Nonessential Genes Research Articles

Refining the balance of attenuation and immunogenicity of respiratory syncytial virus by targeted codon deoptimization of virulence genes.

ABSTRACTRespiratory syncytial virus (RSV) is the most important pathogen for lower respiratory tract illness in children for which there is no licensed vaccine. Live-attenuated RSV vaccines are the most clinically advanced in children, but achieving an optimal balance of attenuation and immunogenicity is challenging. One way to potentially retain or enhance immunogenicity of attenuated virus is to mutate virulence genes that suppress host immune responses. The NS1 and NS2 virulence genes of the RSV A2 strain were codon deoptimized according to either human or virus codon usage bias, and the resulting recombinant viruses (dNSh and dNSv, respectively) were rescued by reverse genetics. RSV dNSh exhibited the desired phenotype of reduced NS1 and NS2 expression. RSV dNSh was attenuated in BEAS-2B and primary differentiated airway epithelial cells but not in HEp-2 or Vero cells. In BALB/c mice, RSV dNSh exhibited a lower viral load than did A2, and yet it induced slightly higher levels of RSV-neutralizing antibodies than did A2. RSV A2 and RSV dNSh induced equivalent protection against challenge strains A/1997/12-35 and A2-line19F. RSV dNSh caused less STAT2 degradation and less NF-κB activation than did A2 in vitro. Serial passage of RSV dNSh in BEAS-2B cells did not result in mutations in the deoptimized sequences. Taken together, RSV dNSh was moderately attenuated, more immunogenic, and equally protective compared to wild-type RSV and genetically stable.

Signs of neutralization in a redundant gene involved in homologous recombination in Wolbachia endosymbionts.

Genomic reduction in bacterial endosymbionts occurs through large genomic deletions and long-term accumulation of mutations. The latter process involves successive steps including gene neutralization, pseudogenization, and gradual erosion until complete loss. Although many examples of pseudogenes at various levels of degradation have been reported, neutralization cases are scarce because of the transient nature of the process. Gene neutralization may occur due to relaxation of selection in nonessential genes, for example, those involved in redundant functions. Here, we report an example of gene neutralization in the homologous recombination (HR) pathway of Wolbachia, a bacterial endosymbiont of arthropods and nematodes. The HR pathway is often depleted in endosymbiont genomes, but it is apparently intact in some Wolbachia strains. Analysis of 12 major HR genes showed that they have been globally under strong purifying selection during the evolution of Wolbachia strains hosted by arthropods, supporting the evolutionary importance of the HR pathway for these Wolbachia genomes. However, we detected signs of recent neutralization of the ruvA gene in a subset of Wolbachia strains, which might be related to an ancestral, clade-specific amino acid change that impaired DNA-binding activity. Strikingly, RuvA is part of the RuvAB complex involved in branch migration, whose function overlaps with the RecG helicase. Although ruvA is experiencing neutralization, recG is under strong purifying selection. Thus, our high phylogenetic resolution suggests that we identified a rare example of targeted neutralization of a gene involved in a redundant function in an endosymbiont genome.

Protein Subcellular Localization Feature of Essential/Nonessential Genes in 28 Prokaryotes

This study aimed to pursue the correlation between essential/nonessential gene and protein subcellular localization. The protein sequences of the essential/nonessential genes of 28 prokaryotes in Database of Essential Genes were analyzed by PSORTb3.0. Results show that proteins of essential genes locate in cytoplasm with relatively high percentage, i.e., in the range of 40% to 55%. Percentages of the proteins of essential genes locate in cytoplasma membrane are lower than that of nonessential genes, which mostly are about 15%. However, the values of proteins of nonessential genes are mostly about 20%, and that of Gram-positive bacteria are close to 30%. The distributions of protein subcellular localization of the essential/nonessential genes are different evidently. This could be used for classification of essential and nonessential genes.

