Donna Parke

Hydroxycinnamate (hca) Catabolic Genes from Acinetobacter sp. Strain ADP1 Are Repressed by HcaR and Are Induced by Hydroxycinnamoyl-Coenzyme A Thioesters

ABSTRACT Hydroxycinnamates are plant products catabolized through the diphenol protocatechuate in the naturally transformable bacterium Acinetobacter sp. strain ADP1. Genes for protocatechuate catabolism are central to the dca-pca-qui-pob-hca chromosomal island, for which gene designations corresponding to catabolic function are dca (dicarboxylic acid), pca (protocatechuate), qui (quinate), pob (p-hydroxybenzoate), and hca (hydroxycinnamate). Acinetobacter hcaC had been cloned and shown to encode a hydroxycinnamate:coenzyme A (CoA) SH ligase that acts upon caffeate, p-coumarate, and ferulate, but genes for conversion of hydroxycinnamoyl-CoA to protocatechuate had not been characterized. In this investigation, DNA from pobS to an XbaI site 5.3 kb beyond hcaC was captured in the plasmid pZR8200 by a strategy that involved in vivo integration of a cloning vector near the hca region of the chromosome. pZR8200 enabled Escherichia coli to convert p-coumarate to protocatechuate in vivo. Sequence analysis of the newly cloned DNA identified five open reading frames designated hcaA, hcaB, hcaK, hcaR, and ORF1. An Acinetobacter strain with a knockout of HcaA, a homolog of hydroxycinnamoyl-CoA hydratase/lyases, was unable to grow at the expense of hydroxycinnamates, whereas a strain mutated in HcaB, homologous to aldehyde dehydrogenases, grew poorly with ferulate and caffeate but well with p-coumarate. A chromosomal fusion of lacZ to the hcaE gene was used to monitor expression of the hcaABCDE promoter. LacZ was induced over 100-fold by growth in the presence of caffeate, p-coumarate, or ferulate. The protein deduced to be encoded by hcaR shares 28% identity with the aligned E. coli repressor, MarR. A knockout of hcaR produced a constitutive phenotype, as assessed in the hcaE::lacZ-Kmr genetic background, revealing HcaR to be a repressor as well. Expression of hcaE::lacZ in strains with knockouts in hcaA, hcaB, or hcaC revealed unambiguously that hydroxycinnamoyl-CoA thioesters relieve repression of the hcaABCDE genes by HcaR.

The Evolution of Induction Mechanisms in Bacteria: Insights Derived from the Study of the β-Ketoadipate Pathway

Publisher Summary The comparative survey of induction mechanisms used by different bacterial genera to govern the synthesis of enzymes for a single pathway can give insight into some of the factors that contributed to the selection of particular compounds as inducers and to the evolution of patterns of inductive control. This chapter presents different types of inductive control and discusses the regulation of inducer levels. Inductive control evolves through the modification of preexisting genetic material. The chapter discusses β-ketoadipate pathway, which is a multistep route for the dissimilation of aromatic compounds. It highlights the regulation of β-ketoadipate pathway in two bacterial species, Acinetobacter calcoaceticus and Pseudomonas putida . This provides a background for tracing the development of concepts about four evolutionary factors that influence the evolution of induction mechanisms: (1) the economy of enzyme synthesis, (2) unification of inductive control, (3) the preexisting genetic material from which genes evolved, and 4) the role of nutritional opportunities in the natural environment in the selection of organisms with specific patterns of induction.

