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Nature Methods Oct 2009Biological pathways are structured in complex networks of interacting genes. Solving the architecture of such networks may provide valuable information, such as how...
Biological pathways are structured in complex networks of interacting genes. Solving the architecture of such networks may provide valuable information, such as how microorganisms cause disease. Here we present a method (Tn-seq) for accurately determining quantitative genetic interactions on a genome-wide scale in microorganisms. Tn-seq is based on the assembly of a saturated Mariner transposon insertion library. After library selection, changes in frequency of each insertion mutant are determined by sequencing the flanking regions en masse. These changes are used to calculate each mutant's fitness. Using this approach, we determined fitness for each gene of Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. A genome-wide screen for genetic interactions of five query genes identified both alleviating and aggravating interactions that could be divided into seven distinct categories. Owing to the wide activity of the Mariner transposon, Tn-seq has the potential to contribute to the exploration of complex pathways across many different species.
Topics: Chromosome Mapping; Computer Simulation; DNA, Bacterial; Models, Genetic; Proteome; Sequence Analysis, DNA; Signal Transduction; Streptococcus pneumoniae
PubMed: 19767758
DOI: 10.1038/nmeth.1377 -
International Journal of Molecular... Oct 2022Mutation is a source of genetic diversity widely used in breeding programs for the acquisition of agronomically interesting characters in commercial varieties of the... (Review)
Review
Mutation is a source of genetic diversity widely used in breeding programs for the acquisition of agronomically interesting characters in commercial varieties of the species, as well as in the rest of crop species. Mutation can occur in nature at a very low frequency or can be induced artificially. Spontaneous or bud sport mutations in somatic cells can be vegetatively propagated to get an individual with the mutant phenotype. Unlike animals, plants have unlimited growth and totipotent cells that let somatic mutations to be transmitted to the progeny. On the other hand, in vitro tissue culture makes it possible to induce mutation in plant material and perform large screenings for mutant's selection and cleaning of chimeras. Finally, targeted mutagenesis has been boosted by the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 and Transcription activator-like effector nuclease (TALEN) editing technologies. Over the last few decades, environmental stressors such as global warming have been threatening the supply of global demand for food based on population growth in the near future. For this purpose, the release of new varieties adapted to such changes is a requisite, and selected or generated mutants by properly regulated mechanisms could be helpful to this task. In this work, we reviewed the most relevant mutations for breeding traits in species such as flowering time, self-compatibility, fruit quality, and disease tolerance, including new molecular perspectives in the present postgenomic era including CRISPR/Cas9 and TALEN editing technologies.
Topics: Animals; Gene Editing; CRISPR-Cas Systems; Transcription Activator-Like Effector Nucleases; Prunus; Plant Breeding; Mutation; Endonucleases; Genome, Plant
PubMed: 36362061
DOI: 10.3390/ijms232113273 -
Journal of the American Society of... Jun 2022C3 glomerulopathy (C3G) is a heterogeneous group of chronic renal diseases characterized predominantly by glomerular C3 deposition and complement dysregulation....
BACKGROUND
C3 glomerulopathy (C3G) is a heterogeneous group of chronic renal diseases characterized predominantly by glomerular C3 deposition and complement dysregulation. Mutations in factor H-related (FHR) proteins resulting in duplicated dimerization domains are prototypical of C3G, although the underlying pathogenic mechanism is unclear.
METHODS
Using and assays, we performed extensive characterization of an FHR-1 mutant with a duplicated dimerization domain. To assess the FHR-1 mutant's association with disease susceptibility and renal prognosis, we also analyzed copy number variations and FHR-1 plasma levels in two Spanish C3G cohorts and in a control population.