Analysis and identification of essential genes in humans using topological properties and biological information

Genes that are indispensable for survival are termed essential genes. The analysis and identification of essential genes are very important for understanding the minimal requirements of cellular survival and for practical purposes. Proteins do not exert their function in isolation of one another but rather interact together in PPI networks. A global analysis of protein interaction networks provides an effective way to elucidate the relationships between proteins. With the recent large-scale identifications of essential genes and the production of large amounts of PPIs in humans, we are able to investigate the topological properties and biological properties of essential genes. However, until recently, no one has ever investigated human essential genes using topological and biological properties. In this study, for the first time, 28 topological properties and 22 biological properties were used to investigate the characteristics of essential and non-essential genes in humans. Most of the properties were statistically discriminative between essential and non-essential genes. The F-score was used to estimate the essentiality of each property. The GO-enrichment analysis was performed to investigate the functions of the essential and non-essential genes. Finally, based on the topological features and the biological characteristics, a machine-learning classifier was constructed to predict the essential genes. The results of the jackknife test and 10-fold cross validation test are encouraging, indicating that our classifier is an effective human essential gene discovery method.

Protein localization analysis of essential genes in prokaryotes.

Essential genes, those critical for the survival of an organism under certain conditions, play a significant role in pharmaceutics and synthetic biology. Knowledge of protein localization is invaluable for understanding their function as well as the interaction of different proteins. However, systematical examination of essential genes from the aspect of the localizations of proteins they encode has not been explored before. Here, a comprehensive protein localization analysis of essential genes in 27 prokaryotes including 24 bacteria, 2 mycoplasmas and 1 archaeon has been performed. Both statistical analysis of localization information in these genomes and GO (Gene Ontology) terms enriched in the essential genes show that proteins encoded by essential genes are enriched in internal location sites, while exist in cell envelope with a lower proportion compared with non-essential ones. Meanwhile, there are few essential proteins in the external subcellular location sites such as flagellum and fimbrium, and proteins encoded by non-essential genes tend to have diverse localizations. These results would provide further insights into the understanding of fundamental functions needed to support a cellular life and improve gene essentiality prediction by taking the protein localization and enriched GO terms into consideration.

Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network.

Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

Mechanisms of plasmid segregation: Have multicopy plasmids been overlooked?

Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

Linking genetics to structural biology: complex heterozygosity screening with actin alanine scan alleles identifies functionally related surfaces on yeast actin.

Previous genome-level genetic interaction screens with the single essential actin gene of yeast identified 238 nonessential genes that upon deletion result in deleterious, digenic complex haploinsufficiences with an actin null allele. Deletion alleles of these 238 genes were tested for complex heterozygous interactions with 32 actin alanine scan alleles, which target clusters of residues on the surface of actin. A total of 891 deleterious digenic combinations were identified with 203 of the 238 genes. Two-dimensional hierarchical cluster analysis of the interactions identified nine distinct groups, and the alleles within clusters tended to affect localized regions on the surface of actin. The mutants in one cluster all affect electrostatic interactions between stacked subunits in the long pitch helix of the actin filament. A second cluster that contains the most highly interactive alleles may disrupt the tropomyosin/myosin system, as one of the mutants in that cluster cannot support Type V myosin-dependent movement of secretory vesicles in haploids and causes processivity defects in heterozygous diploids. These examples suggest the clusters represent mutations with shared protein−protein interaction defects. These results show that complex heterozygous interaction screens have benefit for detecting actin-related genes and suggest that having actin filaments of mixed composition, containing both mutant and wild-type subunits, presents unique challenges to the cell.

ECM stiffness regulates glial migration in Drosophila and mammalian glioma models.

Cell migration is an important feature of glial cells. Here, we used the Drosophila eye disc to decipher the molecular network controlling glial migration. We stimulated glial motility by pan-glial PDGF receptor (PVR) activation and identified several genes acting downstream of PVR. Drosophila lox is a non-essential gene encoding a secreted protein that stiffens the extracellular matrix (ECM). Glial-specific knockdown of Integrin results in ECM softening. Moreover, we show that lox expression is regulated by Integrin signaling and vice versa, suggesting that a positive-feedback loop ensures a rigid ECM in the vicinity of migrating cells. The general implication of this model was tested in a mammalian glioma model, where a Lox-specific inhibitor unraveled a clear impact of ECM rigidity in glioma cell migration.

