Unveiling nonessential gene deletions that confer significant morphological phenotypes beyond natural yeast strains

Background

Phenotypes are variable within species, with high phenotypic variation in the fitness and cell morphology of natural yeast strains due to genetic variation. A gene deletion collection of yeast laboratory strains also contains phenotypic variations, demonstrating the involvement of each gene and its specific function. However, to date, no study has compared the phenotypic variations between natural strains and gene deletion mutants in yeast.

Results

The morphological variance was compared between 110 most distinct gene deletion strains and 36 typical natural yeast strains using a generalized linear model. The gene deletion strains had higher morphological variance than the natural strains. Thirty-six gene deletion mutants conferred significant morphological changes beyond that of the natural strains, revealing the importance of the genes with high genetic interaction and specific cellular functions for species conservation.

Conclusion

Based on the morphological analysis, we discovered gene deletion mutants whose morphologies were not seen in nature. Our multivariate approach to the morphological diversity provided a new insight into the evolution and species conservation of yeast.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-932) contains supplementary material, which is available to authorized users.     

Isolation and properties of genetically defined strains of the methylotrophic yeast Hansenula polymorpha CBS4732

Genetically defined strains of the yeast Hansenula polymorpha were constructed from a clone of H. polymorpha CBS4732 with very low mating and sporulation abilities. Mating, spore viability, and the percentage of four-spore-containing asci were increased to a level at which tetrad analysis was possible. Auxotrophic mutations in 30 genes were isolated and used to construct strains with multiple markers for mapping studies, transformation with plasmid DNA, and mutant screening. Various other types of mutants were isolated and characterized, among them mutants that displayed an altered morphology, methanol-utilization deficient mutants and strains impaired in the biosynthesis of alcohol oxidase and catalase. Also, the mutability of H. polymorpha CBS4732 vs H. polymorpha NCYC495 was compared. The data revealed clear differences in frequencies of appearance and mutational spectra of some mutants isolated. Many of the mutants isolated had good mating abilities, and diploids resulting from their crossing displayed high sporulation frequencies and high spore viability. Most of the markers used revealed normal Mendelian segregation during meiosis.The frequency of tetratype spore formation was lower than in Saccharomyces cerevisiae suggesting a lower frequency of recombination during the second meiotic division. The properties of genetically defined strains of H. polymorpha CBS4732 as well as their advantages for genetics and molecular studies are discussed.     

A rapid method for cloning mutagenic DNA repair genes: isolation of umu-complementing genes from multidrug resistance plasmids R391, R446b, and R471a.

Genetic and physiological experiments have demonstrated that the products of the umu-like operon are directly required for mutagenic DNA repair in enterobacteria. To date, five such operons have been cloned and studied at the molecular level. Given the apparent wide occurrence of these mutagenic DNA repair genes in enterobacteria, it seems likely that related genes will be identified in other bacterial species and perhaps even in higher organisms. We are interested in identifying such genes. However, standard methods based on either DNA or protein cross-hybridization are laborious and, given the overall homology between previously identified members of this family (41 to 83% at the protein level), would probably have limited success. To facilitate the rapid identification of more diverse umu-like genes, we have constructed two Escherichia coli strains that allow us to identify umu-like genes after phenotypic complementation assays. With these two strains, we have cloned novel umu-like genes from three R plasmids, the IncJ plasmid R391 and two IncL/M plasmids, R446b and R471a.     

