Stepwise assembly of virulence-associated traits in the intracellular pathogen Coxiella burnetii

22 Coxiella burnetii is the only member of the order Legionellales known to primarily infect vertebrates. 23 The Q fever pathogen is also unusual in that it replicates within an acidified phagolysosome-like 24 vacuole. The evolutionary origins of the virulence determinants underlying this lifestyle remain 25 unclear. More broadly, little is known about how virulence-related traits arise in specialized 26 intracellular lineages, where access to foreign-origin DNA may be more episodic. To address this 27 question, we used Legionellales-wide comparative phylogenomics to reconstruct the gain and loss of 28 traits affecting host interaction, immune evasion, intracellular survival, and metabolism. We found that 29 many virulence-associated traits in C. burnetii predate the modern pathogen and were assembled 30 stepwise in ancestors that likely occupied niches distinct from the acidified vacuolar niche of modern 31 C. burnetii. The common ancestor shared with soft-tick Coxiella endosymbionts likely encoded most 32 C. burnetii type IVB secretion system effectors, indicating that much of the host-manipulation 33 repertoire in C. burnetii was already present before the emergence of the modern pathogen. Distinctive 34 lipopolysaccharide features associated with immune evasion also appear to have accumulated 35 progressively within the Coxiella lineage, including genes implicated in synthesis of virenose, a unique 36 O-antigen sugar critical for C. burnetii virulence. Traits likely to support replication in the acidic 37 Coxiella-containing vacuole likewise accumulated gradually, with generalized stress-tolerance 38 functions predating acquisition of an Mrp cation/proton antiporter that may have further supported pH 39 homeostasis. Additional changes in sugar transport and catabolism, glycolytic control, and respiratory 40 metabolism likely enhanced metabolic flexibility and access to diverse substrates in this nutrient-rich 41 niche. Together, these findings support a model in which vertebrate pathogenicity in C. burnetii 42 emerged through stepwise remodeling of an ancestral host-associated lineage and provide a framework 43 for understanding how virulence-related traits evolve in specialized intracellular pathogens. 44 45 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 3 AUTHOR SUMMARY 46 Coxiella burnetii is the bacterium that causes Q fever, a disease that can spread from animals to 47 humans. Unlike its close relatives, C. burnetii primarily infects vertebrates and grows inside an acidic 48 compartment within host cells. New bacterial pathogens often evolve by gaining genes from other 49 bacteria, but how virulence evolves in lineages that grow only inside host cells, where opportunities to 50 gain new genes may be infrequent, remains unclear. We wanted to understand how C. burnetii evolved 51 the traits needed for its distinctive intracellular lifestyle. By comparing its genome to those of related 52 bacteria across the order Legionellales, we found that features involved in host manipulation, immune 53 evasion, acid tolerance, and nutrient use appeared at different times in its ancestry rather than being 54 acquired all at once by the modern pathogen. Our findings suggest that specialized intracellular 55 pathogens can emerge through gradual changes in ancestral host-associated lineages, including gene 56 acquisition, gene loss, retention of older traits, and repurposing of existing functions. 57 58 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 4

59 The order Legionellales contains several intracellular bacteria that infect eukaryotic hosts, but Coxiella 60 burnetii, the causative agent of Q fever, is the only known member that primarily infects vertebrates 61 [Santos et al., 2003; Boamah et al., 2017; Eldin et al., 2017; Duron et al., 2018; Celina & Cerný, 2022; 62 Kidane et al., 2022; Floriano et al., 2025; Ingle et al., 2025]. This unusual host range is coupled to a 63 distinctive intracellular niche: C. burnetii replicates within a large Coxiella-containing vacuole (CCV) 64 that matures through the canonical endocytic pathway and acquires phagolysosomal characteristics 65 [van Schaik et al., 2013; Kohler & Roy, 2015; Dragan & Voth, 2020]. As the vacuole acidifies to 66 approximately pH 4.75, dormant small cell variants transition into metabolically active large cell 67 variants and assemble the Dot/Icm type IVB secretion system (T4BSS), which is essential for 68 intracellular replication [Beare et al., 2011; Carey et al., 2011; Beare et al., 2012; Newton et al., 2013; 69 Park et al., 2022]. The mature CCV is therefore both hostile and nutrient rich, exposing C. burnetii to 70 low pH, lysosomal hydrolases, reactive oxygen species, and osmotic stress while also providing access 71 to diverse host-derived substrates [Howe et al., 2010; Mertens & Samuel, 2012; Miller et al., 2019]. 72 Understanding how C. burnetii evolved the ability to survive and replicate in this compartment is 73 central to uncovering the origin of vertebrate pathogenicity in the genus. 74 Several traits are known to be critical to C. burnetii pathogenesis. First, the Dot/Icm T4BSS 75 delivers effector proteins that modulate host trafficking pathways and promote fusion of the CCV with 76 endosomes, lysosomes, and autophagosomes, thereby enabling vacuole expansion and bacterial growth 77 [Carey et al., 2011; van Schaik et al., 2013; Kohler & Roy, 2015; Ruart et al., 2025]. Second, C. 78 burnetii LPS contributes to immune evasion, and loss of the full-length O-antigen prevents productive 79 infection in immunocompetent vertebrates [Vishwanath & Hackstadt, 1988; Hoover et al., 2002; 80 Shannon et al., 2005]. This O-antigen contains the unusual sugars virenose and 81 dihydrohydroxystreptose, which are rarely found in other bacteria and are essential for normal O-82 antigen extension [Amano et al., 1987; Hoover et al., 2002; Toman et al., 2009; Beare et al., 2018]. 83 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 5 Third, C. burnetii is more metabolically flexible relative to other Legionellales and can efficiently use 84 both amino acids and sugars as carbon and energy sources, a feature that may be advantageous in the 85 chemically diverse environment of the CCV [Häuslein et al., 2017; Vallejo-Esquerra et al., 2017; Kuba 86 et al., 2019]. Together, these features suggest that vertebrate pathogenicity in C. burnetii depends on 87 host-cell manipulation, immune evasion, adaptation to acidic intracellular conditions, and metabolic 88 versatility. 89 The evolutionary origins of these traits remain incompletely resolved. Earlier work proposed 90 that C. burnetii arose from a maternally inherited tick endosymbiont that acquired the factors needed to 91 infect vertebrate cells [Duron et al., 2015]. More recent analyses instead indicate that C. burnetii and 92 closely related Coxiella endosymbionts (CEs) descended from pathogenic ancestors and that the 93 common ancestor of C. burnetii and soft-tick endosymbionts likely already possessed T4BSS and the 94 capacity to infect macrophage-like cells [Brenner et al., 2021]. This shift in perspective reframes the 95 problem from the origin of pathogenicity itself to the origin of the more specialized traits that enabled 96 C. burnetii to exploit vertebrate hosts and thrive in an acidic intracellular vacuole. 97 To address this question, we used comparative phylogenomics across Legionellales, together 98 with a newly assembled genome of the CE in Ornithodoros peruvianus, to trace the evolution of traits 99 associated with vertebrate pathogenicity in C. burnetii (Figure 1). This genome is especially 100 informative for reconstructing C. burnetii evolution because Ornithodoros-associated CEs are the 101 closest sampled relatives of the pathogen and preserve a comparatively broad set of orthologous and 102 pseudogenized genes shared with C. burnetii (Table 1). We focus particularly on systems likely to 103 influence host interaction, immune evasion, survival in acidic intracellular conditions, and exploitation 104 of host-derived nutrients. Our results indicate that much of C. burnetii’s virulence-associated 105 machinery predates the modern pathogen, and that key features linked to vertebrate adaptation, 106 including aspects of LPS structure, acid tolerance, and metabolic flexibility, were assembled more 107 gradually. Together, these findings support a model in which modern C. burnetii emerged through 108 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 6 stepwise remodeling of an ancestral host-associated lineage rather than through a recent transition from 109 a benign tick symbiont. 110 111

112 Ancient T4BSS machinery was paired with a more recently assembled C. burnetii effector 113 repertoire 114 The Dot/Icm T4BSS is essential for virulence in Legionellales [Beare et al., 2011; Nagai & Kubori, 115 2011; Leclerque & Kleespies, 2008; Segal et al., 1998]. Analysis of T4BSS components revealed that 116 most are broadly conserved throughout Legionellales, with the main exception being the CEs (Figure 117 2). In contrast to the T4BSS machinery, most effectors were found only in close relatives of Coxiella. 118 Of the 28 C. burnetii effectors with established host interactions [Ruart et al., 2025], 20 have orthologs 119 in one or more Ornithodoros CEs and six have orthologs in Paracoxiella (Table 2). Few effectors 120 could be traced beyond the common ancestor of Coxiella and Paracoxiella: seven lacked any orthologs 121 elsewhere in the Legionellales, and six had no orthologs outside C. burnetii. These data suggest that 122 many C. burnetii effectors arose relatively recently and are tailored to its particular host range and 123 intracellular lifestyle. 124 Our analysis also indicates that the effector MceB (CirC, CBU_0937) and the hypothetical 125 protein CBU_1699 arose through duplication of the LbtP siderophore porin, which is present 126 throughout Legionellales (Figure 3). MceB targets host mitochondria [Fielden et al., 2021; Arunima et 127 al., 2025], but it also resides in the bacterial outer membrane [Yang et al., 2025]. Its retention in CEs 128 that lack functional T4BSS machinery suggests that MceB may primarily function as a siderophore 129 porin, although it may have acquired an additional role after C. burnetii diverged from CEs. 130 CstK (CBU_0175), which affects endosomal trafficking and autophagy [Martinez et al., 2020], 131 may have been acquired through intra-Legionellales horizontal gene transfer (HGT). Outside Coxiella 132 and Paracoxiella, the top-scoring matches to CstK were proteins from Berkiella species. Given the 133 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 7 distant relationship between Coxiella and Berkiella (Figure 1), the high sequence identity (>65%), and 134 the absence of CstK-like proteins in other Legionellales, these proteins are unlikely to be vertically 135 inherited. Instead, the evidence suggests an HGT event either between Coxiella and Berkiella or from 136 an unidentified common donor. The contrast between broadly conserved secretion machinery and more 137 restricted effector distributions supports a model in which the T4BSS was inherited from an ancient 138 Legionellales ancestor, while much of the C. burnetii effector repertoire has undergone lineage-139 specific expansion and innovation, likely reflecting adaptation to its distinct host range and 140 intracellular niche. 141 142 Recent assembly of O-antigen biosynthesis traits shaped a virulence-associated LPS structure 143 C. burnetii LPS contains the O-antigen-sugar virenose (Figure 4), whose loss prevents O-antigen 144 elongation and results in attenuated virulence in mammals [Moos & Hackstadt, 1987; Long et al., 145 2019]. The pathway for virenose synthesis has not been fully characterized, but genes located in a 146 genomic region lost from the avirulent NMII strain are known to participate in this process [Amano et 147 al., 1987; Hoover et al., 2002; Beare et al., 2018]. GDP-4-keto-6-deoxy-D-mannose, an intermediate in 148 the biosynthesis of several O-antigen sugars such as fucose, perosamine, and rhamnose, is also thought 149 to serve as an intermediate in virenose synthesis [Thompson et al., 2003; Narasaki et al., 2011; Flores-150 Ramirez et al., 2012]. We found that the genes responsible for generating GDP-4-keto-6-deoxy-D-151 mannose — CBU_0294 (phosphomannomutase), CBU_0671 (GDP-mannose pyrophosphorylase), and 152 CBU_0689 (GDP-mannose-4,6-dehydratase) — were likely inherited from the common ancestor of 153 Coxiellaceae or an even earlier ancestor (Figure 5). 