin than is typically added to growth media. Thus, we investigated the effects of excess biotin on growth rate and filamentation by C. albicans in different media. At 37 °C, excess biotin (4 µM) enhanced germ tube formation (GTF) ca. 10-fold in both Lee’s medium and a defined glucose-proline medium, and ca. 4-fold in 1% serum. Two biotin precursors, desthiobiotin and 7-keto-8-aminopelargonic acid (KAPA), also stimulated GTF. During these studies we also noted an inverse correlation between the number of times the inoculum had been washed and the concentration of serum needed to stimulate GTF. C. albicans cells that had been washed eight times achieved 80% GTF with only 0.1% sheep serum. The mechanism by which 1–4 µM biotin enhances GTF is still unknown except to note that equivalent levels of biotin are needed to create an internal supply of stored biotin and biotinylated histones. Biotin did not restore filamentation for any of the four known filamentation defective mutants tested. C. albicans is auxotrophic for biotin and this biotin auxotrophy was fulfilled by biotin, desthiobiotin, or KAPA. However, biotin auxotrophy is not temperature dependent or influenced by the presence of 5% CO2. Biotin starvation upregulated the biotin biosynthetic genes BIO2, BIO3, and BIO4 by 11-, 1500-, and 150-fold, respectively, and BIO2p is predicted to be mitochondrion-localized. Based on our findings, we suggest that biotin has two roles in the physiology of C. albicans, one as an enzymatic cofactor and another as a morphological regulator. Finally, we found no evidence supporting prior claims that C. albicans only forms hyphae at very low biotin (0.1 nM) growth conditions.

The Atypical Occurrence of Two Biotin Protein Ligases in Francisella novicida Is Due to Distinct Roles in Virulence and Biotin Metabolism

ABSTRACTThe physiological function of biotin requires biotin protein ligase activity in order to attach the coenzyme to its cognate proteins, which are enzymes involved in central metabolism. The model intracellular pathogen Francisella novicida is unusual in that it encodes two putative biotin protein ligases rather than the usual single enzyme. F. novicida BirA has a ligase domain as well as an N-terminal DNA-binding regulatory domain, similar to the prototypical BirA protein in E. coli. However, the second ligase, which we name BplA, lacks the N-terminal DNA binding motif. It has been unclear why a bacterium would encode these two disparate biotin protein ligases, since F. novicida contains only a single biotinylated protein. In vivo complementation and enzyme assays demonstrated that BirA and BplA are both functional biotin protein ligases, but BplA is a much more efficient enzyme. BirA, but not BplA, regulated transcription of the biotin synthetic operon. Expression of bplA (but not birA) increased significantly during F. novicida infection of macrophages. BplA (but not BirA) was required for bacterial replication within macrophages as well as in mice. These data demonstrate that F. novicida has evolved two distinct enzymes with specific roles; BplA possesses the major ligase activity, whereas BirA acts to regulate and thereby likely prevent wasteful synthesis of biotin. During infection BplA seems primarily employed to maximize the efficiency of biotin utilization without limiting the expression of biotin biosynthetic genes, representing a novel adaptation strategy that may also be used by other intracellular pathogens.

Successful conversion of the Bacillus subtilis BirA Group II biotin protein ligase into a Group I ligase.

Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that allows transcriptional regulation of biotin biosynthetic and transport genes whereas Group I BPLs lack this N-terminal domain. The Bacillus subtilis BPL, BirA, is classified as a Group II BPL based on sequence predictions of an N-terminal helix-turn-helix motif and mutational alteration of its regulatory properties. We report evidence that B. subtilis BirA is a Group II BPL that regulates transcription at three genomic sites: bioWAFDBI, yuiG and yhfUTS. Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function. This is the first example of successful conversion of a Group II BPL to a Group I BPL with retention of full ligase activity.

Conservation of the biotin regulon and the BirA regulatory signal in Eubacteria and Archaea.