P-Element Mutations Affecting Embryonic Peripheral Nervous System Development in Drosophila Melanogaster

The Drosophila embryonic peripheral nervous system (PNS) is an excellent model system to study the molecular mechanisms governing neural development. To identify genes controlling PNS development, we screened 2000 lethal P-element insertion strains. The PNS of mutant embryos was examined using the neural specific marker MAb 22C10, and 92 mutant strains were retained for further analysis. Genetic and cytological analysis of these strains shows that 42 mutations affect previously isolated genes that are known to be required for PNS development: longitudinals lacking (19), mastermind (15), numb (4), big brain (2), and spitz (2). The remaining 50 mutations were classified into 29 complementation groups and the P-element insertions were cytologically mapped. The mutants were classified in five major classes on the basis of their phenotype: gain of neurons, loss of neurons, organizational defects, pathfinding defects and morphological defects. Herein we report the preliminary phenotypic characterization of each of these complementation groups as well as the embryonic lacZ expression pattern of each P-element strain. Our analysis indicates that in most of the P-element insertion strains, the lacZ reporter gene is not expressed in the developing PNS.     

Deletion of ku homologs increases gene targeting frequency in Streptomyces avermitilis

Streptomyces avermitilis is an industrially important soil bacterium known for production of avermectins, which are antiparasitic agents useful in animal health care, agriculture, and treatment of human infections. ku genes play a key role in the non-homologous end-joining pathway for repair of DNA double strand breaks. We identified homologs of eukaryotic ku70 and ku80 genes, termed ku1 and ku2, in S. avermitilis. Mutants with deletion of ku1, ku2, and both genes were constructed and their phenotypic changes were characterized. Deletion of ku genes had no apparent adverse effects on growth, spore formation, or avermectin production. The ku mutants, in comparison to wild-type strain, were slightly more sensitive to the DNA-damaging agent ethyl methanesulfonate, but not to UV exposure or to bleomycin. Gene targeting frequencies by homologous recombination were higher in the ku mutants than in wild-type strain. We conclude that ku-deleted strains will be useful hosts for efficient gene targeting and will facilitate functional analysis of genes in S. avermitilis and other industrially important bacterial strains.     

Genetic analysis of leaf form mutants from the Arabidopsis Information Service collection

Although a vast inventory of morphological mutants of Arabidopsis thaliana is available, only some have been used for genetic studies of leaf development. Such is the case with the Arabidopsis Information Service (AIS) Form Mutants collection, assembled by A. R. Kranz and currently stored at the Nottingham Arabidopsis Stock Centre, which includes a large number of mutant lines, most of which have been little studied. With the aim of contributing to the genetic dissection of leaf ontogeny, we have subjected 57 mutant lines isolated by others to genetic analysis; 47 of which were from the AIS collection. These are characterized by vegetative leaves of abnormal shape or size, and were chosen as candidates for mutations in genes required for leaf morphogenesis. The mutant phenotypes studied were shown to be inherited as single recessive Mendelian traits and were classified into 10 phenotypic classes. These mutant strains were found to fall into 37 complementation groups, 7 of which corresponded to known genes. Results of the phenotypic analysis and data on the genetic interactions of these mutants are presented, and their possible developmental defects discussed.     

Concordance between phenotypical features and PCR-REA for the identification of Malassezia spp

The genus Malassezia has been recently revised and nowadays includes 11 species that cannot always be differentiated from each other by physiological and morphological tests. This study was aimed to evaluate the correlation between a molecular method and conventional phenotypic features in the identification of Malassezia spp. To achieve this aim, 92 Argentinean clinical strains isolated between 2001 and 2005 were analyzed along with three reference strains (Malassezia furfur CBS 7019, Malassezia sympodialis CBS 7222 and Malassezia slooffiae CBS 7956). By using PCR and restriction enzyme analysis with three different DNA endonucleases (PCR-REA), the molecular method consistently identified all three reference strains and all 92 clinical isolates as follows: 63 M. sympodialis, 18 M. furfur, 10 Malassezia globosa and one Malassezia obtusa. Phenotypic studies undentified 85 clinical isolates and two of the reference strains (total agreement > 91%). In particular for M. sympodialis, M. furfur and M. globosa, the species more frequently involved in human pathology, the agreement ranged between 84 and 96%. This result suggests that phenotypic studies are suitable for the presumptive identification of important Malassezia species in the clinical medical mycology laboratories where molecular methodologies are not available.     

Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae. Characterization of mutants in 34 complementation groups

To identify nuclear functions required for cytochrome c oxidase biogenesis in yeast, recessive nuclear mutants that are deficient in cytochrome c oxidase were characterized. In complementation studies, 55 independently isolated mutants were placed into 34 complementation groups. Analysis of the content of cytochrome c oxidase subunits in each mutant permitted the definition of three phenotypic classes. One class contains three complementation groups whose strains carry mutations in the COX4, COX5a, or COX9 genes. These genes encode subunits IV, Va, and VIIa of cytochrome c oxidase, respectively. Mutations in each of these structural genes appear to affect the levels of the other eight subunits, albeit in different ways. A second class contains nuclear mutants that are defective in synthesis of a specific mitochondrial-encoded cytochrome c oxidase subunit (I, II, or III) or in both cytochrome c oxidase subunit I and apocytochrome b. These mutants fall into 17 complementation groups. The third class is represented by mutants in 14 complementation groups. These strains contain near normal amounts of all cytochrome c oxidase subunits examined and therefore are likely to be defective at some step in holoenzyme assembly. The large number of complementation groups represented by the second and third phenotypic classes suggest that both the expression of the structural genes encoding the nine polypeptide subunits of cytochrome c oxidase and the assembly of these subunits into a functional holoenzyme require the products of many nuclear genes.     

Genetic analysis of leaf form mutants from the Arabidopsis Information Service collection.

Although a vast inventory of morphological mutants of Arabidopsis thaliana is available, only some have been used for genetic studies of leaf development. Such is the case with the Arabidopsis Information Service (AIS) Form Mutants collection, assembled by A. R. Kranz and currently stored at the Nottingham Arabidopsis Stock Centre, which includes a large number of mutant lines, most of which have been little studied. With the aim of contributing to the genetic dissection of leaf ontogeny, we have subjected 57 mutant lines isolated by others to genetic analysis; 47 of which were from the AIS collection. These are characterized by vegetative leaves of abnormal shape or size, and were chosen as candidates for mutations in genes required for leaf morphogenesis. The mutant phenotypes studied were shown to be inherited as single recessive Mendelian traits and were classified into 10 phenotypic classes. These mutant strains were found to fall into 37 complementation groups, 7 of which corresponded to known genes. Results of the phenotypic analysis and data on the genetic interactions of these mutants are presented, and their possible developmental defects discussed. Received: 28 October 1998 / Accepted: 21 February 1999     

The genetic basis of cellular morphogenesis in the filamentous fungus Neurospora crassa

Cellular polarity is a fundamental property of every cell. Due to their extremely fast growth rate (>/=1 microm/s) and their highly elongated form, filamentous fungi represent a prime example of polarized growth and are an attractive model for the analysis of fundamental mechanisms underlying cellular polarity. To identify the critical components that contribute to polarized growth, we developed a large-scale genetic screen for the isolation of conditional mutants defective in this process in the model fungus Neurospora crassa. Phenotypic analysis and complementation tests of ca. 950 mutants identified more than 100 complementation groups that define 21 distinct morphological classes. The phenotypes include polarity defects over the whole hypha, more specific defects localized to hyphal tips or subapical regions, and defects in branch formation and growth directionality. To begin converting this mutant collection into meaningful biological information, we identified the defective genes in 45 mutants covering all phenotypic classes. These genes encode novel proteins as well as proteins which 1) regulate the actin or microtubule cytoskeleton, 2) are kinases or components of signal transduction pathways, 3) are part of the secretory pathway, or 4) have functions in cell wall formation or membrane biosynthesis. These findings highlight the dynamic nature of a fungal hypha and establish a molecular model for studies of hyphal growth and polarity.     

Phenotypic characterization of the archaebacterial genus Sulfolobus: comparison of five wild-type strains.

