Page 52 - Read Online

Stuehler et al. Microbiome Res Rep. 2025;4:19 https://dx.doi.org/10.20517/mrr.2024.84 Page 5 of 16

not find a match to NCBI via nucleotide, BLASTx was used instead to compare protein homology. BUSCO
[26]
v5.2.2 was used to assess the transcriptome completeness of obtained Wolbachia orthologs . The CAP3
[27]
long read assembler in UGENE v40 was used to further assemble fragmented Wolbachia sequences. The
contigs that did not assemble any further were grouped with the CAP3 contig assemblies, which were
subject to gene annotation.

Phylogenetic characterization
Two methods were employed to characterize the evolutionary lineage of Wolbachia found in L. fagarae
from Florida. The first approach used a concatenated sequence of nine conserved genes, where sequences
were only gathered and analyzed if they were part of a fully assembled Wolbachia genome on NCBI. The
resulting concatenated sequence alignment, performed with MUSCLE in UGENE v40, totaled 7,655
nucleotides in length, where partial sequences of CoxA, ftsZ, gatB, gltA, groEL, and hcpA were used along
with full-length sequences of fbpA, wsp, and 16S rRNA. The second approach used a portion of the wsp gene
that encodes a transmembrane structure, which plays a role in controlled cell death, cell proliferation,
pathogenicity, and host immune response . The wsp sequences obtained from NCBI, belonging to various
[28]
Wolbachia strains of interest, were aligned with MUSCLE in UGENE v40, and trimmed to the limiting
sequence, resulting in a length of 609 nucleotides. Both phylogenetic trees were built using a bootstrap value
of 1000 and the default Maximum Likelihood parameters in UGENE v40.

Transcriptome functional annotation
Four Wolbachia gene datasets available in the KEGG Automatic Annotation Server (KAAS v2.1) were used
to obtain KEGG Orthology identifiers of wLfag-FL sequences [29-31] . The four datasets added together totaled
4,425 sequences and were entitled Wolbachia endosymbiont of Drosophila melanogaster (wol), Wolbachia
sp. wRi (wri), Wolbachia endosymbiont of Culex quinquefasciatus Pel (wpi), and Wolbachia endosymbiont
strain TRS of Brugia malayi (wbm). Both unassembled and CAP3 assembled sequences were placed into the
KAAS server for annotation via the BLAST method and SBH approach. Identification of sequences
containing ankyrin domains was carried out post CAP3 assembly of identified wLfag-FL contigs and was
initiated by BLASTx analysis to a dataset of 182 Multispecies Wolbachia ankyrin domain-containing
proteins from NCBI. Aligned sequences were then verified by selecting whole ExPASy sequence
translations of the same frames, which had homology to Wolbachia ANK domain proteins predicted by
[32]
BLASTx. The translated sequences were compared against the UniProt database for final confirmation.

RESULTS
Phylogenetic analysis
Both phylogenetic characterization strategies using MLST [Figure 1] and wsp [Figure 2] assigned Wolbachia
strain wLfag-FL to supergroup B, which contains closely related psyllids along with other Hemipterans,
Dipterans, and a Lepidopteran. A wsp gene coding for a surface protein of Wolbachia was specifically
chosen for further analysis as it evolves at a much faster rate than other genes such as 16S or ftsZ, and has
[33]
been previously used in characterizing Wolbachia strains . Moreover, the wsp gene is allotted for further
analysis of strains without a completely assembled genome. Analysis was attempted with a protein
translation of the wsp gene, but given the lack of whole sequence availability for other Wolbachia strains,
incomplete protein translations could not differentiate lineages of closely related strains. The Wolbachia
endosymbiont of B. cockerelli is seemingly the closest relative of wLfag-FL according to wsp analysis. All
sequence information used to build the phylograms is recorded in Table 1. A depiction of the wsp
alignment created in Jalview v2.11.1 is available in Supplementary Figure 1.
[34]