Biosynthesis of riboflavin: cloning, sequencing, and expression of the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli.

3,4-Dihydroxy-2-butanone 4-phosphate is biosynthesized from ribulose 5-phosphate and serves as the biosynthetic precursor for the xylene ring of riboflavin. The gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli has been cloned and sequenced. The gene codes for a protein of 217 amino acid residues with a calculated molecular mass of 23,349.6 Da. The enzyme was purified to near homogeneity from a recombinant E. coli strain and had a specific activity of 1,700 nmol mg-1 h-1. The N-terminal amino acid sequence and the amino acid composition of the protein were in agreement with the deduced sequence. The molecular mass as determined by ion spray mass spectrometry was 23,351 +/- 2 Da, which is in agreement with the predicted mass. The previously reported loci htrP, "luxH-like," and ribB at 66 min of the E. coli chromosome are all identical to the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase, but their role had not been hitherto determined. Sequence homology indicates that gene luxH of Vibrio harveyi and the central open reading frame of the Bacillus subtilis riboflavin operon code for 3,4-dihydroxy-2-butanone 4-phosphate synthase.     

Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus

The gene coding aspartate racemase (EC 5.1.1.13) was cloned from the lactic acid bacteria Streptococcus thermophilus IAM10064 and expressed efficiently in Escherichia coli. The 2.1 kilobase pairs long full length clone had an open reading frame of 729 nucleotides coding for 243 amino acids. The calculated molecular weight of 27,945 agreed well with the apparent molecular weight of 28,000 found in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the aspartate racemase purified from S. thermophilus. The N-terminal amino acid sequence from the purified protein exactly matches the derived sequence. In addition, the amino acid composition compiled from the derived sequence is very similar to that obtained from the purified recombinant protein. No significantly homologous proteins were found in a protein sequence data bank. Even the homology scores with alanine racemases of Salmonella typhimurium and Bacillus stearothermophilus were low. Aspartate racemase was overproduced in Escherichia coli NM522 with plasmid pAG6-2-7, which was constructed from two copies of the gene linked with a tac promoter and plasmid vector pUC18. The amount of aspartate racemase increases with the growth of E. coli and almost no degradation of the enzyme was observed. The maximum amount of the produced enzyme reached approx. 20% of the total protein of E. coli.     

Localization of the T4 phage ribonucleotide reductase B1 subunit gene and the nucleotide sequence of its upstream and 5' coding regions

The nucleotide (nt) sequence in a 757-bp [corrected] segment downstream from the intron-containing T4 phage thymidylate synthase gene (td) has been determined. This region was found to contain two open reading frames (ORFs). The first ORF(ORF2) [corrected] 261 bp [corrected] in length, is 24 [corrected] nt downstream from the td gene. The second ORF(ORF3) [corrected]) is 200 bp long at 558 [corrected] nt from the td gene and extends to the end of the Eco RI fragment. The amino acid (aa) sequence (66 aa residues) deduced from the second truncated ORF shows 59% homology to the sequence of the N-terminal portion of the ribonucleotide reductase large subunit of either Escherichia coli (B1 subunit) or mouse (M1 subunit). This tentatively identifies the truncated gene to be the 5' end of the T4 phage ribonucleotide reductase subunit B1 (nrdA) gene and pinpoints its exact location on the T4 phage genomic map. Southern hybridization analysis suggests good sequence homology among the nrdA genes of various T-even phages.     

Cloning of the biotin synthetase gene from Bacillus sphaericus and expression in Escherichia coli and Bacilli

Biotin synthetase (BS) catalyses the biotransformation of dethiobiotin (DTB) to biotin. Here we report the cloning, characterization and expression of the gene encoding BS of Bacillus sphaericus. A recombinant plasmid pSB01, containing an 8.2-kb DNA fragment from B. sphaericus, was isolated by phenotypic complementation of an Escherichia coli bioB strain. Nucleotide sequence analysis of this fragment and N-terminal sequence determination of the recombinant protein product revealed that the bioB gene of B. sphaericus consists of a 996-bp open reading frame which is closely associated with at least one other gene. E. coli cells transformed with a bioB expression vector performed efficient bioconversion of DTB to biotin under defined culture conditions. Biotin production from transformed Bacillus subtilis and B. sphaericus recombinant strains was also demonstrated. Comparison of the amino acid sequences of BS from E. coli and B. sphaericus revealed extensive similarity.     

