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Preliminary Characterization of Phage-Like Particles from the Male-Killing Mollicute Spiroplasma poulsonii (an Endosymbiont of Drosophila)

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Abstract

Bacteriophages are vastly abundant, diverse, and influential, but with few exceptions (e.g. the Proteobacteria genera Wolbachia and Hamiltonella), the role of phages in heritable bacteria-arthropod interactions, which are ubiquitous and diverse, remains largely unexplored. Despite prior studies documenting phage-like particles in the mollicute Spiroplasma associated with Drosophila flies, genomic sequences of such phage are lacking, and their effects on the Spiroplasma-Drosophila interaction have not been comprehensively characterized. We used a density step gradient to isolate phage-like particles from the male-killing bacterium Spiroplasma poulsonii (strains NSRO and MSRO-Br) harbored by Drosophila melanogaster. Isolated particles were subjected to DNA sequencing, assembly, and annotation. Several lines of evidence suggest that we recovered phage-like particles of similar features (shape, size, DNA content) to those previously reported in Drosophila-associated Spiroplasma strains. We recovered three ~ 19 kb phage-like contigs (two in NSRO and one in MSRO-Br) containing 21–24 open reading frames, a read-alignment pattern consistent with circular permutation, and terminal redundancy (at least in NSRO). Although our results do not allow us to distinguish whether these phage-like contigs represent infective phage-like particles capable of transmitting their DNA to new hosts, their encoding of several typical phage genes suggests that they are at least remnants of functional phage. We also recovered two smaller non-phage-like contigs encoding a known Spiroplasma toxin (Ribosome Inactivating Protein; RIP), and an insertion element, suggesting that they are packaged into particles. Substantial homology of our particle-derived contigs was found in the genome assemblies of members of the Spiroplasma poulsonii clade.

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Data Availability

The raw sequence data used for the present study are available at NCBI under Project Number PRJNA545743; BioSamples SAMN11919470, SAMN11919470, SAMN23459927; and SRA SRR17050036, SRR17063333, SRR17065466. Assembled and annotated contigs are available at NCBI under GenBank Accession Nos. OL689226-OL689230 and OL778852.

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Acknowledgements

Anika Stankov for technical assistance. Paul de Figueiredo provided feedback on the research and early versions of the manuscript. Portions of this research were conducted with high-performance research computing resources provided by Texas A&M University (https://hprc.tamu.edu). This research was conducted in partial fulfillment of the Master’s of Science degree requirements of Paulino Ramirez.

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  1. Paulino Ramirez

    Present address: Department of Cell Systems and Anatomy, University of Texas Health San Antonio, San Antonio, TX, USA

Authors and Affiliations

  1. Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX, USA

    Paulino Ramirez & Mariana Mateos

  2. Department of Animal Science, Texas A&M University, College Station, TX, USA

    Justin C. Leavitt & Jason J. Gill

  3. Department of Ecology and Conservation Biology, Texas A&M University, College Station, TX, USA

    Mariana Mateos

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Contributions

Conceived and designed the analysis: PR, JCL, JJG, MM. Collected the data: PR, JCL, JJG. Contributed materials, data or analysis tools: PR, JCL, JJG, MM. Performed the analyses and interpreted the results: PR, JCL, JJG, MM. Wrote the paper: PR, MM. Edited the paper: PR, MM, JCL, JJG.

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Correspondence to Mariana Mateos.

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Supplementary Information

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284_2022_3099_MOESM1_ESM.jpg

Supplementary file1 (JPG 306 KB) Fig. S1 Original Canu assembly of NSRO phage like contigs. Grey arrows in the reference are imperfect terminal repeats in the contig. A) NSRO-P1, B) NSRO-P2

284_2022_3099_MOESM2_ESM.jpg

Supplementary file2 (JPG 902 KB) Fig. S2 Alignment of Nanopore reads to the original Canu assembly contigs. Grey arrows in the reference are imperfect terminal repeats in the contig. The raw long reads are shown in black. Gray outline boxes in reads represent trimmed regions (i.e., different from reference). A) NSRO-P1, B) NSRO P2

284_2022_3099_MOESM3_ESM.jpg

Supplementary file3 (JPG 595 KB) Fig. S3 Geneious-based alignments with annotated ORFs of (A) NSRO-P1, NSRO-P2 and MSRO-P1 phage-like contigs, and (B) Insertion element identified in the phage particle assembly from NSRO and MSRO-Br. Sites with identical bases in all positions are depicted green in the identity bar. In (B) two ORFs were identified along with inverted repeat sequences (grey). The two sequences have 100% homology, except at terminal ends of the contigs. The yellow ORFs (possible translational frameshift) code for a DDE superfamily endonuclease and mobile element like protein respectively, which can form an Insertion element

284_2022_3099_MOESM4_ESM.png

Supplementary file4 (PNG 386 KB) Fig. S4 Alignment of the nucleotide sequences of NCBI U44405.1 and our particle-derived contigs: NSRO-P1, MSRO-Br-P1, NSRO-P2. Grey = position identical to consensus sequence; black = position different from consensus. Open reading frames (ORFs) are indicated with colored block arrows. The color-coding and labels of our particle-derived contigs follow Fig. 4 (yellow = hypothetical; others assigned a putative function). Yellow annotations in U44405 reflect those in NCBI, whereas the green ones were added as a result of the new findings based on HHPred

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Supplementary file11 (DOCX 37 KB)

Supplementary file12 (DOCX 38 KB)

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Ramirez, P., Leavitt, J.C., Gill, J.J. et al. Preliminary Characterization of Phage-Like Particles from the Male-Killing Mollicute Spiroplasma poulsonii (an Endosymbiont of Drosophila). Curr Microbiol 80, 6 (2023). https://doi.org/10.1007/s00284-022-03099-7

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