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Volume 163, Issue 5

Malonate degradation in ADP1: operon organization and regulation by MdcR Free

Abstract

Transcriptional regulators in the LysR or GntR families are typically encoded in the genomic neighbourhood of bacterial genes for malonate degradation. While these arrangements have been evaluated using bioinformatics methods, experimental studies demonstrating co-transcription of predicted operons were lacking. Here, transcriptional regulation was characterized for a cluster of genes that enable a soil bacterium, ADP1, to use malonate as a carbon source. Despite previous assumptions that the gene set forms one operon, our studies revealed distinct promoters in two different regions of a nine-gene cluster. Furthermore, a single promoter is insufficient to account for transcription of , a regulatory gene that is convergent to other genes. MdcR, a LysR-type transcriptional regulator, was shown to bind specifically to a site where it can activate -gene transcription. Although deletion prevented growth on malonate, a 1 nt substitution in the promoter of enabled MdcR-independent growth on this carbon source. Regulation was characterized by methods including transcriptional fusions, quantitative reverse transcription PCR, reverse transcription PCR, 5′-rapid amplification of cDNA ends and gel shift assays. Moreover, a new technique was developed for transcriptional characterization of low-copy mRNA by increasing the DNA copy number of specific chromosomal regions. MdcR was shown to respond to malonate, in the absence of its catabolism. These studies contribute to ongoing characterization of the structure and function of a set of 44 LysR-type transcriptional regulators in ADP1.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): gene amplification , LysR , malonate , multiple promoters and operon structure
© 2017 The Authors
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2017-05-01
2024-04-25
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Malonate degradation in Acinetobacter baylyi ADP1: operon organization and regulation by MdcR
Microbiology 163, 789 (2017); https://doi.org/10.1099/mic.0.000462
/content/journal/micro/10.1099/mic.0.000462
/content/journal/micro/10.1099/mic.0.000462
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