154 GDP-4-keto-6-deoxy-D-mannose may function as the substrate for CBU_0688, a fucose 155 synthase-like gene, and CBU_0683, a methyltransferase that has been proposed to participate in 156 virenose synthesis [Toman et al., 2013; Seshadri et al., 2003]. Pseudogenized orthologs of CBU_0688 157 were detected in CEs of hard and soft ticks, whereas pseudogenized orthologs of CBU_0683 were 158 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 8 identified only in Ornithodoros CEs, suggesting that at least part of the virenose synthesis pathway 159 was present in the common ancestor of C. burnetii and CEs (Figure 5). Another gene experimentally 160 shown to be essential for virenose synthesis is CBU_0678 [Beare et al., 2018]. Although its precise 161 role remains unclear, CBU_0678 is similar to the bifunctional gene HldE involved in LPS inner-core 162 sugar biosynthesis and our analysis indicate that it was likely acquired from Alphaproteobacteria 163 (Figure 6). These distributions place several genes implicated in virenose production in the recent 164 ancestry of C. burnetii, consistent with stepwise assembly of O-antigen biosynthetic capacity before 165 the emergence of the fully virulent C. burnetii LPS structure. 166 167 LPS inner-core remodeling likely altered envelope properties and host recognition 168 The LPS core oligosaccharide links the lipid A anchor to the repeating O-antigen polysaccharide 169 (Figure 4). It is generally less variable than the O-antigen and commonly contains the sugar L-glycero-170 beta-D-manno-heptose (LD-heptose). GmhD, which catalyzes the final step of the heptose synthesis 171 pathway that converts DD-heptose to LD-heptose, has been lost in C. burnetii and Ornithodoros CEs 172 (Figure 7). GmhD is retained in most hard-tick CEs and is also present in Paracoxiella, indicating that 173 it was present in the common ancestor of Coxiella. Similar to C. burnetii LPS, shift from LD-heptose 174 to DD-heptose appears to have occurred independently in Aquicella and Legionella species (Figure 7). 175 Changes in heptose stereochemistry could alter host recognition because bacterial heptose and heptose-176 derived metabolites are detected by innate immune pathways. The cytosolic ALPK1 pathway responds 177 to bacterial ADP-heptose metabolites, and pulmonary surfactant protein D can bind heptose-containing 178 LPS core structures; in both cases, recognition can differ between LD- and DD-heptose forms [Wang 179 et al., 2008; Zhou et al., 2018]. 180 C. burnetii LPS features a mannose substitution at the 4′-position of inner core heptose I 181 (Figure 4), catalyzed by the glycosyltransferase CBU_1657 [Beare et al., 2018]. CBU_1657 was likely 182 acquired in the common ancestor of Coxiella and Paracoxiella and has been retained in several hard- 183 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 9 and soft-tick CEs. Phylogenetic analysis suggests that CBU_1657 was derived from 184 Thermodesulfobacteriota, a phylum of extremophilic bacteria not known to include pathogens (Figure 185 8). Geobacter sulfurreducens, a member of this group, also contains mannose attached to HepI in its 186 LPS inner core [Vinogradov et al., 2004], suggesting that the mannosyltransferase activity of 187 CBU_1657 originated prior to its acquisition via HGT by the common ancestor of Coxiella and 188 Paracoxiella. Because HepI/HepII substitutions can influence serum sensitivity and interactions with 189 host factors, acquisition of a mannosyltransferase provides a second mechanism by which inner-core 190 remodeling could affect the bacterial surface [Caroff & Karibian, 2003]. The inferred loss of GmhD 191 and acquisition of CBU_1657 support a model in which the Coxiella LPS inner core was reshaped 192 through both gene loss and horizontal gene gain, potentially altering surface properties relevant to host 193 interaction. 194 195 C. burnetii inherited a simplified lipid A anchor with weak TLR4-stimulatory potential 196 C. burnetii and the CEs appear to have the simplest form of lipid A, lipid IVA, which is tetra-acylated 197 and requires only the core cytoplasmic lipid A biosynthesis enzymes (Table 3). In humans, lipid IVA 198 acts as a TLR4-MD2 antagonist and is only weakly agonistic in mice [Saha et al., 2022]. Unlike 199 Coxiella, all other Legionellales encode one or more enzymes that carry out periplasmic modifications 200 of lipid IVA after its synthesis (Table 3). The distribution of these periplasmic modification genes 201 suggests that the common ancestor of Coxiella already used a simplified lipid IVA form as its LPS 202 anchor, although this state may date to an even deeper ancestor. By retaining a simplified lipid IVA-203 like anchor, C. burnetii may have preserved an ancestral LPS feature that limits TLR4 stimulation, 204 linking lipid A evolution to a functional property relevant to host interaction. 205 206 Horizontal acquisition of polyamine biosynthesis expanded stress-tolerance potential 207 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 10 Polyamines such as putrescine and homospermidine contribute to bacterial tolerance of high 208 temperature, reactive oxygen species, and low pH [Michael, 2018; di Martino et al., 2013; Shah & 209 Swiatlo, 2008]. C. burnetii’s polyamine synthesis gene cluster (CBU_0720-CBU_0722) is also present 210 in CEs, Paracoxiella, and Rickettsiella, but not in other Legionellales. This distribution suggests that 211 the cluster was acquired in the common ancestor of Coxiellaceae, and our phylogenetic analysis 212 indicates that it was horizontally acquired from Alphaproteobacteria (Figure 9). Experiments in 213 alphaproteobacterial family Pelagibacteraceae indicate that although the type III PLP-dependent 214 decarboxylase encoded in this cluster (CBU_0722) is structurally similar to an ornithine 215 decarboxylase, it functions predominantly as an arginine decarboxylase [Li et al., 2023] (Figure 10). 216 Assuming this is also true in Coxiella, this gene cluster constitutes a complete pathway for the 217 conversion of arginine to putrescine and its derivative homospermidine. These data point to horizontal 218 acquisition of a polyamine biosynthesis pathway whose predicted products could improve C. burnetii’s 219 tolerance to oxidative or acidic stress in its intracellular niche. 220 221 Acquired ammonia-producing enzymes connect arginine metabolism to pH homeostasis 222 C. burnetii’s pathway for converting arginine to proline consists of two ammonia-producing enzymes 223 (Figure 10). These atypical genes can increase pH in an acidic environment [Lu et al., 2013; Guan & 224 Liu, 2020; Liu et al., 2025]. The first enzyme (CBU_0279) is homologous to the N-terminal domain of 225 arginine dihydrolase, which is characteristically found in cyanobacteria [Zhang et al., 2018] (Figure 226 11). Three Legionella taxa also encode genes homologous to arginine dihydrolase, but the sparse 227 distribution of this function across Legionellales suggests that these genes are unlikely to be 228 orthologous. The second enzyme, ornithine cyclodeaminase (CBU_1727), was also likely acquired in 229 the common ancestor of Coxiella and Paracoxiella. BLASTp analysis identified homologs in several 230 distantly related proteobacteria and a CBU_1727 protein tree placed its closest relative in the archaeal 231 genus Thermofilum (Figure 12). By adding atypical ammonia-producing steps to arginine metabolism, 232 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 11 the C. burnetii lineage may have gained a mechanism that could contribute to pH homeostasis in acidic 233 environments. 234 235 Fatty acid pathway remodeling expanded membrane stress-adaptation capacity 236 Since diverging from Legionella, the lineage leading to C. burnetii has undergone several changes in 237 fatty acid synthesis and modification that likely affect stress tolerance (Figure 13). Coxiella is the only 238 Legionellales lineage predicted to produce monounsaturated fatty acids (UFAs) using FabA 239 (CBU_0037) and FabB (CBU_0035). FabAB is part of a larger lipid metabolism gene cluster whose 240 distribution suggests that it was acquired in the common ancestor of Coxiella, as these genes are found 241 only in C. burnetii and the CEs (Figure 14). Most of these genes have older functional counterparts 242 elsewhere in the genome; thus, C. burnetii encodes two fatty acid synthesis systems: an ancestral 243 pathway for saturated fatty acids and a more recently acquired pathway that can generate UFAs. Low 244 pH typically increases the density and order of lipid bilayers, so genes that can maintain membrane 245 fluidity could be beneficial in this environment [Abhinav et al., 2022]. C. burnetii’s membrane is 246 primarily composed of branched chain fatty acids [Tzianabos et al., 1981; Amano et al., 1984], so the 247 specific role of UFAs is unclear, but likely allows C. burnetii to fine tune membrane fluidity in 248 response to various environmental stressors including pH. 249 The common ancestor of Coxiella and Paracoxiella also gained a delta-9 acyl-CoA desaturase 250 (CBU_0920), which desaturates fatty acyl chains bound to acyl-CoA. That same ancestor lost a DesA-251 family acyl-lipid desaturase that acts on fatty acyl chains already incorporated into membrane lipids. 252 Unlike many other Legionellales, C. burnetii also lacks cyclopropane fatty acid synthase (CfaS), and 253 our reconstruction suggests that this gene was lost in the common ancestor of C. burnetii and 254 Ornithodoros endosymbionts. Collectively, this pattern of gene loss and gain suggests that fatty acid 255 desaturation became more closely coupled to fatty acid synthesis during Coxiella evolution, potentially 256 expanding the capacity to regulate membrane composition under environmental stress. 257 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 12 258 Recent acquisition of the Mrp antiporter may support pH homeostasis 259 C. burnetii encodes a multi-subunit Mrp (Multiple resistance and pH adaptation) antiporter gene 260 cluster that appears to be essential because multiple attempts to generate mutants have been 261 unsuccessful [Martinez et al., 2014; Metters et al., 2023]. Additionally, Mrp genes have been 262 pseudogenized or lost in CEs, indicating that they are not critical in tick symbionts that do not inhabit 263 acidic intracellular vacuoles. The Mrp cluster was likely acquired in the common ancestor of C. 264 burnetii and the Ornithodoros CEs (Figure 15). Mrp antiporters are also present in a small number of 265 Legionella species, but their distribution across Legionellales suggests that the system was acquired 266 independently in Coxiella and Legionella. The function of C. burnetii’s Mrp system has not been 267 experimentally established but is thought to aid in pH homeostasis because it is required for growth in 268 both acidic axenic medium and within host cells [Martinez et al., 2014; Metters et al., 2023; Yadav et 269 al., 2023]. Mrp is typically associated with alkaliphilic and halophilic bacteria, where protons are 270 imported in exchange for Na+/Li+ export [Swartz et al., 2005; Ito et al., 2017]. However, Mrp mutation 271 in Pyrococcus furiosus was found to cause growth impairment under acidic conditions [Haja & 272 Adams, 2021], suggesting that the antiporter likely contributes to pH homeostasis during growth 273 within the acidic CCV. 274 275 An acquired mannose catabolism pathway connects host-derived carbon to central metabolism 276 Our analyses indicate that the common ancestor of Coxiellaceae gained a gene cluster (CBU_1275-277 CBU_1277) that may allow host-derived mannose to be used as a carbon and energy source (Figure 278 16). This pathway resembles hexuronate catabolism pathways found in bacteria and fungi, but it uses 279 an aldohexose dehydrogenase (AldT, CBU_1276) to generate mannonate directly from mannose rather 280 than from a hexuronate. A mannose utilization pathway of this general type has been characterized in 281 the archaeon Thermoplasma [Kopp et al., 2019]. A conserved domain search further indicates that 282 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 13 CBU_1276 is similar to a gluconate 5-dehydrogenase-like short-chain dehydrogenase/reductase (E-283 value 1e-47), which is consistent with the proposed function. Mannose catabolism is widespread in 284 bacteria; however, it generally involves converting mannose to the glycolytic intermediate fructose-6-285 phosphate. The unusual method in Coxiellaceae instead converts mannose to pyruvate and to our 286 knowledge has not been characterized outside of archaea. Although the physiological role of this 287 pathway remains to be experimentally validated, it may enable C. burnetii to utilize host-derived 288 mannose through a distinct metabolic route that feeds directly into central carbon metabolism. 