In JoVE (1)

Other Publications (10)

Articles by Alla Gagarinova in JoVE

Other articles by Alla Gagarinova on PubMed

Association of the Transcriptional Response of Soybean Plants with Soybean Mosaic Virus Systemic Infection

The Journal of General Virology. Apr, 2008  |  Pubmed ID: 18343851

Compatible virus infection induces and suppresses host gene expression at the global level. These gene-expression changes are the molecular basis of symptom development and general stress and defence-like responses of the host. To assess transcriptional changes in soybean plants infected with soybean mosaic virus (SMV), the first soybean trifoliate leaf, immediately above the SMV-inoculated unifoliate leaf, was sampled at 7, 14 and 21 days post-inoculation (p.i.) and subjected to microarray analysis. The identified changes in gene expression in soybean leaves with SMV infection at different time points were associated with the observed symptom development. By using stringent selection criteria (>or=2- or

ESGA: E. Coli Synthetic Genetic Array Analysis

Nature Methods. Sep, 2008  |  Pubmed ID: 18677321

Physical and functional interactions define the molecular organization of the cell. Genetic interactions, or epistasis, tend to occur between gene products involved in parallel pathways or interlinked biological processes. High-throughput experimental systems to examine genetic interactions on a genome-wide scale have been devised for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, but have not been reported previously for prokaryotes. Here we describe the development of a quantitative screening procedure for monitoring bacterial genetic interactions based on conjugation of Escherichia coli deletion or hypomorphic strains to create double mutants on a genome-wide scale. The patterns of synthetic sickness and synthetic lethality (aggravating genetic interactions) we observed for certain double mutant combinations provided information about functional relationships and redundancy between pathways and enabled us to group bacterial gene products into functional modules.

Identification and Molecular Characterization of Two Naturally Occurring Soybean Mosaic Virus Isolates That Are Closely Related but Differ in Their Ability to Overcome Rsv4 Resistance

Virus Research. Dec, 2008  |  Pubmed ID: 18793685

A naturally occurring Rsv4 resistance-breaking isolate (L-RB) and a closely related non-resistance-breaking isolate (L) of Soybean mosaic virus (SMV) were identified in soybean fields in London, Ontario, Canada. The viral genomes of L and L-RB were completely sequenced. Each isolate has a 9585-nucleotide genome with a single open reading frame encoding a polyprotein of approximately 350 kDa. L-RB and L have a very high sequence similarity (99.6%) at both the nucleotide and amino acid levels. Phylogenetic analysis showed that the two isolates belong to the G2 pathotype. Pathogenicity predictions of all virus/soybean combinations, based on the phylogenetic profile, were confirmed by pathogenicity tests using L and L-RB isolates and soybeans carrying different resistance genes, with an exception that L-RB infected a soybean cultivar carrying Rsv4 resistance. The temporal and spatial proximity of L and L-RB and their high sequence similarity suggest L-RB was likely derived from the SMV-L quasispecies. Recombination analysis did not reveal the evidence of genetic recombination for the emergence of L-RB. Mutations introduced by virus-encoded RNA-dependent RNA polymerase during viral genome replication and selection pressure probably contributed to the occurrence of L-RB.

Recombination Analysis of Soybean Mosaic Virus Sequences Reveals Evidence of RNA Recombination Between Distinct Pathotypes

Virology Journal. 2008  |  Pubmed ID: 19036160

RNA recombination is one of the two major factors that create RNA genome variability. Assessing its incidence in plant RNA viruses helps understand the formation of new isolates and evaluate the effectiveness of crop protection strategies. To search for recombination in Soybean mosaic virus (SMV), the causal agent of a worldwide seed-borne, aphid-transmitted viral soybean disease, we obtained all full-length genome sequences of SMV as well as partial sequences encoding the N-terminal most (P1 protease) and the C-terminal most (capsid protein; CP) viral protein. The sequences were analyzed for possible recombination events using a variety of automatic and manual recombination detection and verification approaches. Automatic scanning identified 3, 10, and 17 recombination sites in the P1, CP, and full-length sequences, respectively. Manual analyses confirmed 10 recombination sites in three full-length SMV sequences. To our knowledge, this is the first report of recombination between distinct SMV pathotypes. These data imply that different SMV pathotypes can simultaneously infect a host cell and exchange genetic materials through recombination. The high incidence of SMV recombination suggests that recombination plays an important role in SMV evolution. Obtaining additional full-length sequences will help elucidate this role.

