More Like this

1. Characterization of Ssc, an N -acetylgalactosamine-containing Staphylococcus aureus surface polysaccharide

   https://doi.org/10.1128/jb.00048-24  

   Lei, Mei G; Jorgenson, Matthew A; Robbs, Emily J; Black, Ian M; Archer-Hartmann, Stephanie; Shalygin, Sergei; Azadi, Parastoo; Lee, Chia Y (May 2024, Journal of Bacteriology)

   Henkin, Tina M (Ed.)

   ABSTRACT Whole genome sequencing has revealed that the genome ofStaphylococcus aureuspossesses an uncharacterized 5-gene operon (SAOUHSC_00088–00092 in strain 8325 genome) that encodes factors with functions related to polysaccharide biosynthesis and export, indicating the existence of a new extracellular polysaccharide species. We designate this locus assscfor staphylococcal surface carbohydrate. We found that thesscgenes were weakly expressed and highly repressed by the global regulator MgrA. To characterize Ssc, Ssc was heterologously expressed inEscherichia coliand extracted by heat treatment. Ssc was also conjugated to AcrA fromCampylobacter jejuniinE. coliusing protein glycan coupling technology (PGCT). Analysis of the heat-extracted Ssc and the purified Ssc-AcrA glycoconjugate by tandem mass spectrometry revealed that Ssc is likely a polymer consisting ofN-acetylgalactosamine. We further demonstrated that the expression of thesscgenes inS. aureusaffected phage adsorption and susceptibility, suggesting that Ssc is surface-exposed. IMPORTANCESurface polysaccharides play crucial roles in the biology and virulence of bacterial pathogens.Staphylococcus aureusproduces four major types of polysaccharides that have been well-characterized. In this study, we identified a new surface polysaccharide containing N-acetylgalactosamine (GalNAc). This marks the first report of GalNAc-containing polysaccharide inS. aureus. Our discovery lays the groundwork for further investigations into the chemical structure, surface location, and role in pathogenesis of this new polysaccharide. 

   more » « less
2. CRISPR RNA ‐guided integrase enables high‐efficiency targeted genome engineering in Agrobacterium tumefaciens

   https://doi.org/10.1111/pbi.13872  

   Aliu, Ephraim; Lee, Keunsub; Wang, Kan (July 2022, Plant Biotechnology Journal)

   Summary Agrobacterium tumefaciens, the causal agent of plant crown gall disease, has been widely used to genetically transform many plant species. The inter‐kingdom gene transfer capability madeAgrobacteriuman essential tool and model system to study the mechanism of exporting and integrating a segment of bacterial DNA into the plant genome. However, many biological processes such asAgrobacterium‐host recognition and interaction are still elusive. To accelerate the understanding of this important plant pathogen and further improve its capacity in plant genetic engineering, we adopted a CRISPR RNA‐guided integrase system forAgrobacteriumgenome engineering. In this work, we demonstrate thatINsertion ofTransposableElements byGuideRNA–AssistedTargEting (INTEGRATE) can efficiently generate DNA insertions to enable targeted gene knockouts. In addition, in conjunction with Cre‐loxPrecombination system, we achieved precise deletions of large DNA fragments. This work provides new genetic engineering strategies forAgrobacteriumspecies and their gene functional analyses. 

   more » « less
3. A chromosome scale assembly of the parasitoid wasp Venturia canescens provides insight into the process of virus domestication

   https://doi.org/10.1093/g3journal/jkad137  

   Mao, Meng; Simmonds, Tyler J.; Stouthamer, Corinne M.; Kehoe, Tara M.; Geib, Scott M.; Burke, Gaelen R.; Lawniczak, ed., M. (June 2023, G3: Genes, Genomes, Genetics)