289 290 Xylose utilization was assembled from older transport and newer catabolic functions 291 C. burnetii encodes a xylose isomerase (CBU_0344a) and a xylulose kinase (CBU_0346), which likely 292 convert imported xylose to xylulose-5-phosphate, a pentose phosphate pathway intermediate (Figure 293 16). Pseudogenized versions of these genes were identified in Ornithodoros CEs, whereas no orthologs 294 were detected in other Legionellales, indicating that this cluster was likely acquired in the common 295 ancestor of C. burnetii and Ornithodoros CEs. The adjacent sugar transporter (CBU_0347), annotated 296 as a xylose-proton symporter, appears to be older and likely dates to the common ancestor of 297 Coxiellaceae and Legionella. The evolutionary history of this locus suggests that xylose utilization was 298 assembled in stages, with recent acquisition of catabolic enzymes building on an older sugar transport 299 system and potentially creating a route for host-derived sugars to enter the pentose phosphate pathway 300 in C. burnetii. 301 302 Acquired ATP-dependent phosphofructokinase added regulatory control to glycolysis 303 In addition to the inorganic pyrophosphate phosphofructokinase (PFP, CBU_1273) found throughout 304 the Legionellales, the common ancestor of Coxiella gained an ATP-dependent phosphofructokinase 305 (PFK, CBU_0341). The ancestral bidirectional PFP can function in both glycolysis and 306 gluconeogenesis, whereas the newly-acquired PFK reaction is generally considered irreversible and 307 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 14 therefore only glycolytic (Figure 16). PFKs also typically contain an allosteric site, which allows 308 tighter regulation of pathway flux [Compton & Patrick, 2025], thereby likely enhancing C. burnetii’s 309 regulatory control over glycolysis. Acquisition of ATP-dependent PFK added a second 310 phosphofructokinase with biochemical properties distinct from the ancestral PFP, potentially allowing 311 C. burnetii to direct carbon flux toward glycolysis with greater regulatory control as nutrient 312 availability fluctuates within the CCV. 313 314 Respiratory-chain simplification reduced reliance on ROS-prone cytochrome c systems 315 The common ancestor of Coxiella and Paracoxiella lost all cytochrome c-associated systems, 316 including the cytochrome caa3 terminal oxidase, cytochrome bc1 complex, and cytochrome c 317 maturation machinery that are present in most other Legionellales (Figure 17). These systems can be 318 bypassed because Coxiella and Paracoxiella encode ubiquinol terminal oxidases that accept electrons 319 directly from NADH dehydrogenase and succinate dehydrogenase. Coxiella, Paracoxiella, and 320 Aquicella encode a cytochrome bo3 ubiquinol oxidase (CBU_1035d, CBU_1038-CBU_1040), 321 suggesting that this system was acquired in the common ancestor of Coxiellaceae. Unlike the ancestral 322 cytochrome bd ubiquinol oxidase, cytochrome bo3 acts as a proton pump [Siletsky & Borisov, 2021]. 323 Cytochrome bo3 is the only terminal oxidase retained in all CEs, possibly because proton pumping 324 remains advantageous even in the symbiotic lifestyle. Acquisition of cytochrome bo3 may have 325 reduced the selective pressure to maintain cytochrome c-dependent systems. Loss of those systems 326 may in turn benefit C. burnetii in its present environment because the cytochrome bc1 complex is 327 prone to generating reactive oxygen species when challenged by a steep proton gradient [Lanciano et 328 al., 2013; Pagacz et al., 2025]. Loss of cytochrome c-dependent systems alongside retention of 329 ubiquinol terminal oxidases indicates a shift toward a streamlined respiratory chain that may preserve 330 energy conservation while reducing dependence on complexes that could be costly in the acidic CCV 331 environment. 332 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 15 333

334 Our analyses support a model in which vertebrate pathogenicity in C. burnetii emerged through the 335 stepwise assembly and remodeling of traits within an ancestral pathogenic background. Earlier 336 phylogenomic work showed that soft-tick Coxiella endosymbionts retain remnants of a more virulent 337 ancestor, placing C. burnetii within a broader evolutionary history of host association [Brenner et al., 338 2021]. Building on that framework, our trait-by-trait reconstruction of gene gain and loss shows that 339 key infection-associated traits trace to ancestors at different evolutionary depths. This Legionellales-340 wide analysis indicates that many systems central to C. burnetii pathogenesis, including traits 341 associated with vertebrate infection and replication in the Coxiella-containing vacuole, predate the 342 modern pathogen and accumulated across multiple ancestral lineages. 343 Comparative studies of other bacterial pathogens have shown that virulence evolution 344 commonly involves gene acquisition, gene loss, pseudogenization, genome rearrangement, and 345 modification of preexisting cellular functions. In Yersinia, genomic studies have emphasized how 346 mammalian pathogenesis evolved through a mixture of gene gain, gene loss, and genome 347 rearrangement [McNally et al., 2016]. In Shigella, genomic plasticity and acquisition of a distinctive 348 virulence plasmid enabled the repeated emergence of invasive pathogens from different Escherichia 349 coli ancestors, while accelerated gene loss and pseudogenization accompanied specialization to a 350 restricted host-associated niche [The et al., 2016; Hershberg et al., 2007]. Salmonella provides another 351 example in which horizontally acquired pathogenicity islands encode secretion systems and effectors 352 that are central to host interaction and invasion [Lou et al., 2019]. Work on Vibrio cholerae adds an 353 ecological dimension, showing that environmental populations can harbor virulence-associated alleles, 354 mobile elements, and pathogenic traits before the emergence of successful pathogenic clones [Shapiro 355 et al., 2016; Sakib et al., 2018; López-Pérez et al., 2025]. 356 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 16 These examples largely involve environmental, enteric, or facultatively host-associated 357 bacteria, where access to diverse microbial communities can provide repeated opportunities for gene 358 exchange. In obligately intracellular lineages, opportunities for horizontal gene transfer may be more 359 episodic, depending on rare ecological contexts such as coinfection of the same host cell [Bordenstein 360 and Reznikoff, 2005; Bordenstein and Wernegreen, 2004; Duron, 2013]. C. burnetii therefore provides 361 an opportunity to examine how virulence-related traits accumulate in a specialized intracellular 362 lineage. Our study shows that the modern C. burnetii virulence profile was shaped by familiar 363 evolutionary processes, including HGT, gene loss, retention, duplication, and repurposing, but that 364 these changes were layered across longer evolutionary timescales. 365 The evolutionary history of the Dot/Icm type IVB secretion system is consistent with this 366 model. Most structural components of the secretion apparatus were already widespread across 367 Legionellales, indicating that the machinery itself is ancient within the order. In contrast, the effector 368 repertoire associated with C. burnetii appears to be much younger and concentrated within Coxiella 369 and its closest relatives. The common ancestor shared with Ornithodoros endosymbionts likely 370 encoded most C. burnetii effectors, implying that much of the host-manipulation capacity now 371 associated with vertebrate infection was already present before the emergence of the modern pathogen. 372 At the same time, the limited distribution of many effectors outside Coxiella and Paracoxiella suggests 373 that effector repertoires remain evolutionarily labile and are shaped strongly by lineage-specific host 374 associations. This pattern fits the broader view that secretion systems can be conserved over long 375 periods whereas their cargo proteins turn over more rapidly as intracellular bacteria adapt to different 376 eukaryotic environments. 377 Our reconstruction of lipopolysaccharide evolution similarly suggests that key immune-evasion 378 traits were assembled in stages rather than acquired all at once. Several changes affecting the C. 379 burnetii envelope appear to have accumulated along successive Coxiella ancestors, including lipid A 380 simplification, modifications to the inner core, and acquisition of genes associated with synthesis of 381 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 17 unusual O-antigen sugars. Inner-core remodeling provides one plausible route for altered host 382 recognition. Loss of GmhD has shifted heptose stereochemistry from LD- to DD-heptose, which may 383 reduce recognition by host factors that respond differently to heptose isomers [Amano et al., 1987; 384 Toman & Skultéty, 1996]. In parallel, acquisition of CBU_1657 introduced a mannose substitution at 385 HepI, and substitutions at HepI or HepII are broadly conserved among pathogenic bacteria and can 386 influence serum susceptibility. The specific contribution of the C. burnetii mannose substitution 387 remains unresolved, but these observations suggest that inner-core remodeling could affect both 388 immune recognition and envelope interactions. The O-antigen shows a similar pattern of staged 389 evolution: the common ancestor of C. burnetii and Ornithodoros endosymbionts may already have 390 possessed several genes implicated in virenose synthesis, a distinctive sugar that is critical for full O-391 antigen elaboration and vertebrate virulence in C. burnetii. More generally, the inferred sequence of 392 LPS changes argues against a single decisive innovation and instead supports progressive remodeling 393 of the cell surface, with each step potentially altering resistance to host recognition, antimicrobial 394 factors, or other environmental pressures. 395 Several traits associated with stress tolerance also appear to have accumulated before the 396 emergence of modern C. burnetii. Horizontal acquisition of a polyamine biosynthesis pathway, 397 addition of atypical ammonia-producing steps in arginine metabolism, and changes in fatty acid 398 synthesis and desaturation all have plausible connections to survival under acidic, oxidative, or 399 otherwise stressful conditions. These functions are not specific to the Coxiella-containing vacuole, and 400 some may originally have supported tolerance of broader environmental or host-associated stresses. 401 This is important because the CCV, in addition to being acidic, is rich in lysosomal enzymes, reactive 402 oxygen species, osmotic stress, and host-derived nutrients. The inferred history therefore suggests that 403 C. burnetii acid tolerance may have emerged from a combination of general stress-resilience traits and 404 more niche-relevant systems. In this context, the Mrp cation/proton antiporter is notable because it 405 stands out as a more direct candidate for improved pH homeostasis. Its apparent essentiality in C. 406 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 18 burnetii, coupled with loss or pseudogenization in tick endosymbionts, is consistent with the idea that 407 it became especially important in the acidic intracellular compartment exploited by the pathogen. 408 Together, these patterns suggest that growth in the acidic CCV was enabled by a layered set of traits, 409 combining ancestral general stress-resilience functions with recently acquired systems more directly 410 tied to pH homeostasis. 