Though amenable to routine manipulation and a popular subject of molecular genetic and biochemical studies on archaebacteria, the genus Sulfolobus has remained poorly described in phenotypic terms. To delineate their physiological capabilities and diversity, five laboratory strains, including type strains of the described species Sulfolobus acidocaldarius and S. solfataricus, were compared with respect to a variety of growth and biochemical parameters, including component profile of the surface-layer cell wall, inhibitors of growth, growth rate as a function of temperature and pH, and compounds used as sole sources of carbon or nitrogen. Motility and photoregulated production of an orange pigment were detected in all five strains tested. The results provide new criteria for distinguishing Sulfolobus strains as well as potential tools for the physiological and genetic manipulation of these extreme thermophiles.     

Seeing is believing: imaging techniques to monitor plant health.

Historically, early stress-induced changes in plants have been mainly detected after destructive sampling followed by biochemical and molecular determinations. Imaging techniques that allow immediate detection of stress-situations, before visual symptoms appear and adverse effects become established, are emerging as promising tools for crop yield management. Such monitoring approaches can also be applied to screen plant populations for mutants with increased stress tolerance. At the laboratory scale, different imaging methods can be tested and one or a combination best suited for crop surveillance chosen. The system of choice can be applied under controlled laboratory conditions to guide selective sampling for the molecular characterisation of rapid stress-induced changes. Such an approach permits to isolate presymptomatically induced genes, or to obtain a panoramic view of early gene expression using gene-arrays when plants undergo physiological changes undetected by the human eye. Using this knowledge, plants can be engineered to be more stress resistant, and tested for field performance by the same methodologies. In ongoing efforts of genome characterisation, genes of unknown function are revealed at an ever-accelerating pace. By monitoring changes in phenotypic characteristics of transgenic plants expressing those genes, imaging techniques could help to identify their function.     

Sensitivity to lovastatin of Saccharomyces cerevisiae strains deleted for pleiotropic drug resistance (PDR) genes

The use of statins is well established in human therapy, and model organisms such as Saccharomyces cerevisiae are commonly used in studies of drug action at molecular and cellular levels. The investigation of the resistance mechanisms towards statins may suggest new approaches to improve therapy based on the use of statins. We investigated the susceptibility to lovastatin of S. cerevisiae strains deleted for PDR genes, responsible for exporting hydrophobic and amphiphilic drugs, such as lovastatin. Strains deleted for the genes tested, PDR1, PDR3, PDR5 and SNQ2, exhibited remarkably different phenotypes, with deletion of PDR5 causing the highest sensitivity to lovastatin. The study helped clarifying which pdr mutants to use in studies of physiological actions of statins in yeast.     

Genomic and phenotypic heterogeneity of Acidithiobacillus spp. strains isolated from diverse habitats in China

The genetic variability among 32 Chinese Acidithiobacillus spp. environmental isolates and four reference strains representing three recognized species of the genus Acidithiobacillus was characterized by using a combination of molecular methods, namely restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers, repetitive element PCR, arbitrarily primed PCR and 16S rRNA gene sequence analyses. 16S rRNA gene sequences revealed that all Acidithiobacillus spp. strains could be assigned to seven groups, three of which encompassed the Acidithiobacillus ferrooxidans strains from various parts of the world. A comparative analysis of the phylogenetic Group 1 and 2 was undertaken. Restriction fragment length polymorphism results allowed us to separate the 35 Acidithiobacillus strains into 15 different genotypes. An integrated phenotypic and genotypic analysis indicated that the distribution of A. ferrooxidans strains among the physiological groups were in agreement with their distribution among the genomic groups, and that no clear correlation was found between the genetic polymorphism of the Acidithiobacillus spp. strains and either the geographic location or type of habitats from which the strains were isolated. In addition, five unidentified sulfur-oxidizing isolates may represent one or two novel species of the genus Acidithiobacillus. The results showed that the Chinese Acidithiobacillus spp. isolates exhibited a high degree of genomic and phenotypic heterogeneity.     

Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum

The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum.