Regulation of tricarboxylate transport and metabolism in Acinetobacter baylyi ADP1.

Despite the significant presence of plant-derived tricarboxylic acids in some environments, few studies detail the bacterial metabolism of trans-aconitic acid (Taa) and tricarballylic acid (Tcb). In a soil bacterium, Acinetobacter baylyi ADP1, we discovered interrelated pathways for the consumption of Taa and Tcb. An intricate regulatory scheme tightly controls the transport and catabolism of both compounds and may reflect that they can be toxic inhibitors of the tricarboxylic acid cycle. The genes encoding two similar LysR-type transcriptional regulators, TcuR and TclR, were clustered on the chromosome with tcuA and tcuB, genes required for Tcb consumption. The genetic organization differed from that in Salmonella enterica serovar Typhimurium, in which tcuA and tcuB form an operon with a transporter gene, tcuC. In A. baylyi, tcuC was not cotranscribed with tcuAB. Rather, tcuC was cotranscribed with a gene, designated pacI, encoding an isomerase needed for Taa consumption. TcuC appears to transport Tcb and cis-aconitic acid (Caa), the presumed product of PacI-mediated periplasmic isomerization of Taa. Two operons, tcuC-pacI and tcuAB, were transcriptionally controlled by both TcuR and TclR, which have overlapping functions. We investigated the roles of the two regulators in activating transcription of both operons in response to multiple effector compounds, including Taa, Tcb, and Caa.IMPORTANCEIngestion of Taa and Tcb by grazing livestock can cause a serious metabolic disorder called grass tetany. The disorder, which results from Tcb absorption by ruminants, focuses attention on the metabolism of tricarboxylic acids. Additional interest stems from efforts to produce tricarboxylic acids as commodity chemicals. Improved understanding of bacterial enzymes and pathways for tricarboxylic acid metabolism may contribute to new biomanufacturing strategies.

Engineering CatM, a LysR-Type Transcriptional Regulator, to Respond Synergistically to Two Effectors.

The simultaneous response of one transcriptional regulator to different effectors remains largely unexplored. Nevertheless, such interactions can substantially impact gene expression by rapidly integrating cellular signals and by expanding the range of transcriptional responses. In this study, similarities between paralogs were exploited to engineer novel responses in CatM, a regulator that controls benzoate degradation in Acinetobacter baylyi ADP1. One goal was to improve understanding of how its paralog, BenM, activates transcription in response to two compounds (cis,cis-muconate and benzoate) at levels significantly greater than with either alone. Despite the overlapping functions of BenM and CatM, which regulate many of the same ben and cat genes, CatM normally responds only to cis,cis-muconate. Using domain swapping and site-directed amino acid replacements, CatM variants were generated and assessed for the ability to activate transcription. To create a variant that responds synergistically to both effectors required alteration of both the effector-binding region and the DNA-binding domain. These studies help define the interconnected roles of protein domains and extend understanding of LysR-type proteins, the largest family of transcriptional regulators in bacteria. Additionally, renewed interest in the modular functionality of transcription factors stems from their potential use as biosensors.

Malonate degradation in Acinetobacter baylyi ADP1: operon organization and regulation by MdcR.

Transcriptional regulators in the LysR or GntR families are typically encoded in the genomic neighbourhood of bacterial genes for malonate degradation. While these arrangements have been evaluated using bioinformatics methods, experimental studies demonstrating co-transcription of predicted operons were lacking. Here, transcriptional regulation was characterized for a cluster of mdc genes that enable a soil bacterium, Acinetobacter baylyi ADP1, to use malonate as a carbon source. Despite previous assumptions that the mdc-gene set forms one operon, our studies revealed distinct promoters in two different regions of a nine-gene cluster. Furthermore, a single promoter is insufficient to account for transcription of mdcR, a regulatory gene that is convergent to other mdc genes. MdcR, a LysR-type transcriptional regulator, was shown to bind specifically to a site where it can activate mdc-gene transcription. Although mdcR deletion prevented growth on malonate, a 1 nt substitution in the promoter of mdcA enabled MdcR-independent growth on this carbon source. Regulation was characterized by methods including transcriptional fusions, quantitative reverse transcription PCR, reverse transcription PCR, 5'-rapid amplification of cDNA ends and gel shift assays. Moreover, a new technique was developed for transcriptional characterization of low-copy mRNA by increasing the DNA copy number of specific chromosomal regions. MdcR was shown to respond to malonate, in the absence of its catabolism. These studies contribute to ongoing characterization of the structure and function of a set of 44 LysR-type transcriptional regulators in A. baylyi ADP1.

Analysis of IS1236-mediated gene amplification events in Acinetobacter baylyi ADP1.

Recombination between insertion sequence copies can cause genetic deletion, inversion, or duplication. However, it is difficult to assess the fraction of all genomic rearrangements that involve insertion sequences. In previous gene duplication and amplification studies of Acinetobacter baylyi ADP1, an insertion sequence was evident in approximately 2% of the characterized duplication sites. Gene amplification occurs frequently in all organisms and has a significant impact on evolution, adaptation, drug resistance, cancer, and various disorders. To understand the molecular details of this important process, a previously developed system was used to analyze gene amplification in selected mutants. The current study focused on amplification events in two chromosomal regions that are near one of six copies of the only transposable element in ADP1, IS1236 (an IS3 family member). Twenty-one independent mutants were analyzed, and in contrast to previous studies of a different chromosomal region, IS1236 was involved in 86% of these events. IS1236-mediated amplification could occur through homologous recombination between insertion sequences on both sides of a duplicated region. However, this mechanism presupposes that transposition generates an appropriately positioned additional copy of IS1236. To evaluate this possibility, PCR and Southern hybridization were used to determine the chromosomal configurations of amplification mutants involving IS1236. Surprisingly, the genomic patterns were inconsistent with the hypothesis that intramolecular homologous recombination occurred between insertion sequences following an initial transposition event. These results raise a novel possibility that the gene amplification events near the IS1236 elements arise from illegitimate recombination involving transposase-mediated DNA cleavage.

Genome-wide selection for increased copy number in Acinetobacter baylyi ADP1: locus and context-dependent variation in gene amplification.

Renewed interest in gene amplification stems from its importance in evolution and a variety of medical problems ranging from drug resistance to cancer. However, amplified DNA segments (amplicons) are not fully characterized in any organism. Here we report a novel Acinetobacter baylyi system for genome-wide studies. Amplification mutants that consume aromatic compounds were selected under conditions requiring high-level expression from three promoters in a linked set of chromosomal genes. Tools were developed to relocate these catabolic genes to any non-essential chromosomal position, and 49 amplification mutants from five genomic contexts were characterized. Amplicon size (18-271 kb) and copy number (2-105) indicated that 30% of mutants carried more than 1 Mb of amplified DNA. Amplification features depended on genomic position. For example, amplicons from one locus were similarly sized but displayed variable copy number, whereas those from another locus were differently sized but had comparable copy number. Additionally, the importance of sequence context was highlighted in one region where amplicons differed depending on the presence of a promoter mutation in the strain from which they were selected. DNA sequences at amplicon boundaries in 19 mutants reflected illegitimate recombination. Furthermore, steady-state duplication frequencies measured under non-selective conditions (10(-4) to 10(-5) ) confirmed that spontaneous gene duplication is a major source of genetic variation.