411 Carbon metabolism and respiration provide a parallel example of sequential trait assembly. The 412 mature CCV is nutrient rich, and C. burnetii is able to use both amino acids and sugars as carbon and 413 energy sources. Our analyses suggest that this flexibility reflects several distinct evolutionary events 414 rather than one broad metabolic expansion. Coxiellaceae acquired an unusual mannose catabolism 415 pathway predicted to route mannose-derived carbon into central metabolism. Xylose utilization 416 appears to have been assembled in stages, with recent acquisition of catabolic enzymes building on an 417 older sugar transporter that may have broader substrate specificity, including glucose transport [Kuba 418 et al., 2019]. The addition of ATP-dependent phosphofructokinase alongside the ancestral 419 pyrophosphate-dependent enzyme may also have increased regulatory control over carbon flux through 420 glycolysis. These changes occurred against a background of respiratory chain remodeling, including 421 loss of cytochrome c-associated systems and reliance on ubiquinol terminal oxidases. This respiratory 422 architecture eliminated dependence on cytochrome c-associated complexes that can generate reactive 423 oxygen species under steep proton gradients but also reduces the overall proton pumping efficiency of 424 the respiratory chain, which may have increased reliance on glycolysis as an energy source [Noda et 425 al., 2006; Kihira et al., 2012; Cordero et al., 2022]. Together, these metabolic changes suggest that C. 426 burnetii both expanded its capacity to use host-derived carbon sources and refined control over central 427 carbon metabolism, consistent with adaptation to a chemically complex intracellular compartment. 428 Several limitations should guide interpretation of these results. Comparative genomics can 429 identify plausible gains, losses, duplications, and horizontal transfers, but targeted experiments will be 430 needed to determine how these evolutionary changes affect C. burnetii physiology, intracellular 431 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 19 growth, and virulence. For instance, some functional assignments, including parts of the virenose 432 pathway, the mannose catabolism route, and the role of Mrp, require biochemical or genetic validation. 433 Inferred evolutionary timing also depends on taxon sampling, orthology detection, and the availability 434 of high-quality genomes from close relatives. Additional Coxiella-like bacteria, especially from 435 understudied hosts, may refine the placement of individual acquisitions and losses. Even with these 436 caveats, the major pattern is consistent across multiple systems: traits relevant to C. burnetii 437 pathogenesis have different evolutionary depths and were combined gradually within a host-associated 438 lineage, with ancestral functions later co-opted for survival and growth in the modern CCV. 439 Overall, our findings refine the evolutionary narrative of C. burnetii. Rather than emerging 440 through abrupt acquisition of pathogenicity, C. burnetii is better understood as the product of a longer 441 process in which ancestral host-interaction machinery was retained while envelope structure, stress 442 tolerance, and metabolism were repeatedly modified. This framework helps explain how a member of 443 Legionellales came to specialize in a vertebrate-associated, acidic intracellular niche and identifies 444 specific evolutionary steps that can now be tested experimentally for their contributions to C. burnetii 445 virulence and intracellular growth. More broadly, our results suggest that specialized intracellular 446 pathogens can emerge through long-term remodeling of ancestral host-associated lineages, with older 447 traits retained, repurposed, or combined with later gains and losses. 448 449

450 Genome Sequencing and Assembly 451 A female O. peruvianus tick was collected from a cave inhabited by Desmodus rotundus bats on Pan 452 de Azucar Island. DNA was extracted from the tick using the DNeasy Blood & Tissue Kit (Qiagen) 453 and submitted to the Yale Center for Genome Analysis for Illumina (NovaSeq) sequencing. The 454 resulting 150-bp paired-end reads were trimmed with Trimmomatic [Bolger et al., 2014] using the 455 following parameters: ILLUMINACLIP:2:30:10 SLIDINGWINDOW:5:25 LEADING:20 456 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 20 TRAILING:20 MINLEN:50. After trimming, approximately 490 million read pairs were retained. 457 Reads were assembled into contigs with SPAdes v3.13.0 [Nurk et al., 2017] using default parameters. 458 Open reading frames were identified with Prodigal [Hyatt et al., 2010]. Contigs were then binned with 459 CONCOCT [Alneberg et al., 2014] on the basis of coverage and k-mer composition. The Coxiella-460 containing bin was identified by BLASTn and BLASTp using a database of Coxiellaceae genomes. 461 Approximately 13 million paired reads mapped to the Coxiella bin and were used for a final assembly, 462 yielding 112 contigs. Read mapping was performed with Bowtie2 [Langmead and Salzberg, 2012] 463 using a database of Coxiellaceae genome sequences. The O. peruvianus CE genome contains 106 464 highly conserved single-copy genes as defined by Albertsen et al. (2013), comparable to the number 465 typically found in C. burnetii strains. Final genome annotation was performed with the NCBI 466 Prokaryotic Genome Annotation Pipeline [Tatusova et al., 2016]. Sequence data have been deposited 467 under BioProject PRJNA1189154, BioSample SAMN44862134, SRA accession SRR31639864, and 468 WGS master accession JBLIZD000000000. Assembly and annotation statistics for the O. peruvianus 469 CE are provided in Table 1. 470 471 Orthogroups 472 OrthoFinder v3.1.0 [Emms & Kelly, 2019] was used to infer protein orthogroups for both tree 473 construction and ancestral state reconstruction. The program was run multiple times with MCL 474 inflation parameters of 1.2, 2.0, 2.5, and 3.0 to assess whether orthogroup assignments for focal genes 475 were robust to clustering granularity. Orthogroup counts reported in Table 1 were generated using an 476 inflation parameter of 2.0. Protein-coding genes annotated as pseudogenized were translated into 477 amino acid sequences and included in the orthogroup analysis. The full genome set used for 478 orthogroup inference is listed in Table S1. 479 480 481 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 21 Phylogenomic Analyses 482 The Coxiella genus-level phylogenomic tree was constructed from protein sequences encoded by 78 483 genes (Table S2), whereas the Legionellales tree was constructed from 152 genes (Table S3). In both 484 cases, single-copy genes were identified from the orthogroup results, and genes annotated as 485 pseudogenized were excluded from tree building. Individual protein sequences were aligned with 486 MAFFT v7.490 [Katoh & Standley, 2013] using the L-INS-i/globalpair iterative refinement strategy 487 and then trimmed with trimAL v1.5.1 using the gappyout method [Capella-Gutiérrez et al., 2009]. The 488 resulting alignments were concatenated and partition boundaries were recorded. Maximum-likelihood 489 trees were inferred with IQ-TREE v3.0.1 [Wong et al., 2026]. ModelFinder was used for model 490 selection, with the candidate set restricted to amino acid substitution matrices appropriate for bacteria. 491 Branch support was estimated using 100 nonparametric bootstrap replicates. 492 Because many CEs have highly reduced genomes, including them in the order-level 493 phylogenomic analysis substantially reduced the number of single-copy protein-coding genes available 494 for tree construction. We therefore analyzed the Coxiella genus tree separately from the broader 495 Legionellales tree to maintain robust gene sampling in both datasets. Coxiellaceae MAGs lacking 496 species-level classification and originating from multi-isolate projects were evaluated for completeness 497 before inclusion. MAGs without annotations were first annotated with Prokka v1.14.5 [Seemann, 498 2014]. Genomes were excluded if they contained fewer than 100 of the highly conserved single-copy 499 genes defined by Albertsen et al. (2013), a threshold that excluded more than half of the available 500 genomes. After preliminary tree construction, additional genomes were removed if their placements 501 were unstable across analyses using different gene sets (70 to 152 genes) or if they consistently showed 502 low branch support. The genomes retained in the final analyses, together with their accession numbers 503 and host/source information are provided in Table S1. 504 Ancestral reconstruction and HGT inference were performed by applying Orthofinder results 505 and the Legionellales and Coxiella ML trees (Figure 1) to PastML using the MPPA and F81 settings 506 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 22 [Emms & Kelly, 2019; Ishikawa et al., 2019]. Pseudogenized orthologs were coded as present for 507 ancestral-state reconstruction but interpreted as evidence of subsequent loss of function in extant taxa. 508 For each C. burnetii gene, the origin node was determined to be the oldest node at which posterior 509 probability was ≥0.7, with all subsequent nodes also having posterior probability of ≥0.7, as described 510 previously [Peer & Margalit, 2014]. Metabolic pathways and supplemental annotations were collected 511 from KEGG using BlastKOALA [Kanehisa & Goto, 2000; Kanehisa et al., 2016], RAST Subsystems 512 [Aziz et al., 2008], and the Conserved Domain Database using RPS-BLAST [Marchler-Bauer et al., 513 2015]. Findings for focal genes were further confirmed using individual protein trees as described 514 below. 515 For individual protein trees, homologs from taxa outside Legionellales were retrieved from the 516 NCBI nonredundant protein (nr) database using BLASTp. The top 50 to 70 hits ranked by E-value 517 were retained; the exact number varied when multiple hits shared the same E-value so that equivalent 518 matches were not excluded arbitrarily. No more than five representatives from the same genus and no 519 more than one representative from the same species were included. Alignments and maximum-520 likelihood trees were generated as described above. Branch support was evaluated with 1,000 ultrafast 521 bootstrap replicates. Figures were prepared with iTOL v7.5.1 [Letunic & Bork, 2024]. 522 523 DATA AVAILABILITY 524 Sequence data have been deposited under accessions JBLIZD000000000 (WGS master), PRJNA1189154 525 (BioProject), SAMN44862134 (BioSample), and SRR31639864 (SRA). 526 527 ACKNOWLEDGMENTS 528 We thank Sebastian Munoz-Leal and Daniel Gonzalez-Acuna for collecting Ornithodoros peruvianus 529 ticks. This work was supported in part by the University of Texas at San Antonio and National Institute 530 of Allergy and Infectious Diseases grant AI126385. 531 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 23 532