A Dual Function of the CRISPR-Cas System in Bacterial Antivirus Immunity and DNA Repair

Molecular Microbiology. Jan, 2011  |  Pubmed ID: 21219465

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.

Ribosome-dependent ATPase Interacts with Conserved Membrane Protein in Escherichia Coli to Modulate Protein Synthesis and Oxidative Phosphorylation

PloS One. 2011  |  Pubmed ID: 21556145

Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.

Array-based Synthetic Genetic Screens to Map Bacterial Pathways and Functional Networks in Escherichia Coli

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21815091

Cellular processes are carried out through a series of molecular interactions. Various experimental approaches can be used to investigate these functional relationships on a large-scale. Recently, the power of investigating biological systems from the perspective of genetic (gene-gene or epistatic) interactions has been evidenced by the ability to elucidate novel functional relationships. Examples of functionally related genes include genes that buffer each other's function or impinge on the same biological process. Genetic interactions have traditionally been investigated in bacteria by combining pairs of mutations (e.g., gene deletions) and assessing deviation of the phenotype of each double mutant from an expected neutral (or no interaction) phenotype. Fitness is a particularly convenient phenotype to measure: when the double mutant grows faster or slower than expected, the two mutated genes are said to show alleviating or aggravating interactions, respectively. The most commonly used neutral model assumes that the fitness of the double mutant is equal to the product of individual single mutant fitness. A striking genetic interaction is exemplified by the loss of two nonessential genes that buffer each other in performing an essential biological function: deleting only one of these genes produces no detectable fitness defect; however, loss of both genes simultaneously results in systems failure, leading to synthetic sickness or lethality. Systematic large-scale genetic interaction screens have been used to generate functional maps for model eukaryotic organisms, such as yeast, to describe the functional organization of gene products into pathways and protein complexes within a cell. They also reveal the modular arrangement and cross talk of pathways and complexes within broader functional neighborhoods (Dixon et al., Annu Rev Genet 43:601-625, 2009). Here, we present a high-throughput quantitative Escherichia coli Synthetic Genetic Array (eSGA) screening procedure, which we developed to systematically infer genetic interactions by scoring growth defects among large numbers of double mutants in a classic Gram-negative bacterium. The eSGA method exploits the rapid colony growth, ease of genetic manipulation, and natural efficient genetic exchange via conjugation of laboratory E. coli strains. Replica pinning is used to grow and mate arrayed sets of single gene mutant strains and to select double mutants en masse. Strain fitness, which is used as the eSGA readout, is quantified by the digital imaging of the plates and subsequent measuring and comparing single and double mutant colony sizes. While eSGA can be used to screen select mutants to probe the functions of individual genes, using eSGA more broadly to collect genetic interaction data for many combinations of genes can help reconstruct a functional interaction network to reveal novel links and components of biological pathways as well as unexpected connections between pathways. A variety of bacterial systems can be investigated, wherein the genes impinge on a essential biological process (e.g., cell wall assembly, ribosome biogenesis, chromosome replication) that are of interest from the perspective of drug development (Babu et al., Mol Biosyst 12:1439-1455, 2009). We also show how genetic interactions generated by high-throughput eSGA screens can be validated by manual small-scale genetic crosses and by genetic complementation and gene rescue experiments.

