Single Gene Deletion Research Articles

Genome-wide screens reveal Escherichia coli genes required for growth of T1-like phage LL5 and V5-like phage LL12

The host factor requirements of phages and mechanisms of mutational phage insensitivity must be characterized for rational design of phage cocktails. To characterize host dependencies of two novel Escherichia coli phages, the T1-like siphophage LL5 and the V5-like myophage LL12, forward genetic screens were conducted against the Keio collection, a library of single non-essential gene deletions in E. coli str. BW25113. These screens and subsequent experiments identified genes required by phages LL5 and LL12. E. coli mutants deficient in heptose II and the phosphoryl substituent of heptose I of the inner core lipopolysaccharide (LPS) were unable to propagate phage LL5, as were mutants deficient in the outer membrane protein TolC. Mutants lacking glucose I of the LPS outer core failed to propagate LL12. Two additional genes encoding cytoplasmic chaperones, PpiB and SecB, were found to be required for efficient propagation of phage LL5, but not LL12. This screening approach may be useful for identifying host factors dependencies of phages, which would provide valuable information for their potential use as therapeutics and for phage engineering.

The contribution of large genomic rearrangements in BRCA1 and BRCA2 to South African familial breast cancer

BackgroundPathogenic variants that occur in the familial breast cancer genes (BRCA1/2) lead to truncated ineffective proteins in the majority of cases. These variants are mostly represented by small deletions/insertions, nonsense- and splice-site variants, although some larger pathogenic rearrangements occur. Currently, their contribution to familial breast cancer (BC) and ovarian cancer (OVC) in South Africa (SA) is unknown.MethodsSeven hundred and forty-four patients affected with BC or OVC were screened for larger genomic rearrangements (LGRs) by means of multiplex ligation-dependent probe amplification or Next Generation Sequencing using the Oncomine™ BRCA research assay.ResultsThe patients represented mostly medium to high-risk families, but also included lower risk patients without a family history of the disease, diagnosed at an early age of onset (< 40 years). Eight LGRs were detected (1.1%); seven in BRCA1 with a single whole gene deletion (WGD) detected for BRCA2. These eight LGRs accounted for 8.7% of the 92 BRCA1/2 pathogenic variants identified in the 744 cases. The pathogenic LGRs ranged from WGDs to the duplication of a single exon.ConclusionsLarger rearrangements in BRCA1/2 contributed to the overall mutational burden of familial BC and OVC in SA. Almost a quarter of all pathogenic variants in BRCA1 were LGRs (7/30, 23%). The spectrum observed included two WGDs, one each for BRCA1 and BRCA2.

Multiple cellular responses guarantee yeast survival in presence of the cell membrane/wall interfering agent sodium dodecyl sulfate

Sodium dodecyl sulfate (SDS), a representative anionic surfactant, is a commonly used reagent in studies of the cell membrane and cell wall. However, the mechanisms through which SDS affects cellular functions have not yet been fully examined. Thus, to gain further insights into the cellular functions and responses to SDS, we tested a haploid library of Saccharomyces cerevisiae single-gene deletion mutants to identify genes required for tolerance to SDS. After two rounds of screening, we found 730 sensitive and 77 resistant mutants. Among the sensitive mutants, mitochondrial gene expression; the mitogen-activated protein kinase signaling pathway; the metabolic pathways involved in glycoprotein, lipid, purine metabolic process, oxidative phosphorylation, cellular amino acid biosynthesis and pentose phosphate pathway were found to be enriched. Additionally, we identified a set of transcription factors related to SDS responses. Among the resistant mutants, disruption of ribosome biogenesis and translation alleviated SDS-induced cytotoxicity. Collectively, our results provided new insights into the mechanisms through which SDS regulates the cell membrane or cell wall.

Zfp36 family members redundantly protect against T cell-mediated autoinflammation and premature mortality

