Ellen L. Neidle

Key aromatic-ring-cleaving enzyme, protocatechuate 3,4-dioxygenase, in the ecologically important marine Roseobacter lineage.

ABSTRACT Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the α-proteobacteria was investigated. Initial screens suggested that isolates in theRoseobacter lineage can degrade aromatic compounds via the β-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six Roseobacter isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the β-ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate, p-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the Roseobacter group isolates had inducible protocatechuate 3,4-dioxygenase activity in cell extracts when grown onp-hydroxybenzoate. The pcaGH genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates,Sagittula stellata E-37 and isolate Y3F, and in both cases the genes could be expressed in Escherichia coli to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the β-ketoadipate pathway (pcaC,pcaQ, and pobA) were found to cluster withpcaGH in these two isolates. Pairwise sequence analysis of the pca genes revealed greater similarity between the twoRoseobacter group isolates than between genes from eitherRoseobacter strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of pcaH from two additionalRoseobacter group isolates, and Southern hybridization indicated the presence of pcaH in the remaining two isolates. This evidence of protocatechuate 3,4-dioxygenase and the β-ketoadipate pathway was found in all six Roseobacterisolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.

Genome plasticity in Acinetobacter : new degradative capabilities acquired by the spontaneous amplification of large chromosomal segments

In Acinetobacter sp. ADP1, growth on benzoate requires regulation of the cat genes by two transcriptional activators. Here, mutants were obtained from a strain lacking both activators by selecting for growth on benzoate medium. The mutants, which arose within 3 weeks at a frequency of approximately 10−8, carried amplified chromosomal regions (amplicons) encompassing the cat genes. Multiple occurrences of low‐level expression of catA and the catBCIJFD operon provided sufficient transcription for growth. The amplicons of four independently isolated mutants varied in size from approximately 30–100 kbp of the normally 3.8 Mbp chromosome. Mutants had approximately 10–20 copies of an amplicon in adjacent head‐to‐tail orientations. At the amplicons chromosomal endpoint, an atypical junction juxtaposed normally distant DNA regions from opposite sides of the cat genes. The sequences of these junctions revealed the precise recombination sites underlying amplification. Additionally, amplicon stability was evaluated in the absence of selective pressure. The natural competence of Acinetobacter for transformation by linear DNA has allowed the development of a powerful new model system for investigating chromosomal rearrangements and for engineering DNA amplifications for wide‐ranging applications. The frequent spontaneous amplification of these large chromosomal segments demonstrated the importance of supra‐operonic gene clustering in the evolution of catabolic pathways.

Synergistic transcriptional activation by one regulatory protein in response to two metabolites

BenM is a LysR-type bacterial transcriptional regulator that controls aromatic compound degradation in Acinetobacter sp. ADP1. Here, in vitro transcription assays demonstrated that two metabolites of aromatic compound catabolism, benzoate and cis,cis-muconate, act synergistically to activate gene expression. The level of BenM-regulated benA transcription was significantly higher in response to both compounds than the combined levels due to each alone. These compounds also were more effective together than they were individually in altering the DNase I footprint patterns of BenM-DNA complexes. This type of dual-inducer synergy provides great potential for rapid and large modulations of gene expression and may represent an important, and possibly widespread, feature of transcriptional control.

Diversity of the Ring-Cleaving Dioxygenase Gene pcaH in a Salt Marsh Bacterial Community

ABSTRACT Degradation of lignin-related aromatic compounds is an important ecological process in the highly productive salt marshes of the southeastern United States, yet little is known about the mediating organisms or their catabolic pathways. Here we report the diversity of a gene encoding a key ring-cleaving enzyme of the β-ketoadipate pathway, pcaH, amplified from bacterial communities associated with decaying Spartina alterniflora, the salt marsh grass that dominates these coastal systems, as well as from enrichment cultures with aromatic substrates (p-hydroxybenzoate, anthranilate, vanillate, and dehydroabietate). Sequence analysis of 149 pcaH clones revealed 85 unique sequences. Thirteen of the 53 amino acid residues compared were invariant in the PcaH proteins, suggesting that these residues have a required catalytic or structural function. Fifty-eight percent of the clones matched sequences amplified from a collection of 36 bacterial isolates obtained from seawater, marine sediments, or senescent Spartina. Fifty-two percent of thepcaH clones could be assigned to the roseobacter group, a marine lineage of the class α-Proteobacteria abundant in coastal ecosystems. Another 6% of the clones matched genes retrieved from isolates belonging to the genera Acinetobacter, Bacillus, and Stappia, and 42% of the clones could not be assigned to a cultured bacterium based on sequence identity. These results suggest that the diversity of the genes encoding a single step in aromatic compound degradation in the coastal marsh examined is high.

X-ray crystal structure of benzoate 1,2-dioxygenase reductase from Acinetobacter sp. strain ADP1.

