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Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum.

ABSTRACT: The groESL operon of Clostridium acetobutylicum was cloned in Escherichia coli by using a gene probe of E. coli groESL. Sequencing of a positively reacting 2.2-kbp HindIII fragment contained in the recombinant plasmid pFN1 and a 2.5-kbp XbaI fragment present in pFN4 revealed that both fragments partially overlapped and together spanned 3,493 bp of the clostridial chromosome. Two complete open reading frames (288 and 1632 bp) were found and identified as the groES- and groEL-homologous genes of C. acetobutylicum, respectively. The 3' end of a third gene (orfZ), which was divergently transcribed, showed no significant homology to other sequences available in the EMBL and GenBank data bases. The length of the groESL-specific mRNA (2.2 kb), a transcription terminator downstream of groEL, and a transcription start site upstream of groES, identified by primer extension analysis, indicated that groES and groEL of C. acetobutylicum are organized in a bicistronic operon. From the transcription start site, the promoter structure 5'-TTGCTA (17 bp) TATTAT that shows high homology to the consensus promoter sequence of gram-positive bacteria as well as E. coli was deduced. Transcription of the groESL operon was strongly heat inducible, and maximum levels of mRNA were detected 15 min after heat shock from 30 to 42 degrees C. An 11-bp inverted repeat, located between promoter and translation start sites of groES and partially identical with similar structures in front of several heat shock genes of other bacteria, may play an important role in the regulation of heat shock gene expression in this organism.

SUBMITTER: Narberhaus F

PROVIDER: S-EPMC205997 | biostudies-other | 1992 May

REPOSITORIES: biostudies-other

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Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum.

Narberhaus F F Bahl H H

Journal of bacteriology 19920501 10

The groESL operon of Clostridium acetobutylicum was cloned in Escherichia coli by using a gene probe of E. coli groESL. Sequencing of a positively reacting 2.2-kbp HindIII fragment contained in the recombinant plasmid pFN1 and a 2.5-kbp XbaI fragment present in pFN4 revealed that both fragments partially overlapped and together spanned 3,493 bp of the clostridial chromosome. Two complete open reading frames (288 and 1632 bp) were found and identified as the groES- and groEL-homologous genes of C ...[more]

PMID: 1349602

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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.

Dataset Information

Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum.

Publications

Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum.

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