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Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis.

ABSTRACT: A DNA region of Clostridium acetobutylicum contiguous with the adc operon has been cloned and sequenced. Structural genes encoding the acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase (ctfB and ctfA) and an alcohol/aldehyde dehydrogenase (adhE) could be identified. These three genes together with a small open reading frame (ORF) of unknown function (upstream of adhE) formed an operon (sol operon), as shown by mRNA analyses. The complete sol operon was transcriptionally induced or derepressed before the onset of solventogenesis, thus confirming earlier results of Northern hybridizations with a ctfB gene probe (U. Gerischer and P. Dürre, J. Bacteriol. 174:426-433, 1992). Upstream of the sol operon, we identified two putative promoters that were located in regions with possible stem-loop structures formed by several inverted repeats. The distal promoter P1 showed only minor transcription initiation in solventogenic C. acetobutylicum cells but was recognized in Escherichia coli, presumably because of its high similarity to the sigma 70 consensus sequence. The adhE-proximal promoter P2 directed the major transcription start point in solventogenic C. acetobutylicum but was not recognized in E. coli. The clostridial AdhE showed high similarity to a novel family (type III) of alcohol dehydrogenases. Two other ORFs (ORF 5 and ORF 6) were found on the cloned DNA region that showed no significant similarity to sequences in various available data bases. mRNA studies revealed that ORF 5 formed a monocistronic operon and showed increased expression before onset of solventogenesis.

SUBMITTER: Fischer RJ

PROVIDER: S-EPMC206823 | biostudies-other | 1993 Nov

REPOSITORIES: biostudies-other

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Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis.

Fischer R J RJ Helms J J Dürre P P

Journal of bacteriology 19931101 21

A DNA region of Clostridium acetobutylicum contiguous with the adc operon has been cloned and sequenced. Structural genes encoding the acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase (ctfB and ctfA) and an alcohol/aldehyde dehydrogenase (adhE) could be identified. These three genes together with a small open reading frame (ORF) of unknown function (upstream of adhE) formed an operon (sol operon), as shown by mRNA analyses. The complete sol operon was transcriptionally induced or d ...[more]

PMID: 8226639

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Project description:Acetoacetate decarboxylase (ADC) (EC4.1.1.4) of Clostridium acetobutylicum DSM 792 was purified to homogeneity, and its first 25 N-terminal amino acids were determined. Oligonucleotide probes deduced from this sequence were used to detect positive clones in partial gene banks derived from Sau3A and HaeIII digests with following ligation into the vector pUC9. In Escherichia coli, the 2.1-kbp HaeIII clones expressed high levels of ADC activity. The expression was independent of the orientation of the insert with respect to the lac promoter of the vector and also of the addition of isopropyl-beta-D-thiogalactopyranoside, thus indicating that sequences located on the clostridial DNA controlled transcription and translation. From the E. coli clone with the recombinant plasmid pUG93 containing the 2.1-kbp HaeIII fragment, the ADC protein was purified and compared with the native enzyme. Both were indistinguishable with respect to the molecular mass of subunits and native protein as well as to activity stain. The 2.9-kbp Sau3A fragment could be shown to contain the amino terminus of the acetoacetate decarboxylase (adc) gene but did not express enzyme activity. It partially overlapped with the HaeIII fragment, spanning together 4,053 bp of the clostridial genome that were completely sequenced. Four open reading frames (ORFs) could be detected, one of which was unambiguously assigned to the acetoacetate decarboxylase (adc) gene. Amino acid sequences of the N terminus and the catalytic center as deduced from the nucleotide sequence were identical to sequences obtained from direct analysis of the protein. Typical procaryotic transcriptional and translational start and stop signals could be found in the DNA sequence. Together with these regulatory sequences, the adc gene formed a single operon. The carboxyl terminus of the enzyme proved to be rather hydrophobic. In vitro transcription-translation assays resulted in formation of ADC and ORF3 gene product; the other two ORFs were not expressed. Whereas no homology of the adc gene and ORF2 could be detected with sequences available in the EMBL or GenBank data bases, the obviously truncated ORF1 showed significant similarity to alpha-amylase of Bacillus subtilis. The restriction pattern and N-terminal amino acid sequence (as deduced from the nucleotide sequence) of ORF3 proved to be identical to those of the large subunit of acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase.

Project description:Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser-->L-Asn, while in iturin A these amino acids are inverted (i.e., D-Asn-->L-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.

Project description:Haemophilus somnus undergoes antigenic and structural phase variation in its lipooligosaccharide (LOS). A gene (lob-1) containing repetitive 5'-CAAT-3' sequences that may, in part, contribute to phase variation was cloned and sequenced (T. J. Inzana et al., Infect. Immun. 65:4675-4681, 1997). We have now identified another putative gene (lob-2A) immediately upstream from lob-1. Lob-2A contained homology to several LOS biosynthesis proteins of the family Pasteurellaceae and the LgtB and LgtE galactosyltransferases of Neisseria meningitidis and N. gonorrhoeae. Unlike lob-1, lob-2A contained 18 to 20 5'-GA-3' repeats 141 bp upstream of the termination codon as determined by PCR amplification of DNA from individual colonies. Twenty repeats were most common, but when 19 5'-GA-3' repeats were present a stop codon would occur 1 bp after the last 5'-GA-3' repeat. A 630-bp SalI-BsgI fragment within lob-2A was deleted, and a kanamycin resistance (Km(r)) gene was inserted into this site to create pCAATDeltalob2A. Following electroporation of pCAATDeltalob2A into H. somnus 738, several allelic exchange mutants were isolated. The LOS electrophoretic profile of one mutant, strain 738-lob2A1::Km, was altered, and the phase variation rate was reduced but phase variation was not eliminated. A variant with 19 5'-GA-3' repeats in lob-2A had an LOS profile similar to that of 738-lob2A1::Km, suggesting that lob-2A was turned off in this phase. Nanoelectrospray mass spectrometry (nES-MS) and nuclear magnetic resonance spectroscopy showed that 738-lob2A1::Km was deficient in the terminal betaGal(1-3)betaGlcNAc residue present in parent strain 738. Mutant 738-lob2A1::Km was significantly more sensitive to the bactericidal action of normal bovine serum and was less virulent in mice than was parent strain 738. When H. somnus 129Pt was electrotransformed with shuttle vector pLS88 containing lob-2A, its LOS electrophoretic profile was modified and additional N-acetylhexosamine residues were present, as determined by nES-MS analysis. These results indicated that lob-2A may be an N-acetylglucosamine transferase involved in LOS biosynthesis and phase variation and that LOS structure is important to H. somnus virulence.

Dataset Information

Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis.

Publications

Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis.

Similar Datasets

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