Structure-function studies of Escherichia coli biotin synthase via a chemical modification and site-directed mutagenesis approach.

In Escherichia coli, biotin synthase (bioB gene product) catalyzes the key step in the biotin biosynthetic pathway, converting dethiobiotin (DTB) to biotin. Previous studies have demonstrated that BioB is a homodimer and that each monomer contains an iron-sulfur cluster. The purified BioB protein, however, does not catalyze the formation of biotin in a conventional fashion. The sulfur atom in the iron-sulfur cluster or from the cysteine residues in BioB have been suggested to act as the sulfur donor to form the biotin molecule, and yet unidentified factors were also proposed to be required to regenerate the active enzyme. In order to understand the catalytic mechanism of BioB, we employed an approach involving chemical modification and site-directed mutagenesis. The properties of the modified and mutated BioB species were examined, including DTB binding capability, biotin converting activity, and Fe(2+) content. From our studies, four cysteine residues (Cys 53, 57, 60, and 97) were assigned as the ligands of the iron-sulfur cluster, and Cys to Ala mutations completely abolished biotin formation activity. Two other cysteine residues (Cys 128 and 188) were found to be involved mainly in DTB binding. The tryptophan and histidine residues were suggested to be involved in DTB binding and dimer formation, respectively. The present study also reveals that the iron-sulfur cluster with its ligands are the key components in the formation of the DTB binding site. Based on the current results, a refined model for the reaction mechanism of biotin synthase is proposed.

Molecular cloning and characterization of a unique 60 kDa/72 kDa antigen gene encoding enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) of Mycoplasma hyopneumoniae.

The recombinant clone expressing a 60 kDa (P60) antigen was isolated from Escherichia coli by screening a lambda EMBL3 genomic library using rabbit produced antiserum against Mycoplasma hyopneumoniae. Sequence analysis revealed that an interrupted (by a UGA codon) open reading frame coding for a 72 kDa protein (P72) may contain the P60 antigen gene. Western blot analysis with an anti-P60 monospecific antibody confirmed the presence of a P72 antigen from the total protein of M. hyopneumoniae, and a 72 kDa protein was also expressed in E. coli after changing the codon (UGA to UGG) by site-directed mutagenesis. BLAST (Basic Local Alignment Search Tool) comparison showed that the amino acid sequences of P72 share approximately 70% homology with the phosphotransferase enzyme I (PTSI) of bacteria and other mycoplasma species. The biological function of the P72 cytosolic protein was further confirmed by complementation using an E. coli ptsI mutant. The bacterial phosphoenolpyruvate-sugar phosphotransferase system (PTS) is known to mediate the uptake and phosphorylation of carbohydrates and to be involved in signal transduction. The immune responses of specific pathogen free (SPF) pigs and farm animals toward this unique antigen were observed. The transcription start positions of the PTSI gene were determined in M. hyopneumoniae and E. coli by primer extension experiments and the promoter site was also predicted.

Lysozyme association with nucleic acids.

Lysozyme is well known for the ability to hydrolyze the cell wall of bacteria. Based on the similarity of structure between lysozyme and histones as seen from the results of X-ray crystal structure determinations, we have postulated that binding to nucleic acids may be another biological function of lysozyme. We have therefore begun a systematic study of the interactions of lysozyme and related molecules with nucleic acids, and present here a preliminary report. Binding to DNA and RNA has been demonstrated from gel electrophoresis, enzyme activity, and coprecipitation studies. We suggest that this function of lysozyme will provide an explanation why Lee-Huang et al. (1999) [Proc. Natl. Acad. Sci. USA 96, 2678-2681] were able to call lysozyme a "killer protein" against the AIDS virus, and may provide a new avenue of research on AIDS therapy.

Bioassay of biotin concentration with a Escherichia coli bio deletion mutant.

Biotin concentration was determined unequivocally with the E. coli bio mutant. The results demonstrate that this simple and efficient method can determine biotin concentration in the range of 10 pg to 50 ng/ml. The present method can also clearly distinguish biotin from its precursor and analog, dethiobiotin.

Molecular structure of the amyloid-forming protein kappa I Bre.