Measuring error rates in genomic perturbation screens: gold standards for human functional genomics

Technological advancement has opened the door to systematic genetics in mammalian cells. Genome-scale loss-of-function screens can assay fitness defects induced by partial gene knockdown, using RNA interference, or complete gene knockout, using new CRISPR techniques. These screens can reveal the basic blueprint required for cellular proliferation. Moreover, comparing healthy to cancerous tissue can uncover genes that are essential only in the tumor; these genes are targets for the development of specific anticancer therapies. Unfortunately, progress in this field has been hampered by off-target effects of perturbation reagents and poorly quantified error rates in large-scale screens. To improve the quality of information derived from these screens, and to provide a framework for understanding the capabilities and limitations of CRISPR technology, we derive gold-standard reference sets of essential and nonessential genes, and provide a Bayesian classifier of gene essentiality that outperforms current methods on both RNAi and CRISPR screens. Our results indicate that CRISPR technology is more sensitive than RNAi and that both techniques have nontrivial false discovery rates that can be mitigated by rigorous analytical methods.

Genes found essential in other mycoplasmas are dispensable in Mycoplasma bovis.

Mycoplasmas are regarded to be useful models for studying the minimum genetic complement required for independent survival of an organism. Mycoplasma bovis is a globally distributed pathogen causing pneumonia, mastitis, arthritis, otitis media and reproductive tract disease, and genome sequences of three strains, the type strain PG45 and two strains isolated in China, have been published. In this study, several Tn4001 based transposon constructs were generated and used to create a M. bovis PG45 insertional mutant library. Direct genome sequencing of 319 independent insertions detected disruptions in 129 genes in M. bovis, 48 of which had homologues in Mycoplasma mycoides subspecies mycoides SC and 99 of which had homologues in Mycoplasma agalactiae. Sixteen genes found to be essential in previous studies on other mycoplasma species were found to be dispensable. Five of these genes have previously been predicted to be part of the core set of 153 essential genes in mycoplasmas. Thus this study has extended the list of non-essential genes of mycoplasmas from that previously generated by studies in other species.

Genome evolution in yeast reveals connections between rare mutations in human cancers.

Cancer cells are riddled with mutations. Less than one percent of these are thought to be mutations that drive cancer phenotypes. However, a recent study conducted on the yeast knockout collections by Teng et al. [Mol. Cell (2013) 52: 485-494] provides hard evidence that single gene deletions/mutations in most non-essential genes can drive the selection for cancer-like mutations.

A Strategy to Identify Dominant Point Mutant Modifiers of a Quantitative Trait

A central goal in the analysis of complex traits is to identify genes that modify a phenotype. Modifiers of a cancer phenotype may act either intrinsically or extrinsically on the salient cell lineage. Germline point mutagenesis by ethylnitrosourea can provide alleles for a gene of interest that include loss-, gain-, or alteration-of-function. Unlike strain polymorphisms, point mutations with heterozygous quantitative phenotypes are detectable in both essential and nonessential genes and are unlinked from other variants that might confound their identification and analysis. This report analyzes strategies seeking quantitative mutational modifiers of ApcMin in the mouse. To identify a quantitative modifier of a phenotype of interest, a cluster of test progeny is needed. The cluster size can be increased as necessary for statistical significance if the founder is a male whose sperm is cryopreserved. A second critical element in this identification is a mapping panel free of polymorphic modifiers of the phenotype, to enable low-resolution mapping followed by targeted resequencing to identify the causative mutation. Here, we describe the development of a panel of six “isogenic mapping partner lines” for C57BL/6J, carrying single-nucleotide markers introduced by mutagenesis. One such derivative, B6.SNVg, shown to be phenotypically neutral in combination with ApcMin, is an appropriate mapping partner to locate induced mutant modifiers of the ApcMin phenotype. The evolved strategy can complement four current major initiatives in the genetic analysis of complex systems: the Genome-wide Association Study; the Collaborative Cross; the Knockout Mouse Project; and The Cancer Genome Atlas.

