The Shewanella Federation: Functional Genomic Investigations of Dissimilatory Metal-Reducing Shewanella
- Univ. of Oklahoma, Norman, OK (United States)
Generation and validation of a Shewanella oneidensis MR-1 clone set for protein expression and phage display. An ORF clone set for S. oneidensis was created using the lambda recombinase system. ORFs within entry vectors in this system can be readily transferred into multiple destination vectors, making the clone set a useful resource for research groups studying this microorganism. To establish that the S. oneidensis clone set could be used for protein expression and functional studies, three sets of ORFs were examined for expression of His-tag proteins, expression of His/GST-tag proteins, or for effective display on phage. A total of 21 out of 30 (70%) predicted two-component transcriptional regulators from S. oneidensis were successfully expressed in the His-tag format. The use of the S. oneidensis clone set for functional studies was tested using a phage display system. The method involves the fusion of peptides or proteins to a coat protein of a bacteriophage. This results in display of the fused protein on the exterior of the phage, while the DNA encoding the fusion resides within the virion. The physical linkage between the displayed protein and the DNA encoding it allows screening of vast numbers of proteins for ligand-binding properties. With this technology, a phage clone encoding thioredoxin TrxA was isolated from a sub-library consisting of 80 clones. It is evident that the S. oneidensis clone set can be used for expression of functional S. oneidensis proteins in E. coli using the appropriate destination vectors. Characterization of ArcA. In Escherichia coli, metabolic transitions between aerobic and anaerobic growth states occur when cells enter an oxygen-limited condition. Many of these metabolic transitions are controlled at the transcriptional level by the activities of the global regulatory proteins ArcA (aerobic respiration control) and Fnr (fumarate nitrate regulator). A homolog of ArcA (81% amino acid sequence identity) was identified in S. oneidensis MR-1, and arcA mutants with MR-1 as the parental strain were generated. Phenotype characterization showed the arcA deletion mutant grew slower than the wild-type and was hypersensitive to H2O2 stress. Microarray analysis indicated that S. oneidensis ArcA regulates a large number of different genes from that in E. coli although they do have overlapping regulatory functions on a small set of genes. The S. oneidensis arcA gene was also cloned and expressed in E. coli. The ArcA proteins from the wild-type and a point mutant strains (D54N) were purified and their DNA binding properties were analyzed by electrophoretic motility shift (EMS) and DNase I footprinting assays. The results indicate that phosphorylated ArcA proteins bind to a DNA site similar in sequence to the E. coli ArcA binding site. The common feature of the binding sites is the presence of a conserved 15 base pair motif that contains 2-3 mismatches when compared to the E. coli ArcA-P consensus binding motif. Genome scale computational predictions of binding sites were also performed and 331 putative ArcA regulatory targets were identified. Therefore, the regulation of aerobic/anaerobic respiration may be more complex than it was expected in S. oneidensis. A high-throughput percentage-of-binding strategy to measure binding energies in DNA–protein interactions. Based on results of studies on ArcA of S. oneidensis, we developed a high-throughput approach to measure binding energies in DNA-protein interactions, which enables a more precise prediction for DNA-binding sites in genomes. With this approach, the importance of each position within the ArcA-P binding site was quantitatively established by characterizing the interaction between Shewanella ArcA-P and a series of mutant promoter DNAs, whereby each position in the binding site was systematically mutated to all possible single nucleotide changes. The results of the fine mapping were used to create a position-specific energy matrix (PEM) that was used for a genome-scale prediction of 45 ArcA-P sites in Shewanella. A further examination suggests that this prediction is >81% consistency with in vivo gene regulation according to microarray studies and >92% (13/14) accuracy in comparison with published in vitro gel shift validation binding assays. In addition, this study predicted 27 ArcA-P sites for 15 published E. coli ArcA-P footprinted DNAs, and 24 of them were found exactly within the footprinting protected regions and the other three sites fall into the regions that were not examined by footprinting assays. This is the first report showing that footprinting protected regions can be effectively predicted by starting from a single known transcription factor binding site. Finally, the predicted H. influenzae ArcA-P sites correlate well with in vivo regulation determined by a microarray analysis in that the eight predicted binding sites with the most favorable ΔG scores all exhibit ArcA dependent gene regulation.
- Research Organization:
- Univ. of Oklahoma, Norman, OK (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC)
- DOE Contract Number:
- FG02-06ER64200
- OSTI ID:
- 946694
- Report Number(s):
- DOE/ER/64200-Final Report; TRN: US201108%%407
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
60 APPLIED LIFE SCIENCES
AMINO ACID SEQUENCE
DNA
ESCHERICHIA COLI
FUNCTIONALS
GENE REGULATION
GENES
IN VITRO
IN VIVO
MUTANTS
NITRATES
NUCLEOTIDES
PEPTIDES
PHENOTYPE
PROTEINS
REGULATIONS
RESPIRATION
STRAINS
TRANSCRIPTION FACTORS
VECTORS
Functional genomics
Shewanella
metal reduction