This project focuses on a new bacterial system for studying chromosomal rearrangements involving gene duplication and further amplification. Gene amplification, which is a common process in all organisms, has significant consequences. It is essential to evolution, genetic diversity, and the ability of organisms to adapt to variable environments. Gene amplification also contributes to serious problems such as drug resistance, cancer, and microbial virulence. Despite its importance, many aspects of gene amplification remain poorly understood. Unlike other types of genetic change, amplification is dynamic and reversible. It may leave no evidence of its position or frequency. In this research project, an unusual characteristic of a soil bacterium, Acinetobacter baylyi ADP1, facilitates the systematic study of gene amplification. The critical characteristic is that A. baylyi naturally takes up DNA from the environment with exceptionally high efficiency and incorporates it into the genome via homologous recombination. This natural competence for DNA uptake permits the use of a transformation assay to detect the precise endpoints of duplicated chromosomal regions in mutants that arise spontaneously. The DNA sequence of such duplication sites provides information about the underlying genetic recombination event. This project builds on intriguing results from initial studies in which a new type of position specific illegitimate recombination (PSIR) process was discovered. The PSIR events lack DNA features that characterize typical site-specific recombination. One objective of this project is to determine the mechanism of PSIR, which appears to be novel. Additionally, a genome-wide approach will be used to characterize spontaneous duplications. Important features of DNA that contribute to gene duplication will be investigated such as DNA sequence and genomic context. This strategy will improve our understanding of a fundamental, common and important genetic process.
Broader Impacts. Students will be trained in important multidisciplinary areas that bridge genetics, physiology, biochemistry, and computation. The project is accessible to students at all levels and will involve undergraduate and graduate students. Additionally, a postdoctoral researcher will conduct related investigations and help mentor the project participants. Ongoing programs at the University of Georgia will enhance the opportunity to train a diverse group of scientists. Such programs include an NSF-supported Research Experiences for Undergraduate (REU) site program in prokaryotic biology. The long-term impact of this research has the potential to offset harmful effects of gene amplification through a better understanding of the underlying mechanisms. Moreover, gene amplification can be developed for beneficial biotechnology applications. Ideally, chromosomal gene amplification could be used for desired manipulations to avoid the problematic use of plasmids and antibiotic selections in genetic engineering. Furthermore, computational analyses of chromosomal rearrangements have predictive value that will expand the utility of DNA sequences deposited in databases.
Gene amplification contributes significantly to important processes such as adaptation, evolution, virulence, and drug resistance. Yet despite its importance, the systematic study of gene amplification remains challenging. It has long been known that the duplication of chromosomal segments in bacteria can occur at a much high frequency than other types of genetic mutation. After gene duplication, the copy number of a duplicated segment may increase or decrease due to homologous recombination or other mechanisms. In this fashion, genomic DNA dynamically expands and contracts. However, this type of chromosomal flexibility is difficult to characterize at a detailed molecular level because the genetic changes are typically unstable in the absence of selective pressure. In this project, methods were developed to characterize chromosomal variation in a soil bacterium, Acinetobacter baylyi ADP1. Since strain ADP1 is naturally competent to take up DNA, it was possible to exploit the high efficiency with which transforming DNA gets incorporated into the chromosome by homologous recombination. A transformation assay enabled us to identify the precise endpoints of amplified chromosomal segments (amplicons) in mutants that were isolated by selective growth conditions. Strains were selected that each carry multiple chromosomal copies of genes needed for benzoate consumption (the cat-genes). Independently isolated amplification mutants were examined to determine the size and copy number of their amplicons. Furthermore, several parent strains were engineered such that the cat-gene region was placed in various chromosomal locations. With this approach it was possible to characterize genetic duplication events that occurred in different chromosomal regions. These studies revealed variation in the size of the selected amplicon, which ranged from 18 to approximately 300 kb. The number of amplicon copies also varied, ranging from 2 to 105. The mutants were able to sustain a large increase in the amount of chromosomal DNA. Whereas the wild-type chromosome is 3.6 Mb, approximately 30% of the isolated mutants carried more than 1Mb of additional amplified DNA. Features of the duplication and amplification events were context dependent such that the characteristics varied in different chromosomal regions. The spontaneous duplication of a specific gene was measured in different chromosomal regions and found to be approximately 10-4 to 10-5 under non-selective conditions. Thus, this project helped confirm that duplication and amplification events occur frequently in bacteria and revealed new information about the extent, underlying mechanisms, and features of these events. This project also addressed the types of duplication events that occur in the vicinity of a copy of the only transposable element in ADP1, IS1236 (an IS3 family member). The percent of the duplication events that involved a transposable element appeared to increase when studies focused on events in chromosomal regions near one of the six copies of this insertion sequence. A simple explanation for this observation would be transposition of the insertion sequence followed by intramolecular homologous recombination. However, this possible mechanism for IS1236-mediated duplication was ruled out. Instead, our results raised a novel possibility that the gene amplification events near the IS1236 elements arise from illegitimate recombination involving transposase-mediated DNA cleavage. Collectively these studies showed that A. baylyi serves as a good experimental model for systematic studies of chromosomal gene amplification. Moreover, the transformation assay used to identify the endpoints of amplicons has many practical applications. The new genetic tools that were developed created a selectable cassette that can be specifically amplified by transformation with an appropriate DNA fragment. Amplification mutants that are so generated can be readily selected by growth on benzoate as a sole carbon source. The ability to induce amplification of specific chromosomal segments lays the groundwork for using gene amplification for applications in biotechnology and bioengineering. The ease with which genetic studies can be done in A. baylyi makes it an ideal experimental organism for integrating teaching and research. This project was used to train many students, including one high school student, three undergraduates, and four graduate students. Two of the graduate students earned doctoral degrees based on their studies of gene amplification. In addition, one postdoctoral researcher was involved in this project. Several other work-study students learned basic lab skills in conjunction with this research. This project dovetailed with a summer undergraduate research program and an undergraduate course in which students conduct authentic research studies that address molecular biology and metabolism in A. baylyi. Efforts to increase diversity and broaden participation in scientific endeavors led to the training of several students who are members of underrepresented groups.