A Chromatin-Based Mechanism for Limiting Divergent Noncoding Transcription

In addition to their annotated transcript, many eukaryotic mRNA promoters produce divergent noncoding transcripts. To define determinants of divergent promoter directionality, we used genomic replacement experiments. Sequences within noncoding transcripts specified their degradation pathways, and functional protein-coding transcripts could be produced in the divergent direction. To screen for mutants affecting the ratio of transcription in each direction, a bidirectional fluorescent protein reporter construct was introduced into the yeast nonessential gene deletion collection. We identified chromatin assembly as an important regulator of divergent transcription. Mutations in the CAF-I complex caused genome-wide derepression of nascent divergent noncoding transcription. In opposition to the CAF-I chromatin assembly pathway, H3K56 hyperacetylation, together with the nucleosome remodeler SWI/SNF, facilitated divergent transcription by promoting rapid nucleosome turnover. We propose that these chromatin-mediated effects control divergent transcription initiation, complementing downstream pathways linked to early termination and degradation of the noncoding RNAs.

Enhanced specialized transduction using recombineering in Mycobacterium tuberculosis.

ABSTRACTGenetic engineering has contributed greatly to our understanding of Mycobacterium tuberculosis biology and has facilitated antimycobacterial and vaccine development. However, methods to generate M. tuberculosis deletion mutants remain labor-intensive and relatively inefficient. Here, methods are described that significantly enhance the efficiency (greater than 100-fold) of recovering deletion mutants by the expression of mycobacteriophage recombineering functions during the course of infection with specialized transducing phages delivering allelic exchange substrates. This system has been successfully applied to the CDC1551 strain of M. tuberculosis, as well as to a ΔrecD mutant generated in the CDC1551 parental strain. The latter studies were undertaken as there were precedents in both the Escherichia coli literature and mycobacterial literature for enhancement of homologous recombination in strains lacking RecD. In combination, these measures yielded a dramatic increase in the recovery of deletion mutants and are expected to facilitate construction of a comprehensive library of mutants with every nonessential gene of M. tuberculosis deleted. The findings also open up the potential for sophisticated genetic screens, such as synthetic lethal analyses, which have so far not been feasible for the slow-growing mycobacteria.

A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium tetraurelia.

In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction. These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia.

Development of an engineered bioluminescent reporter phage for the sensitive detection of viable Salmonella typhimurium.

Because foodborne illnesses continuously threaten public health, rapid and sensitive detection of pathogens in food has become an important issue. As an alternative to time-consuming and laborious conventional detection methods, a technique using recombinant reporter phages has been developed. Here, we developed an advanced bioluminescent reporter phage SPC32H-CDABE by inserting a bacterial luxCDABE operon into the Salmonella temperate phage SPC32H genome. Whole SPC32H genome sequencing enabled the selection of nonessential genes, which can be replaced with approximately 6-kb luxCDABE operon, which provides both luciferase (LuxAB) and its substrate, fatty aldehyde, as generated by fatty acid reductase (LuxCDE). Thus, the SPC32H-CDABE detection assay is simpler and more efficient compared to the luxAB-based assay because the substrate addition step is excluded. At least 20 CFU/mL of pure S. Typhimurium culture was detectable using SPC32H-CDABE within 2 h, and the signals increased proportionally to the number of cells contaminated in lettuce, sliced pork, and milk. These results thereby demonstrate that this phage successfully detects live Salmonella without appreciable interference from food components. Furthermore, the presented data suggest that SPC32H-CDABE represents a promising easy-to-use diagnostic tool for the detection of Salmonella contamination in food.

