A complete collection of single‐gene deletion mutants of Acinetobacter baylyi ADP1

We have constructed a collection of single‐gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2‐3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.     

Two-component systems of Corynebacterium glutamicum: deletion analysis and involvement of the PhoS-PhoR system in the phosphate starvation response

Corynebacterium glutamicum contains genes for 13 two-component signal transduction systems. In order to test for their essentiality and involvement in the adaptive response to phosphate (Pi) starvation, a set of 12 deletion mutants was constructed. One of the mutants was specifically impaired in its ability to grow under Pi limitation, and therefore the genes lacking in this strain were named phoS (encoding the sensor kinase) and phoR (encoding the response regulator). DNA microarray analyses with the C. glutamicum wild type and the DeltaphoRS mutant supported a role for the PhoRS system in the adaptation to Pi starvation. In contrast to the wild type, the DeltaphoRS mutant did not induce the known Pi starvation-inducible (psi) genes within 1 hour after a shift from Pi excess to Pi limitation, except for the pstSCAB operon, which was still partially induced. This indicates an activator function for PhoR and the existence of at least one additional regulator of the pst operon. Primer extension analysis of selected psi genes (pstS, ugpA, phoR, ushA, and nucH) confirmed the microarray data and provided evidence for positive autoregulation of the phoRS genes.     

A fast neutron deletion mutagenesis-based reverse genetics system for plants

A new reverse genetics method has been developed to identify and isolate deletion mutants for targeted plant genes. Deletion mutant libraries are generated using fast neutron bombardment. DNA samples extracted from the deletion libraries are used to screen for deletion mutants by polymerase chain reaction (PCR) using specific primers flanking the targeted genes. By adjusting PCR conditions to preferentially amplify the deletion alleles, deletion mutants were identified in pools of DNA samples, each pool containing DNA from 2592 mutant lines. Deletion mutants were obtained for 84% of targeted loci from an Arabidopsis population of 51 840 lines. Using a similar approach, a deletion mutant for a rice gene was identified. Thus we demonstrate that it is possible to apply this method to plant species other than Arabidopsis. As fast neutron mutagenesis is highly efficient, it is practical to develop deletion mutant populations with more complete coverage of the genome than obtained with methods based on insertional mutagenesis. Because fast neutron mutagenesis is applicable to all plant genetic systems, this method has the potential to enable reverse genetics for a wide range of plant species.     

Deleteagene: a fast neutron deletion mutagenesis-based gene knockout system for plants

Deleteagene(trade mark) (Delete-a-gene) is a deletion-based gene knockout system for plants. To obtain deletion mutants for a specific gene, random deletion libraries created by fast neutron mutagenesis are screened by polymerase chain reaction (PCR) using primers flanking the target gene. By adjusting the PCR extension time to preferentially amplify the deletion alleles, deletion mutants can be identified in pools of DNA samples with each sample representing more than a thousand mutant lines. In Arabidopsis, knockout plants for greater than 80% of targeted genes have been obtained from a population of 51 840 lines. A large number of deletion mutants have been identified and multiple deletion alleles are often recovered for targeted loci. In Arabidopsis, the method is very useful for targeting small genes and can be used to find deletion mutants mutating two or three tandem homologous genes. In addition, the method is demonstrated to be effective in rice as a deletion mutant for a rice gene was obtained with a similar approach. Because fast neutron mutagenesis is applicable to all plant genetic systems, Deleteagene(trade mark) has the potential to enable reverse genetics for a wide range of plant species.     

Overlapping functions of the yeast oxysterol-binding protein homologues

The Saccharomyces cerevisiae genome encodes seven homologues of the mammalian oxysterol-binding protein (OSBP), a protein implicated in lipid trafficking and sterol homeostasis. To determine the functions of the yeast OSBP gene family (OSH1-OSH7), we used a combination of genetics, genomics, and sterol lipid analysis to characterize OSH deletion mutants. All 127 combinations and permutations of OSH deletion alleles were constructed. Individual OSH genes were not essential for yeast viability, but the elimination of the entire gene family was lethal. Thus, the family members shared an essential function. In addition, the in vivo depletion of all Osh proteins disrupted sterol homeostasis. Like mutants that affect ergosterol production, the viable combinations of OSH deletion alleles exhibited specific sterol-related defects. Although none of the single OSH deletion mutants was defective for growth, gene expression profiles revealed that each mutant had a characteristic molecular phenotype. Therefore, each gene performed distinct nonessential functions and contributed to a common essential function. Our findings indicated that OSH genes performed a multitude of nonessential roles defined by specific subsets of the genes and that most shared at least one essential role potentially linked to changes in sterol lipid levels.     