Biotin is a necessary cofactor of numerous biotin-dependent carboxylases in a variety of microorganisms. The strict control of biotin biosynthesis in Escherichia coli is mediated by the bifunctional BirA protein, which acts both as a biotin-protein ligase and as a transcriptional repressor of the biotin operon. Little is known about regulation of biotin biosynthesis in other bacteria. Using comparative genomics and phylogenetic analysis, we describe the biotin biosynthetic pathway and the BirA regulon in most available bacterial genomes. Existence of an N-terminal DNA-binding domain in BirA strictly correlates with the presence of putative BirA-binding sites upstream of biotin operons. The predicted BirA-binding sites are well conserved among various eubacterial and archaeal genomes. The possible role of the hypothetical genes bioY and yhfS-yhfT, newly identified members of the BirA regulon, in the biotin metabolism is discussed. Based on analysis of co-occurrence of the biotin biosynthetic genes and bioY in complete genomes, we predict involvement of the transmembrane protein BioY in biotin transport. Various nonorthologous substitutes of the bioC-coupled gene bioH from E. coli, observed in several genomes, possibly represent the existence of different pathways for pimeloyl-CoA biosynthesis. Another interesting result of analysis of operon structures and BirA sites is that some biotin-dependent carboxylases from Rhodobacter capsulatus, actinomycetes, and archaea are possibly coregulated with BirA. BirA is the first example of a transcriptional regulator with a conserved binding signal in eubacteria and archaea.

Cloning and characterization of biotin biosynthetic genes of Kurthia sp.

The biotin biosynthesis genes of Kurthia sp., which is an aerobic gram-positive bacterium, were cloned from Kurthia sp. 538-KA26 and characterized. Eleven biotin biosynthetic genes have been identified in Kurthia sp. Kurthia sp. has two genes coding for KAPA synthase, bioF and bioFII, and also has two genes coding for BioH protein, bioH and bioHII. In addition, three genes, orf1, orf2, and orf3, whose functions are unknown, were found in the biotin gene clusters of Kurthia sp. The bioA, bioD, and orf1 genes are arranged in a gene cluster in the order orf1bioDA, and the bioB, bioF, and orf2 genes are arranged in a gene cluster in the order orf2bioFB. These gene clusters proceed to both directions; the face to face promoters and two 40-bp of palindrome sequences exist upstream of the orf1 and orf2 genes. The bioC, bioFII, and bioHII genes are arranged in a gene cluster in the order bioFIIHIIC; a 40-bp of palindrome sequence exists upstream of the bioFII gene. The bioH and orf3 genes are arranged in a gene cluster in the order bioHorf3; a palindrome sequence was not found upstream of the bioH gene. These palindrome sequences are extremely similar to each other, suggesting that the orf1bioDA, orf2bioFB, and bioFIIHIIC gene clusters are regulated by biotin. Kurthia sp. does not have the bioW gene coding pimeloyl-CoA synthase, suggesting that pimeloyl-CoA may be produced by a different pathway than that of gram-positive bacterium B.subtilis or B.sphaericus, further suggesting a modified fatty acid synthesis pathway via acetyl-CoA instead as E.coli has.

Is plant biotin holocarboxylase synthetase a bifunctional enzyme?

Holocarboxylase synthetases (HCSs) catalyse the biotinylation of biotin-dependent carboxylases in both prokaryotes and eukaryotes. In Escherichia coli and Bacillus subtilis, the protein also acts as a transcriptional repressor that regulates the synthesis of biotin. Previously, we isolated and characterized a cDNA encoding an Arabidopsis thaliana HCS and subsequently assigned this enzyme form to the chloroplast compartment. To investigate whether or not the Arabidopsis protein may function as a regulator in E. coli, we have expressed the functional plant HCS in a birA-derepressed mutant strain of E. coli devoid of the corresponding E. coli protein and carrying a promoter-less LacZ gene marker inserted into the biotin operon, such that the bio promoter drives the synthesis of β-galactosidase. Our data demonstrate that although the expressed plant HCS efficiently complemented the function of apo-carboxylase biotinylation in E. coli, it proved unable to regulate the expression of the biotin biosynthetic genes.