RESULTS
Duplication of the dimerization domain conferred FHR-1 with an increased capacity to interact with C3-opsonized surfaces, which resulted in an excessive activation of the alternative pathway. This activation does not involve C3b binding competition with factor H. These findings support a scenario in which mutant FHR-1 binds to C3-activated fragments and recruits native C3 and C3b; this leads to formation of alternative pathway C3 convertases, which increases deposition of C3b molecules, overcoming FH regulation. This suggests that a balanced FHR-1/FH ratio is crucial to control complement amplification on opsonized surfaces. Consistent with this conceptual framework, we show that the genetic deficiency of FHR-1 or decreased FHR-1 in plasma confers protection against developing C3G and associates with better renal outcome.
CONCLUSIONS
Our findings explain how FHR-1 mutants with duplicated dimerization domains result in predisposition to C3G. They also provide a pathogenic mechanism that may be shared by other diseases, such as IgA nephropathy or age-related macular degeneration, and identify FHR-1 as a potential novel therapeutic target in C3G.
Topics: Blood Proteins; Complement C3; Complement C3b Inactivator Proteins; Complement Factor H; DNA Copy Number Variations; Disease Susceptibility; Glomerulonephritis, IGA; Humans; Prognosis
PubMed: 35545301
DOI: 10.1681/ASN.2021101318 -
Biomolecules May 2022The human (h) transporter hZIP4 is the primary Zn importer in the intestine. hZIP4 is also expressed in a variety of organs such as the pancreas and brain. Dysfunction...
The human (h) transporter hZIP4 is the primary Zn importer in the intestine. hZIP4 is also expressed in a variety of organs such as the pancreas and brain. Dysfunction of hZIP4 can result in the Zn deficiency disease acrodermatitis enteropathica (AE). AE can disrupt digestive and immune system homeostasis. A limited number of hZIP4 expression strategies have hindered increasing knowledge about this essential transmembrane protein. Here, we report the heterologous expression of hZIP4 in . Both a wild-type and a mutant strain, in which the endogenous Zn transporters were deleted, were used to test the expression and localization of an hZIP4-GFP fusion protein. A full-length hZIP4-GFP and a truncated membrane-domain-only (mhZIP4-GFP) protein were observed to be present in the plasma membrane in yeast.
Topics: Acrodermatitis; Carrier Proteins; Cation Transport Proteins; Humans; Saccharomyces cerevisiae; Zinc
PubMed: 35625653
DOI: 10.3390/biom12050726 -
Frontiers in Plant Science 2020Gametophytic mutants share very small proportion of the total mutants generated by any mutagenic approach; even rarer are the fertilization-defective gametophytic... (Review)
Review
Gametophytic mutants share very small proportion of the total mutants generated by any mutagenic approach; even rarer are the fertilization-defective gametophytic mutants. They require an efficient and targeted strategy instead of 'brute force' screening approach. The classical gametophyte mutant screening method, mainly based on the segregation distortion, can distinguish gametophytic mutants from the others. However, the mutants pooled after the screening constitute both fertilization-defective and developmental-defective gametophytic mutants. Until recently, there has not been any straightforward way to screen the former from the latter. Additionally, most of the mutations affecting both gametes are lost during the screening process. The novel gametophyte screening approach tends to circumvent those shortcomings. This review discusses on the classical approach of gametophytic mutant screening and focuses on the novel approach on distinguishing fertilization-/developmental-defective gametophytic mutants (both male and female). It offers an empirical basis of screening such mutants by taking in the consideration of earlier studies on fertilization failure, initiation of seed coat formation, and fertilization recovery system in plants.
PubMed: 32714355
DOI: 10.3389/fpls.2020.00967 -
PLoS Pathogens May 2022Ebola virus (EBOV) VP35 is a polyfunctional protein involved in viral genome packaging, viral polymerase function, and host immune antagonism. The mechanisms regulating...