Sequence and functional analysis of the cloned Neisseria meningitidis CMP-NeuNAc synthetase

The CMP-N-acetylneuraminic acid (CMP-NeuNAc) synthetase gene of Neisseria meningitidis group B is located on a 2.3-kb EcoRI fragment within the cps gene cluster. Nucleotide sequence determination of the gene encoding the CMP-NeuNAc synthetase revealed a 515-bp open reading frame that can encode a 18.9-kDA protein. A computer data base scan revealed a 59.4% identity to the CMP-NeuNAc synthetase gene of E. coli K1. Enzymatic activity was confirmed in vitro and in vivo. Transformation of the CMP-NeuNAc defective E. coli K1 strain EV5 with the meningococcal CMP-NeuNAc synthetase could complement the defect in E. coli.     

Escherichia coli YrbI is 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase

3-Deoxy-d-manno-octulosonate 8-phosphate (KDO 8-P) phosphatase, which catalyzes the hydrolysis of KDO 8-P to KDO and inorganic phosphate, is the last enzyme in the KDO biosynthetic pathway for which the gene has not been identified. Wild-type KDO 8-P phosphatase was purified from Escherichia coli B, and the N-terminal amino acid sequence matched a hypothetical protein encoded by the E. coli open reading frame, yrbI. The yrbI gene, which encodes for a protein of 188 amino acids, was cloned, and the gene product was overexpressed in E. coli. The recombinant enzyme is a tetramer and requires a divalent metal cofactor for activity. Optimal enzymatic activity is observed at pH 5.5. The enzyme is highly specific for KDO 8-P with an apparent K(m) of 75 microm and a k(cat) of 175 s(-1) in the presence of 1 mm Mg(2+). Amino acid sequence analysis indicates that KDO 8-P phosphatase is a member of the haloacid dehalogenase hydrolase superfamily.     

Characterization of methylated neutral amino acids from Escherichia coli ribosomes.

The methylated neutral amino acids from both 30S and 50S ribosomal subunits of an Escherichia coli K strain were characterized. The 50S ribosomal subunit contains three methylated neutral amino acids: N-monomethylalanine, N-monomethylmethionine, and an as yet unidentified methylated amino acid found in protein L11. Both N-monomethylalanine and N-monomethylmethionine were found in protein L33. The amount of N-monomethylmethionine in this protein, however, is variable but not more than 0.25 molecules per protein. Thus protein L33 from this E. coli K strain has heterogeneity in its N-terminal amino acid and can start with either N-monomethylalanine or N-monomethylmethionine. The N-monomethylmethionine residue was not derived from the reduction of N-formylmethionine in the protein. The 30S ribosomal subunit contains only one methylated neutral amino acid: N-monomethylalanine.     

Cloning and sequencing of a Bacteroides ruminicola B(1)4 endoglucanase gene.

Bacteroides ruminicola B(1)4, a noncellulolytic rumen bacterium, produces an endoglucanase (carboxymethylcellulase [CMCase]) that is excreted into the culture supernatant. Cultures grown on glucose, fructose, maltose, mannose, and cellobiose had high specific activities of CMCase (greater than 3 mmol of reducing sugar per mg of protein per min), but its synthesis was repressed by sucrose. B. rumincola did not grow on either ball-milled or acid-swollen cellulose even though the CMCase could hydrolyze swollen cellulose. The CMCase gene was cloned into Escherichia coli, and its nucleotide sequence contained a single open reading frame coding for a protein of 40,481 daltons. The enzyme was overproduced in E. coli under the control of the tac promoter and purified to homogeneity. The N-terminal sequence, amino acid composition, and molecular weight of the purified enzyme were similar to the values predicted from the open reading frame of the DNA sequence. However, the CMCase present in B. ruminicola was found to have a monomer molecular weight of 88,000 by Western immunoblotting. This discrepancy appeared to have resulted from our having cloned only part of the CMCase gene into E. coli. The amino acid sequence of the CMCase showed homology to sequences of beta-glucanases from Ruminococcus albus and Clostridium thermocellum.     