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Nature Chemical Biology, 14(6), 850 575–581. doi:10.1038/s41589-018-0038-z 851 Zhou, P., She, Y., Dong, N., Li, P., He, H., Borio, A., Wu, Q., Lu, S., Ding, X., Cao, Y., Xu, Y., Gao, 852 W., Dong, M., Ding, J., Wang, D. C., Zamyatina, A., & Shao, F. (2018). Alpha-kinase 1 is a 853 cytosolic innate immune receptor for bacterial ADP-heptose. Nature, 561(7721), 122–126. 854 doi:10.1038/s41586-018-0433-3 855 856 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 31 FIGURE LEGENDS 857 Figure 1. Legionellales members are found in diverse hosts or environments and have varied genome 858 sizes. Tips of the tree are colored to indicate the host or environment from which they were collected. 859 Circles represent the range of genome sizes within the genus. The Legionella sp. clade has been 860 collapsed for brevity. All nodes have bootstrap values of 100 except for those with listed values. The 861 Coxiella ML tree (top) was generated using 78 protein sequences (Table S2). The Legionellales ML 862 tree (bottom) was generated using 152 protein sequences (Table S3). 863 864 Figure 2. Most type IVB secretion system components are present throughout Legionellales. (A) 865 Conservation of T4BSS genes present in C. burnetii: genes shown in color have homologs in Tra/Trb 866 conjugation system (shown below), genes highlighted in grey boxes are absent in one or more 867 Legionellales taxa, and genes without highlights have been maintained throughout Legionellales, 868 excluding the CEs. (B) Summary of major gains (red arrows) and losses (red crosses, red asterisks) in 869 T4BSS evolution, dashed gray lines indicate branches where T4BSS has been lost or pseudogenized.870 871 872 Figure 3. Protein phylogeny of LbtP-like sequences. The clade in red contains C. burnetii MceB and 873 its orthologs found in Paracoxiella and all of the CEs. The clade in green contains C. burnetii 874 CBU_1699 and its orthologs found in Paracoxiella and few CEs. Rickettsiella and Aquicella each have 875 only one LptP-type gene. Bootstrap values are labeled for major branches. 876 877 Figure 4. Summary of LPS structural changes in Coxiellaceae. (A) Components of LPS. (B) Gain and 878 loss events. 879 880 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 32 Figure 5. Distribution of genes likely required for virenose production. (A) Putative pathway for 881 virenose synthesis. (B) Distribution of virenose synthesis genes across Legionellales. PMM: 882 phosphomannomutase / phosphoglucomutase, GMP: GDP-mannose pyrophosphorylase, GMD: GDP-883 mannose-4,6-dehydratase. 884 885 Figure 6. CBU_0678 was likely acquired from Alphaproteobacteria. Bootstrap values are labeled for 886 major branches. Mauve: Alphaproteobacteria; Blue: C. burnetii and Ornithodoros CEs. 887 888 Figure 7. Synthesis of LD-heptose was lost in the common ancestor of C. burnetii and Ornithodoros 889 CEs. (A) Pathway for generating L,D-Heptose. (B) GmhD, the enzyme responsible for converting DD-890 heptose to LD-heptose, is absent in C. burnetii. 891 892 Figure 8. b-1,4 manosyltransferase (CBU_1657) may have been acquired from 893 Thermodesulfobacteria. Bootstrap values are labeled for major branches. Blue: Coxiella and 894 Paracoxiella; Red: Thermodesulfobacteria; Green: Proteobacteria. 895 896 Figure 9. Coxiellaceae appears to have acquired a polyamine synthesis gene cluster (CBU_0720-22) 897 from Alphaproteobacteria. Coxiella, Paracoxiella, and Rickettsiella are highlighted in blue while 898 Alphaproteobacteria are highlighted in mauve. A representative ML tree of CBU_0722 is shown here. 899 ML trees of CBU_0720 and CBU_0721 produced similar results. Bootstrap values are labeled for 900 major branches. 901 902 Figure 10. Pathways for polyamine synthesis and arginine metabolism in C. burnetii. 903 904 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 33 Figure 11. CBU_0279 was likely acquired in the common ancestor of Coxiella and Paracoxiella 905 (blue) from cyanobacteria (green). Homologs were also found in three Legionella species (purple). 906 Bootstrap values are labeled for major branches. 907 908 Figure 12. Ornithine cylcodeaminase (CBU_1727) may have been horizontally acquired. Coxiella and 909 Paracoxiella are shown in blue. The closest homolog was found in an archaeon (green), while most 910 others were in various proteobacteria (orange). Bootstrap values are labeled for major branches. 911 912 Figure 13. Summary of changes to fatty acid synthesis/modification in the lineage leading to C. 913 burnetii. 914 915 Figure 14. Potential horizontal acquisition of FabA (CBU_0037) in the common ancestor of Coxiella. 916 Closest homologs of Coxiella’s FabA (blue) are found in a diverse group of bacteria including 917 Proteobacteria (green), spirochetes (yellow), and Thermodesulfobacteriota (red). Bootstrap values are 918 labeled for major branches. 919 920 Figure 15. Distribution of Mrp cation/proton transporter across Legionellales. 921 922 Figure 16. Changes in sugar catabolism, glycolytic control, and respiratory metabolism. (A) Summary 923 of gene gain events that have affected sugar utilization in the lineage leading to C. burnetii. (B) Sugar 924 and sugar alcohol catabolism pathways in C. burnetii. AldT: aldohexose dehydrogenase; DHAP: 925 dihydroxyacetone phosphate; eda: 2-dehydro-3-deoxyphosphogluconate aldolase; F-1,6-diP: Fructose-926 1,6-bisphosphatase; F6P: Fructose-6-phosphatase; G3P: glyceraldehyde 3-phosphate; KDG: 2-keto-3-927 deoxygluconate; KdgK: 2-dehydro-3-deoxygluconokinase; PFK: ATP-dependent 928 phosphofructokinase; PFP: inorganic pyrophosphate-phosphofructokinase; UQ: ubiquinone; UQH2: 929 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 34 ubiquinol; UxuA: mannonate dehydratase; X5P: xylulose 5-phosphate; XI: xylose isomerase; XK: 930 xylulokinase 931 932 Figure 17. Distribution of cytochrome genes in Legionellales. bo3: cytochrome bo3 ubiquinol oxidase; 933 bd: cytochrome bd ubiquinol oxidase; caa3: cytochrome caa3 oxidase; bc1: cytochrome bc1 complex; 934 Ccm: cytochrome c maturation machinery. 935 936 937 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 35 TABLES 938 Table 1. Coxiella genome characteristics. 939 Taxa Genome Size (Mb) Protein Coding Genes Pseudogenes Orthogroups Assembly Accession Coxiella burnetii RSA 493 2.033 1,833 207 1758 GCF_000007765.2 CE Ornithodoros peruvianus 1.672 1,250 638 1523 CE Ornithodoros amblus 1.561 1,049 534 1352 GCF_019425495.1 CE Ornithodoros maritimus 1.671 1,225 622 1475 GCF_907164965.1 CE Amblyomma nuttalli 1.003 668 16 657 GCF_018107685.1 CE Haemaphysalis longicornis 0.987 716 26 718 GCA_048491735.1 CE Rhipicephalus microplus 1.565 638 163 684 GCF_002871095.1 CE Rhipicephalus appendiculatus 1.449 1,222 206 1190 GCF_030643785.1 CE Rhipicephalus sanguineus 1.716 1,535 104 1272 GCF_002804145.1 CE Rhipicephalus turanicus 1.734 1,626 151 1329 GCF_001077715.1 CE Haemaphysalis japonica 0.878 679 16 666 GCA_048491875.1 CE Haemaphysalis qinghaiensis 0.884 689 18 674 GCA_048492375.1 CE Dermacentor marginatus 0.901 637 18 630 GCF_907164955.1 CE Dermacentor silvarum 0.887 637 17 633 GCA_048493135.1 CE Dermacentor nuttalli 0.888 641 18 639 GCA_048494595.1 CE Amblyomma americanum 0.657 559 7 557 GCF_000815025.1 CE Amblyomma americanum 0.657 559 7 557 GCF_000815025.1 CE Amblyomma sculptum 0.623 526 9 528 GCF_009883795.1 940 941 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 36 Table 2. Presence of C. burnetii effector orthologs in CEs or Paracoxiella. 942 locus Acronym Coxiella Endosymbionts Paracoxiella cheracis CBU_0175 CstK ✓ ✓ CBU_0513 CinF ✓ ✓ CBU_0937 MceB/CirC ✓ ✓ CBU_1425 MceC ✓ ✓ CBU_1863 CvpE ✓ ✓ CBU_0021 CvpB/Cig2 ✓ CBU_0041 CirA/CoxCC1 ✓ CBU_0077 MceA ✓ CBU_0122 CvpM ✓ CBU_0388 CetCb2 ✓ CBU_0447 AnkF ✓ CBU_0635 - ✓ CBU_0781 AnkG ✓ CBU_1370 CbEPF1 ✓ CBU_1387 EmcA/Cem6 ✓ CBU_1751 Cig57 ✓ CBU_2007 Vice ✓ CBU_2013 EmcB ✓ CBUD_0462 CaeA ✓ CBUA0013 CpeB ✓ CBU_0425 CirB CBU_0626 CvpF CBU_0665 CvpA CBU_1217 NopA CBU_1314 coxCC6 CBU_1531 CaeB CBU_1823 IcaA 943 944 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 37 Table 3. Distribution of periplasmic lipid A modification genes across Legionellales 945 Cellular Localization Coxiella burnetii CEs Paracoxiella cheracis Rickettsiella isopodorum Aquicella siphonis Legionella pneumophila Berkiella cookevillensis LpxA Cytosolic 1 1 1 1 1 2 1 LpxC Cytosolic 1 1 1 1 1 1 1 LpxD Cytosolic 1 1 1 1 1 3 1 LpxH Cytosolic 1 1 1 1 1 1 1 LpxB Cytosolic 1 1 1 1 1 2 1 LpxK Cytosolic 1 1 1 1 1 1 1 KdtA Cytosolic 1 1 1 1 1 1 1 lpxL/P Periplasmic 0 0 0 1 0 3 0 LpxO Periplasmic 0 0 0 1 0 0 0 ArnTa Periplasmic 0 0 0 1 1 0 0 PagP Periplasmic 0 0 0 0 0 1 0 PagL Periplasmic 0 0 2 1 1 0 1 a in addition to other arn genes 946 947 948 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint 38 SUPPLEMENTAL INFORMATION 949 Table S1. Taxa used in analysis. 950 Table S2. Proteins used to build Coxiella phylogenomic tree. 951 Table S3. Proteins used to build Legionellales phylogenomic tree. 952 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 1. Legionellales members are found in diverse hosts or environments and have varied genome sizes. Tips of the tree are colored to indicate the host or environment from which they were collected. Circles represent the range of genome sizes within the genus. The Legionella sp. clade has been collapsed for brevity. All nodes have bootstrap values of 100 except for those with listed values. The Coxiella ML tree (top) was generated using 78 protein sequences (Table S2). The Legionellales ML tree (bottom) was generated using 152 protein sequences (Table S3). Piscirickettsia salmonis ASM970895v1GCA 947478905.1B aquae HT99GCA 963976875.1B cookevillensis CC99 GCA 964647825.1GCA 964612855.1GCA 964466595.1GCA 964449835.1GCA 964597435.1GCA 964492045.1GCA 964527435.1GCA 964648015.1A siphonisA lusitana SGT-39GCA 964341295.1RLE Dermanyssus gallinaeR viridisRLE OperuvianusGCA 964417555.1Aquirickettsiella gammari ASM229064v2R grylliR isopodorumRLE Litargus connexusGCA 965662625.1GCA 964341305.1GCA 049797115.1RLE Miltochrista miniataRLE Aleochara curtulaGCA 037439325.1GCA 036495935.1RLE Xylota segnisRLE Rhagonycha lignosaParacoxiella cheraxiCLE Rsanguineus CRS-CATCLE Operuvianus 90 89 98 98 79 88 Rickettsiella 1.5 - 1.9 Mb Aquicella 1.8 - 3.2 Mb Berkiella Amoeboid protists / Freshwater / Human infection Activated sludge Amoeboid protist (Acanthamoeba polyphaga) Amoeboid protist (Acanthamoeba polyphaga) Freshwater lake Frog gut contents Frog gut contents Frog gut contents Salmon (Salmo salar) Marine plankton Arctic Ocean seawater Crayfish (Cherax quadricarinatus) Soldier beetle (Rhagonycha lignosa) Hover fly (Xylota segnis) Rove beetle (Aleochara curtula) Moth (Miltochrista miniata) Hard tick (Ixodes ricinus) Hairy fungus beetle (Litargus connexus) Woodlouse (suborder: Oniscidea) Pill bug (family: Armadillidiidae) Amphipod (Gammarus fossarum) Soft tick (Ornithodoros peruvianus) Soft tick (Ornithodoros phacochoerus) Rove beetle (Aleochara curtula) Red mite (Dermanyssus gallinae) Hard tick (Ixodes ricinus) Grassland soil hydrothermal sediments soil from urban greenspace geothermal spa Effluent from manure-amended soils Agricultural soil Agricultural soil Effluent from manure-amended soils geothermal spa Host: arthropod environmental: ground water, soil, subsurface environmental: marine Host: protist Host: vertebrate environmental: other Paracoxiella cheracis 2.3 Mb Coxiella burnetii 2.0 - 2.2 Mb Legionella Piscirickettsia salmonis Coxiella endosymbionts 0.6 - 1.7 Mb Soft tick (Ornithodoros amblus) Hard tick (Amblyomma nutalli) Hard tick (Haemophilus longicornis) Soft tick (Ornithodoros maritimus) Mammals Soft tick (Ornithodoros peruvianus) Paracoxiella cheraxi CLE Asculptum CLE Aamericanum C904 CLE Dnuttalli NX032 CLE Dsilvarum NX090 1 CLE Dmarginatus CLE Hqinghaiensis NX126 CLE Hjaponica NX147 1 CLE Rturanicus CLE Rsanguineus CRS-CAT CLE Rappendiculatus CLE Rmicroplus CLERM CLE Hlongicornis NX151 CLE Anuttalli CLE Oamblus CLE Omaritimus CLE Operuvianus Cburn Dugway 5J108-111 Hard tick (Rhipicephalus microplus) Hard tick (Rhipicephalus appendiculatus) Hard tick (Rhipicephalus sanguineus) Hard tick (Rhipicephalus turanicus) Hard tick (Haemophilus japonica) Hard tick (Haemophilus qinghaiensis) Hard tick (Dermacentor marginatus) Hard tick (Dermacentor silvarum) Hard tick (Dermacentor nuttalli) Hard tick (Amblyomma americanum) Hard tick (Amblyomma sculptum) 88 92 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 2. Most type IVB secretion system components are present throughout Legionellales. (A) Conservation of T4BSS genes present in C. burnetii: genes shown in color have homologs in Tra/Trb conjugation system (shown below), genes highlighted in grey boxes are absent in one or more Legionellales taxa, and genes without highlights have been maintained throughout Legionellales, excluding the CEs. (B) Summary of major gains (red arrows) and losses (red crosses, red asterisks) in T4BSS evolution, dashed gray lines indicate branches where T4BSS has been lost or pseudogenized. * Coxiella bu rnetii str. RSA493 Co xiella bu rne tii str. Dugwa y Legionella CEs of soft ticks Paracoxiella CEs of hard ticks Rickettsiella Aquicella Berkiella Piscirickettsia CE_of_soft_t icks Coxiella_burnetii_ 1 Legionell a CE_o f_hard_ ticks Piscirickettsi a Aquicell a Berkiell a Paracoxiell a Coxiella_burnetii_ 2 Rickettsiell aIcmX and IcmQ gain Tra/Trb chromosomal integration IcmF and IcmH lost CoxigA gain * * † † IcmX lost IcmS gain B Dot/Icm T4BSS C. burnetii Tra/Trb orthologs IncI R64 plasmid YUTRQPONMtraH I J K W X trbA CB Fx3HBJDCGEKLx2NOPQCoxigATSVWX dotA dotB dotDdotC A Absent in full genera Absent in specific taxa .