Ebola virus (EBOV) VP35 is a polyfunctional protein involved in viral genome packaging, viral polymerase function, and host immune antagonism. The mechanisms regulating VP35's engagement in different functions are not well-understood. We previously showed that the host E3 ubiquitin ligase TRIM6 ubiquitinates VP35 at lysine 309 (K309) to facilitate virus replication. However, how K309 ubiquitination regulates the function of VP35 as the viral polymerase co-factor and the precise stage(s) of the EBOV replication cycle that require VP35 ubiquitination are not known. Here, we generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35/K309 (rEBOV-VP35/K309G/-R) and show that both mutations prohibit VP35/K309 ubiquitination. The K309R mutant retains dsRNA binding and efficient type-I Interferon (IFN-I) antagonism due to the basic residue conservation. The rEBOV-VP35/K309G mutant loses the ability to efficiently antagonize the IFN-I response, while the rEBOV-VP35/K309R mutant's suppression is enhanced. The replication of both mutants was significantly attenuated in both IFN-competent and -deficient cells due to impaired interactions with the viral polymerase. The lack of ubiquitination on VP35/K309 or TRIM6 deficiency disrupts viral transcription with increasing severity along the transcriptional gradient. This disruption of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. In addition, lack of ubiquitination on K309 results in enhanced interactions with the viral nucleoprotein and premature nucleocapsid packaging, leading to dysregulation of virus assembly. Overall, we identified a novel role of VP35 ubiquitination in coordinating viral transcription and assembly.
Topics: Ebolavirus; Hemorrhagic Fever, Ebola; Humans; Lysine; Nucleocapsid Proteins; Ubiquitination; Viral Regulatory and Accessory Proteins; Viral Transcription
PubMed: 35533195
DOI: 10.1371/journal.ppat.1010532 -
Trends in Plant Science Jan 2022Plants have evolved complex defence mechanisms to avoid invasion of potential pathogens. Despite this, adapted pathogens deploy effector proteins to manipulate host... (Review)
Review
Plants have evolved complex defence mechanisms to avoid invasion of potential pathogens. Despite this, adapted pathogens deploy effector proteins to manipulate host susceptibility (S) genes, rendering plant defences ineffective. The identification and mutation of plant S genes exploited by bacterial pathogens are important for the generation of crops with durable and broad-spectrum resistance. Application of mutant S genes in the breeding of resistant crops is limited because of potential pleiotropy. New genome editing techniques open up new possibilities for the modification of S genes. In this review, we focus on S genes manipulated by bacteria and propose ways for their identification and precise modification. Finally, we propose that genes coding for transporter proteins represent a new group of S genes.
Topics: Bacteria; CRISPR-Cas Systems; Crops, Agricultural; Disease Resistance; Genome, Plant; Plant Breeding; Plant Diseases; Plants, Genetically Modified
PubMed: 34400073
DOI: 10.1016/j.tplants.2021.07.018 -
Journal of Bacteriology Jul 2022The symbiont Vibrio fischeri uses motility to colonize its host. In numerous bacterial species, motility is negatively controlled by cyclic-di-GMP (c-di-GMP), which is...
The symbiont Vibrio fischeri uses motility to colonize its host. In numerous bacterial species, motility is negatively controlled by cyclic-di-GMP (c-di-GMP), which is produced by diguanylate cyclases (DGCs) with GGDEF domains and degraded by phosphodiesterases with either EAL or HD-GYP domains. To begin to decode the functions of the 50 Vibrio fischeri genes with GGDEF, EAL, and/or HD-GYP domains, we deleted each gene and assessed each mutant's migration through tryptone broth salt (TBS) soft agar medium containing or lacking magnesium (Mg) and calcium (Ca), which are known to influence V. fischeri motility. We identified 6, 13, and 16 mutants with altered migration in TBS-Mg, TBS, and TBS-Ca soft agar, respectively, a result that underscores the importance of medium conditions in assessing gene function. A biosensor-based assay revealed that Mg and Ca affected c-di-GMP levels negatively and positively, respectively; the severe decrease in c-di-GMP caused by Mg addition correlates with its strong positive impact on bacterial migration. A mutant defective for , a homolog of V. cholerae , exhibited a severe defect in migration across all conditions. Motility of a double mutant was also severely defective and could be restored by expression of "c-di-GMP-blind" alleles of master flagellar regulator . Together, this work sheds light on the genes and conditions that influence c-di-GMP-mediated control over motility in V. fischeri and provides a foundation for (i) assessing roles of putative c-di-GMP-binding proteins, (ii) evaluating other c-di-GMP-dependent phenotypes in V. fischeri, (iii) uncovering potential redundancy, and (iv) deciphering signal transduction mechanisms. Critical bacterial processes, including motility, are influenced by c-di-GMP, which is controlled by environment-responsive synthetic and degradative enzymes. Because bacteria such as Vibrio fischeri use motility to colonize their hosts, understanding the roles of c-di-GMP-modulating enzymes in controlling motility has the potential to inform on microbe-host interactions. We leveraged recent advances in genetic manipulation to generate 50 mutants defective for putative c-di-GMP synthetic and degradative enzymes. We then assessed the consequences on motility, manipulating levels of magnesium and calcium, which inversely influenced motility and levels of c-di-GMP. Distinct subsets of the 50 genes were required under the different conditions. Our data thus provide needed insight into the functions of these enzymes and environmental factors that influence them.