One of the major processes for aerobic biodegradation of aromatic compounds is initiated by Rieske dioxygenases. Benzoate dioxygenase contains a reductase component, BenC, that is responsible for the two-electron transfer from NADH via FAD and an iron-sulfur cluster to the terminal oxygenase component. Here, we present the structure of BenC from Acinetobacter sp. strain ADP1 at 1.5 A resolution. BenC contains three domains, each binding a redox cofactor: iron-sulfur, FAD and NADH, respectively. The [2Fe-2S] domain is similar to that of plant ferredoxins, and the FAD and NADH domains are similar to members of the ferredoxin:NADPH reductase superfamily. In phthalate dioxygenase reductase, the only other Rieske dioxygenase reductase for which a crystal structure is available, the ferredoxin-like and flavin binding domains are sequentially reversed compared to BenC. The BenC structure shows significant differences in the location of the ferredoxin domain relative to the other domains, compared to phthalate dioxygenase reductase and other known systems containing these three domains. In BenC, the ferredoxin domain interacts with both the flavin and NAD(P)H domains. The iron-sulfur center and the flavin are about 9 A apart, which allows a fast electron transfer. The BenC structure is the first determined for a reductase from the class IB Rieske dioxygenases, whose reductases transfer electrons directly to their oxygenase components. Based on sequence similarities, a very similar structure was modeled for the class III naphthalene dioxygenase reductase, which transfers electrons to an intermediary ferredoxin, rather than the oxygenase component.

Full-Length Structures of BenM and Two Variants Reveal Different Oligomerization Schemes for LysR-Type Transcriptional Regulators

BenM, a LysR-type transcriptional regulator (LTTR) from the bacterium Acinetobacter baylyi, responds synergistically to benzoate and cis,cis-muconate. With these effectors, BenM activates gene expression during benzoate consumption. Without effectors, BenM represses transcription. Here, X-ray crystallography was used to determine the full-length structures of BenM and two variants that activate transcription without benzoate or cis,cis-muconate: BenM(R156H) and BenM(E226K). Previous studies indicate that these regulators function as tetramers. Here, interconnections between subunits in the crystals prevented the formation of a closed oligomer and highlighted the inherent flexibility of this multidomain regulator. Nevertheless, analysis of subunit interfaces suggested the functional significance of key interactions. The structures of BenM and its variants were nearly identical, implying that transcriptional differences rely on factors beyond major conformational changes defined solely by sequence. Comparisons of BenM with other LTTRs, including unpublished structures in the Protein Data Bank, revealed extensive variation in the relative orientations of DNA-binding domains (DBDs) and effector-binding domains (EBDs). To form dimers, different LTTRs used similar interfaces between two EBDs, each containing two subdomains: EBD-I and EBD-II. Surprisingly, the dimers used three substantially different schemes to form higher-order oligomers. In one scheme used by BenM, oligomer assembly involved contacts between the EBD-II regions and the DBD regions of adjacent subunits. In another scheme, there were no contacts between the EBDs; only the DBDs were involved in tetramer formation. In the third scheme, the oligomer interface involved DBD and EBD-I/EBD-II contacts. These diverse schemes demonstrate novel variation in the oligomeric structures of individual LTTRs within this large and important family.

Directed introduction of DNA cleavage sites to produce a high-resolution genetic and physical map of the Acinetobacter sp. strain ADP1 (BD413UE) chromosome.

The natural transformability of the soil bacterium Acinetobacter sp. ADP1 (BD413UE), formerly classified as A. calcoaceticus, has facilitated previous physiological and biochemical investigations. In the present studies, the natural transformation system was exploited to generated a physical and genetic map of this strains 3780 +/- 191 kbp circular chromosome. Previously isolated Acinetobacter genes were modified in vitro to incorporate a recognition sequence for the restriction endonuclease NotI. Following transformation of the wild-type strain by the modified DNA, homologous recombination placed each engineered NotI cleavage site at the chromosomal location of the corresponding gene. This allowed precise gene localization and orientation of more than 40 genes relative to a physical map which was constructed with transverse alternating field electrophoresis (TAFE) and Southern hybridization methods. The positions of NotI, AscI and I-CeuI recognition sites were determined, and the latter enzyme identified the presence of seven ribosomal RNA operons. Multiple chromosomal copies of insertion sequence IS1236 were indicated by hybridization. Several of these copies were concentrated in one region of the chromosome in which a spontaneous deletion of approximately 100 kbp occurred. Moreover, contrary to previous reports, ColE1-based plasmids appeared to replicate autonomously in Acinetobacter sp. ADP1.