Abstract Cytokine production must be tightly regulated in order to prevent auto-inflammatory diseases. The zinc finger 36 (Zfp36) family of RNA-binding proteins, including Zfp36, Zfp36l1, and Zfp36l2, are known to negatively regulate mRNA stability or translation of many transcripts, including cytokines. Polymorphisms in ZFP36L1 and ZFP36L2 have been identified in GWAS studies of a variety of human autoimmune diseases, necessitating understanding the functions of these genes. While there are reports of all three family members controlling cytokine production from T cells, delineating the exact functions of these genes has been challenging due to spontaneous phenotypes or mortality upon global deletion of single genes and potential redundancy in their functions. To overcome this, we generated Cd4-Cre+ Zfp36fl/fl Zfp36l1fl/fl Zfp36l2fl/fl mice. Only upon triple deletion, but not individual or various paired deletions, do mice spontaneously develop an inflammatory disease characterized by early mortality and immune cell infiltration into various organs, including the central nervous system, kidneys, and liver. These mice have drastically elevated levels of many cytokines in their sera. Our findings demonstrate a novel redundancy of the Zfp36 family members in regulating T cell homeostasis and suppressing cytokine-driven inflammation. We are currently investigating the specific mechanisms and mRNA targets that contribute to this phenotype and whether disease is primarily driven by CD4+ or CD8+ T cells. Understanding the individual and redundant functions of the Zfp36 family members may lead to opportunities to target them for suppression of T cell-driven autoimmunity or for activating anti-tumor or anti-pathogen T cell responses.

Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining.

One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

Dimensionality reduction by UMAP to visualize physical and genetic interactions

Dimensionality reduction is often used to visualize complex expression profiling data. Here, we use the Uniform Manifold Approximation and Projection (UMAP) method on published transcript profiles of 1484 single gene deletions of Saccharomyces cerevisiae. Proximity in low-dimensional UMAP space identifies groups of genes that correspond to protein complexes and pathways, and finds novel protein interactions, even within well-characterized complexes. This approach is more sensitive than previous methods and should be broadly useful as additional transcriptome datasets become available for other organisms.

Suppression of antibiotic resistance evolution by single-gene deletion

Antibiotic treatment generally results in the selection of resistant bacterial strains, and the dynamics of resistance evolution is dependent on complex interactions between cellular components. To better characterize the mechanisms of antibiotic resistance and evaluate its dependence on gene regulatory networks, we performed systematic laboratory evolution of Escherichia coli strains with single-gene deletions of 173 transcription factors under three different antibiotics. This resulted in the identification of several genes whose deletion significantly suppressed resistance evolution, including arcA and gutM. Analysis of double-gene deletion strains suggested that the suppression of resistance evolution caused by arcA and gutM deletion was not caused by epistatic interactions with mutations known to confer drug resistance. These results provide a methodological basis for combinatorial drug treatments that may help to suppress the emergence of resistant pathogens by inhibiting resistance evolution.

Pulling the Hood off Genetic Susceptibility to Hypertensive Renal Disease

Pulling the Hood off Genetic Susceptibility to Hypertensive Renal Disease

Genome-Scale Metabolic Reconstruction and in Silico Perturbation Analysis of the Polar Diatom Fragilariopsis cylindrus Predicts High Metabolic Robustness.

Diatoms are major primary producers in polar environments where they can actively grow under extremely variable conditions. Integrative modeling using a genome-scale model (GSM) is a powerful approach to decipher the complex interactions between components of diatom metabolism and can provide insights into metabolic mechanisms underlying their evolutionary success in polar ecosystems. We developed the first GSM for a polar diatom, Fragilariopsis cylindrus, which enabled us to study its metabolic robustness using sensitivity analysis. We find that the predicted growth rate was robust to changes in all model parameters (i.e., cell biochemical composition) except the carbon uptake rate. Constraints on total cellular carbon buffer the effect of changes in the input parameters on reaction fluxes and growth rate. We also show that single reaction deletion of 20% to 32% of active (nonzero flux) reactions and single gene deletion of 44% to 55% of genes associated with active reactions affected the growth rate, as well as the production fluxes of total protein, lipid, carbohydrate, DNA, RNA, and pigments by less than 1%, which was due to the activation of compensatory reactions (e.g., analogous enzymes and alternative pathways) with more highly connected metabolites involved in the reactions that were robust to deletion. Interestingly, including highly divergent alleles unique for F. cylindrus increased its metabolic robustness to cellular perturbations even more. Overall, our results underscore the high robustness of metabolism in F. cylindrus, a feature that likely helps to maintain cell homeostasis under polar conditions.

