Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

Genomic analyses have revealed how the gut microbiota impacts human health. However, knowledge about the physiology of most gut commensals is largely lacking. Here, we sorted cells from a pooled library to construct an ordered collection of transposon-insertion mutants in the model commensal Bacteroides thetaiotaomicron. We applied a pooling strategy with barcode sequencing to locate mutants and created a condensed collection with single insertions in 2,565 genes. This effort enabled the development of an accurate model for progenitor-collection assembly, which identified strain-abundance biases and multi-insertion strains as important factors that limit coverage. To demonstrate the potential for phenotypic screening, we analyzed growth dynamics and morphology of the condensed collection and identified growth defects and altered cell shape in the sphingolipid-synthesis gene BT0870 and the thiamine-scavenging gene BT2397. Analyses of this collection and utilization of the platform described herein to construct future ordered libraries will increase understanding of gut commensal physiology and colonization strategies.

Identification of an Exopolysaccharide Biosynthesis Gene in Bradyrhizobium diazoefficiens USDA110

Exopolysaccharides (EPS) play critical roles in rhizobium-plant interactions. However, the EPS biosynthesis pathway in Bradyrhizobium diazoefficiens USDA110 remains elusive. Here we used transposon (Tn) mutagenesis with the aim to identify genetic elements required for EPS biosynthesis in B. diazoefficiens USDA110. Phenotypic screening of Tn5 insertion mutants grown on agar plates led to the identification of a mutant with a transposon insertion site in the blr2358 gene. This gene is predicted to encode a phosphor-glycosyltransferase that transfers a phosphosugar onto a polyprenol phosphate substrate. The disruption of the blr2358 gene resulted in defective EPS synthesis. Accordingly, the blr2358 mutant showed a reduced capacity to induce nodules and stimulate the growth of soybean plants. Glycosyltransferase genes related to blr2358 were found to be well conserved and widely distributed among strains of the Bradyrhizobium genus. In conclusion, our study resulted in identification of a gene involved in EPS biosynthesis and highlights the importance of EPS in the symbiotic interaction between USDA110 and soybeans.

Identification of the Natural Transformation Genes in Riemerella anatipestifer by Random Transposon Mutagenesis

In our previous study, it was shown that Riemerella anatipestifer, a Gram-negative bacterium, is naturally competent, but the genes involved in the process of natural transformation remain largely unknown. In this study, a random transposon mutant library was constructed using the R. anatipestifer ATCC11845 strain to screen for the genes involved in natural transformation. Among the 3000 insertion mutants, nine mutants had completely lost the ability of natural transformation, and 14 mutants showed a significant decrease in natural transformation frequency. We found that the genes RA0C_RS04920, RA0C_RS04915, RA0C_RS02645, RA0C_RS04895, RA0C_RS05130, RA0C_RS05105, RA0C_RS09020, and RA0C_RS04870 are essential for the occurrence of natural transformation in R. anatipestifer ATCC11845. In particular, RA0C_RS04895, RA0C_RS05130, RA0C_RS05105, and RA0C_RS04870 were putatively annotated as ComEC, DprA, ComF, and RecA proteins, respectively, in the NCBI database. However, RA0C_RS02645, RA0C_RS04920, RA0C_RS04915, and RA0C_RS09020 were annotated as proteins with unknown function, with no homology to any well-characterized natural transformation machinery proteins. The homologs of these proteins are mainly distributed in the members of Flavobacteriaceae. Taken together, our results suggest that R. anatipestifer encodes a unique natural transformation machinery.

Capsid-E2 interactions rescue core assembly in viruses that cannot form cytoplasmic nucleocapsid cores

Alphavirus capsid proteins (CPs) have two domains: the N-terminal domain (NTD) that interacts with the viral RNA, and the C-terminal domain (CTD) that forms CP-CP interactions and interacts with the cytoplasmic domain of the E2 spike protein (cdE2). In this study, we examine how mutations in the CP NTD affect CP CTD interactions with cdE2. We changed the length and/or charge of the NTD of Ross River virus CP and found that changing the charge of the NTD has a greater impact on core and virion assembly than changing the length of the NTD. The NTD CP insertion mutants are unable to form cytoplasmic cores during infection but they do form cores or core-like structures in virions. Our results are consistent with cdE2 having a role in core maturation during virion assembly and rescuing core formation when cytoplasmic cores are not assembled. We go on to find that the isolated cores from some mutant virions are now assembly competent in that they can be disassembled and reassembled back into cores. These results show how the two domains of CP may have distinct yet coordinated roles. IMPORTANCE: Structural viral proteins have multiple roles during entry and assembly. The capsid protein (CP) of alphaviruses has one domain that interacts with the viral genome and another domain that interacts with the E2 spike protein. In this work we determine that the length and/or charge of the CP affects cytoplasmic core formation. However, defects in cytoplasmic core formation can be overcome by E2-CP interactions, thus assembling a core or core-like complex in the virion. In the absence of both cytoplasmic cores and CP-E2 interactions, CP is not even packaged in the released virions, but some infectious particles are still released presumably as RNA packaged in a glycoprotein containing membrane shell. This suggests that the virus has multiple mechanisms in place to ensure the viral genome is surrounded by a capsid core during its lifecycle.

