Dataset Information

RpoN (sigma 54) is required for conversion of phenol to catechol in Acinetobacter calcoaceticus.

ABSTRACT: Members of the sigma 54 protein family, encoded by rpoN, are required for the transcription of genes associated with specialized metabolic functions. The ability to grow with phenol appears to be a specialized trait because it is expressed by few of the microorganisms that grow with catechol, the metabolic product of phenol monooxygenase. A mutation preventing the expression of phenol monooxygenase in the bacterial strain Acinetobacter calcoaceticus NCIB8250 was complemented by wild-type DNA segments containing an open reading frame encoding a member of the sigma 54 protein family. DNA sequencing revealed a second open reading frame, designated ORF2, directly downstream of A. calcoaceticus rpoN. The locations of both ORF2 and the 113-residue amino acid sequence of its product are highly conserved in other bacteria. The mutation preventing the expression of rpoN results in an opal codon that terminates the translation of RpoN at a position corresponding to Trp-91 in the 483-residue amino acid sequence of the wild-type protein. Negative autoregulation of rpoN was suggested by the fact that the mutation inactivating RpoN enhanced the transcription of rpoN. Primer extension revealed independent transcription start sites for rpoN and ORF2.

SUBMITTER: Ehrt S

PROVIDER: S-EPMC205536 | biostudies-other | 1994 Jun

REPOSITORIES: biostudies-other

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RpoN (sigma 54) is required for conversion of phenol to catechol in Acinetobacter calcoaceticus.

Ehrt S S Ornston L N LN Hillen W W

Journal of bacteriology 19940601 12

Members of the sigma 54 protein family, encoded by rpoN, are required for the transcription of genes associated with specialized metabolic functions. The ability to grow with phenol appears to be a specialized trait because it is expressed by few of the microorganisms that grow with catechol, the metabolic product of phenol monooxygenase. A mutation preventing the expression of phenol monooxygenase in the bacterial strain Acinetobacter calcoaceticus NCIB8250 was complemented by wild-type DNA seg ...[more]

PMID: 8206826

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Project description:On the basis of the constitutive phenotypes of two catM mutants of Acinetobacter calcoaceticus, the CatM protein was proposed to repress expression of two different loci involved in catechol degradation, catA and catBCIJFD (E. Neidle, C. Hartnett, and L. N. Ornston, J. Bacteriol. 171:5410-5421, 1989). In spite of its proposed negative role as a repressor, CatM is similar in amino acid sequence to positive transcriptional activators of the LysR family. Investigating this anomaly, we found that insertional inactivation of catM did not cause the phenotype expected for the disruption of a repressor-encoding gene: in an interposon-generated catM mutant, no cat genes were expressed constitutively, but rather catA and catB were still inducible by muconate. Moreover, this catM mutant grew poorly on benzoate, a process requiring the expression of all cat genes. The inducibility of the cat genes in this catM mutant was completely eliminated by a 3.5-kbp deletion 10 kbp upstream of catM. In this double mutant, catM in trans restored muconate inducibility to both catA and catB. These results suggested the presence of an additional regulatory locus controlling cat gene expression. The ability of CatM to function as an activator was also suggested by these results. In support of this hypothesis, in vivo methylation protection assays showed that CatM protects two guanines in a dyad 65 nucleotides upstream of the catB transcriptional start site, in a location and pattern typical of LysR-type transcriptional activators. Gel mobility shift assays indicated that CatM also binds to a region upstream of catA. DNA sequence analysis revealed a nucleotide near the 3' end of catM not present in the published sequence. Translation of the corrected sequence resulted in the deduced CatM protein being 52 residues longer than previously reported. The size, amino acid sequence, and mode of action of CatM now appear similar to, and typical of, what has been found for transcriptional activators in the LysR family. Analysis of one of the constitutive alleles of catM previously thought to encode a dysfunctional repressor indicated instead that it encodes an inducer-independent transcriptional activator.

Project description:Some bacteria are capable of forming flocs, in which bacterial cells become self-flocculated by secreted extracellular polysaccharides and other biopolymers. The floc-forming bacteria play a central role in activated sludge, which has been widely utilized for the treatment of municipal sewage and industrial wastewater. Here, we use a floc-forming bacterium, Aquincolatertiaricarbonis RN12, as a model to explore the biosynthesis of extracellular polysaccharides and the regulation of floc formation. A large gene cluster for exopolysaccharide biosynthesis and a gene encoding the alternative sigma factor RpoN1, one of the four paralogues, have been identified in floc formation-deficient mutants generated by transposon mutagenesis, and the gene functions have been further confirmed by genetic complementation analyses. Interestingly, the biosynthesis of exopolysaccharides remained in the rpoN1-disrupted flocculation-defective mutants, but most of the exopolysaccharides were secreted and released rather than bound to the cells. Furthermore, the expression of exopolysaccharide biosynthesis genes seemed not to be regulated by RpoN1. Taken together, our results indicate that RpoN1 may play a role in regulating the expression of a certain gene(s) involved in the self-flocculation of bacterial cells but not in the biosynthesis and secretion of exopolysaccharides required for floc formation.IMPORTANCE Floc formation confers bacterial resistance to predation of protozoa and plays a central role in the widely used activated sludge process. In this study, we not only identified a large gene cluster for biosynthesis of extracellular polysaccharides but also identified four rpoN paralogues, one of which (rpoN1) is required for floc formation in A. tertiaricarbonis RN12. In addition, this RpoN sigma factor regulates the transcription of genes involved in biofilm formation and swarming motility, as previously shown in other bacteria. However, this RpoN paralogue is not required for the biosynthesis of exopolysaccharides, which are released and dissolved into culture broth by the rpoN1 mutant rather than remaining tightly bound to cells, as observed during the flocculation of the wild-type strain. These results indicate that floc formation is a regulated complex process, and other yet-to-be identified RpoN1-dependent factors are involved in self-flocculation of bacterial cells via exopolysaccharides and/or other biopolymers.

Dataset Information

RpoN (sigma 54) is required for conversion of phenol to catechol in Acinetobacter calcoaceticus.

Publications

RpoN (sigma 54) is required for conversion of phenol to catechol in Acinetobacter calcoaceticus.

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