Biomedical subjects

Ellen L Neidle

Publications and source records attributed to Ellen L Neidle.

13 recordsLinked to original sources

PubMed2006

CatM regulation of the benABCDE operon: functional divergence of two LysR-type paralogs in Acinetobacter baylyi ADP1.

Two LysR-type transcriptional regulators, BenM and CatM, control benzoate consumption by the soil bacterium Acinetobacter baylyi ADP1. These homologs play overlapping roles in the expression of multiple genes. This study focuses on the benABCDE operon, which initiates benzoate catabolism. At this locus, BenM and CatM each activate transcription in response to the catabolite cis,cis-muconate. BenM, but not CatM, additionally responds to benzoate as an effector. Regulation by CatM alone is insufficient for growth on benzoate as the sole carbon source. However, three point mutations independently increased CatM-activated benA transcription and enabled growth on benzoate without BenM. Two mutations generate variants with one amino acid change in the 303-residue CatM, CatM(V158M) and CatM(R156H). These substitutions affected regulation of benA differently than that of catB, another CatM-regulated gene involved in benzoate catabolism. In relation to CatM, CatM(V158M) increased cis,cis-muconate-dependent transcription of benA but decreased that of catB. CatM(R156H) increased effector-independent expression of catB compared to CatM. In contrast, cis,cis-muconate was required with CatM(R156H) to activate unusually high benA expression. Thus, induction by cis,cis-muconate depends on both the sequence of CatM and the promoter. A point mutation at position -40 of the benA promoter enhanced CatM-activated gene expression and altered regulation by CatM(R156H). BenM and CatM bound to the same locations on ben region DNA. The frequency with which spontaneous mutations allow CatM to substitute for BenM might predict that one regulator would be sufficient for controlling benzoate consumption. This prediction is discussed in light of current and previous studies of the BenM-CatM regulon.

Acinetobacter↗

PubMed2004

Gene amplification involves site-specific short homology-independent illegitimate recombination in Acinetobacter sp. strain ADP1.

A system for studying gene amplification in the bacterium Acinetobacter sp. strain ADP1 was used to isolate 105 spontaneous mutants. The method selects for the elevated expression of neighboring transcriptional units in a parent strain lacking its normal transcriptional activators. Gene amplification can compensate for the activator loss by increasing the copy number of seven weakly expressed genes. Mutant colonies arose from the parent strain at a frequency of 10(-8) within three weeks. All but one of these mutants carried tandem head-to-tail repeats of a chromosomal segment (amplicon). These amplicons varied in size from approximately 12-290 kb and ranged in copy number from 3 to more than 30. Gene amplification involved a two-step process in which duplications formed independently of recA. Illegitimate recombination fused normally distant chromosomal regions to create novel DNA duplication junctions. These junctions were isolated from amplification mutants using an assay that exploits Acinetobacter natural transformability. Sequence analysis of 72 junctions revealed little identity in the recombining regions. Furthermore, multiple independently isolated mutants contained identical junctions. Six different junctions, each found in two to six mutants, revealed that some recombination events are site-specific. Several recurring junctions were studied using PCR. In each case, the identical duplication present in the mutant was estimated to have occurred in as many as one in a million cells in populations of strains never exposed to selective conditions. These duplications appeared to form spontaneously by a novel type of short homology-independent, site-specific process. However, in the absence of recA, mutant colonies were not selected from parent cells containing these duplications. Thus, the second gene amplification step most likely depends on homologous recombination to increase amplicon copy number. These studies support the theory that gene amplification is a driving force in the evolution of functionally related gene clusters.

Acinetobacter↗

PubMed2004

Diverse organization of genes of the beta-ketoadipate pathway in members of the marine Roseobacter lineage.

Members of the Roseobacter lineage, an ecologically important marine clade within the class alpha-Proteobacteria, harbor genes for the protocatechuate branch of the beta-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 beta-ketoadipate pathway.

Adipates↗

PubMed2004

Benzoate decreases the binding of cis,cis-muconate to the BenM regulator despite the synergistic effect of both compounds on transcriptional activation.

Fluorescence emission spectroscopy was used to investigate interactions between two effectors and BenM, a transcriptional regulator of benzoate catabolism. BenM had a higher affinity for cis,cis-muconate than for benzoate as the sole effector. However, the presence of benzoate increased the apparent dissociation constant (reduced the affinity) of the protein for cis,cis-muconate. Similar results were obtained with truncated BenM lacking the DNA-binding domain. High-level transcriptional activation may require that some monomers within a BenM tetramer bind benzoate and others bind cis,cis-muconate.

Bacterial Proteins↗

PubMed2004

Selection for gene clustering by tandem duplication.

In prokaryotic genomes, related genes are frequently clustered in operons and higher-order arrangements that reflect functional context. Organization emerges despite rearrangements that constantly shuffle gene and operon order. Evidence is presented that the tandem duplication of related genes acts as a driving evolutionary force in the origin and maintenance of clusters. Gene amplification can be viewed as a dynamic and reversible regulatory mechanism that facilitates adaptation to variable environments. Clustered genes confer selective benefits via their ability to be coamplified. During evolution, rearrangements that bring together related genes can be selected if they increase the fitness of the organism in which they reside. Similarly, the benefits of gene amplification can prevent the dispersal of existing clusters. Examples of frequent and spontaneous amplification of large genomic fragments are provided. The possibility is raised that tandem gene duplication works in concert with horizontal gene transfer as interrelated evolutionary forces for gene clustering.