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 3. Protein phylogeny of LbtP-like sequences. The clade in red contains C. burnetii MceB and its orthologs found in Paracoxiella and all of the CEs. The clade in green contains C. burnetii CBU_1699 and its orthologs found in Paracoxiella and few CEs. Rickettsiella and Aquicella each have only one LptP-type gene. Bootstrap values are labeled for major branches. A siphon is AQULUS R S0756 0 WP 14833 9464.1 NZ LR 699119.1 LbtU fami ly sidero phore p orin Aquic ella GC A 96459 7435.1 P MCMDPGO 017 80 hypothetica l protein Aquic ella GC A 96449 2045.1 M KGPPE BM 0270 8 hypothetical p rotein Aquic ella GC A 96452 7435.1 NE NKCLC M 02427 hyp othetical pr otein Aquicella GC A 96464 8015.1 AMCDIMJA 00272 hyp othetical prote in A lusitana SGT-39 AQUSI P RS040 00 WP 114834496.1 NZ LR 699114.1 LbtU fami ly sidero phore porin Aquicella GCA 964612855.1 GPCGICCG 000 94 hypothetical p rotein Aquicella GCA 964647825.1 HECNCAJE 01286 hypoth etical protein Aquicella GCA 964449835.1 NJHPCBPC 00976 hypothetical protein Aquicella GCA 964466595.1 CEKOKCPA 00500 hypothetical protein CLE Dn uttalli NX0 32 AB 8V08 02 850 MFV 999090 5.1 JBGDBH0 100000 01.1 LbtU family si derop hore po rin CLE Dsi lvaru m NX090 1 AB 8W12 01355 M FW006757 8.1 JBGDED0 100000 01.1 LbtU family si derop hore po rin CLE Dsi lvaru m NX123 1 AB 8Y17 023 85 MFW007 4739.1 JBGDF K0100 00006.1 LbtU fa mily side ropho re porin CLE Dn uttalli NX0 79 AB 8V81 00 025 MFW004 9658.1 JBGDD R01000 0001.1 LbtU fam ily sider ophor e porin CLE D margi natus ONG85 RS00 140 W P 26443 5572.1 NZ OU01 5521.1 LbtU fami ly sidero phor eporin CLE Ra ppendic ulatus MRH 55 RS04 590 W P 30498 5093.1 NZ CP 094378.1 C BU 0937 fa mily por in CLE R microp lus CLER M CLER M RS03 480 W P 102157 200.1 NZ NS HJ01000 150.1 LbtU fami ly sidero phore p orin CLE Rsa nguine us CRS- CAT CVD1 3 RS044 60 W P 157843 747.1 NZ CP 024961.1 L btU family side ropho re porin nirop erohporedis ylimaf UtbL 1.621110PC ZN 1.538078751 P W 59340SR tRelC sucinarutR ELC nirop erohporedis ylimaf UtbL 1.110000010NGDGBJ 1.4788900 WFM 08030 30Z8BA 1 641XN acinopajH ELCCLE Hq inghai ensis NX1 38 AB 8Y84 017 85 MFW008 0387.1 JBGDGG010 000012.1 L btU family side ropho re por in CLE Hq inghai ensis NX1 26 AB 8Y58 028 60 MFW008 1268.1 JBGDFO0 100000 20.1 LbtU family si derop hore po rin CLE Hj aponica N X147 1 AB8 Z13 03005 M FW01073 82.1 JBGDGP01 000000 9.1 LbtU family sid eroph ore po rin CLE Aam erican um C904 Z 664 RS02 435 W P 15696 2725.1 NZ C P00754 1.1 LbtU family sid eroph ore por in CLE Asculptu m EGQ50 RS0 2315 W P 1597482 00.1 NZ CP0 33868.1 LbtU fa m ily side ropho re porin CLE Hl ongico rnis NX1 51 AB 8Z32 0221 0 MFW0104 352.1 JBGDGU01 000000 2.1 LbtU family sid eroph ore po rin CLE Hl ongico rnis NX1 55 AB 8Z33 0267 5 MFW0100 857.1 JBGDGY0 100000 04.1 LbtU family si derop hore po rin nirop erohporedis yli maf UtbL 1.438460PC ZN 1.344131044 P W 04910SR nAEC illattunA ELC Cburn BRASOV NYQ13 RS04680 WP 010957902.1 NZ CP103435.1 Dot/Icm type IV secretion system effector CoxDFB1 Cburn RMSFV PDI63 RS04940 WP 005768494.1 NZ CP115461.1 CBU 0937 family porin Cburn CB68 NYQ14 RS04700 WP 005768494.1 NZ CP103427.1 CBU 0937 family porinCburn CB170 NYQ18 RS04710 WP 005768494.1 NZ CP103429.1 CBU 0937 family porin Cburn Dugway 5J108-111 CBUD RS05680 WP 010957902.1 NC 009727.1 Dot/Icm type IV secreti on system effector CoxDF B1 Cburn Schperling AYO29 RS04770 WP 005768494.1 NZ CP 014563.1 C BU 0937 fam ily porin Cburn MSU Goat Q177 A35 RS04970 W P 00576 8494.1 NZ C P018150.1 CBU 093 7 family po rin Cburn nine mile phase II EP 112 RS06820 WP 005768 494.1 NZ CP 035112.1 CBU 0 937 family p orin Cburn VS42 QJ12 0 RS049 40 WP 005768 494.1 NZ AP019759.1 C BU 0937 fam ily porin Cburn CB155 N YQ16 RS0 4700 W P 005768 494.1 NZ CP 103430.1 C BU 0937 fam ily porin Cbur n KZQ3 OHM78 R S0469 0 WP 00576 8494.1 NZ C P10724 7.1 CBU 0937 fa mily por in Cbur n RSA 439 B7L74 R S0478 0 WP 01095 7902.1 NZ C P02061 6.1 Dot/Icm type IV secretion syste m effector CoxD FB1 Cbur n CbuG Q212 CBUG R S05430 W P 012570 067.1 NC 0 11527.1 D ot/Icm type IV secretion system e ffector CoxD FB1 Cbur n Scurry Q217 AYM1 7 RS055 00 W P 284698 268.1 NZ CP 014565.1 C BU 0937 fam ily porin Cbur n DogUtad N YQ19 RS05 430 W P 28469 8268.1 NZ C P103425.1 C BU 0937 fa mily por in CLE Oam blus FIV31 R S0756 5 WP 28012 4348.1 NZ V FIV0100 0099.1 LbtU fami ly sidero phore p orin CLE Oma ritimus OWO34 RS 04425 W P 26725 6685.1 NZ C AJRAM 0100000 38.1 LbtU family si derop hore po rin CLE Ope ruvianus ACJL XJ 00980 n ode003 LbtU fa mily side ropho re porin nirop erohporedis yli maf UtbL 1.584681PC ZN 1.160460324 P W 58050SR RK MJCA ixarehc alleixocaraP Par acoxiell a cheraxi ACJ M KR RS1 0910 W P 423064 246.1 NZ CP 186485.1 L btU family side ropho re porin Cbur n RS A 439 B7 L74 RS0 8735 W P 01095 8380.1 NZ C P02061 6.1 LbtU family sid eroph ore po rin Cbur n VS42 QJ12 0 RS091 35 W P 010958 380.1 NZ AP0 19759.1 LbtU fa m ily side ropho re porin Cbur n CbCVIC1 AYM0 2 RS037 55 W P 010958 380.1 NZ CP 014549.1 L btU family side ropho re porin Cbur n 701Cb B1 AYM9 4 RS067 25 W P 010958 380.1 NZ CP 014553.1 LbtU fa m ily side ropho re porin Cbur n 406 QMM2 8 RS015 70 W P 010958 380.1 NZ AP0 19757.1 LbtU fa mily side ropho re porin Cbur n RMSF V PDI63 R S09135 W P 010958 380.1 NZ CP 11546 1.1 LbtU family sid eroph ore po rin Cbur n Z3055 TY29 R S0862 5 W P 01095 8380.1 NZ LK 937696.1 L btU family side ropho re por in Cbur n 14160 -002 AYM 11 RS0 3495 W P 01095 8380.1 NZ C P01483 6.1 LbtU family sid eroph ore po rin Cbur n nine mi le phase II EP 112 RS 10795 W P 010958 380.1 NZ CP 035 112.1 LbtU fami ly sidero phore p orin Cbur n RS A 331 COX BURS A331 RS0 9355 W P 010958 380.1 NC 010 117.1 LbtU family si derop hore po rin Cbur n CbuG Q212 CBUG R S01610 W P 012569 710.1 NC 0 11527.1 L btU family side ropho re por in Cbur n DogUtad N YQ19 RS01 560 W P 01256 9710.1 NZ C P103425.1 L btU family side ropho re por in Cbur n CB170 N YQ18 RS0 8690 W P 005772 234.1 NZ CP 103429.1 LbtU fa mily side ropho re porin Cbur n CB68 N YQ14 RS08 685 W P 00577 2234.1 NZ C P103427.1 L btU family side ropho re por in Cbur n CB149 N YQ15 RS0 8545 W P 005772 234.1 NZ CP 103431.1 LbtU fa mily side ropho re porin Cbur n CB155 N YQ16 RS0 8710 W P 005772 234.1 NZ CP 103430.1 LbtU fa mily side ropho re porin Cbur n Schpe rling AYO29 R S01655 W P 005772 234.1 NZ CP 014563.1 LbtU fa mily side ropho re porin Cbur n Scur ry Q217 AYM1 7RS0 1665 W P 0125697 10.1 NZ CP0 14565.1 LbtU fa mily side ropho re porin Cbur n Dugway 5J1 08-111 CBUD R S0152 0 WP 011996 514.1 N C00972 7.1 LbtU family sid eropho re por in Cbur n KZQ3 pseud o OHM78 RS 08705 NZ C P1072 47.1 LbtU family si derop hore po rin Cbur n MSU Goat Q177 pse udo A35 R S01890 N Z CP0181 50.1 LbtU family si derop hore po rin CLE Ope ruvianus ps eudo ACJL XJ 08815 n ode074 LbtU fa mily side ropho re porin CLE Rsa nguine us CRS- CAT CVD1 3 RS010 40 WP 100622 623.1 NZ CP 024961.1 hy pothetical pr otein CLE Rtu ranicus Cl eRt RS 11205 W P 05309 7815.1 NZ C P011126.1 hypoth etical prote in Rickettsiell a GCA 964341 305.1 LKJGOED K 00084 hy pothetical pr otein Rickettsiell a GCA 965662625.1 EIC BIGED 00792 hy pothetical p rotein RLE Lita rgus conn exus ACICP9 RS00 410 WP 395497 944.1 NZ OZ195 516.1 LbtU fami ly sidero phore porin R isopod orum A1D18 RS01035 WP 071661 970.1 NZ LU KY01000029.1 LbtU fa mily siderophore porin R grylli RICGR RS064 65 WP 006035924.1 NZ AAQJ02000001.1 L btU family side rophore porin Rickettsiella GCA 049797115.1 Rickettsie lla GCA 049797115.1 WA659 05605 MGC1854827.1 JBARFJ010000023.1 LbtU family si derophore porin RLE Rhagonycha lignosa AAHI99 RS00450 WP 342227732.1 NZ OZ035011.1 LbtU family siderophore porin RLE Xylota segnis AACL18 RS00425 WP 339050614.1 NZ OZ026451.1 LbtU family siderophore porin Rickettsiella GCA 036495935.1 Rickettsiella GCA 036495935.1 VGH95 06165 HEY2567268.1 DASXLX010000054.1 LbtU family siderophore porin RLE Aleochara curtula AAHF87 RS04450 WP 342147294.1 NZ OZ034990.1 LbtU family siderophore porin RLE Miltochrista miniata AAHH40 RS00430 WP 342220159.1 NZ OZ035017.1 LbtU family siderophore porin Rickettsiella GCA 037439325.1 LFOLLN AC 01441 hypothetical p rotein Aquirickettsiella gammari ASM229064v2 CFE62 005225 R DH40151.1 NMOS02 000014.1 L btU family side rophore porin R viridis DMP0 2 RS06715 WP 126323371.1 NZ AP018005.1 LbtU fa mily siderophore porin RLE De rmanyss us gallin ae KX723 RS091 55 WP 218814 029.1 NZ CP 079094.1 L btU family side rophore porin Rickettsiell a GCA 964417 555.1 DJLDOFH A 00080 hy pothetical p rotein RLE m assiliens is 20B S3O R S01045 20 WP 156792 744.1 NZ AJGC010 00001.1 hyp othetical pr otein RLE m assiliens is 20B S3O R S01045 25 WP 010597 882.1 NZ AJGC010 00001.1 LbtU fa mily side ropho re porin Rickettsiell a GCA 964341 295.1 JPFIB ADB 00264 hy pothetical p rotein L mor avica DYH 62 RS15 600 W P 02838 2878.1 NZ UGOG01000 001.1 LbtU fami ly sidero phore p orin L quatei rensis DX Z53 RS1 6255 W P 0584748 75.1 NZ UGOW010000 01.1 LbtU family si derop hore po rin L bonon iensis IZU94 R S1693 0 WP 203 11352 9.1 NZ JADOBG01 000002 3.1 LbtU family sid eroph ore por in L worsl eiensis D X131 RS 07030 W P 058493 050.1 NZ UG PA010 00001.1 LbtU fa mily side rophor e porin L antarctica H RS36 R S17395 W P 173238 343.1 NZ AP0 22839.1 LbtU fa mily side ropho re porin L maio ricensis LO X96 RS0 3125 W P 2504205 65.1 NZ JAJK BJ0100 00002.1 Flx A-lik e family pr otein L falloni i LLAP -10 L FA RS17 180 W P 04509 7250.1 NZ LN 614827.1 LbtU fa mily side ropho re porin L pneu mophil a AS M194158v 1 AVR58 R S1530 0WP 010948 644.1 NZ CP0 13742.1 Flx A-lik e family pr otein L pneu mophil a A9E 85 RS148 60 W P 010948 644.1 NZ CP 015941.1 F lxA-l ike family pr otein L nor rlandic a OK13 RS1 2420 W P 0358912 64.1 NZ JNCF0 100007 8.1 LbtU family sid eroph ore po rin L walters ii CKW04 R S16975 W P 058480 802.1 NZ LT9064 42.1 LbtU family si derop hore po rin L shakespe arei D SM 2308 7 Lsha RS0 3245 W P 01857 8422.1 NZ LN YW01000 019.1 LbtU fami ly sidero phore p orin L israe lensis FOG18 RS 02490 W P 14386 5990.1 NZ CP 041668.1 L btU family side ropho re por in L impl etisoli E1O4 1 RS086 35 W P 131776 915.1 NZ CA AAI A01 000000 7.1 LbtU family sid eroph ore po rin L yabuuch iae E1O47 R S0750 0 W P 13313 0646.1 NZ CA AAIW0100 00017.1 LbtU fa mily side ropho re porin L septentri onalis km 542 E LY14 R S0143 5 W P 37192 3196.1 NZ RZGT 010000 02.1 LbtU family si deroph ore po rin L septentri onalis ELY11 RS00 325 W P 37192 3196.1 NZ RZGS 010000 01.1 LbtU family si derop hore po rin L nagasak iensis E1O5 2 RS077 50 W P 133128 139.1 NZ C AAAID010 000007.1 L btU family side ropho re por in nirop erohporedisyli maf UtbL 1.92000010PYNL ZN 1.686483520 P W 09590SR kaoL sisnegdirkao L L adela idensis La de RS10 440 W P 05846 3102.1 NZ LN KA010 00019.1 LbtU fa m ily side ropho re porin L clemso nensis cle m RS005 95 W P 094089 830.1 NZ CP0 16397.1 Flx A-lik e family pr otein L jamesto wniensis D SM 192 15 BM2 59 RS02 490 W P 058449 716.1 NZ FOTZ0 100000 2.1 FlxA- like family p rotein L hackeli ae LH A RS14 550 W P 045107 187.1 NZ LN6 81225.1 LbtU fa m ily side ropho re porin nietorp yli maf ekil-AxlF 1.80000010VXNL ZN 1.373144850 P W 53560SR urbL sisnenurb L nietorp yli maf ekil-AxlF 1.870911PC ZN 1.938880572 P W 07141SR 50XXP acaidrac L L jord anis EL2 03 RS13 525 W P 05847 1716.1 NZ LR1 34383.1 LbtU fa m ily side ropho re porin L lansin gensis CK V79 RS 12970 W P 028372 822.1 NZ LT906 451.1 FlxA -like fam ily protein L feeleii N CTC 11978 D X470 R S15455 W P 11517 6283.1 NZ UGNY 010000 01.1 LbtU family si derop hore po rin L feeleii E1O60 RS0 1130 W P 13175 3131.1 NZ C AAAH T01000 0002.1 LbtU fam ily sider ophore p orin L donal dsonii D YC89 RS 15085 W P 115222 532.1 NZ UGOA01 000001.1 L btU family side ropho re porin L droza nskii LLA P-1 E 1O80 RS06 535 W P 058497 061.1 NZ C AAAIU01 000000 4.1 LbtU family sid eroph ore po rin L nautar um Lnau R S0286 0 W P 05850 3650.1 NZ LN YO010000 08.1 LbtU family si derop hore po rin L maceac herni i DYE4 3 RS004 65 W P 0584537 36.1 NZ UHIB0 100000 1.1 LbtU family sid eroph ore po rin L micda dei LMI RS 14165 W P 04510 0366.1 NZ LN6 14830.1 LbtU fa mily side ropho re porin L massil iensis BN1094 RS 01565 W P 043872 665.1 NZ CC VW010000 01.1 FlxA -like fami ly protein L fairfiel densis E1O4 8 RS098 25 W P 028388 951.1 NZ CA AAIZ010 000021.1 L btU family side ropho re por in L bir mingha mensis D YH42 RS 01815 W P 058523 326.1 NZ UGNW010 00001.1 LbtU fa mily side ropho re porin L quinl ivanii Lq ui RS05 985 W P 05850 7257.1 NZ LN YS010 00006.1 LbtU fa mily side ropho re porin L dresd enensis ACFOR L RS122 40 W P 382344 447.1 NZ JBH SAB 0100000 29.1 LbtU family si derop hore po rin L geestian a E4T54 R S0623 0 W P 02838 5567.1 NZ CP 038271.1 L btU family side ropho re por in L busane nsisDYH3 0 RS007 55 W P 11532 9590.1 NZ UGOD01 000001.1 L btU family side ropho re porin L gresi lensis E1 P04 RS 06370 W P 131782 082.1 NZ CA AAH X01000 0004.1 Flx A-like fa mily prote in L belia rdensis D YE47 R S0062 0 W P 115301 419.1 NZ UGNV 010000 01.1 LbtU family si deroph ore po rin L rub rilucens E1O83 RS0 7795 W P 0585325 41.1 NZ CA AAIN010 000004.1 L btU family side ropho re por in L taurin ensis DY E45 RS1 3875 W P 11530 1009.1 NZ UGOZ01 000001. 