Genome-wide identification for genes involved in sodium dodecyl sulfate toxicity in Saccharomyces cerevisiae

BackgroundSodium dodecyl sulfate (SDS) is one of the most widely used anionic alkyl sulfate surfactants. Toxicological information on SDS is accumulating, however, mechanisms of SDS toxicity regulation remain poorly understood. In this study, the relationship between the SDS-sensitive mutants and their intracellular ROS levels has been investigated.ResultsThrough a genome-scale screen, we have identified 108 yeast single-gene deletion mutants that are sensitive to 0.03% SDS. These genes were predominantly related to the cellular processes of metabolism, cell cycle and DNA processing, cellular transport, transport facilities and transport routes, transcription and the protein with binding function or cofactor requirement (structural or catalytic). Measurement of the intracellular ROS (reactive oxygen species) levels of these SDS-sensitive mutants showed that about 79% of SDS-sensitive mutants accumulated significantly higher intracellular ROS levels than the wild-type cells under SDS stress. Moreover, SDS could generate oxidative damage and up-regulate several antioxidant defenses genes, and some of the SDS-sensitive genes were involved in this process.ConclusionThis study provides insight on yeast genes involved in SDS tolerance and the elevated intracellular ROS caused by SDS stress, which is a potential way to understand the detoxification mechanisms of SDS by yeast cells.

Identification of the Genetic Requirements for Zinc Tolerance and Toxicity in Saccharomyces cerevisiae.

Zinc is essential for almost all living organisms, since it serves as a crucial cofactor for transcription factors and enzymes. However, it is toxic to cell growth when present in excess. The present work aims to investigate the toxicity mechanisms induced by zinc stress in yeast cells. To this end, 108 yeast single-gene deletion mutants were identified sensitive to 6 mM ZnCl2 through a genome-wide screen. These genes were predominantly related to the biological processes of vacuolar acidification and transport, polyphosphate metabolic process, cytosolic transport, the process utilizing autophagic mechanism. A result from the measurement of intracellular zinc content showed that 64 mutants accumulated higher intracellular zinc under zinc stress than the wild-type cells. We further measured the intracellular ROS (reactive oxygen species) levels of 108 zinc-sensitive mutants treated with 3 mM ZnCl2. We showed that the intracellular ROS levels in 51 mutants were increased by high zinc stress, suggesting their possible involvement in regulating ROS homeostasis in response to high zinc. The results also revealed that excess zinc could generate oxidative damage and then activate the expression of several antioxidant defenses genes. Taken together, the data obtained indicated that excess zinc toxicity might be mainly due to the high intracellular zinc levels and ROS levels induced by zinc stress in yeast cells. Our current findings would provide a basis to understand the molecular mechanisms of zinc toxicity in yeast cells.

Genetic Analysis of Four Sexual Differentiation Process Proteins (isp4/SDPs) in Chaetomium thermophilum and Thermomyces lanuginosus Reveals Their Distinct Roles in Development

Fungal sexual development requires the involvement of a large number of functional genes. Fungal genes encoding sexual differentiation process proteins (SDPs), isps, have been known for decades. isp4/SDP and its homologs function as oligopeptide transporters (OPTs), yet their roles in reproduction are unknown. Here, we genetically analyzed all four isp4/SDP homologs in the sexual species Chaetomium thermophilum and asexual species Thermomyces lanuginosus. Using single gene deletion mutants, we found that T. lanuginosus SDP (TlSDP) participated in asexual sporulation, whereas the other homologs participated in sexual morphogenesis. In complementary tests, C. thermophilum SDPs (CtSDP1-3) restored sporulation defects in TlSDP deletion strains (ΔTlSDP), and their translated proteins, which were localized onto the cytomembrane, possessed OPT activity. Interestingly, CtSDP2 accumulated at the top of the hyphae played a distinct role in determining the sexual cycle, glutathione transport, and lifespan shortening. A unique 72nt-insertion fragment (72INS) was discovered in CtSDP2. Biological analysis of the 72INS deletion and DsRED-tagged fusion strains implied the involvement of 72INS in fungal growth and development. In contrast to TlSDP, which only contributes to conidial production, the three CtSDPs play important roles in sexual and asexual reproduction, and CtSDP2 harbors a unique functional 72INS that initiates sexual morphogenesis.