Subcloning and nucleotide sequence of the 3,4-dihydroxyphenylacetate (homoprotocatechuate) 2,3-dioxygenase gene from Escherichia coli C

A cloned gene encoding the Escherichia coli C homoprotocatechuate (HPC) dioxygenase, an aromatic ring cleavage enzyme, was used to produce large amounts of the protein. Preparations of E. coli C HPC dioxygenase, whether expressed from the cloned gene or produced by the bacterium, lost activity very rapidly. The pure protein showed one type of subunit of Mr 33,000. The first 21 N-terminal amino acids were sequenced and the data used to confirm that the open reading frame of 831 bp, identified from the nucleotide sequence, encoded HPC dioxygenase. Comparison of the derived amino acid sequence with those of other extradiol and intradiol dioxygenases showed no obvious similarity to any of them.     

Sequence of the Clostridium thermocellum mannanase gene man26B and characterization of the translated product

The man26B gene of Clostridium thermocellum strain F1 was found in pKS305, which had been selected as a recombinant plasmid conferring endoglucanase activity on Escherichia coli. The open reading frame of man26B consists of 1,773 nucleotides encoding a protein of 591 amino acids with a predicted molecular weight of 67,047. Man26B is a modular enzyme composed of an N-terminal signal peptide and three domains in the following order: a mannan-binding domain, a family 26 mannanase domain, and a dockerin domain responsible for cellulosome assembly. We found that this gene was a homologue of the man26A gene of C. thermocellum strain YS but that there were insertion or deletion mutations that caused a frame-shift mutation affecting a stretch of 26 amino acids in the catalytic domain. Man26B devoid of the dockerin domain was constructed and purified from a recombinant E. coli, and its enzyme properties were examined. Immunological analysis indicated that Man26B was a catalytic component of the C. thermocellum F1 cellulosome.     

Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum

The nucleotide sequence of the cellulase gene celC, encoding endoglucanase C of Clostridium thermocellum, has been determined. The coding region of 1032 bp was identified by comparison with the N-terminal amino acid (aa) sequence of endoglucanase C purified from Escherichia coli. The ATG start codon is preceded by an AGGAGG sequence typical of ribosome-binding sites in Gram-positive bacteria. The derived amino acid sequence corresponds to a protein of Mr 40,439. Amino acid analysis and apparent Mr of endoglucanase C are consistent with the amino acid sequence as derived from the DNA sequencing data. A proposed N-terminal 21-aa residue leader (signal) sequence differs from other prokaryotic signal peptides and is non-functional in E. coli. Most of the protein bears no resemblance to the endoglucanases A, B, and D of the same organism. However, a short region of homology between endoglucanases A and C was identified, which is similar to the established active sites of lysozymes and to related sequences of fungal cellulases.     

Cloning and sequencing of the gene encoding a 125-kilodalton surface-layer protein from Bacillus sphaericus 2362 and of a related cryptic gene.

Using the vector pGEM-4-blue, a 4,251-base-pair DNA fragment containing the gene for the surface (S)-layer protein of Bacillus sphaericus 2362 was cloned into Escherichia coli. Determination of the nucleotide sequence indicated an open reading frame (ORF) coding for a protein of 1,176 amino acids with a molecular size of 125 kilodaltons (kDa). A protein of this size which reacted with antibody to the 122-kDa S-layer protein of B. sphaericus was detected in cells of E. coli containing the recombinant plasmid. Analysis of the deduced amino acid sequence indicated a highly hydrophobic N-terminal region which had the characteristics of a leader peptide. The first amino acid of the N-terminal sequence of the 122-kDa S-layer protein followed the predicted cleavage site of the leader peptide in the 125-kDa protein. A sequence characteristic of promoters expressed during vegetative growth was found within a 177-base-pair region upstream from the ORF coding for the 125-kDa protein. This putative promoter may account for the expression of this gene during the vegetative growth of B. sphaericus and E. coli. The gene for the 125-kDa protein was followed by an inverted repeat characteristic of terminators. Downstream from this gene (11.2 kilobases) was an ORF coding for a putative 80-kDa protein having a high sequence similarity to the 125-kDa protein. Evidence was presented indicating that this gene is cryptic.     

Molecular cloning of actinohivin, a novel anti-HIV protein from an actinomycete, and its expression in Escherichia coli

Syncytium-inducing variants of the HIV-1 virus are correlated with poor diagnosis and rapid disease progression. We have recently discovered a novel anti-HIV protein, referred to as actinohivin, that inhibits syncytium formation. Here we describe the cloning and sequencing of the gene encoding actinohivin from the actinomycete strain K97-0003, and its expression in Escherichia coli. The actinohivin gene was located on a 0.8-kb BamHI fragment of genomic DNA. The fragment contained an open reading frame of 480 bp, which encoded a protein of 160 amino acids with calculated molecular weight of 17492.7. The N-terminal region was found to be a typical signal peptide of prokaryotes, and actinohivin was located at amino acid positions 46-160. The actinohivin gene could be expressed in E. coli using a pET30Xa/LIC expression vector and the purified recombinant actinohivin was found to inhibit syncytium formation to a similar extent as actinohivin from its natural source.     

Primary structure of the oligo-1,6-glucosidase of Bacillus cereus ATCC7064 deduced from the nucleotide sequence of the cloned gene

The gene coding for Bacillus cereus ATCC7064 (mesophile) oligo-1,6-glucosidase was cloned within a 2.8-kb SalI-EcoRI fragment of DNA, using the plasmid pUC19 as a vector and Escherichia coli C600 as a host. E. coli C600 bearing the hybrid plasmid pBCE4 accumulated oligo-1,6-glucosidase in the cytoplasm. The cloned enzyme coincided absolutely with B. cereus oligo-1,6-glucosidase in its Mr (65,000), in its electrophoretic behavior on a polyacrylamide gel with or without sodium dodecyl sulfate, in its isoelectric point (4.5), in the temperature dependence of its stability and activity, and in its antigenic determinants. The nucleotide sequence of B. cereus oligo-1,6-glucosidase gene and its flanking regions was determined with both complementary strands of DNA (each 2838 nucleotides). The gene consisted of an open reading frame of 1674 bp commencing with a ATG start codon and followed by a TAA stop codon. The amino acid sequence deduced from the nucleotide sequence predicted a protein of 558 amino acid residues with a Mr of 66,010. The amino acid composition and Mr were comparable with those of B. cereus oligo-1,6-glucosidase. The predicted N-terminal sequence of 10 amino acid residues agreed completely with that of the cloned ligo-1,6-glucosidase. The deduced amino acid sequence of B. cereus oligo-1,6-glucosidase was 72% and 42% similar to those from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile) oligo-1,6-glucosidase and from Saccharomyces carlsbergensis CB11 alpha-glucosidase, respectively. Predictions of protein secondary structures along with amino acid sequence alignments demonstrated that B. cereus oligo-1,6-glucosidase may take the similar (alpha/beta)8-barrel super-secondary structure, a barrel of eight parallel beta-strands surrounded by eight alpha-helices, in its N-terminal active site domain as S. carlsbergensis alpha-glucosidase and Aspergillus oryzae alpha-amylase.