Genome-wide screening for genes associated with FK506 sensitivity in fission yeast

We have been studying calcineurin signal transduction pathway in fission yeast Schizosaccharomyces pombe (S. pombe) by developing a genetic screen for mutants that show hypersensitivity to the immunosuppressive calcineurin inhibitor FK506 (tacrolimus). In the present study, to identify nonessential genes that are functionally related to the calcineurin signaling pathway, we performed a genome-wide screen of 3004 haploid deletion strains and confirmed 72 deletion strains to be FK506 sensitive. These 72 genes are classified into nine functional groups to include membrane trafficking (16 genes), signal transduction (10 genes), ubiquitination (8 genes), chromatin remodeling (6 genes), cytokinesis (4 genes), ribosomal protein (3 genes), RNA binding protein (3 genes), and a variety of other known functions (17 genes) or still unknown functions (5 genes) in the biological system. In our previous screening of FK506-sensitive mutants we isolated several membrane-trafficking mutants showing defective cell wall integrity. Here, we further examined the vacuolar fusion, the v-SNARE synaptobrevin Syb1 localization, and the sensitivity to the β-glucan synthase inhibitor micafungin in these 72 FK506-sensitive strains. Results showed that 25 deletion strains exhibited abnormal vacuole fusion, 19 deletion strains exhibited Syb1 mislocalization, and 14 deletion strains exhibited both abnormal vacuole fusion and Syb1 mislocalization, while 42 deletion strains showed both normal vacuole fusion and Syb1 localization. Likewise, 16 deletion strains showed sensitivity to micafungin. Altogether, our present study indicates that calcineurin mediates a plethora of physiological processes in fission yeast, and that calcineurin is extensively involved in cross-talk between signaling pathways.     

A Comprehensive Analysis of the Importance of Translation Initiation Factors for Haloferax volcanii Applying Deletion and Conditional Depletion Mutants

Translation is an important step in gene expression. The initiation of translation is phylogenetically diverse, since currently five different initiation mechanisms are known. For bacteria the three initiation factors IF1 – IF3 are described in contrast to archaea and eukaryotes, which contain a considerably higher number of initiation factor genes. As eukaryotes and archaea use a non-overlapping set of initiation mechanisms, orthologous proteins of both domains do not necessarily fulfill the same function. The genome of Haloferax volcanii contains 14 annotated genes that encode (subunits of) initiation factors. To gain a comprehensive overview of the importance of these genes, it was attempted to construct single gene deletion mutants of all genes. In 9 cases single deletion mutants were successfully constructed, showing that the respective genes are not essential. In contrast, the genes encoding initiation factors aIF1, aIF2γ, aIF5A, aIF5B, and aIF6 were found to be essential. Factors aIF1A and aIF2β are encoded by two orthologous genes in H. volcanii. Attempts to generate double mutants failed in both cases, indicating that also these factors are essential. A translatome analysis of one of the single aIF2β deletion mutants revealed that the translational efficiency of the second ortholog was enhanced tenfold and thus the two proteins can replace one another. The phenotypes of the single deletion mutants also revealed that the two aIF1As and aIF2βs have redundant but not identical functions. Remarkably, the gene encoding aIF2α, a subunit of aIF2 involved in initiator tRNA binding, could be deleted. However, the mutant had a severe growth defect under all tested conditions. Conditional depletion mutants were generated for the five essential genes. The phenotypes of deletion mutants and conditional depletion mutants were compared to that of the wild-type under various conditions, and growth characteristics are discussed.     

The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols

A set of homozygous diploid deletion mutants of the yeast Saccharomyces cerevisiae was screened for the genes required for tolerance to aliphatic alcohols. The screen identified 137, 122 and 48 deletion mutants sensitive to ethanol, 1-propanol and 1-pentanol, respectively. A number of the genes required for ethanol tolerance were those also required for tolerance to other alcohols. Numerous mutants with defective genes encoding for vacuolar H+ -ATPase (V-ATPase) were cosensitive to these alcohols. A global screening approach of yeast deletion library mutants was useful in elucidating the mechanisms of alcohol tolerance based on different lipophilicities.     

Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B

Twelve populations of Escherichia coli B all lost D-ribose catabolic function during 2,000 generations of evolution in glucose minimal medium. We sought to identify the population genetic processes and molecular genetic events that caused these rapid and parallel losses. Seven independent Rbs(-) mutants were isolated, and their competitive fitnesses were measured relative to that of their Rbs(+) progenitor. These Rbs(-) mutants were all about 1 to 2% more fit than the progenitor. A fluctuation test revealed an unusually high rate, about 5 x 10(-5) per cell generation, of mutation from Rbs(+) to Rbs(-), which contributed to rapid fixation. At the molecular level, the loss of ribose catabolic function involved the deletion of part or all of the ribose operon (rbs genes). The physical extent of the deletion varied between mutants, but each deletion was associated with an IS150 element located immediately upstream of the rbs operon. The deletions apparently involved transposition into various locations within the rbs operon; recombination between the new IS150 copy and the one upstream of the rbs operon then led to the deletion of the intervening sequence. To confirm that the beneficial fitness effect was caused by deletion of the rbs operon (and not some undetected mutation elsewhere), we used P1 transduction to restore the functional rbs operon to two Rbs(-) mutants, and we constructed another Rbs(-) strain by gene replacement with a deletion not involving IS150. All three of these new constructs confirmed that Rbs(-) mutants have a competitive advantage relative to their Rbs(+) counterparts in glucose minimal medium. The rapid and parallel evolutionary losses of ribose catabolic function thus involved both (i) an unusually high mutation rate, such that Rbs(-) mutants appeared repeatedly in all populations, and (ii) a selective advantage in glucose minimal medium that drove these mutants to fixation.     

Involvement of Streptococcus gordonii beta-glucoside metabolism systems in adhesion, biofilm formation, and in vivo gene expression

Streptococcus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite (sHA). To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these induced genes were analyzed. To confirm the function of genes in each locus, strains were constructed with gene inactivation, deletion, and/or reporter gene fusions. Four novel systems responsible for beta-glucoside metabolism were identified, including three phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides. Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants. Esculin and oligochitosaccharides induced genes in one of the three beta-glucoside metabolism PTS and in four other genetic loci. Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic surfaces, and/or growth rate in liquid medium. Therefore, genes associated with beta-glucoside metabolism may regulate S. gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization.     

Characterization of a yeast mitochondrial locus necessary for tRNA biosynthesis. Deletion mapping and restriction mapping studies

Yeast mitochondrial DNA codes for a complete set of tRNAs. Although most components necessary for the biosynthesis of mitochondrial tRNA are coded by nuclear genes, there is one genetic locus on mitochondrial DNA necessary for the synthesis of mitochondrial tRNAs other than the mitochondrial tRNA genes themselves. Characterization of mutants by deletion mapping and restriction enzyme mapping studies has provided a precise location of this yeast mitochondrial tRNA synthesis locus. Deletion mutants retaining various segments of mitochondrial DNA were examined for their ability to synthesize tRNAs from the genes they retain. A subset of these strains was also tested for the ability to provide the tRNA synthesis function in complementation tests with deletion mutants unable to synthesize mature mitochondrial tRNAs. By correlating the tRNA synthetic ability with the presence or absence of certain wild-type restriction fragments, we have confined the locus to within 780 base pairs of DNA located between the tRNAMetf gene and tRNAPro gene, at 29 units on the wild-type map. Heretofore, no genetic function or gene product had been localized in this area of the yeast mitochondrial genome.     

Identification and classification of genes required for tolerance to high-sucrose stress revealed by genome-wide screening of Saccharomyces cerevisiae

Yeasts used in bread making are exposed to high concentrations of sucrose during sweet dough fermentation. Despite its importance, tolerance to high-sucrose stress is poorly understood at the gene level. To clarify the genes required for tolerance to high-sucrose stress, genome-wide screening was undertaken using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 273 deletions that yielded high sucrose sensitivity, approximately 20 of which were previously uncharacterized. These 273 deleted genes were classified based on their cellular function and localization of their gene products. Cross-sensitivity of the high-sucrose-sensitive mutants to high concentrations of NaCl and sorbitol was studied. Among the 273 sucrose-sensitive deletion mutants, 269 showed cross-sensitivities to sorbitol or NaCl, and four (i.e. ade5,7, ade6, ade8, and pde2) were specifically sensitive to high sucrose. The general stress response pathways via high-osmolarity glycerol and stress response element pathways and the function of the invertase in the ade mutants were similar to those in the wild-type strain. In the presence of high-sucrose stress, intracellular contents of ATP in ade mutants were at least twofold lower than that of the wild-type cells, suggesting that depletion of ATP is a factor in sensitivity to high-sucrose stress. The genes identified in this study might be important for tolerance to high-sucrose stress, and therefore should be target genes in future research into molecular modification for breeding of yeast tolerant to high-sucrose stress.     

Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum

As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study.     

Genome-Wide Screen in Saccharomyces cerevisiae Identifies Vacuolar Protein Sorting,Autophagy, Biosynthetic,and tRNA Methylation Genes Involved in Life Span Regulation

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.     

大肠杆菌侵袭基因缺失突变株与人脑微血管内皮细胞的相互作用

ibeA,ibeB,ibeC是与大肠杆菌侵袭人脑微血管内皮细胞(HBMEC)密切相关的基因,但迄今各基因的功能并不清楚。应用侵袭分析和免疫荧光技术分析了各基因的缺失突变型及野生型大肠杆菌对HBMEC的侵袭、细胞骨架与细胞间紧密连接的影响。结果显示：野生型菌大肠杆菌侵袭率为3.46％,而ibeA,ibeB,ibeC缺失突变株分别为0.54％、0．82％和0.73％：ibeA缺失突变型与野生型大肠杆菌作用相似,可引起HBMEC的细胞骨架蛋白分布改变,在细胞膜处呈明显的聚集,而ibeB和ibeC缺失突变株并未引起细胞骨架的明显改变;野生型和ibeA缺失突变型大肠杆菌可引起紧密连接结构的明显改变,而ibeB和ibeC缺失突变株对紧密连接结构的影响不明显。这些观察到的结果提示：ibeB和ibeC基因产物可能在调节细胞骨架和影响细胞紧密连接中起重要作用,而ibeA基因产物在其中的作用较小。     

Improved detection of small deletions in complex pools of DNA

About 40% of the genes in the nematode Caenorhabditis elegans have homologs in humans. Based on the history of this model system, it is clear that the application of genetic methods to the study of this set of genes would provide important clues to their function in humans. To facilitate such genetic studies, we are engaged in a project to derive deletion alleles in every gene in this set. Our standard methods make use of nested PCR to hunt for animals in mutagenized populations that carry deletions at a given locus. The deletion bearing animals exist initially in mixed populations where the majority of the animals are wild type at the target. Therefore, the production of the PCR fragment representing the deletion allele competes with the production of the wild type fragment. The size of the deletion fragment relative to wild type determines whether it can compete to a level where it can be detected above the background. Using our standard conditions, we have found that when the deletion is <600 bp, the deletion fragment does not compete effectively with the production of the wild type fragment in PCR. Therefore, although our standard methods work well to detect mutants with deletions >600 bp, they do not work well to detect mutants with smaller deletions. Here we report a new strategy to detect small deletion alleles in complex DNA pools. Our new strategy is a modification of our standard PCR based screens. In the first round of the nested PCR, we include a third PCR primer between the two external primers. The presence of this third primer leads to the production of three fragments from wild type DNA. We configure the system so that two of these three fragments cannot serve as a template in the second round of the nested PCR. The addition of this third primer, therefore, handicaps the amplification from wild type template. On the other hand, the amplification of mutant fragments where the binding site for the third primer is deleted is unabated. Overall, we see at least a 500-fold increase in the sensitivity for small deletion fragments using our new method. Using this new method, we report the recovery of new deletion alleles within 12 C.elegans genes.     

Srs2 overexpression reveals a helicase-independent role at replication forks that requires diverse cell functions

Srs2 is a 3'-5' DNA helicase that regulates many aspects of DNA metabolism in Saccharomyces cerevisiae. It is best known for its ability to counteract homologous recombination by dismantling Rad51 filaments, but is also involved in checkpoint activation, adaptation and recovery, and in resolution of late recombination intermediates. To further address its biological roles and uncover new genetic interactions, we examined the consequences of overexpressing SRS2 as well as two helicase-dead mutants, srs2-K41A and srs2-K41R, in the collection of 4827 yeast haploid deletion mutants. We identified 274 genes affecting a large variety of cellular functions that are required for cell growth when SRS2 or its mutants are overexpressed. Further analysis of these interactions reveals that Srs2 acts independently of its helicase function at replication forks likely through its recruitment by the sumoylated PCNA replication clamp. This helicase-independent function is responsible for the negative interactions with DNA metabolism genes and for the toxicity of SRS2 overexpression in many of the diverse cellular pathways revealed in our screens.