Association between Different Polymorphic Markers and β-Thalassemia Intermedia in Central Iran

β-Thalassemia intermedia (β-TI) is a clinical condition characterized by moderate, non transfusional anemia and hepatosplenomegaly. The main objective of this study was to determine the molecular basis of the clinical phenotype of β-TI in Iran. To elucidate the mild phenotype of many patients with β-TI, we screened for three prevalent β-globin gene mutations [IVS-II-1 (G>A) HBB: c.315+1G>A, IVS-I-110 (G>A) HBB: c.93-21G>A and IVS-I-5 (G>C) [HBB: c.92+5G>C], deletions on the α-globin genes, XmnI polymorphisms and restriction fragment length polymorphism (RFLP) haplotypes on the β-globin gene cluster in 50 β-TI patients. Fifty-eight percent of the patients (29 cases) were associated with the mentioned mutations. We showed that the HBB: c.315+1G>A mutation is linked to haplotype [+ – + +] (57.69%). This haplotype is in linkage disequilibrium with the XmnI polymorphism (NG_000007.3: g.42677C>T) and has been associated with increased expression of Hb F in β-TI patients. The XmnI polymorphism is defined in association with this prevalent mutation. Two patients had a single α-globin gene deletion [–α3.7 (rightward) deletion]. The main genetic factor in mild phenotype β-TI patients is the linkage of an XmnI polymorphism (NG_000007.3: g.42677C>T) with the HBB: c.315+1G>A (80.76%), which is associated with increased production of Hb F and coinheritance of haplotype [+ – + +] with β-TI, especially with the homozygous HBB: c.315+1G>A mutation. Molecular basis of β-TI could be explained by the involvement of different factors that tend to develop the disease phenotype.

Pseudorabies virus (PRV) strain with defects in gE, gC, and TK genes protects piglets against an emerging PRV variant.

The prevalence of an emerging variant of the pseudorabies virus (PRV) has been causing serious losses to farmers in China. Moreover, the commercially available PRV vaccine often fails to provide thorough protection. Therefore, in this study, we generated a PRV-∆gC\\gE∆TK strain with defects in gC, gE, and TK of PRV. Compared to the parental PRV strain and the single gene deletion strains (PRV-∆gC, PRV-∆gE, and PRV-∆TK), PRV-∆gC\\gE∆TK grew slowly, and exhibited fewer and smaller plaques on swine testis (ST) cells. Furthermore, animal experiment results showed that mice that were immunized intramuscularly with PRV-∆gC\\gE∆TK, survived throughout the experiment with no observed clinical symptoms, and were completely protected against PRV challenge. Additionally, deletion of the gC, gE, and TK genes significantly alleviated viral damage in the brain. Furthermore, one-day-old weaned piglets immunized intramuscularly with PRV-∆gC\\gE∆TK elicited higher levels of gB antibodies against both the emerging PRV variant and the parental PRV, exhibited full protection against challenge with both variants, and showed neutralization capacity against PRV. These data suggest that PRV-∆gC\\gE∆TK is a promising vaccine candidate for the control of pseudorabies.

Porphyromonas gingivalis genes conferring fitness in a tobacco-rich environment.

Smokers are more likely than non-smokers to harbour Porphyromonas gingivalis, they are more susceptible to destructive periodontal disease and smokers may, ultimately, benefit from tobacco-specific preventive and treatment strategies. A Mariner transposon insertion library for P. gingivalis ATCC 33277 was exploited to define 256 genes as essential for P. gingivalis survival in a tobacco-rich environment. Genes whose products play roles in protein transport and catabolism, nicotinamide processing, protection against oxidative stress, drug resistance, and transcriptional regulation have all been identified as essential for CSE survival. Many of these tobacco-essential genes are also requisite for epithelial colonization and abscess formation, suggestive of a core stress-related P. gingivalis genome. Single-gene deletions in several of the TnSeq-implicated genes led to significantly reduced P. gingivalis fitness upon competition with the parent strain, under conditions of cigarette smoke extract-induced stress (1,000ng/ml nicotine equivalents). This study identifies, for the first time, a subset of P. gingivalis genes required for surviving the plethora of insults present in cigarette smoke. Such conditionally essential genes may delineate bacterial persistence strategies and represent novel therapeutic foci for the prevention of P. gingivalis infection and related diseases in smokers and in general.

A Small Horizontally Transferred Gene Cluster Contributes to the Sporulation of Alternaria alternata.

Horizontal gene transfer (HGT) has been identified as an important source of genomic innovation in fungi. However, how HGT drove the evolution of Alternaria alternata, a necrotrophic fungus which can be ubiquitously isolated from soil and various plants and decaying plant materials is largely known. In this study, we identified 12 protein-encoding genes that are likely acquired from lineages outside Pezizomycotina. Phylogenetic trees and approximately unbiased comparative topology tests strongly supported the evolutionary origin of these genes. According to their predicted functions, these HGT candidates are involved in nitrogen and carbohydrate metabolism. Especially, five genes of them were likely transferred as a physically linked cluster from Tremellales (Basidiomycota). Functionally knocking out the five-gene cluster in an A. alternata isolate causing citrus brown spot resulted in an 80% decrease in asexual spore production in the deletion mutant. We further knocked out each of these five genes in this cluster and the resultant single-gene deletion mutants exhibited a various degree of reduction in spore production. Except for conidiation, functions of these genes associated with vegetative growth, stress tolerance, and virulence are very limited. Our results provide new evidence that HGT has played important roles over the course of the evolution of filamentous fungi.