Benzoates↗

PubMed2003

Histidine ligand protonation and redox potential in the rieske dioxygenases: role of a conserved aspartate in anthranilate 1,2-dioxygenase.

The Rieske dioxygenase, anthranilate 1,2-dioxygenase, catalyzes the 1,2-dihydroxylation of anthranilate (2-aminobenzoate). As in all characterized Rieske dioxygenases, the catalytic conversion to the diol occurs within the dioxygenase component, AntAB, at a mononuclear iron site which accepts electrons from a proximal Rieske [2Fe-2S] center. In the related naphthalene dioxygenase (NDO), a conserved aspartate residue lies between the mononuclear and Rieske iron centers, and is hydrogen-bonded to a histidine ligand of the Rieske center. Engineered substitutions of this aspartate residue led to complete inactivation, which was proposed to arise from elimination of a productive intersite electron transfer pathway [Parales, R. E., Parales, J. V., and Gibson, D. T. (1999) J. Bacteriol. 181, 1831-1837]. Substitutions of the corresponding aspartate, D218, in AntAB with alanine, asparagine, or glutamate also resulted in enzymes that were completely inactive over a wide pH range despite retention of the hexameric quaternary structure and iron center occupancy. The Rieske center reduction potential of this variant was measured to be approximately 100 mV more negative than that for the wild-type enzyme at neutral pH. The wild-type AntAB became completely inactive at pH 9 and exhibited an altered Rieske center absorption spectrum which resembled that of the D218 variants at neutral pH. These results support a role for this aspartate in maintaining the protonated state and reduction potential of the Rieske center. Both the wild-type and D218A variant AntABs exhibited substrate-dependent rapid phases of Rieske center oxidations in stopped-flow time courses. This observation does not support a role for this aspartate in a facile intersite electron transfer pathway or in productive substrate gating of the Rieske center reduction potential. However, since the single turnovers resulted in anthranilate dihydroxylation by the wild-type enzyme but not by the D218A variant, this aspartate must also play a crucial role in substrate dihydroxylation at or near the mononuclear iron site.

Acinetobacter↗

PubMed2003

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 amplicon's 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.

Acinetobacter↗

PubMed2003

Transcriptional cross-regulation of the catechol and protocatechuate branches of the beta-ketoadipate pathway contributes to carbon source-dependent expression of the Acinetobacter sp. strain ADP1 pobA gene.

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 beta-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 beta-ketoadipate pathway.

Acinetobacter↗

PubMed2002

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.

Bacterial Proteins↗

PubMed2002

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.

Acinetobacter↗

PubMed2002

The benPK operon, proposed to play a role in transport, is part of a regulon for benzoate catabolism in Acinetobacter sp. strain ADP1.

BenM and CatM are distinct, but similar, LysR-type transcriptional regulators of the soil bacterium Acinetobacter sp. strain ADP1. Together, the two regulators control the expression of at least 14 genes involved in the degradation of aromatic compounds via the catechol branch of the beta-ketoadipate pathway. In these studies, BenM and CatM were each purified to homogeneity to test the possibility that they regulate the expression of two additional genes, benP and benK, that are adjacent to benM on the chromosome. Each regulator bound to a DNA fragment containing the benP promoter region. Additional transcriptional studies suggested that benP and benK are co-transcribed as an operon, and a site of transcription initiation was identified. Alignment of this initiation site with those of several CatM- and BenM-regulated genes revealed common regulatory motifs. Mutants lacking both CatM and BenM failed to activate benP transcription. The ability of each protein to regulate gene expression was inferred from strains lacking either CatM or BenM that were still capable of increasing benP expression in response to cis,cis-muconate. This compound has previously been shown to induce all enzymes of the catechol branch of the beta-ketoadipate pathway through a complex transcriptional circuit involving CatM and BenM. Thus, the regulated expression of the benPK operon in concert with other genes of the regulon is consistent with the model that BenP, a putative outer-membrane porin, and BenK, an inner-membrane permease, transport aromatic compounds in strain ADP1.

Acinetobacter↗

PubMed2002

Redox-dependent structural changes in archaeal and bacterial Rieske-type [2Fe-2S] clusters.

Proteins containing Rieske-type [2Fe-2S] clusters play important roles in many biological electron transfer reactions. Typically, [2Fe-2S] clusters are not directly involved in the catalytic transformation of substrate, but rather supply electrons to the active site. We report herein X-ray absorption spectroscopic (XAS) data that directly demonstrate an average increase in the iron-histidine bond length of at least 0.1 A upon reduction of two distantly related Rieske-type clusters in archaeal Rieske ferredoxin from Sulfolobus solfataricus strain P-1 and bacterial anthranilate dioxygenases from Acinetobacter sp. strain ADP1. This localized redox-dependent structural change may fine tune the protein-protein interaction (in the case of ARF) or the interdomain interaction (in AntDO) to facilitate rapid electron transfer between a lower potential Rieske-type cluster and its redox partners, thereby regulating overall oxygenase reactions in the cells.

Acinetobacter↗

PubMed1997

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 strain's 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.

Acinetobacter↗