Transcriptional Cross-Regulation of the Catechol and Protocatechuate Branches of the β-Ketoadipate Pathway Contributes to Carbon Source-Dependent Expression of the Acinetobacter sp. Strain ADP1 pobA Gene

ABSTRACT Transcriptional control of carbon source preferences by Acinetobacter sp. strain ADP1 was assessed with a pobA::lacZ fusion during growth on alternative substrates. The pobA-encoded enzyme catalyzes the first step in the degradation of 4-hydroxybenzoate, a compound consumed rapidly as a sole carbon source. If additional aromatic carbon sources are available, 4-hydroxybenzoate consumption is inhibited by unknown mechanisms. As reported here, during growth on aromatic substrates, pobA was not expressed despite the presence of 4-hydroxybenzoate, an inducer that normally causes the PobR regulator to activate pobA transcription. Growth on organic acids such as succinate, fumarate, and acetate allowed higher levels of pobA expression. In each case, pobA expression increased at the end of the exponential growth phase. Complex transcriptional regulation controlled 4-hydroxybenzoate catabolism in multisubstrate environments. Additional studies focused on the wild-type preference for benzoate consumption prior to 4-hydroxybenzoate consumption. These compounds are degraded via the catechol and protocatechuate branches of the β-ketoadipate pathway, respectively. Here, mutants were characterized that degraded benzoate and 4-hydroxybenzoate concurrently. These mutants lacked the BenM and CatM transcriptional regulators that normally activate genes for benzoate catabolism. A model is presented in which BenM and CatM prevent pobA expression indirectly during growth on benzoate. These regulators may affect pobA expression by lowering the PcaK-mediated uptake of 4-hydroxybenzoate. Consistent with this model, BenM and CatM bound in vitro to an operator-promoter fragment controlling the expression of several pca genes, including pcaK. These studies provide the first direct evidence of transcriptional cross-regulation between the distinct but analogous branches of the β-ketoadipate pathway.

Diverse Organization of Genes of the β-Ketoadipate Pathway in Members of the Marine Roseobacter Lineage

ABSTRACT Members of the Roseobacter lineage, an ecologically important marine clade within the class α-Proteobacteria, harbor genes for the protocatechuate branch of the β-ketoadipate pathway, a major catabolic route for lignin-related aromatic compounds. The genes of this pathway are typically clustered, although gene order varies among organisms. Here we characterize genes linked to pcaH and -G, which encode protocatechuate 3,4-dioxygenase, in eight closely related members of the Roseobacter lineage (pairwise 16S rRNA gene sequence identities, 92 to 99%). Sequence analysis of genomic fragments revealed five unique pca gene arrangements. Identical gene organization was found for isolates demonstrating species-level identity (i.e., >99% 16S rRNA gene similarity). In one isolate, six functionally related genes were clustered: pcaQ, pobA, pcaD, pcaC, pcaH, and pcaG. The remaining seven isolates lacked at least one of these genes in their clusters, although the relative order of the remaining genes was preserved. Three genes (pcaC, -H, and -G) were physically linked in all isolates. A highly conserved open reading frame (ORF) was found immediately downstream of pcaG in all eight isolates. Reverse transcription-PCR analysis of RNA from one isolate, Silicibacter pomeroyi DSS-3, provides evidence that this ORF is coexpressed with upstream pca genes. The absence of this ORF in similar bacterial pca gene clusters from diverse microbes suggests a niche-specific role for its protein product in Roseobacter group members. Collectively, these comparisons of bacterial pca gene organization illuminate a complex evolutionary history and underscore the widespread ecological importance of the encoded β-ketoadipate pathway.

Characterization and evolution of anthranilate 1,2-dioxygenase from Acinetobacter sp. strain ADP1.

The two-component anthranilate 1,2-dioxygenase of the bacterium Acinetobacter sp. strain ADP1 was expressed in Escherichia coli and purified to homogeneity. This enzyme converts anthranilate (2-aminobenzoate) to catechol with insertion of both atoms of O(2) and consumption of one NADH. The terminal oxygenase component formed an alpha(3)beta(3) hexamer of 54- and 19-kDa subunits. Biochemical analyses demonstrated one Rieske-type [2Fe-2S] center and one mononuclear nonheme iron center in each large oxygenase subunit. The reductase component, which transfers electrons from NADH to the oxygenase component, was found to contain approximately one flavin adenine dinucleotide and one ferredoxin-type [2Fe-2S] center per 39-kDa monomer. Activities of the combined components were measured as rates and quantities of NADH oxidation, substrate disappearance, product appearance, and O(2) consumption. Anthranilate conversion to catechol was stoichiometrically coupled to NADH oxidation and O(2) consumption. The substrate analog benzoate was converted to a nonaromatic benzoate 1,2-diol with similarly tight coupling. This latter activity is identical to that of the related benzoate 1, 2-dioxygenase. A variant anthranilate 1,2-dioxygenase, previously found to convey temperature sensitivity in vivo because of a methionine-to-lysine change in the large oxygenase subunit, was purified and characterized. The purified M43K variant, however, did not hydroxylate anthranilate or benzoate at either the permissive (23 degrees C) or nonpermissive (39 degrees C) growth temperatures. The wild-type anthranilate 1,2-dioxygenase did not efficiently hydroxylate methylated or halogenated benzoates, despite its sequence similarity to broad-substrate specific dioxygenases that do. Phylogenetic trees of the alpha and beta subunits of these terminal dioxygenases that act on natural and xenobiotic substrates indicated that the subunits of each terminal oxygenase evolved from a common ancestral two-subunit component.