The molecular structure of the amyloid-forming Bence-Jones protein kappa I Bre has been determined by X-ray crystallography at 2.0 A resolution. The fragment from the kappa chain of immunoprotein contains 107 amino acid residues, and polymerizes in the crystal form into a giant helical spiral, surrounding a cylinder of water 50 A in diameter with a repeat of 77.56 A, containing 12 kappa molecules, plus another 12 molecules from neighboring parallel spirals. The resulting structure has many features which have been found or suggested from studies on the protein fibrils found in amyloid deposits. From the results of the X-ray crystal structure a hypothesis is presented for the structure and formation of the amyloid fibril.

Nucleotide sequencing of S-RNA segment and sequence analysis of the nucleocapsid protein gene of the newly isolated Akabane virus PT-17 strain.

The nucleotide sequences of the S-RNA of Akabane viruses JaGAr-39, OBE-1, Iriki and the newly isolated PT-17 strains and the Aino virus were determined and compared. The results reveal that the S-RNAs of the four Akabane strains share 96.9% homology in nucleotide sequences. Only one amino acid difference out of the 233 amino acids of the nucleocapsid protein (N) and three amino acid differences in the 91 amino acids of the nonstructural protein (NSs) were found among the Akabane viruses. Amino acid sequences of N and NSs proteins of the Aino virus have approximately 80% identity as compared with the Akabane viruses. The results also demonstrate that the four Akabane viruses and the Aino virus can be clearly differentiated by RFLP (restriction fragments length polymorphism) analysis using RT-PCR generated nucleocapsid protein genes and digested with HaeIII and HindIII. The phylogenetic tree based on the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) analysis of the sequences of nucleocapsid protein genes and the S-DNAs revealed that the newly isolated PT-17 strain is most closely related to Iriki strain, than the JaGAr-39 or OBE-1 strains.

Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome.

To study the variation in spontaneous mutation frequencies in different chromosomal domains, a mini-Mu-kan-lacZ- transposable element was constructed to insert the lacZ-(Trp570 --> Opal) allele into many different loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop --> LacZ+; candidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14000 colonies, and named plm-1 (preferential lacZ mutagenesis) and plm-2. The frequency of LacZ- --> LacZ+ mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac+ reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan(r) gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ+ --> LacZ- and Kan(r) --> Kan(s) mutant frequencies of these Lac+ revertants were in the range of 10(-3) to 10(-2), indicating that this putative localized mutagenesis is neither allele nor gene specific. To identify the plm loci, the chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome.

Construction and characterization of a biotin-regulated gene expression system in Escherichia coli.

An autoregulated gene expression system in Escherichia coli was designed such that the cloned genes on the vector were not expressed until biotin was depleted during cell growth. The expression vectors were constructed by assembling the DNA fragments containing the regulatory region of the E. coli biotin operon (bio operon), the universal ribosome-binding site (RBS) and the strong transcription terminator rrnBT1T2. The promoter region was further modified by site-directed mutagenesis to create promoters of varied strength. The feasibility of this system was examined in E. coli strain R901 (with bio operon deleted) using various marker genes, including the E. coli birA gene, T7 RNA polymerase gene and yellowfin-porgy growth-hormone gene. The results demonstrated that the induction of marker-gene expression can be triggered as the biotin concentration drops to a threshold value of approximately 2 ng/mL by metabolic utilization.

Molecular cloning and analysis of a HSP (heat shock protein)-like 42 kDa antigen gene of Mycoplasma hyopneumoniae.

The recombinant clone expressing the 42 kDa protein (P42) of Mycoplasma hyopneumoniae in Escherichia coli was analyzed. The 4.4 kb HindIII-Xmal DNA fragment expressing the p42 gene product encodes three ORFs: p42 and p16 in the forwarding strand, p24 in the reverse strand. Sequence comparisons revealed that p42 could be part of a p65 gene, and has 62% identities with Mycoplasma genitalium HSP70 gene and 56% identities with Bacillus subtilis dnaK gene; p16 and p24 genes share 73% and 47% identities with Erysipelothrix rhusiopathiae dnaJ gene and Pseudomonas fluorescens uvrC gene, respectively. Further analysis demonstrated that P42 is indeed a heat shock protein and the monospecific antibodies against P42 can block the growth of Mycoplasma hyopneumoniae.

Molecular cloning and nucleotide sequencing of bioF (7-keto-8-amino pelargonic acid synthetase), bioC and bioD (dethiobiotin synthetase) genes of Erwinia herbicola.