1LbtU family sid eroph ore po rin L erythr a Lery RS 13725 W P 05852 7794.1 NZ LN YA010 00034.1 LbtU fa mily side rophor e porin L spirite nsis CKW05 R S14815 W P 058482 001.1 NZ LT9064 57.1 LbtU family si derop hore po rin Coxiella & Paracoxiella CBU_1699 Coxiella & Paracoxiella MceB (CBU_0937) Aquicella Rickettsiella Legionella LbtP 100 88 100 90 97 5545 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 4. Summary of LPS structural changes in Coxiellaceae. (A) Components of LPS. (B) Gain and loss events. Hep Hep Man Man GlcN GlcN Lipid A Inner Core Outer Core O-antigen Vir Man DHHS Hep N GlcNAc Glu GalNAc Man Kdo KdoKdo A B Coxiella burnetii str. RSA493 Coxiella burnetii str. Dugway Legionella CEs of soft ticks Paracoxiella CEs of hard ticks Rickettsiella Aquicella Lipid IVA usage Virenose synthesis gain L,D-heptose loss β-1,4-mannosyltransferase gain .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 5. Distribution of genes likely required for virenose production. (A) Putative pathway for virenose synthesis. (B) Distribution of virenose synthesis genes across Legionellales. PMM: phosphomannomutase / phosphoglucomutase, GMP: GDP-mannose pyrophosphorylase, GMD: GDP-mannose-4,6-dehydratase. Ricke tt siell a Paracoxiell a Pisc irick etts ia Legionell a Coxiella_burneti i CE_of_hard_tick s Aquicell a CE_o f_so ft _tick s Berkiell a CBU_0678 GMP CBU_0671 EC 2.7.7.13 GMD CBU_0689 EC 4.2.1.- CBU_0688 EC 1.1.1.- CBU_0678 EC 2.7.7.- Mannose-6p Vir Virenose PMM CBU_0294 EC 5.4.2.- CBU_0683 EC 2.1.1.- CBU_0683 Rickettsiella isopodorum Paracoxiella cheracis Piscirickettsia salmonis Legionella pneumophila Coxiella burnetii CE of R.sanguineus Aquicella lusitana CE of O.peruvianus Berkiella aquae CBU_0688GMDGMPPMM X X X X X X Functional Pseudogenized Absent A B .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 6. CBU_0678 was likely acquired from Alphaproteobacteria. Bootstrap values are labeled for major branches. Mauve: Alphaproteobacteria; Blue: C. burnetii and Ornithodoros CEs. WP 068738 298.1 Tardiphaga r obiniae WP 439068941.1 Tardiphaga sp. 367 B4 N1 1WP 320447599.1 Tardiphaga sp. 42S5 WP 245285516.1 Bradyrhizobium japonicum WP 284280899.1 Bradyrhizobium liaoningense WP 374631501.1 Ferrovibrio sp. WP 151114676.1 Hy pericibacter adh aerens WP 15117554 5.1 Hyperic ibacter ter rae WP 431574 872.1 Hype ricibacte r sp. WP 194214 444.1 Ko rdiim onas pum ila WP 237383 582.1 Sulfi dibacter co rallic ola WP 317340 225.1 Thalass ospira l ucentensis WP 233925 513.1 Poly nucleob acter sp. IMCC 291 46 W P 233990 459.1 Poly nucleob acter sp. IMCC 302 28 WP 300539 193.1 Pol arom onas sp. WP 216177 528.1 Poly nucleob acter sp. AP -Feld -500C -C5 W P 211099 496.1 Ske rmanel la aero lata W P 379899 332.1 Ma rinibac ulum pu m ilum W P 119834 222.1 Azosp irill um caver nae W P 209768 450.1 Azospi rillu m rugosum .ps retcabitnalP 1.119668743 P W W P 373071 246.1 Gemm atimonas sp. W P 348136 058.1 Pyr uvatibacter sp. W P 353560 065.1 MU LTISP ECIES unc lassified Py ruvatibacter W P 043949 999.1 Cand idatus Pha eoma rinibacte r ectocarpi W P 350103 801.1 Thalass obaculu m sp. WP 189992 705.1 Thalass obaculu m fulvum WP 322334 103.1 Thalass obaculu m sp. OXR-137 WP 115515 186.1 Undi bacter mob ilis WP 155153 072.1 Curv ivirga a plysinae WP 115694 158.1 Pseu dolabrys tai wanensis WP 137045 751.1 Pseu dolab rys sp. FHR47 WP 423955 900.1 Br adyrhiz obium sp. WP 020592 370.1 Kil oniella lamin ariae WP 256033 132.1 Methyl ocystis suflitae WP 434495835.1 Methyl ocystis sp. WP 014891561.1 Methylocystis hydrogenophila WP 284315665.1 Labrys miyagiensis WP 413992688.1 Labrys okinawensisWP 305528100.1 Methyl obacterium amylolyticum WP 379722 398.1 Fer rovibrio xuzhou ensis WP 108045 956.1 Allobosea sp. 124 WP 376984 662.1 Allobosea sp. R86505 WP 256813 345.1 Man grovib revibacte r kandel iae WP 244432 020.1 Rhod opseudo monas sp. B29 WP 109 11745 0.1 Azosp irillum sp. TSO22 -1 WP 169627 110.1 Pacific ispira spo ngiicola WP 291005 292.1 Hypho micro bium sp.W P 168606 728.1 Cand idatus Pel agibacte r giovann onii W P 262590 203.1 Cand idatus Pel agibacte r commu nis W P 428079 397.1 Cand idatus Pel agibacte r sp. W P 340380 912.1 Daej eonell a sp. W P 332895 024.1 Mag netovibr io sp. PR-2 W P 331896 743.1 Mag netovibr io sp. W P 028466 552.1 Nisae a denitrific ans W P 350052 453.1 Nisae a sp. W P 339854 202.1 uncultu red Nisa ea sp. W P 173977 771.1 Mag netospir illum sp. U T-4 W P 291720 071.1 Mag netospir illum sp. 64 -120 W P 372522 809.1 Sulfu ricaul is sp. WP 037445 975.1 Ske rmane lla stibii resistens WP 012570 209.1 Coxie lla bur netii WP 280123 410.1 Coxie lla endosy mbiont of Ornith odoros a mblus WP 040947 780.1 Coxie lla bur netii WP 005772 293.1 Coxie lla bur netii WP 042526 057.1 Coxie lla bur netii WP 005771 856.1 Coxie lla bur netii 100 75 88 9397 Coxiella Alphaproteobacteria .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 7. Synthesis of LD-heptose was lost in the common ancestor of C. burnetii and Ornithodoros CEs. (A) Pathway for generating L,D-Heptose. (B) GmhD, the enzyme responsible for converting DD-heptose to LD-heptose, is absent in C. burnetii. GmhDGmhBHldEGmhA Ricke tt siell a Paracoxiell a Pisc irick etts ia Legionell a Coxiella_burneti i CE_of_hard_tick s Aquicell a CE_o f_so ft _tick s Berkiell a Rickettsiella Paracoxiella Piscirickettsia Legionella Coxiella burnetii CE of hard ticks Aquicella CE of soft ticks Berkiella Pentose Phosphate Pathway GmhD EC 5.1.3.20 GmhA CBU_0674 CBU_1743 EC 5.3.1.28 HldE CBU_1655 EC 2.7.1.167 GmhB CBU_0673 CBU_1996 EC 3.1.3.82 HldE CBU_1655 EC 2.7.7.70 L,D-Heptose OH D,D-Heptose HO B A Functional Absent .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 8. b-1,4 manosyltransferase (CBU_1657) may have been acquired from Thermodesulfobacteria. Bootstrap values are labeled for major branches. Blue: Coxiella and Paracoxiella; Red: Thermodesulfobacteria; Green: Proteobacteria. WP 4179 11412.1 Ca ndidatus E lectrone ma sp. TJ WP 4202 08758.1 Ca ndidatus E lectrone ma sp. JC WP 417914 674.1 Cand idatus Elect ronem a sp. JM WP 339134 663.1 Cand idatus Elect rothrix sp. GW3-4 WP 446009 500.1 Cand idatus Elect rothrix sp. WP 205222 645.1 Desulfo granu m mar inum WP 028583 065.1 Desulfo granu m medite rran eum WP 028578 975.1 Desulfo granu m japon icum W P 306546 614.1 Desulfo bulbus sp. W P 028319 158.1 Desulfo bulbus el ongatus W P 205243 349.1 Desulfo bulbus alk aliphi lus W P 373499 834.1 Desulfoc occus sp. W P 178365 875.1 Desulfo bacter latus W P 289020 751.1 Desulfo bacter postgatei W P 320043 687.1 uncultu red Desu lfobacter sp. W P 299980 347.1 Desulfo bacula sp. W P 006965 860.1 Desulfoti gnum ph osphitoxid ans W P 024334 045.1 Desulfoti gnum ba lticum W P 022664 364.1 Desulfos pira jo ergens enii W P 245809 226.1 Desulfa mplus m agnetoval limo rtis WP 005770 612.1 Coxie lla bur netii WP 061301 443.1 Coxie lla bur netii WP 012569 726.1 Coxie lla bur netii WP 103093 787.1 Coxie lla bur netii WP 129545 630.1 Coxie lla bur netii WP 267257 068.1 Coxie lla endosy mbiont of Ornith odoros maritim us WP 280124 138.1 Coxie lla endosy mbiont of Ornith odoros a mblus WP 102157 003.1 Coxie lla endosy mbiont of Rhi picepha lus micro plus WP 304985 520.1 Coxie lla-like endosy mbiont WP 244897097.1 Cand idatus Coxie lla mudrowiaeMFW0104592.1 MAG Coxiella-like endosymbiont WP 423064273.1 Candidiatus Paracoxiella cheracis WP 023525522.1 MULTISPECIES Leptospirillum WP 143461649.1 Leptospirillum ferriphilum WP 062487100.1 Cand idatus Nitros pira inopinata WP 413934 863.1 Nitros pira sp. BLG 1 WP 086425 448.1 Nitros pira cf. moscovi ensis SB R1015 WP 115694 149.1 Pseu dolabrys tai wanensis WP 198164 519.1 Rugos italea oryz ae WP 424619 649.1 Br adyrhiz obium sp. WP 127997 033.1 MU LTISP ECIES P iscinibacter WP 245909 561.1 Sph aerotil us hippei WP 196984 825.1 Caen imonas a quaedu lcis WP 422834 292.1 Vari ovorax sp. HJS M1 2 W P 086489 594.1 Thioflex ithrix pseku psensis W P 029707 513.1 Rhod oferax said enbache nsis W P 214124 090.1 Curv ibacter sp. CHR R-16 W P 304768 303.1 parti al Undib acteriu m sp. W P 057675 972.1 Curv ibacter sp. PAE- UM W P 315678 532.1 Curv ibacter sp. APW13 W P 413861 428.1 Cand idatus Aalbo rgicol a defluviih abitans W P 296507 192.1 Rhod oferax sp. W P 313880 556.1 Rhod oferax pota m i W P 226403 366.1 Fer ribacte rium li mneticum W P 310493 605.1 Dechl orom onas sp. W P 138120 046.1 Bathy modiol us heckera e thiotroph ic gill symbi ont WP 318844 625.1 Cand idatus Thiog lobus autotr ophicus WP 082345 029.1 Cand idatus Pseu dothiogl obus singu laris WP 416695 849.1 Cand idatus Pseu dothiogl obus sp. Uisw 050 01 WP 435189 482.1 Pseu dothiogl obus sp. nBU S 23 WP 13608 1149.1 P ontiella d esulfatans WP 416430 792.1 Pisci rickettsia sal monis WP 127131 442.1 Pseu doflavitale a rhizosp haerae WP 176957 723.1 Ma riprofu ndus sp. KV WP 176963 083.1 Ma riprofundus sp. NF WP 018295298.1 Mariprofundus ferrooxydans WP 013820620.1 Methylomonas methanica WP 292569019.1 Methylomonas sp. WP 064030292.1 Methylomonas koyamae WP 119333045.1 Geobacte r sulfurreducensWP 039643782.1 Geobacte r anodireducensWP 199384 836.1 Geomes ophilobacter sedi minis 100 53 60 94 64 69 Coxiella & Paracoxiella Thermodesulfobacteria Proteobacteria .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 9. Coxiellaceae appears to have acquired a polyamine synthesis gene cluster (CBU_0720-22) from Alphaproteobacteria. Coxiella, Paracoxiella, and Rickettsiella are highlighted in blue while Alphaproteobacteria are highlighted in mauve. A representative ML tree of CBU_0722 is shown here. ML trees of CBU_0720 and CBU_0721 produced similar results. Bootstrap values are labeled for major branches. W P 188445 574.1 Sph ingom onas psychr olutea W P 332793 753.1 Pa rasphin gomon as frigor is W P 093223 862.1 MU LTISP ECIE S S phingo m onas W P 183982 561.1 Sph ingom onas jinj uensis sisneupez sano mognihpsaraP 1.385517233 P W W P 431849 154.1 Allos phingos inicella sp. W P 284053 178.1 Stakel ama ma rina W P 219236 619.1 Stakel ama flava W P 264578 680.1 MU LTISP ECIES unc lassified S phingo bium W P 264587 024.1 Sph ingobi um sp. B2D3B W P 181559 376.1 MU LTISP ECIES S phingo bium W P 264570 056.1 MU LTISP ECIES unc lassified S phingo bium WP 307749 431.1 Sph ingobi um sp. DEH P117 WP 237364 797.1 Rhizo rhapis sp. S PR117 WP 135245 097.1 Glaciei bacteriu m arsha anense WP 324375 811.1 Sph ingomo nas sp. WP 442681 304.1 Sph ingom onas sp. ASY06-1R WP 165324 690.1 Rhizo rhabd us phycospha erae WP 340315 390.1 Rhizo rhabd us argentea WP 184020 306.1 Sph ingobi um boeckii WP 157216 959.1 Flavis phingomonas for mosensis WP 068878 552.1 MU LTISPECIES unc lassified Phenylobacterium WP 305598166.1 Phenylobacterium sp. WP 444745738.1 Phenylobacterium sp. WP 304171323.1 Phenylobacterium aquaticumWP 374576499.1 Phenylobacterium sp.WP 307352272.1 Caulobacter ginsengisoli WP 116489253.1 MU LTISPECIES Ca ulobacter WP 116569 523.1 Caul obacter ra dicis WP 419318 874.1 Caul obacter sp. E rkDOM-E WP 242919 313.1 Caul obacter sp. CCUG 60 055 WP 419468 160.1 Br evundi monas sp. WP 335624 957.1 Br evundi monas sp. WP 286692 957.1 MU LTISP ECIES unc lassified B revund imonas WP 123287 963.1 Br evundi monas hal otolera ns WP 305487 281.1 Br evundi monas sp. WP 302109 860.1 Pei ella sedi menti W P 158916 321.1 Caul obacter sp. S45 W P 291841 916.1 Ma ricaulis sp. W P 138379 084.1 Luteithe rmob acter gelati nilyticus W P 017252 947.1 Coxie lla bur netii W P 080775 105.1 Coxie lla bur netii W P 