Relationship of Host Genetic Factors with Severe Malaria in Nigerian Children

Host genetic factors that influence the outcome of Plasmodium falciparum malaria infection are not fully understood. These include alpha-thalassemia, sickle cell disease, glucose-6-phosphate dehydrogenase (G6PD) deficiency and cytochrome b5 reductase 3 (CYB5R3) T117S and ferroportin Q248H polymorphisms. G6PD, an X-linked gene, encodes the sole enzyme in red blood cells that produces NADPH for protection from reactive oxygen species. Hemi- or homozygosity for G6PD A+ (G6PD c. 376G) is an African specific polymorphism with reduced activity (PMID:26633385) but no apparent phenotype; hemi- or homozygosity for G6PD A- (G6PD c 202A/376G) is associated with both decreased activity and increased risk for oxidant-induced hemolysis (PMID:27040960). A Malaria Genomic Epidemiology Network (MalariaGEN) study with almost 30,000 participants reported that G6PD A- increases the risk for severe malarial anemia (PMID:25261933). CYB5R3 in red blood cells transfers electrons from NADH to cytochrome b5, which in turn converts methemoglobin to hemoglobin. The African-specific CYB5R3 T117S variant, is not associated with methemoglobinemia (PMID:9048929) but may afford some protection from malarial anemia in Zambian children. A prevalent African-specific Q248H mutation of the iron exporter, ferroportin, may protect from high malarial parasitemia (PMID: 29599243). We genotyped 212 Nigerian children (age 4 months - 16 years) for these genetic factors: 117 were asymptomatic children who were studied in the community and 95 were children who were studied at the time of admission to hospital with severe malaria (severe anemia and/or cerebral malaria). We isolated DNA from dried blood spots and crude DNA lysate was used for Taqman genotyping to screen for G6PD A+, G6PD A-, FPN Q248H, sickle cell disease and CYB5R3 T117S and multiplex PCR was used to identify the 3.7 kb and 4.2 kb α-globin gene deletions. The clinical characteristics of the two groups of children are shown in Table 1. Children with severe malaria were significantly younger than the children from the community. The gene frequency of CYB5R3 T117S was 0.303, of hemoglobin S was 0.094, and of FPN Q248H was 0.062. The prevalences of hemizygous and homozygous G6PD A+ and A- were 13.9% and 16.0%, respectively. In addition, the prevalences of single and double alpha globin gene deletions were 34.7 % and 11.7%, respectively. Table 2 compares the prevalence of these genotypes in asymptomatic children versus those admitted with severe malaria. Alpha-thalassemia tended to be more prevalent in the asymptomatic children while ferroportin Q248H and G6PD A- were more prevalent in the children with severe malaria. Some genes seemed to have heterogenous associations: sickle trait and homozygous CYB5R3 T117S tended to be more prevalent in the asymptomatic children while hemoglobin SS and heterozygous T117S tended to be more prevalent in the children with severe malaria. In a logistic regression model that adjusted for age, alpha thalassemia (odds ratio 0.6, P=0.038), hemoglobin S trait (odds ratio 0.5, P=0.203), and CYB5R3 T117S homozygous state (odds ratio 0.5, P=0.211) independently decreased the risk of severe malaria, while ferroportin Q248H (odds ratio 5.1, P=0.008) and G6PD deficiency (1.6, P=0.049) independently increased the risk of severe malaria. In conclusion, our results suggest that common genetic factors affect the risk of severe malaria in children. Research to illuminate the mechanisms of these effects may enhance our understanding of the pathogenesis of severe malaria and point to novel preventive and therapeutic measures. Disclosures Gordeuk: Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Modus Therapeutics: Consultancy, Honoraria; Pfizer: Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding.