Overexpression of HOP2 induces developmental defects and compromises growth in Arabidopsis

HOMOLOGOUS PAIRING 2 (HOP2) is a predominantly meiotic protein that plays a pivotal role in homologous chromosome pairing in organisms as diverse as yeast and mammals. While generating HOP2::GFP reporter lines, we identified two Arabidopsis T-DNA insertion mutants, stunted1(std1) and stunted2 (std2) that exhibit pleiotropic phenotypes, including fasciated stems, altered phyllotaxy, floral organ defects, reduced fecundity, and an overall reduction in growth properties. TAIL-PCR followed by sequencing revealed several insertions near genes, but genotyping showed that none of the insertions are causal. Analysis the std mutants by qRT-PCR, and analysis of dexamethasone inducible HOP2 transgenic plants demonstrated that the std phenotypes are associated with ectopic/overexpression of HOP2. Based on the postulated mechanisms of HOP2 action, we speculate on how overexpression leads to these developmental/growth defects.

Modeling site-specific nucleotide biases affecting Himar1 transposon insertion frequencies in TnSeq datasets

In bacterial TnSeq experiments, a library of transposons insertion mutants is generated, selected under various growth conditions, and sequenced to determine the profile of insertions at different sites in the genome, from which the fitness of mutant strains can be inferred. The widely used Himar1 transposon is known to be restricted to insertions at TA dinucleotides, but otherwise, few site-specific biases have been identified. As a result, most analytical approaches assume that insertion counts are expected a priori to be randomly distributed among TA sites in non-essential regions. However, recent analyses of independent Himar1 Tn libraries in M. tuberculosis have identified a local sequence pattern that is non-permissive for Himar1 insertion. This suggests there are site-specific biases that affect the frequency of insertions of the Himar1 transposon at different TA sites. In this paper, we use statistical and machine learning models to characterize patterns in the nucleotides surrounding TA sites associated with high and low insertion counts. We not only affirm that the previously discovered non-permissive pattern (CG)GnTAnC(CG) suppresses insertions, but conversely show that an A in the -3 position or T in the +3 position from the TA site encourages them. We demonstrate that these insertion preferences exist in Himar1 TnSeq datasets other than M. tuberculosis, including mycobacterial and non-mycobacterial species. We build predictive models of Himar1 insertion preferences as a function of surrounding nucleotides. The final predictive model explains about half of the variance in insertion counts, presuming the rest comes from stochastic variability between libraries or due to sampling differences during sequencing. Based on this model, we present a new method, called the TTN-Fitness method, to improve the identification of conditionally essential genes or genetic interactions, i.e., to better distinguish true biological fitness effects by comparing the observed counts to expected counts using a site-specific model of insertion preferences. Compared to previous methods like Hidden Markov Models, the TTN-Fitness method is able to classify the essentiality of many small genes (with few TA sites) that were previously characterized as Uncertain.

Capsid-E2 interactions rescue core assembly in viruses that cannot form cytoplasmic nucleocapsid cores

Alphavirus capsid proteins (CPs) have two domains: the N-terminal domain (NTD) that interacts with the viral RNA, and the C-terminal domain (CTD) that forms CP-CP interactions and interacts with the cytoplasmic domain of the E2 spike protein (cdE2). In this study, we examine how mutations in the CP NTD affect CP CTD interactions with cdE2. We changed the length and/or charge of the NTD of Ross River virus CP and found that changing the charge of the NTD has a greater impact on core and virion assembly than changing the length of the NTD. The NTD CP insertion mutants are unable to form cytoplasmic cores during infection but they do form cores or core-like structures in virions. Our results are consistent with cdE2 having a role in core maturation during virion assembly and rescuing core formation when cytoplasmic cores are not assembled. We go on to find that the isolated cores from some mutant virions are now assembly competent in that they can be disassembled and reassembled back into cores. These results show how the two domains of CP may have distinct yet coordinated roles.

Species-specific gene duplication in Arabidopsis thaliana evolved novel phenotypic effects on morphological traits under strong positive selection

Gene duplication is increasingly recognized as an important mechanism for the origination of new genes, as revealed by comparative genomic analysis. However, the ways in which new duplicate genes contribute to phenotypic evolution remain largely unknown, especially in plants, owing to a lack of experimental and phenotypic data. In this study, we identified the new gene Exov, derived from a partial gene region duplication of its parental gene Exov-L, which is a member of an exonuclease family, into a different chromosome in Arabidopsis thaliana. We experimentally investigated the phenotypic effects of Exov and Exov-L in an attempt to understand how the new gene diverged from the parental copy and contributes to phenotypic evolution. Evolutionary analysis demonstrated that Exov is a species-specific gene that originated within the last 3.5 million years and shows strong signals of positive selection. Unexpectedly, RNAseq analyses reveal that the new gene, despite its young age, has acquired a large number of novel direct and indirect interactions in which the parental gene does not engage. This is consistent with a high, selection-driven substitution rate in the protein sequence encoded by Exov in contrast to the slowly evolving Exov-L, suggesting an important role for Exov in phenotypic evolution. We analyzed phenotypic effects of exov and exov-l single T-DNA-insertion mutants; double exov, exov-l T-DNA insertion mutants; and CRISPR/Cas9-mediated exovcrp and exov-lcrp knockouts on seven morphological traits in both the new and parental genes. We detected significant segregation of morphological changes for all seven traits when assessed in terms of single mutants, as well as morphological changes for seven traits associated with segregation of double exov, exov-l mutants. Substantial divergence of phenotypic effects between new and parental genes was revealed by principal component analyses, suggesting neofunctionalization in the new gene. These results reveal a young gene that plays critical roles in biological processes that underlie morphological and developmental evolution in Arabidopsis thaliana.