   Abstract The parasitoid wasp Venturia canescens is an important biological control agent of stored products moth pests and serves as a model to study the function and evolution of domesticated endogenous viruses (DEVs). The DEVs discovered in V. canescens are known as virus-like particles (VcVLPs), which are produced using nudivirus-derived components and incorporate wasp-derived virulence proteins instead of packaged nucleic acids. Previous studies of virus-derived components in the V. canescens genome identified 53 nudivirus-like genes organized in six gene clusters and several viral pseudogenes, but how VcVLP genes are organized among wasp chromosomes following their integration in the ancestral wasp genome is largely unknown. Here, we present a chromosomal scale genome of V. canescens consisting of 11 chromosomes and 56 unplaced small scaffolds. The genome size is 290.8 Mbp with a N50 scaffold size of 24.99 Mbp. A high-quality gene set including 11,831 protein-coding genes were produced using RNA-Seq data as well as publicly available peptide sequences from related Hymenoptera. A manual annotation of genes of viral origin produced 61 intact and 19 pseudogenized nudivirus-derived genes. The genome assembly revealed that two previously identified clusters were joined into a single cluster and a total of 5 gene clusters comprising of 60 intact nudivirus-derived genes were located in three chromosomes. In contrast, pseudogenes are dispersed among 8 chromosomes with only 4 pseudogenes associated with nudivirus gene clusters. The architecture of genes encoding VcVLP components suggests it originates from a recent virus acquisition and there is a link between the processes of dispersal and pseudogenization. This high-quality genome assembly and annotation represents the first chromosome-scale assembly for parasitoid wasps associated with VLPs, and is publicly available in the National Center for Biotechnology Information Genome and RefSeq databases, providing a valuable resource for future studies of DEVs in parasitoid wasps. 

   more » « less
4. Molecular identification of a peroxidase gene controlling body size in the entomopathogenic nematode Steinernema hermaphroditum

   https://doi.org/10.1093/genetics/iyad209  

   Schwartz, Hillel_T; Tan, Chieh-Hsiang; Peraza, Jackeline; Raymundo, Krystal_Louise_T; Sternberg, Paul_W; Greenstein, ed., D. (December 2023, GENETICS)

   Abstract The entomopathogenic nematode Steinernema hermaphroditum was recently rediscovered and is being developed as a genetically tractable experimental system for the study of previously unexplored biology, including parasitism of its insect hosts and mutualism with its bacterial endosymbiont Xenorhabdus griffiniae. Through whole-genome re-sequencing and genetic mapping we have for the first time molecularly identified the gene responsible for a mutationally defined phenotypic locus in an entomopathogenic nematode. In the process we observed an unexpected mutational spectrum following ethyl methansulfonate mutagenesis in this species. We find that the ortholog of the essential Caenorhabditis elegans peroxidase gene skpo-2 controls body size and shape in S. hermaphroditum. We confirmed this identification by generating additional loss-of-function mutations in the gene using CRISPR-Cas9. We propose that the identification of skpo-2 will accelerate gene targeting in other Steinernema entomopathogenic nematodes used commercially in pest control, as skpo-2 is X-linked and males hemizygous for loss of its function can mate, making skpo-2 an easily recognized and maintained marker for use in co-CRISPR. 

   more » « less
5. F‐box protein CFK1 interacts with and degrades de novo DNA methyltransferase in Arabidopsis

   https://doi.org/10.1111/nph.17103  

   Chen, Jiani; Liu, Jie; Jiang, Jianjun; Qian, Shuiming; Song, Jingwen; Kabara, Rachel; Delo, Isabel; Serino, Giovanna; Liu, Fengquan; Hua, Zhihua; et al (December 2020, New Phytologist)

   Summary DNA methylation plays crucial roles in cellular development and stress responses through gene regulation and genome stability control. Precise regulation of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), thede novoArabidopsis DNA methyltransferase, is crucial to maintain DNA methylation homeostasis to ensure genome integrity. Compared with the extensive studies on DRM2 targeting mechanisms, little information is known regarding the quality control of DRM2 itself.Here, we conducted yeast two‐hybrid screen assay and identified an E3 ligase, COP9 INTERACTING F‐BOX KELCH 1 (CFK1), as a novel DRM2‐interacting partner and targets DRM2 for degradation via the ubiquitin‐26S proteasome pathway inArabidopsis thaliana. We also performed whole genome bisulfite sequencing (BS‐seq) to determine the biological significance of CFK1‐mediated DRM2 degradation.Loss‐of‐functionCFK1leads to increased DRM2 protein abundance and overexpression of CFK1 showed reduced DRM2 protein levels. Consistently, CFK1 overexpression induces genome‐wide CHH hypomethylation and transcriptional de‐repression at specific DRM2 target loci.This study uncovered a distinct mechanism regulatingde novoDNA methyltransferase by CFK1 to control DNA methylation level. 

   more » « less