Page 58 - Read Online

Stuehler et al. Microbiome Res Rep. 2025;4:19 https://dx.doi.org/10.20517/mrr.2024.84 Page 11 of 16

sequence was further utilized to depict relations to other Wolbachia strains without full genome assemblies.
In both analyses, wLfag-FL consistently grouped with Wolbachia from Supergroup B, which contains other
closely related psyllid species known to house disease-causing Liberibacteria. We also found that wLfag-FL
is closely related to the Wolbachia resident of B. cockerelli, further supporting that both insects originated
from Central America. One other important insect pest of South and Central America is the coffee berry
borer Hypothenemus hampei, which is credited for causing losses of more than a half billion USD in the
coffee-producing industry . H. hampei origins are traced to tropical biomes of Africa and its Wolbachia
[43]
endosymbiont grouped with Drosophila melanogaster, another insect of African origin. The placement of
other Wolbachia strains into the correct supergroups strengthens the interpretation of our MLST analysis.

Regarding endosymbiotic Wolbachia, one of the most intriguing components for host interaction is the
substantial number of genes encoding proteins with ANK domains. As few as five ANK domain-containing
proteins were identified in the Wolbachia endosymbiont of Brugia malayi, and as many as 60 were
[44]
identified in the Wolb-pip strain of Wolbachia from the Culex pipiens group . Annotation of the
Wolbachia endosymbiont of the Diaphorina citri genome revealed 54 predicted proteins containing ANK
domains . ANK genes in Wolbachia have been proposed as major factors in the endosymbiont’s functional
[45]
role and unique symbiotic relationship. Functions of ANK-containing proteins acting as binding molecules
in Wolbachia are difficult to predict since few have sequence similarity outside the ankyrin domains to other
[46]
proteins of known functions . Nevertheless, many studies suggest that Wolbachia ankyrin domain proteins
are involved in many symbiotic pathways, expanding the understanding of how intertwined Wolbachia-host
interaction truly is. Biological processes such as the assembly of cation channels, transcriptional regulation,
cell differentiation, apoptosis, cell signaling, and most importantly, secretion systems are all thought to
contain some sort of protein embodying the ANK motif [47-49] . Further analysis of ANK domain proteins of
wLfag-FL must be conducted to obtain a clearer understanding of their roles in this specific Wolbachia-host
symbiosis, as our recovery of only 5 transcripts is not enough to draw extensive conclusions.

By influencing host metabolic processes, Wolbachia enhance their selection process as the reproductive
assistance they provide may not benefit all hosts selected for infection. Provided the extensive gene loss in
endosymbiotic Wolbachia, it is inferred that these strains are nutritionally dependent upon their hosts.
Bacterioferritin, the bacterial homolog of eukaryotic ferritin, has been inferred to help eukaryotic hosts
[50]
capture excess Fe(2+) to avoid iron toxicity and allow the ferric mineral to have increased bioavailability .
The three coordination environments (A, B, C) in the ferroxidase center of bacterioferritin are highly
conserved, consisting of four alpha helices (A, B, C, and D). Helix A is characterized by two highly
conserved αα’s Glu18, and Tyr25, Helix B by αα’s Glu47, Glu51, and His54, Helix C by αα Glu94, and Helix
D by αα’s Asp126, Glu127, Gly129, and His130 . All the listed conserved residues were shown to exist in
[51]
wLfag-FL bacterioferritin, except the residues of Helix D, as the translated protein sequence obtained from
wLfag-FL accounts only for the first 95 residues of the Wolbachia bacterioferritin protein sequence.

Other predicted metabolites and transporters have been identified in wLfag-FL, inferring the provision of
biological molecules for the metabolism of riboflavin, glutathione, glucose, carbon, pentose phosphates,
pyrimidines, purines, fatty acids, and various amino acids. Transporters associated with various substrates
including pyruvate, carbohydrates, amino acid catabolism, and inorganic cations were additionally
identified [46,52,53] . Analysis of the wLfag-FL transcriptome identified a sequence encoding a phytanoyl-CoA
dioxygenase family protein, which catalyzes conversions of alpha-ketoglutarate to succinate, and phytanoyl-
CoA to 2-hydroxyphytanoyl-CoA . The generation of succinate by phytanoyl-CoA dioxygenase, a family
[54]
member of iron(II)-dependent oxygenases, has been suggested to aid in energy production under
conditions of nutritional stress [55,56] . It has been proposed that in predicted operons combining 2OGFeDOs