Array-based Synthetic Genetic Screens to Map Bacterial Pathways and Functional Networks in Escherichia Coli

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21877280

Cellular processes are carried out through a series of molecular interactions. Various experimental approaches can be used to investigate these functional relationships on a large-scale. Recently, the power of investigating biological systems from the perspective of genetic (gene-gene, or epistatic) interactions has been evidenced by the ability to elucidate novel functional relationships. Examples of functionally related genes include genes that buffer each other's function or impinge on the same biological process. Genetic interactions have traditionally been investigated in bacteria by combining pairs of mutations (for example, gene deletions) and assessing deviation of the phenotype of each double mutant from an expected neutral (or no interaction) phenotype. Fitness is a particularly convenient phenotype to measure: when the double mutant grows faster or slower than expected, the two mutated genes are said to show alleviating or aggravating interactions, respectively. The most commonly used neutral model assumes that the fitness of the double mutant is equal to the product of individual single mutant fitness. A striking genetic interaction is exemplified by the loss of two nonessential genes that buffer each other in performing an essential biological function: deleting only one of these genes produces no detectable fitness defect; however, loss of both genes simultaneously results in systems failure, leading to synthetic sickness or lethality. Systematic large-scale genetic interaction screens have been used to generate functional maps for model eukaryotic organisms, such as yeast, to describe the functional organization of gene products into pathways and protein complexes within a cell. They also reveal the modular arrangement and cross-talk of pathways and complexes within broader functional neighborhoods (Dixon et al. Annu Rev Genet 43:601-625, 2009). Here, we present a high-throughput quantitative Escherichia coli synthetic genetic array (eSGA) screening procedure, which we developed to systematically infer genetic interactions by scoring growth defects among large numbers of double mutants in a classic gram-negative bacterium. The eSGA method exploits the rapid colony growth, ease of genetic manipulation, and natural efficient genetic exchange via conjugation of laboratory E. coli strains. Replica pinning is used to grow and mate arrayed sets of single-gene mutant strains as well as to select double mutants en mass. Strain fitness, which is used as the eSGA readout, is quantified by the digital imaging of the plates and subsequent measuring and comparing single and double mutant colony sizes. While eSGA can be used to screen select mutants to probe the functions of individual genes; using eSGA more broadly to collect genetic interaction data for many combinations of genes can help reconstruct a functional interaction network to reveal novel links and components of biological pathways as well as unexpected connections between pathways. A variety of bacterial systems can be investigated, wherein the genes impinge on a essential biological process (e.g., cell wall assembly, ribosome biogenesis, chromosome replication) that are of interest from the perspective of drug development (Babu et al. Mol Biosyst 12:1439-1455, 2009). We also show how genetic interactions generated by high-throughput eSGA screens can be validated by manual small-scale genetic crosses and by genetic complementation and gene rescue experiments.

Genetic Interaction Maps in Escherichia Coli Reveal Functional Crosstalk Among Cell Envelope Biogenesis Pathways

PLoS Genetics. Nov, 2011  |  Pubmed ID: 22125496

As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among > 235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target.

Genome-scale Genetic Manipulation Methods for Exploring Bacterial Molecular Biology

Molecular BioSystems. Jun, 2012  |  Pubmed ID: 22517266

Bacteria are diverse and abundant, playing key roles in human health and disease, the environment, and biotechnology. Despite progress in genome sequencing and bioengineering, much remains unknown about the functional organization of prokaryotes. For instance, roughly a third of the protein-coding genes of the best-studied model bacterium, Escherichia coli, currently lack experimental annotations. Systems-level experimental approaches for investigating the functional associations of bacterial genes and genetic structures are essential for defining the fundamental molecular biology of microbes, preventing the spread of antibacterial resistance in the clinic, and driving the development of future biotechnological applications. This review highlights recently introduced large-scale genetic manipulation and screening procedures for the systematic exploration of bacterial gene functions, molecular relationships, and the global organization of bacteria at the gene, pathway, and genome levels.

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