Cloning and expression in Escherichia coli of the gene for 10-formyltetrahydrofolate synthetase from Clostridium acidiurici ("Clostridium acidi-urici")

The gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from the purinolytic anaerobic bacterium Clostridium acidiurici ("Clostridium acidi-urici") was cloned into Escherichia coli JM83 with plasmid pUC8. A C. acidiurici genomic library was prepared in E. coli from a partial Sau3A digest and screened with antibody against the synthetase. Of 10 antibody-positive clones, 1 expressed a high level of synthetase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis demonstrated that the protein synthesized in E. coli had the same subunit molecular weight as the C. acidiurici enzyme. The gene was located on an 8.3-kilobase genomic insert and appeared to be transcribed from its own promoter. Analysis of genomic digests with a fragment of the synthetase gene indicated that one copy of the gene was present in the C. acidiurici chromosome.     

Nucleotide sequence and analysis of the purA gene encoding adenylosuccinate synthetase of Escherichia coli K12

Adenylosuccinate synthetase (EC 6.3.4.4), encoded by the purA gene of Escherichia coli K12, catalyzes the synthesis of adenylosuccinate (SAMP) from IMP, the first committed step in AMP biosynthesis. The E. coli K12 purA gene and flanking DNA was cloned by miniMu-mediated transduction, and the nucleotide sequence was determined. The mature SAMP synthetase subunit, as deduced from the DNA sequence, contains 427 amino acid residues and has a calculated Mr of 47,277. The size of the purA mRNA was determined by Northern blotting to be approximately 1.5 kilobase pairs. The 5'-end of the purA mRNA was identified by primer extension and is located 23 nucleotides upstream of the ATG translational initiation codon. Comparison of the purA control region with the guaBA control region revealed a common region of dyad symmetry which may suggest mutual elements of regulation. The purA control region did not resemble the control regions of the other known pur loci.     

Molecular analysis and regulation of the glnA gene of the gram-positive anaerobe Clostridium acetobutylicum.

The nucleotide sequence of a 2.0-kilobase DNA segment containing the Clostridium acetobutylicum glnA gene was determined. The upstream region of the glnA gene contained two putative extended promoter consensus sequences (p1 and p2), characteristic of gram-positive bacteria. A third putative extended gram-positive promoter consensus sequence (p3), oriented towards the glnA gene, was detected downstream of the structural gene. The sequences containing the proposed promoter regions p1 and p2 or p3 were shown to have promoter activity by subcloning into promoter probe vectors. The complete amino acid sequence (444 residues) of the C. acetobutylicum glutamine synthetase (GS) was deduced, and comparisons were made with the reported amino acid sequences of GS from other organisms. To determine whether the putative promoter p3 and a downstream region with an extensive stretch of inverted repeat sequences were involved in regulation of C. acetobutylicum glnA gene expression by nitrogen in Escherichia coli, deletion plasmids were constructed lacking p3 and various downstream sequences. Deletion of the putative promoter p3 and downstream inverted repeat sequences affected the regulation of GS and reduced the levels of GS approximately fivefold under nitrogen-limiting conditions but did not affect the repression of GS levels in cells grown under nitrogen-excess conditions.     