36] Analysis of biotin biosynthesis pathway in coryneform bacteria: Brevibacterium flavum

Publisher Summary This chapter examines the coryneform bacterium, Brevibacterium flavum MJ233, for use as a bioconverter to produce amino acids and organic acids. The chapter also develops a new bioprocess using biotin removal from a minimal medium as a means to repress cellular division. The biosynthetic pathway of biotin is investigated mainly by using strains of Escherichia coli ( E. Coli ). The biotin biosynthetic genes are organized in an operon in E. coli , but in two different clusters in Bacillus sphaericus. Among the enzymes of the pathway, all that are involved in dethiobiotin synthesis from pimelic acid are presented—pimelyl–CoA synthetase, 7-keto-8-aminopelargonic acid synthetase, 7,8-diaminopelargonic acid aminotransferase, and dethiobiotin synthase. However, the final step in biotin synthesis, the conversion of dethiobiotin to biotin involves a flavodoxin, ferredoxin in addition to the bioB gene product. The lack of biotin biosynthesis is a taxonomical characteristic of the glutamic acid-producing group of coryneform bacteria. However, there have been no practical data regarding the deleted steps of biotin biosynthesis. The chapter reveals the deleted steps of the biotin biosynthetic pathway in several coryneform bacteria by using cross-feeding experiments with E. coil bio mutants. In addition, biotin biosynthetic genes are cloned and sequenced from a strain of coryneform bacterium, Brevibacterium flavum, MJ233.

Evidence for distinct ligand-bound conformational states of the multifunctional Escherichia coli repressor of biotin biosynthesis.

The Escherichia coli repressor of biotin biosynthesis (BirA) is a unique transcriptional repressor which catalyzes synthesis of its own corepressor and catalyzes attachment of a cofactor to an essential metabolic enzyme. BirA both catalyzes synthesis of biotinyl-5'-AMP from the substrates ATP and biotin and transfer of the biotin moiety from the adenylate to a lysine residue of a subunit of the acetyl-CoA carboxylase. BirA-bio-5'-AMP, moreover, binds sequence specifically to the biotin operator to repress transcription of the biotin biosynthetic genes. Using a combination of kinetic measurements of binding of the two ligands, biotin and bio-5'-AMP, to BirA as well as proteolytic digestion experiments, we have found evidence for at least three discrete conformational states of BirA. Results of stopped-flow fluorescence measurements of association of both ligands with BirA indicate that the process involves initial formation of a collision complex followed by a slow conformational change. The kinetics of the conformational change are distinct for the two ligands and are the basis for the difference in the thermodynamic stabilities of the two protein-ligand complexes. Different rates of proteolytic digestion of apoBirA and complexes of BirA with the two ligands were also observed. Results of the combined approaches indicate that apoBirA, and the BirA-bio-5'-AMP and BirA-biotin complexes are conformationally distinct.

Integration of the mutated biotin biosynthetic genes to the chromosome of a d-biotin-producing strain of Serratia marcescens

To construct a genetically stable strain of Serratia marcescens for d-biotin production, we integrated the mutated biotin-biosynthetic ( bio) genes into the chromosome of a d-biotin-producing strain of S. marcescens Sr41. Temperature-sensitive suicide vectors consisting of the Tn5 element were used as the integration tool. Extra bio genes carried between the Tn5 termini were integrated into the chromosome of strain ETA23, a d-biotin-producing mutant. Resultant integrants produced 120 mg of d-biotin per l of a medium containing sucrose and urea whereas ETA23 produced d-biotin at 55 mg/ l in the same medium. The integrated bio genes were stably maintained on the chromosome of ETA23 for 100 generations.

A genetic approach to the generation of antibodies with enhanced catalytic activities.

A hydrolytic catalytic antibody, generated against a nitrophenyl phosphonate transition state analogue, has been cloned and expressed in Escherichia coli for use as a model system to demonstrate the feasibility of using genetic selections to enhance catalytic activity. Conditions were found that permit the secretion of active recombinant antibody into the periplasm of a strain of E. coli deficient in the biotin biosynthetic genes (delta bio-gal). A number of substrates were synthesized that, upon hydrolysis by the antibody, yield free biotin, which is required for cell growth. The substrates and selections can be used to identify mutants of the antibody with altered activities. This approach should be generalizable to a wide number of hydrolytic reactions including the selective cleavage of peptide, polysaccharide, phosphodiester, and ester bonds.