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Page 12 of 16 Stuehler et al. Microbiome Res Rep. 2025;4:19 https://dx.doi.org/10.20517/mrr.2024.84

of phytanoyl CoA oxygenase with sulfotransferases, there are collagen-like domain proteins thought to act
[57]
as substrates of the related co-encoded enzymes . A collagen-like protein coding sequence, containing 20
[58]
copies of a collagen triple helix repeat identified by Pfam v34.0 , was found in wLfag-FL and has homology
to another collagen-like protein identified in Wolbachia endosymbionts containing the beforementioned
phytanoyl-CoA dioxygenase family protein. However, this specific collagen-like protein had no homology
to collagen-like proteins identified in wDcit endosymbionts, suggesting collaboration with a hydroxylation
system no longer present in the Wolbachia resident of Diaphorina citri. Other protein coding regions of
interest involved in various processes were identified in wLfag-FL that had no homology to coding regions
within wDcit genome assemblies. These sequences encoded for S-adenosylmethionine uptake transporter,
UDP-glucose 6-dehydrogenase, and an IS66 family transposase. S-adenosylmethionine uptake transporter
of the DMT-superfamily contributes to the transport of S-adenosylmethionine required by various
biological pathways. UDP-glucose 6-dehydrogenase is associated with outer cell surface glycostructures
such as lipopolysaccharide biosynthesis. IS66 family transposase is necessary for DNA transposition and
may cause structural variations in bacterial plasmids. Further research is necessary to identify other gene
products involved with the three mentioned proteins, as around 10% of the transcriptome was recovered.
Enrichment for this new Wolbachia strain and deeper sequencing must be conducted to detect other genetic
elements, enabling full transcriptome or genome comparisons. As Wolbachia exhibit important roles with
an insect’s microbiome, identification of genes necessary to uphold the symbiotic relationship may provide
targets for RNAi.

Fundamental pathways used to assess the combativeness of an infection are those containing secreted
proteins. KEGG analysis revealed proteins from the T1SS, T4SS, and Sec pathways in wLfag-FL. From the
Sec-pathway, SRP ffh was identified and has been shown to work in cohorts with a ribosome-associated
chaperone and peptidyl-prolyl isomerase, which determine whether an actively synthesized protein will be
[59]
inserted into the outer membrane or exported through T2SS . Provided the conservation of the ffh coding
region between Wolbachia strains, it may serve as a component in future MLST studies to assist in the
further classification of newly sequenced Wolbachia strains. The T4SS system is one of several types of
secretion systems utilized by microorganisms for the transport of macromolecules across the cell envelope,
and disruption of this pathway, preventing protein oligomerization, may invoke lower infection rates of
various bacterial species. Lastly, the identification of T4SS Vir proteins suggests that both operons within
the virB-virD4 loci are present in wLfag-FL .
[60]
Limitations
Recovery of the complete Wolbachia transcriptome from the four cDNA datasets was not successful, as
determined by our low BUSCO scores and low percentage of unique transcripts mapped to transcripts of
related Wolbachia. We believe this low number of complete transcripts is due to the fragmented nature of
the transcript assembly and the inability of BUSCO to identify suitable protein alignments. Our N50 value is
relatively low compared to N50s of 1,215 for wDcit dawsonii and 1,236 for wSpic. We attempted to further
assemble the transcripts using CAP3, which negligibly increased our N50 to 751; however, this resulted in a
lower complete BUSCO score. Figures 3 and 4 show a complete distribution of transcripts throughout
whole reference genomes, and not just 15% of the genomes, which would be expected considering our
BUSCO score. Short transcripts, around 150-250 bp, still mapped regularly throughout entire reference
genomes, suggesting their presence, although complete annotation was not possible through BUSCO or
KEGG analysis. This assessment was not surprising as Wolbachia was not the target of initial sequencing
efforts and, therefore, sequencing of nucleic acids enriched for microorganisms by a pretreatment DNase
and filtration protocol must be performed for a complete assessment of the wLfag-FL transcriptome.
[61]
Furthermore, long-read sequencing of the genomic DNA will reveal a more complete assessment of the
total CDS encoded in the wLfag genome.