Topics: Agar; Aliivibrio fischeri; Bacterial Proteins; Biofilms; Calcium; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Magnesium; Vibrio cholerae
PubMed: 35758751
DOI: 10.1128/jb.00109-22 -
Frontiers in Microbiology 2022and species oxidize methanol pyrroloquinoline quinone-methanol dehydrogenases (MDHs). MDHs can be classified into two major groups, Ca-dependent MDH (MxaF) and...
and species oxidize methanol pyrroloquinoline quinone-methanol dehydrogenases (MDHs). MDHs can be classified into two major groups, Ca-dependent MDH (MxaF) and lanthanide (Ln)-dependent MDH (XoxF), whose expression is regulated by the availability of Ln. A set of a siderophore, TonB-dependent receptor, and an ABC transporter that resembles the machinery for iron uptake is involved in the solubilization and transport of Ln. The transport of Ln into the cytosol enhances XoxF expression. A unique protein named lanmodulin from strain AM1 was identified as a specific Ln-binding protein, and its biological function was implicated to be an Ln shuttle in the periplasm. In contrast, it remains unclear how Ln levels in the cells are maintained, because Ln is potentially deleterious to cellular systems due to its strong affinity to phosphate ions. In this study, we investigated the function of a lanmodulin homolog in strain 22A. The expression of a gene encoding lanmodulin () was induced in response to the presence of La. A recombinant LanM underwent conformational change upon La binding. Phenotypic analyses on deletion mutant and overexpressing strains showed that LanM is not necessary for the wild-type and XoxF-dependent mutant's methylotrophic growth. We found that expression was regulated by MxcQE (a two-component regulator for MxaF) and TonB_Ln (a TonB-dependent receptor for Ln). The expression level of was altered to be negatively dependent on Ln concentration in ∆ whereas it was constant in the wild type. Furthermore, when exposed to La, ∆ showed an aggregating phenotype, cell membrane impairment, La deposition in the periplasm evidenced by electron microscopy, differential expression of proteins involved in membrane integrity and phosphate starvation, and possibly lower La content in the membrane vesicle (MV) fractions. Taken together, we concluded that lanmodulin is involved in the complex regulation mechanism of MDHs and homeostasis of cellular Ln levels by facilitating transport and MV-mediated excretion.
PubMed: 35814700
DOI: 10.3389/fmicb.2022.921636 -
Biomolecules Nov 2020The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research,... (Review)
Review
The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β', and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, mutants lacking (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ' subassembly. We observed that the interaction between α2ββ' and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in . We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code's position in the reading frame of plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ'. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β' subunit, and it is the same for both types of mutants.
Topics: Bacterial Proteins; DNA-Directed RNA Polymerases; Mutant Proteins; Protein Subunits; Structure-Activity Relationship; Transcription Factors
PubMed: 33238579
DOI: 10.3390/biom10111588