The biotin operon of Erwinia herbicola Eho 10 was cloned and characterized by complementation of E. coli biotin mutants. The operon was found to contain five genes arranged in the order, bioABFCD. The nucleotide sequences of bioF (7-keto-8-aminopelargonic acid synthetase), bioC and bioD (dethiobiotin synthetase) were determined and analyzed. The nucleotide sequences and deduced amino acid sequences of bioFCD were compared with the corresponding sequences from Escherichia coli, Bacillus sphaericus, Serratia marcescens and Brevibacterium flavum.

Isolation and characterization of the Erwinia herbicola bio operon and the sequences of the bioA and bioB genes.

The biotin operon of Erwinia herbicola was cloned and characterized. The operon consists of five genes arranged in the order, bioABFCD. The operon is negatively regulated via the interaction of a proposed biotin repressor with an operator sequence that lies between the bioA and bioB genes. The nucleotide sequences of bioA (7,8-diaminopelargonic acid transferase), bioB (biotin synthetase) and the regulatory region were determined and analyzed. The deduced amino acid sequences of bioA and bioB are also aligned with currently available homologs to obtain the UPGMA (unweighted pair group method with arithmetic mean) evolutionary tree.

A simple method for DNaseI footprinting analysis.

DNaseI footprinting technique has been a very useful method in gaining direct and immediate information about the location of a protein binding site in the DNA sequence. In this report, we present a method that overcomes many of the inconveniences of previous methods. This method was based on the combination of PCR technique and the dideoxy DNA sequencing reaction. It avoids the need of the secondary restriction sites that any candidate DNA sequence for DNaseI footprinting analysis can be prepared efficiently as long as its flanking sequences are known. Replacing the difficult Maxam-Gilbert DNA sequencing with the dideoxy-mediated DNA sequencing method makes the footprinting experiments even safer and easier.

Determination of biotin concentration by a competitive enzyme-linked immunosorbent assay (ELISA) method.

A method based on competitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of biotin concentrations which takes advantage of the extraordinarily high affinity between biotin and avidin. The biotin assay consisted of two steps, (i) a competition reaction between excess streptavidin-conjugated horseradish peroxidase (streptavidin-HRP) and solutions of known biotin concentrations or sample solutions and (ii) the measurement of the residual activities of the free form streptavidin-HRP which were correlated with the initial biotin concentrations. The procedure was modified by including an extra step of antibody-antigen interaction to assay biotin concentration unambiguously in more complex media. The entire assay was completed within 6 with sensitivities of approximately 1 pg/ml for biotin in a simple aqueous medium and 5 pg/ml in complex media. The method offers significant advantages in time, sensitivity and simplicity for determinations of biotin concentrations in various solutions.

A single Ser-180 mutation desensitizes feedback inhibition of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase in Escherichia coli.

The Escherichia coli phenylalanine-sensitive DAHP (3-deoxy-D-arabino-heptulosonate 7-phosphate) synthetase (aroG product) is one of the DAHP synthetase isozymes that catalyze the first committed step in the biosynthesis of aromatic amino acids and vitamins. Through target-directed mutagenesis of the cloned aroG on a plasmid vector, followed by screening of phenylalanine-resistant colonies, we isolated a clone (pG908) showing feedback-insensitive mutation of DAHP synthetase. The mutations were identified as a T-->A mutation at nucleotide 22, and a C-->T mutation at nucleotide 539, causing a Leu-8 to Ile-8 mutation and a Ser-180 to Phe-180 substitution, respectively. The resulting enzyme exhibited comparable enzymatic activity to the wild type, but the degree of feedback inhibition had declined from approximately 60% to less than 10% in the presence of 20 mM phenylalanine in the assay medium. Replacement of Ile-8 by Leu, and substitution of Phe-180 with Ser, Asn and Cys, using site-directed mutagenesis, demonstrated that Ser-180 is a critical residue in the feedback inhibition of AroG. The result suggests that the major role played by Ser-180 may not involve a simple charge-size effect in the Phe-binding site of the enzyme molecule, but rather may involve more complicated molecular interactions occurring in the feedback inhibition mechanisms.

Transcriptional analysis of the Escherichia coli bio operon.

