Positive Selection for Mutations Affecting Bioconversion of Aromatic Compounds in Agrobacterium tumefaciens : Analysis of Spontaneous Mutations in the Protocatechuate 3,4-Dioxygenase Gene

Abstract

A positive selection method for mutations affecting bioconversion of aromatic compounds was applied to a mutant strain of Agrobacterium tumefaciens A348. The nucleotide sequence of the A348 pcaHGB genes, which encode protocatechuate 3,4-dioxygenase (PcaHG) and ␤-carboxy-cis,cis-muconate cycloisomerase (PcaB) for the first two steps in catabolism of the diphenolic protocatechuate, was determined. An omega element was introduced into the pcaB gene of A348, creating strain ADO2077. In the presence of phenolic compounds that can serve as carbon sources, growth of ADO2077 is inhibited due to accumulation of the tricarboxylate intermediate. The toxic effect, previously described for Acinetobacter sp., affords a powerful selection for suppressor mutations in genes required for upstream catabolic steps. By monitoring loss of the marker in pcaB, it was possible to determine that the formation of deletions was minimal compared to results obtained with Acinetobacter sp. Thus, the tricarboxylic acid trick in and of itself does not appear to select for large deletion mutations. The power of the selection was demonstrated by targeting the pcaHG genes of A. tumefaciens for spontaneous mutation. Sixteen strains carrying putative second-site mutations in pcaH or -G were subjected to sequence analysis. All single-site events, their mutations revealed no particular bias toward multibase deletions or unusual patterns: five (؊1) frameshifts, one (؉1) frameshift, one tandem duplication of 88 bp, one deletion of 92 bp, one nonsense mutation, and seven missense mutations. PcaHG is considered to be the prototypical ferric intradiol dioxygenase. The missense mutations served to corroborate the significance of active site amino acid residues deduced from crystal structures of PcaHG from Pseudomonas putida and Acinetobacter sp. as well as of residues in other parts of the enzyme.

Analysis of Hydroxycinnamic Acid Degradation in Agrobacterium fabrum Reveals a Coenzyme A-Dependent, Beta-Oxidative Deacetylation Pathway

“…The involvement of the pcaHGBCD and pcaIJF operons (Atu4538 to Atu4542 and Atu4547 to Atu4549) encoding proteins in C58 was previously shown by Parke (29). MH4F, methylene-tetrahydrofolate; H4F, methyl-tetrahydrofolate.…”

“…strain HR199 (16) is presented below. ate 3-monooxygenase, PobA (Atu4544), is involved in the direct conversion of p-hydroxybenzoic acid from pCA into protocatechuic acid (29).…”

“…The Pseudomonas pathway involves several steps, including (i) the conversion of FA into feruloyl-CoA by a feruloyl-CoA synthetase (encoded by fcs), (ii) the hydration and subsequent retroaldol cleavage of feruloyl-CoA to produce vanillin and acetyl-CoA through an enoyl-CoA hydratase/aldolase (Ech, encoded by ech), (iii) the NAD ϩ -dependent oxidation of vanillin into vanillic acid (VA) by a vanillin dehydrogenase (encoded by vdh), and (iv) the O-demethylation of VA into protocatechuic acid by an O-demethylase (encoded by vanAB). The protocatechuic acid is trans-formed into succinate and acetyl-CoA, which are carbon and energy sources in numerous bacteria, including A. fabrum C58 (11,16,(24)(25)(26)(27)(28)(29).…”

Toxicity Caused by Hydroxycinnamoyl-Coenzyme A Thioester Accumulation in Mutants of Acinetobacter sp. Strain ADP1

“…Thus, upon their exposure to an appropriate precursor, pcaB-blocked cells give rise to secondary mutants that are protected by their inability to generate the tricarboxylate compound. The application of a positiveselection procedure based on the toxic effect has resulted in the isolation of informative structure-function mutations in upstream enzymes and their regulators (7,14,26,27,34). However, this approach had met with limited success in the case of the initial steps of the hydroxycinnamate pathway because the numerically predominant secondary mutants were those blocked in steps towards the end of the catabolic sequence.…”

Excess Production ofBacillus subtilisQuercetin 2,3-Dioxygenase Affects Cell Viability in the Presence of Quercetin

“…ADP1 and Agrobacterium tumefaciens feed on protocatechuic acid and catabolize it to succinyl-CoA and acetyl-CoA, and that disruption of the pcaB gene, involved in a downstream step of the pathway, causes accumulation of an intermediate, -carboxy-cis,cis-muconic acid, which exerts growth inhibition in minimal medium containing succinate as a carbon source. 28,29) Our homology search of the B. subtilis genome showed that there is no homolog to the Acinetobacter sp. and A. tumefaciens pcaHG genes encoding protocatechuate 3,4-dioxygenase catalyzing the conversion of protocatechuic acid tocarboxy-cis,cis-muconic acid, which suggests that the protocatechuic-acid catabolic pathway of B. subtilis is different from those of Acinetobacter sp.…”