Prioritizing drug targets in Clostridium botulinum with a computational systems biology approach

A computational and in silico system level framework was developed to identify and prioritize the antibacterial drug targets in Clostridium botulinum (Clb), the causative agent of flaccid paralysis in humans that can be fatal in 5 to 10% of cases. This disease is difficult to control due to the emergence of drug-resistant pathogenic strains and the only available treatment antitoxin which can target the neurotoxin at the extracellular level and cannot reverse the paralysis. This study framework is based on comprehensive systems-scale analysis of genomic sequence homology and phylogenetic relationships among Clostridium, other infectious bacteria, host and human gut flora. First, the entire 2628-annotated genes of this bacterial genome were categorized into essential, non-essential and virulence genes. The results obtained showed that 39% of essential proteins that functionally interact with virulence proteins were identified, which could be a key to new interventions that may kill the bacteria and minimize the host damage caused by the virulence factors. Second, a comprehensive comparative COGs and blast sequence analysis of these proteins and host proteins to minimize the risks of side effects was carried out. This revealed that 47% of a set of C. botulinum proteins were evolutionary related with Homo sapiens proteins to sort out the non-human homologs. Third, orthology analysis with other infectious bacteria to assess broad-spectrum effects was executed and COGs were mostly found in Clostridia, Bacilli (Firmicutes), and in alpha and beta Proteobacteria. Fourth, a comparative phylogenetic analysis was performed with human microbiota to filter out drug targets that may also affect human gut flora. This reduced the list of candidate proteins down to 131. Finally, the role of these putative drug targets in clostridial biological pathways was studied while subcellular localization of these candidate proteins in bacterial cellular system exhibited that 68% of the proteins were located in the cytoplasm, out of which 6% was virulent. Finally, this framework may serve as a general computational strategy for future drug target identification in infectious diseases.

Genetic dissection of the mamAB and mms6 operons reveals a gene set essential for magnetosome biogenesis in Magnetospirillum gryphiswaldense.

Biosynthesis of bacterial magnetosomes, which are intracellular membrane-enclosed, nanosized magnetic crystals, is controlled by a set of >30 specific genes. In Magnetospirillum gryphiswaldense, these are clustered mostly within a large conserved genomic magnetosome island (MAI) comprising the mms6, mamGFDC, mamAB, and mamXY operons. Here, we demonstrate that the five previously uncharacterized genes of the mms6 operon have crucial functions in the regulation of magnetosome biomineralization that partially overlap MamF and other proteins encoded by the adjacent mamGFDC operon. While all other deletions resulted in size reduction, elimination of either mms36 or mms48 caused the synthesis of magnetite crystals larger than those in the wild type (WT). Whereas the mms6 operon encodes accessory factors for crystal maturation, the large mamAB operon contains several essential and nonessential genes involved in various other steps of magnetosome biosynthesis, as shown by single deletions of all mamAB genes. While single deletions of mamL, -P, -Q, -R, -B, -S, -T, and -U showed phenotypes similar to those of their orthologs in a previous study in the related M. magneticum, we found mamI and mamN to be not required for at least rudimentary iron biomineralization in M. gryphiswaldense. Thus, only mamE, -L, -M, -O, -Q, and -B were essential for formation of magnetite, whereas a mamI mutant still biomineralized tiny particles which, however, consisted of the nonmagnetic iron oxide hematite, as shown by high-resolution transmission electron microscopy (HRTEM) and the X-ray absorption near-edge structure (XANES). Based on this and previous studies, we propose an extended model for magnetosome biosynthesis in M. gryphiswaldense.

Characterization of essential genes by topological properties in the perturbation sensitivity network

Genes that are indispensable for survival are called essential genes. In recent years, the analysis of essential genes has become extremely important for understanding the way a cell functions. With the advent of large-scale gene expression profiling technologies, it is now possible to profile transcriptional changes in the entire genome of Saccharomyces cerevisiae. Notwithstanding the accumulation of gene expression profiling in recent years, only a few studies have used these data to construct the network for S. cerevisiae. In this paper, based on the transcriptional profiling of the S. cerevisiae genome in hundreds of different gene disruptions, the perturbation sensitivity (PS) network is constructed. A scale-free topology with node degree following a power-law distribution is shown in the PS network. Twelve topological properties are used to investigate the characteristics of essential and non-essential genes in the PS network. Most of the properties are found to be statistically discriminative between essential and non-essential genes. In addition, the F-score is used to estimate the essentiality of each property, and the core number demonstrates the highest F-score among all properties.