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

ABSTRACT Hydroxycinnamates, aromatic compounds that play diverse roles in plants, are dissimilated by enzymes encoded by the hca genes in the nutritionally versatile, naturally transformable bacterium Acinetobacter sp. strain ADP1. A key step in the hca-encoded pathway is activation of the natural substrates caffeate, p-coumarate, and ferulate by an acyl:coenzyme A (acyl:CoA) ligase encoded by hcaC. As described in this paper, Acinetobacter cells with a knockout of the next enzyme in the pathway, hydroxycinnamoyl-CoA hydratase/lyase (HcaA), are extremely sensitive to the presence of the three natural hydroxycinnamate substrates; Escherichia coli cells carrying a subclone with the hcaC gene are hydroxycinnamate sensitive as well. When the hcaA mutation was combined with a mutation in the repressor HcaR, exposure of the doubly mutated Acinetobacter cells to caffeate, p-coumarate, or ferulate at 10−6 M totally inhibited the growth of cells. The toxicity of p-coumarate and ferulate to a ΔhcaA strain was found to be a bacteriostatic effect. Although not toxic to wild-type cells initially, the diphenolic caffeate was itself converted to a toxin over time in the absence of cells; the converted toxin was bactericidal. In an Acinetobacter strain blocked in hcaA, a secondary mutation in the ligase (HcaC) suppresses the toxic effect. Analysis of suppression due to the mutation of hcaC led to the development of a positive-selection strategy that targets mutations blocking HcaC. An hcaC mutation from one isolate was characterized and was found to result in the substitution of an amino acid that is conserved in a functionally characterized homolog of HcaC.

Structural comparison of γ-carboxymuconolactone decarboxylase and muconolactone isomerase from pseudomonas putida

Abstract Muconolactone isomerase and γ-carboxymuconolactone decarboxylase, two Pseudomonas putida enzymes that catalyze analogous biochemical reactions in the dissimilation of aromatic compounds, were compared structurally and chemically. A revised procedure for the purification of the decarboxylase gave higher yields of homogeneous protein in less time than previously published methods. Earlier investigations with sodium dodecyl sulfate gel electrophoresis had indicated that the subunit sizes of the isomerase and the decarboxylase were 11 300 and 13 000, respectively. In the present study sedimentation equilibrium ultracentrifugation of the isomerase at pH 7.0 gave an apparent molecular weight of 111 000, suggesting that the protein is a decamer or a dodecamer. Ultracentrifugation of the decarboxylase under similar conditions indicated that the protein is a hexamer of 82 000 daltons. Dimethyl suberimidate cross-linking of the lysines of the isomerase created two species of cross-linked proteins as revealed by sodium dodecyl sulfate gel electrophoresis; a major species of 62 000–65 000 and a minor amount of a 124 000 molecular weight species. The results suggest that the oligomer of the isomerase may be composed of two stacked discs of pentamers or hexamers. In contrast, the evidence indicated no significant intra-oligomeric cross-linking of the lysines of the decarboxylase. The sequence of the first twelve amino terminal amino acids of the decarboxylase was determined by dansyl-Edman degradation. Comparison of the amino terminal amino acid sequence of the decarboxylase with that of the isomerase demonstrated that these portions of the two proteins have no more than three identical residues in the first twelve positions. The results indicate that either the two proteins had an independent evolutionary origin or they have diverged considerably since a common ancestry.

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

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 beta-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.

Genome Organization, Mutation, and Gene Expression in Acinetobacter

Early investigations of Acinetobacter genetics focused upon Acinetobacter baylyi because the extraordinary competence of this species for natural transformation (Carr et al., 2003; Vaneechoutte et al., 2006) allowed convenient genetic analysis (Young et al., 2005). This background fostered determination of the annotated genomic sequence of A. baylyi (Barbe et al., 2004), which will serve as a useful reference for comparison with other genomic sequences as they become available. Immediately notable was the close similarity ofAcinetobacter and Pseudomonas genes although the two genera have diverged markedly with respect to genome size, gene organization, and the GþC content of their DNA (Barbe et al., 2004). Comparisons of Acinetobacter and Pseudomonas genomes as well as those of divergent Pseudomonas genomes are informative because they may provide an indication of mechanisms that contributed to the divergence and determined the individuality of Acinetobacter species. Contributions of such mechanisms will become clearer as additional Acinetobacter genome sequences become available (Fournier et al., 2006; Smith et al., 2007). This review opens with an overview of relevant genomic structures and a discussion of their differences because these observations may provide a glimpse of what will be revealed by additional genomic information. Progress in this area will provide important practical information likely to facilitate the pressing task of unambiguous typing of newly isolated Acinetobacter strains. The genomic structure ofAcinetobacter species directs attention to mechanisms underlying genetic variation in the genus, and this will be the next topic considered. As described in the following section, mutations influence gene expression, and the complexity of physiological responses of Acinetobacter to environmental signals will be addressed. The review concludes with a statement of promising future directions for investigation of the genetic basis for variation among Acinetobacter species.