Regulation of the tryptophan synthetic enzymes in Clostridium butyricum

Experiments concerned with the regulation of the tryptophan synthetic enzymes in anaerobes were carried out with a strain of Clostridium butyricum. Enzyme activities for four of the five synthetic reactions were readily detected in wild-type cells grown in minimal medium. The enzymes mediating reactions 3, 4, and 5 were derepressed 4- to 20-fold, and the data suggest that these enzymes are coordinately controlled in this anaerobe. The first enzyme of the pathway, anthranilate synthetase, could be derepressed approximately 90-fold under these conditions, suggesting that this enzyme is semicoordinately controlled. Mutants resistant to 5-methyl tryptophan were isolated, and two of these were selected for further analysis. Both mutants retained high constitutive levels of the tryptophan synthetic enzymes even in the presence of repressing concentrations of tryptophan. The anthranilate synthetase from one mutant was more sensitive to feedback inhibition by tryptophan than the enzyme from wild-type cells. The enzyme from the second mutant was comparatively resistant to feedback inhibition by tryptophan. Neither strain excreted tryptophan into the culture fluid. Tryptophan inhibits anthranilate synthetase from wild-type cells noncompetitively with respect to chorismate and uncompetitively with respect to glutamine. The Michaelis constants calculated for chorismate and glutamine are 7.6 x 10(-5)m and 6.7 x 10(-5)m, respectively. The molecular weights of the enzymes estimated by zonal centrifugation in sucrose and by gel filtration ranged from 24,000 to 89,000. With the possible exception of a tryptophan synthetase complex, there was no evidence for the existence of other enzyme aggregates. The data indicate that tryptophan synthesis is regulated by repression control of the relevant enzymes and by feedback inhibition of anthranilate synthetase. That this enzyme system more closely resembles that found in Bacillus than that found in enteric bacteria is discussed.     

Sequence analysis of the Clostridium cellulolyticum endoglucanase-A-encoding gene, celCCA

The nucleotide sequence of a Clostridium cellulolyticum endo-beta-1,4- glucanase (EGCCA)-encoding gene (celCCA) and its flanking regions, was determined. An open reading frame (ORF) of 1425 bp was found, encoding a protein of 475 amino acids (aa). This ORF began with an ATG start codon and ended with a TAA ochre stop codon. The N-terminal region of the EGCCA protein resembled a typical signal sequence of a Gram-positive bacterial extracellular protein. A putative signal peptidase cleavage site was determined. EGCCA, without a signal peptide, was found to be composed of more than 35% hydrophobic aa and to have an Mr of 50715. Comparison of the encoded sequence with other known cellulase sequences showed the existence of various kinds of aa sequence homologies. First, a strong homology was found between the C-terminal region of EGCCA, containing a reiterated stretch of 24 aa, and the conserved reiterated region previously found to exist in four Clostridium thermocellum endoglucanases and one xylanase from the same organism. This region was suspected of playing a role in organizing the cellulosome complex. Second, an extensive homology was found between EGCCA and the N-terminal region of the large endoglucanase, EGE, from C. thermocellum, which suggests that they may have a common ancestral gene. Third, a region, which extended for 21 aa residues beginning at aa + 127, was found to be homologous with regions of cellulases belonging to Bacilli, Clostridia and Erwinia chrysanthemi.     

Nucleotide sequence of the Clostridium thermocellum laminarinase gene.

The sequence presented (1022 bp) shows the Clostridium thermocellum laminarinase gene (lam1) and its flanking regions. The gene lam1 comprises an open reading frame of 726 nt, encoding a 242-aa protein with predicted Mr 27661. The ORF startswith the translation initiation codon ATG. This ATG codon is preceded at a spacing of 7 bp by a potential ribosome binding site (GGAGGT). A putative signal peptide was identified (the potential cleavage site is between position 27-28 aa). The comparison of the primary protein sequence with other beta-1, 3-1, 4-glucanases showed extensive homology for Bacillus amyloliqefaciens and Bacillus subtilis glucanases (identity-46.7%; similarity-57.0%).     

Structure and transcription of genes within the β-hbd-adh1 region of Clostridium acetobutylicum P262

Abstract The 1.2-kb DNA fragment upstream of the linked β- hbd (3-hydroxybutyryl-CoA dehydrogenase) and adh1 (NADPH-dependent alcohol dehydrogenase) genes from Clostridium acetobutylicum P262 was sequenced. The upstream region contained an open reading frame (ORFB) which was found to have 44% amino acid identity to the fixB gene products of yRhizobium and Azorhizobium . The β- hbd and ORFB genes were expressed during the acidogenic and solventogenic phases. The β- hbd gene was transcribed on a single mRNA species of 2.0 kb, whereas the ORFB gene was transcribed on two species of mRNA of 2.0 and 3.5 kb, respectively. The adh1 gene was induced or derepressed at the pH breakpoint before the onset of solventogenesis and was transcribed on a single species of mRNA of 2.4 kb.     

Primary structure of the thermostable formyltetrahydrofolate synthetase from Clostridium thermoaceticum

The complete nucleotide sequence of the Clostridium thermoaceticum formyltetrahydrofolate synthetase (FTHFS) was determined and the primary structure of the protein predicted. The gene was 1680 nucleotides long, encoding a protein of 559 amino acid residues with a calculated subunit molecular weight of 59,983. The initiation codon was UUG, with a probable ribosome binding site 11 bases upstream. A putative ATP binding domain was identified. Two Cys residues likely to be involved in subunit aggregation were tentatively identified. No characterization of the tetrahydrofolate (THF) binding domain was possible on the basis of the sequence. A high level of amino acid sequence conservation between the C. thermoaceticum FTHFS and the published sequences of C. acidiurici FTHFS and the FTHFS domains of the Saccharomyces cerevisiae C1-THF synthases was found. Of the 556 residues shared between the two clostridial sequences, 66.4% are identical. If conservative substitutions are allowed, this percentage rises to 75%. Over 47% of the residues shared between the C. thermoaceticum FTHFS and the yeast C1-THF synthases are identical, 57.4% if conservative substitutions are allowed. Hydrophobicity profiles of the C. acidiurici and C. thermoaceticum enzymes were very similar and did not support the idea that large hydrophobic domains play an important role in thermostabilizing the C. thermoaceticum FTHFS.     

The enzymes of phospholipid synthesis in Clostridium butyricum

We have examined extracts of Clostridium butyricum for several enzymes of phospholipid synthesis. Membrane particles were shown to catalyze the formation of CDP-diglyceride from [3H]CTP and phosphatidic acid. The reaction was dependent on Mg2+ and stimulated by monovalent cations. CDP-diglyceride formed in vitro was found to be a substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. The formation of phosphatidylglycerophosphate from added CDP-diglyceride and [U-14C]sn-glycerol-3-phosphate was dependent on Mg2+ and Triton X-100. The dephosphorylation of endogenously-generated phosphatidylglycerophosphate to yield phosphatidylglycerol was observed to be pH-dependent. The formation of phosphatidylserine from CDP-diglyceride and L-[3-14C]serine was stimulated by Mg2+ and Triton X-100. dCDP-diglyceride was a suitable substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. Phosphatidylserine decarboxylase activity was barely detectable in membrane particles from C. butyricum. The addition of E. coli membrane particles provided efficient phosphatidylserine decarboxylase activity in this system. Although plasmalogens are the principal lipids of C. butyricum, none of the products of phospholipid synthesis formed in vitro contained measurable amounts of plasmalogens. The subcellular distribution of both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase in C. butyricum was also studied. Both were found to be membrane-associated.     

alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.     

Cloning and whole nucleotide sequence of the gene for the light chain component of botulinum type E toxin from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike.

Chromosomal DNAs were extracted from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike. The 6.0 Kbp fragment coding for the entire light chain (L) component and the N-terminus of heavy chain (H) component of botulinum type E toxin was obtained from each extracted DNAs after digestion with HindIII. The entire nucleotide sequences for the light chain components of these cloned genes were determined, and the derived amino acid sequences were compared to each other, and with those of botulinum type A, C1, D, and tetanus toxins reported previously. The cleavage site of L and H components of type E toxin was presumed to be Arg-422. In a total of 422 amino acid residues of L component, 17 residues were different between butyricum and type E toxins, and all these differences were found within 200 residues of N-terminus of L component. On the contrary, five regions showing highly homologous sequences were found in L components among these six toxins, and one more region between botulinum type E and tetanus toxins.