Using primer extension, two in vivo transcription start points (tsp) were identified for rightward transcription of the Escherichia coli biotin operon, at nucleotides (nt) +20 and +29. The strongest leftward transcript begins at +9, with a tenfold less abundant transcript starting at +3. The activity of segments cloned into promoter probe vectors locates the major leftward promoter between nt +1 and +105, as expected for the +9 tsp. Although the activity of a chromosomal operator is reduced about 300-fold by point mutation in either arm of the palindrome extending from nt -20 to +20, either a half-operator segment or a full operator bearing the same point mutation in one arm is substantially repressed when cloned into pKB2000, as though cellular location strongly affects the operator's affinity for the repressor.

Rapid purification of antiserum against Mycoplasma hyopneumoniae by an efficient absorption method.

A simple and efficient method for the removal of unwanted cross-reactive antibodies has been developed. The antiserum purification method was based on treatment of the antiserum with both sonicated extracts and boiling extracts of the Escherichia coli host cells used in immunoscreening the lambda EMBL3 library. We have demonstrated unambiguously that through this simple treatment, the rabbit anti-Mycoplasma hyopneumoniae antiserum can be effectively purified so that the amount of antibodies cross-reacted with Escherichia coli lysate proteins is drastically reduced. Compared with the traditional absorption methods, which require the chemical coupling of an absorbing agent to an insoluble support, and affinity purification methods, which have harsh denaturing condition, this method should greatly facilitate a successful immunoscreening experiment.

DNaseI footprinting studies of Escherichia coli biotin repressor-operator interactions.

Transcription of Escherichia coli biotin operon is repressed by biotin repressor in the presence of biotinyl-5'-adenylate as a corepressor. To determine precisely the site of action of biotin repressor on the operator sequence, DNaseI footprinting experiments were performed on the PCR-produced biotin operator and its mutants. The results indicate that the repressor binds to the wild-type operator as well as mutated operator sequence at +15 position or -15 position, and protects the 39-base region from nucleotide -19 to +20 of the upper strand, and the 40-base region from nucleotide -22 to +18 of the lower strand, with a few hypersensitive sites. This is consistent with previous speculation that the biotin operator is an approximately 40 bp imperfect palindromic DNA sequence capable of binding with two molecules of biotin repressor. However, the protection pattern of the mutant operator which lacks half of the palindromic structure is quite different from the corresponding region of the wild type. Though two repressor monomers bound to the mutant operator, as revealed by parallel binding studies [Lin, Shiuan, and Campbell (1991) Biochim. Biophys. Acta 1090, 317-325], only 12 to 13 bp on the DNA sequence was protected, suggesting that one monomer of the repressor dimer is hanging near the DNA backbone of the mutant operator even though the biotin repressor is functioning as a dimer.

Binding characteristics of Escherichia coli biotin repressor-operator complex.

The genes of the operon bioA.BFCD are transcribed divergently from a single regulatory region between the bioA and, bioB genes. Transcription in both directions is co-repressed by biotinyl-5'-adenylate and the biotin repressor. The multimeric state of the biotin repressor bound to DNA and how it affects transcription have not been fully characterized. Therefore, we isolated the BirA protein from a recombinant strain which overproduced the biotin repressor and studied the repressor-operator binding characteristics through restriction enzyme site protection experiments and the mobility shift assay. We also measured the stoichiometry of the biotin repressor-operator complex directly. The results of restriction enzyme site protection studies are consistent with the postulation that the biotin operator is approx. 40 bp long. Only one retarded DNA band appeared in the mobility shift experiments, suggesting that the repressor-operator binding could be a single step reaction. The repressor-operator binding stoichiometry determination revealed that two repressor monomers may occupy the wild type or half palindromic biotin operator sequences.

Transcriptional regulation and gene arrangement of Escherichia coli, Citrobacter freundii and Salmonella typhimurium biotin operons.

The bio operons of Citrobacter freundii and Escherichia coli K-12 (strain C600) were isolated by screening lambda banks for complementation of E. coli bio mutants. These were compared with the previously isolated bio operon of Salmonella typhimurium and previous data on E. coli K-12. The restriction maps of the operon are very different in the three species, but no difference in gene order was found. Operator-promoter DNA, identified by repressible titration and by biotin-repressible transcription in E. coli, was sequenced and compared to the published E. coli K-12 sequence. In the segment previously identified as operator/bioB promoter, C. freundii and S. typhimurium DNA are identical and differ from E. coli only by 2 bp. The DNA to the right of this segment (indicated by previous data to be the bioA promoter of E. coli) has diverged in all three species, and only E. coli has a sequence resembling a consensus promoter.