Improving l-phenylacetylcarbinol production in Saccharomyces cerevisiae by in silico aided metabolic engineering

L-Phenylacetylcarbinol (L-PAC) which is used as a precursor for the production of ephedrine and pseudoephedrine is the first reported biologically produced α-hydroxy ketone compound. l-PAC is commercially produced by the yeast Saccharomyces cerevisiae. Yeast cells transform exogenously added benzaldehyde into l-PAC by using the action of pyruvate decarboxylase (PDC) enzyme. In this work, genome-scale model and flux balance analysis were used to identify novel target genes for the enhancement of l-PAC production in yeast. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using OptGene and minimization of metabolic adjustments. Six single gene deletion strains, namely Δrpe1, Δpda1, Δadh3, Δadh1, Δzwf1 and Δpdc1, were predicted in silico and further tested in vivo by using knock-out strains cultivated semi-anaerobically on glucose and benzaldehyde as substrates. Δzwf1 mutant exhibited the highest l-PAC formation (2.48 g/L) by using 2 g/L of benzaldehyde which is equivalent to 88 % of the theoretical yield.

Complete chloroplast genomes of all six Hosta species occurring in Korea: molecular structures, comparative, and phylogenetic analyses

BackgroundThe genus Hosta is a group of economically appreciated perennial herbs consisting of approximately 25 species that is endemic to eastern Asia. Due to considerable morphological variability, the genus has been well recognized as a group with taxonomic problems. Chloroplast is a cytoplasmic organelle with its own genome, which is the most commonly used for phylogenetic and genetic diversity analyses for land plants. To understand the genomic architecture of Hosta chloroplasts and examine the level of nucleotide and size variation, we newly sequenced four (H. clausa, H. jonesii, H. minor, and H. venusta) and analyzed six Hosta species (including the four, H. capitata and H. yingeri) distributed throughout South Korea.ResultsThe average size of complete chloroplast genomes for the Hosta taxa was 156,642 bp with a maximum size difference of ~ 300 bp. The overall gene content and organization across the six Hosta were nearly identical with a few exceptions. There was a single tRNA gene deletion in H. jonesii and four genes were pseudogenized in three taxa (H. capitata, H. minor, and H. jonesii). We did not find major structural variation, but there were a minor expansion and contractions in IR region for three species (H. capitata, H. minor, and H. venusta). Sequence variations were higher in non-coding regions than in coding regions. Four genic and intergenic regions including two coding genes (psbA and ndhD) exhibited the largest sequence divergence showing potential as phylogenetic markers. We found compositional codon usage bias toward A/T at the third position. The Hosta plastomes had a comparable number of dispersed and tandem repeats (simple sequence repeats) to the ones identified in other angiosperm taxa. The phylogeny of 20 Agavoideae (Asparagaceae) taxa including the six Hosta species inferred from complete plastome data showed well resolved monophyletic clades for closely related taxa with high node supports.ConclusionsOur study provides detailed information on the chloroplast genome of the Hosta taxa. We identified nucleotide diversity hotspots and characterized types of repeats, which can be used for developing molecular markers applicable in various research area.

Metabolic Modeling of Streptococcus mutans Reveals Complex Nutrient Requirements of an Oral Pathogen.

Streptococcus mutans is a Gram-positive bacterium that thrives under acidic conditions and is a primary cause of tooth decay (dental caries). To better understand the metabolism of S. mutans on a systematic level, we manually constructed a genome-scale metabolic model of the S. mutans type strain UA159. The model, called iSMU, contains 675 reactions involving 429 metabolites and the products of 493 genes. We validated iSMU by comparing simulations with growth experiments in defined medium. The model simulations matched experimental results for 17 of 18 carbon source utilization assays and 47 of 49 nutrient depletion assays. We also simulated the effects of single gene deletions. The model's predictions agreed with 78.1% and 84.4% of the gene essentiality predictions from two experimental data sets. Our manually curated model is more accurate than S. mutans models generated from automated reconstruction pipelines and more complete than other manually curated models. We used iSMU to generate hypotheses about the S. mutans metabolic network. Subsequent genetic experiments confirmed that (i) S. mutans catabolizes sorbitol via a sorbitol-6-phosphate 2-dehydrogenase (SMU_308) and (ii) the Leloir pathway is required for growth on complex carbohydrates such as raffinose. We believe the iSMU model is an important resource for understanding the metabolism of S. mutans and guiding future experiments.IMPORTANCE Tooth decay is the most prevalent chronic disease in the United States. Decay is caused by the bacterium Streptococcus mutans, an oral pathogen that ferments sugars into tooth-destroying lactic acid. We constructed a complete metabolic model of S. mutans to systematically investigate how the bacterium grows. The model provides a valuable resource for understanding and targeting S. mutans' ability to outcompete other species in the oral microbiome.