010957 762.1 Coxie lla bur netii W P 0 11996 709.1 Coxie lla burn etii iitenrub alleixoC 1.948075210 P W sulb ma sorodohtinrO fo tnoib mysodne alleixoC 1.451421082 P W W P 267256 556.1 Coxie lla endosy m biont of Ornith odoros m aritim us W P 012570 193.1 Coxie lla bur netii W P 258267 417.1 Coxie lla-l ike endosy mbiont W P 211923 728.1 Coxie lla endosy mbiont of Amb lyomm a nuttalli W P 048875 076.1 Cand idatus Coxie lla mud rowiae W P 100622 816.1 Cand idatus Coxie lla mud rowiae WP 102157 241.1 Coxie lla endosy mbiont of Rhi picepha lus micro plus WP 039669 876.1 Coxie lla endosy mbiont of Amb lyomm a amer icanum WP 159747 787.1 Coxie lla endosy mbiont of Amb lyomm a sculptum WP 264435 462.1 Coxie lla endosy mbiont of De rmacento r marg inatus WP 423063 657.1 Cand idiatus Pa racoxie lla cher acis WP 339051 650.1 Rickettsiel la endosy mbiont of Xylota se gnis WP 342228 379.1 Rickettsiel la endosy mbiont of Rhag onycha lig nosa WP 342220 820.1 Rickettsiel la endosy mbiont of Miltoc hrista mi niata WP 342147 406.1 Rickettsiel la endosy mbiont of Aleochara cu rtula WP 071662 918.1 MU LTISPECIES Rick ettsiella WP 071661337.1 Rickettsiel la grylli WP 395690072.1 Aestuariivirga sp. WP 130122179.1 Rickettsiales endosymbiont of Peranema trichophorum WP 203095368.1 Skermanella rosea WP 201080528.1 Skermanella cutis WP 202679 643.1 Ske rmanella mucosa WP 326881 344.1 Aliidongia sp. WP 189045 837.1 Aliidongia di nghuens is WP 283524 686.1 Roseo monas sp. E05 WP 160937 690.1 Teichococc us coralli WP 370764 874.1 Teichococc us vastitatis WP 440912 693.1 Cand idatus Pel agibacte r sp. WP 42808 1146.1 Ca ndidatus P elagibacte r sp. WP 440693 270.1 Cand idatus Pel agibacte r sp. HIMB1695 WP 075534 871.1 Cand idatus Pel agibacte r commu nis W P 099339 815.1 Cand idatus Fonsi bacter ubi quis W P 013694 954.1 Cand idatus Pelagibacte rsp. IMCC9063 W P 440679 827.1 Cand idatus Pel agibacte r sp. HIMB1517 100 85 75 97 9770 100 100 Coxiella, Paracoxiella, & Rickettsiella Alphaproteobacteria .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 10. Pathways for polyamine synthesis and arginine metabolism in C. burnetii. Arginine H2O NH3 CO22x + Ornithine 2x Proline NH3 Arginine Decarboxylase CBU_0722 CO2 H+ Agmatine Agmatinase CBU_0720 H2O Urea Putrescine Homospermidine Synthase CBU_0721 Homospermidine Ornithine Decarboxylase CBU_0722 CO2 H+ Arginine Dihydrolase CBU_0279 Ornithine Cylcodeaminase CBU_1727 NH3 +NAD+ NADH .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 11. CBU_0279 was likely acquired in the common ancestor of Coxiella and Paracoxiella (blue) from cyanobacteria (green). Homologs were also found in three Legionella species (purple). Bootstrap values are labeled for major branches. WP 062290812.1 Nostoc piscinale WP 190871158.1 Aulosira sp. FACHB-615 WP 190702981.1 MULTISPECIES unclassified Nostoc WP 193888752.1 Fortiea sp. LEGE X X443 WP 236140500.1 Nostoc sp. CM AA1605 WP 427159 394.1 Aliinostoc sp. HNIBRC Y26 WP 066375 836.1 MU LTISPECIES unc lassified Anabaena WP 420759 928.1 Nostoc sp. CAL U 546 WP 069068 313.1 Nostoc sp. KVJ 20 WP 190880 717.1 Desm onostoc musco rum WP 194002 964.1 a ff. Roholtiell a sp. LEGE 124 11 WP 089091 606.1 Nodu laria sp. NIE S-3585 WP 323194 888.1 Nodu laria h arveyana WP 280651 735.1 Umez akia ovalis porum WP 271731 227.1 MU LTISP ECIES Anab aenopsis elangats mumrepsordnilyC 1.702802510 PWWP 214438 326.1 Atlanticoth rix silvestris WP 198127 108.1 Amaz onocrin is nigrite rrae WP 214434 011.1 Dend ronali um phyll ospher icum WP 017653 699.1 Forti ea contorta WP 190602 992.1 Riche lia sinica WP 323340 502.1 Caloth rix sp. UHCC 01 71 WP 095723 841.1 Br unnivag ina elsteri WP 015200 314.1 Caloth rix sp. PCC 63 03 WP 096626 461.1 Caloth rix sp. NIES -3974 WP 290882 919.1 Fische rella sp. WP 026733337.1 Fische rella sp. PCC 9605 WP 016869671.1 MULTISPECIES Fischerella WP 038079521.1 MULTISPECIES Nostocales WP 407888358.1 Scytonema sp. NUACC26 WP 039713488.1 Scytonema milleiWP 438553642.1 Chroococcidiopsis sp.WP 015157287.1 MULTISPECIES Chroococcidiopsis WP 192153708.1 Chroococcidiopsis spFACHB-1243 WP 317109495.1 Chroococcidiopsis sp. SAG 2025 WP 190641 021.1 Oculatell a sp. FACHB-28 WP 190798 018.1 Leptoly ngbya sp. FACHB-541 WP 190508 220.1 Leptoly ngbya sp. FACH B-321 WP 190449 447.1 MU LTISP ECIES Cya nophyceae WP 223046 070.1 Leptothe rmofons ia sichuan ensis WP 421657 134.1 Leptothe rmofons ia sp. ETS -13 WP 428358 473.1 Leptod esmis sp. WP 233743 066.1 Leptod esmis sichu anensis WP 068817 821.1 Pho rmidesmis pr iestleyi WP 439342 225.1 Vacuol onema ib erom arroc anum aybgnylotpeL deifissalcnu SEICEPSITLUM 1.922297091 PW WP 011056 358.1 Ther mosynechoc occus vestitus WP 297051 028.1 MU LTISP ECIES unc lassified Ther mosynech ococcus WP 156812 671.1 Legi onella tun isiensis WP 242604 155.1 Legi onella fee leii WP 425169 243.1 Legi onella sp. WP 045095 377.1 Legi onella fal lonii WP 423063 478.1 Cand idiatus Pa racoxie lla cher acis WP 244896 995.1 Cand idatus Coxie lla mud rowiae CRS-C AT WP 235378 992.1 Cand idatus Coxie lla mud rowiae CRt WP 005771 471.1 Coxie lla burnetii WP 012220150.1 Coxie lla burnetii WP 039669228.1 Coxie lla burnetii CE of O. amblus pse udo FIV31 RS05115CE of O. maritimus pseudo OWO34 RS07315 CE of O. peruvianus pseudo ACJLXJ 06765 99 99 100 100 Coxiella & Paracoxiella Cyanobacteria Legionella .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 12. Ornithine cylcodeaminase (CBU_1727) may have been horizontally acquired. Coxiella and Paracoxiella are shown in blue. The closest homolog was found in an archaeon (green), while most others were in various proteobacteria (orange). Bootstrap values are labeled for major branches. WP 014714799.1 Francisella orientalis WP 200151208.1 Francisella philomiragia WP 112870448.1 Fra ncisella ad eliensis WP 071664 632.1 Fra ncisella fr igiditu rris WP 119329 999.1 Pseu dofrancise lla aestua rii WP 133940 537.1 Allof rancisel la inopin ata WP 172106 545.1 Allof rancisell a frigida quae WP 039124 966.1 Allof rancisel la guangzh ouensis WP 102951 712.1 Aque lla oligot rophica WP 15 119426 7.1 Cysteiniph ilum sp. JM-1 W P 440993 449.1 Cysteini philu m litorale W P 440617 547.1 MU LTISP ECIES unc lassified Cystei niphil um W P 203249 869.1 MU LTISP ECIES Cystei niphil um W P 208123 139.1 Cysteini philu m halobi um W P 204723 380.1 Fastidi osibacter l acustris sisnegnokgnoh aignaF 1.964470150 P W W P 208123 291.1 Facil ibium su bflavum W P 076083 822.1 Pose idonib acter parv us W P 121627 504.1 Pose idonib acter antarctic us W P 093241 971.1 Psych roflexus hal ocasei W P 024954 883.1 Sulfu rospi rillu m arcacho nense WP 424427 419.1 Pose idonib acter sp. WP 015464 386.1 Psych romon as sp .CNPT3 WP 331774 358.1 Sulfu rospi rillu m sp. 1612 WP 315385 031.1 Mic rovirg ula aer odenitr ificans WP 028499 101.1 MU LTISP ECIES M icrovi rgula WP 273959 359.1 Devosi a sp. ZB163 WP 263595 004.1 Br achybacter ium hug uangmaarense WP 292436031.1 Methyl obacter sp. WP 006889832.1 Methylobacter tundripaludum WP 411725263.1 Methyloglobulus sp. WP 036306482.1 Methyloglobulus morosusWP 442497374.1 Methyl obacter sp. sgz30 2048 WP 228779 209.1 Methyl obacter sp. Bl B1 WP 434482 907.1 Methyl obacter sp. WP 090572 162.1 Nitros omonas sp. N m33 WP 317537 012.1 Nitros omonas sp. Is37 WP 04685 1133.1 Nit rosomo nas commu nis WP 090575 228.1 Nitros omonas sp. N m58 WP 277267 869. 1Nitros omonas n itrosa W P 024297 335.1 Methyl omicr obium l acus W P 374089 109.1 Methyl omicr obium l acus W P 202052 273.1 Cand idatus Methyl omicr obium o ryzae W P 435686 548.1 Sed imentico la selenati reduce ns W P 288106 786.1 Sed imentico la sp. sucihportinegordyh alocitne mideS 1.102292062 P W W P 0 11751 883.1 Ther m ofilum p endens W P 423064 243.1 Cand idiatus Pa racoxie lla cher acis W P 048874 856.1 Cand idatus Coxie lla mud rowiae W P 100622 607. 1Cand idatus Coxie lla mud rowiae W P 251366 210.1 Coxie lla-l ike endosy mbiont of Rhip icephalus sa nguine us WP 304985 545.1 Coxie lla-l ike endosy mbiont WP 005772 857.1 Coxie lla bur netii WP 005770 463.1 Coxie lla bur netii WP 052471 759.1 Coxie lla bur netii WP 012569 595.1 Coxie lla bur netii WP 010958 398.1 Coxie lla bur netii WP 2594 11872.1 Cox iella bu rnetii WP 267257 121.1 Coxie lla endosy mbiont of Ornith odoros maritimus WP 280124219.1 Coxiella endosymbiont of Ornith odoros amblus CE of O. peruvianus ACJLXJ 04935 93 100 99 90 88 100 Coxiella & Paracoxiella Proteobacteria .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 13. Summary of changes to fatty acid synthesis/modification in the lineage leading to C. burnetii. Coxiella bu rnetii str. RSA493 Co xiella bu rne tii str. Dugwa y Legionella CEs of soft ticks Paracoxiella CEs of hard ticks Rickettsiella Aquicella Δ9 family acyl-CoA desaturase gain UFA synthesis gene cluster gain cyclopropane fatty acid synthase loss DesA family acyl-lipid desaturase loss .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 14. Potential horizontal acquisition of FabA (CBU_0037) in the common ancestor of Coxiella. Closest homologs of Coxiella’s FabA (blue) are found in a diverse group of bacteria including Proteobacteria (green), spirochetes (yellow), and Thermodesulfobacteriota (red). Bootstrap values are labeled for major branches. WP 046305 054.1 Bloc hmannia endosy mbiont of Cam ponotus Colo bopsis obl iquus WP 192380 381.1 Bloc hmann ia endosy mbiont of Colo bopsis nipp onica WP 348353 850.1 Pa raglaci ecola sp. WP 018981 762.1 Sal inimo nas chungw hensis WP 2076 11774.1 Alte romon as sp. 5E99-2 WP 308363 795.1 MU LTISP ECIES unc lassified M icrobu lbifer WP 250464 249.1 Mic robulb ifer litor alis WP 067157 329.1 Mic robulb ifer ther motoler ans W P 091391 200.1 Mic robulb ifer ma rinus W P 323847 351.1 Mic robulb ifer mag nicolon ia W P 116302 335.1 Alka lilim nicola eh rlichii W P 093985 411.1 Pseu domonas fluv ialis W P 196137 502.1 Aliik angiell a sp. G2MR2-5 sira m alleignakiilA 1.956698353 P W W P 109762 582.1 Ple ionea m editer ranea W P 438951 566.1 Po rticoccus sp. W P 340676 448.1 MU LTISP ECIES P arape rlucid ibaca W P 116208 595.1 Pa raperl ucidibac a baekdon ensis W P 068858 474.1 Pe rlucidi baca aquatica WP 107864 867.1 Agitococc us lubricus WP 430881 580.1 Granu losicoccus sp. 3- 233 WP 013147 794.1 Methyl otenera ve rsatilis WP 300387 625.1 Methyl otenera sp. WP 300479876.1 Methylotenera sp. WP 020181620.1 Methylotenera sp. 1P/1 WP 029933 703.1 MU LTISP ECIES Thio microsp ira WP 331864 116.1 Thiom icrospi ra sp. WP 185977 729.1 MU LTISP ECIES Thio micro rhabdus WP 029938 894.1 MU LTISP ECIES P iscirickettsiac eae WP 018634136.1 Neomegalonema perideroedes WP 256617829.1 Parvularcula maris WP 189570 294.1 Pa rvularcula lutaon ensisWP 300553 202.1 Ma ricaulis sp. WP 300530 616.1 Ma ricaulis sp.WP 189399 735.1 Are nicella ch itinivora ns WP 353413 086.1 Are nicella sp. 4NH 20-0 111 Candi datus Coxiel la mud rowiae C Rt pseudo Cle Rt RS10900 Candi datus Coxiel la mud rowiae C RS-C AT pseudo C VD13 RS 12120 CE of O. peruvia nus pseudo ACJL XJ 06050 CE of O. mariti mus pseudo OWO34 RS 00180 W P 017253 441.1 Coxie lla bur netii W P 042525 287.1 Coxie lla bur netii W P 005770 369.1 Coxie lla bur netii W P 032074 772.1 Coxie lla bur netii W P 0 11996 435.1 Coxie lla burn etii W P 272534 590.1 Leptosp ira sp. GIMC2001 W P 002770 665.1 Lepton ema ill ini W P 367360 570.1 MU LTISP ECIES Sy ntrophus W P 093884 353.1 Syntr ophus genti anae WP 011418 881.1 Syntr ophus acidit rophicus WP 410269 354.1 Desulfos alsimo nas sp. WP 181550 911.1 Desulfos alsimon as propi onicica WP 141734 457.1 Oligoflex us tunisiensis WP 325145 509.1 Oligoflex us sp. WP 267182 490.1 Ma rinicel la gelatin ilytica WP 188366 134.1 Ma rinicel la pacifica WP 154224 396.1 Ma rinicella rhab doformisWP 099019187.1 Marinicella litoralis WP 014456299.1 Spirochaeta africana WP 400167237.1 Fidelibacter multiformis WP 146655498.1 Labilithrix luteola Coxiella Thermodesulfobacteria Proteobacteria Spirochaetota 100 84 100 53 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 15. Distribution of Mrp cation/proton transporter across Legionellales. Rickettsiella GCA 036495935.1 Aquicella GCA_964647825.1Aquicella GCA964612855.1Aquicella GCA_964466595.1Aquicella GCA_964449835.1Aquicella GCA_964597435.1Aquicella GCA_964527435.1Aquicella GCA_964492045.1Aquicella GCA_964648015.1Aquicella siphonisAquicella lusitanaRickettsiella GCA_964341295.1RE Dermanyssus gallinaeRickettsiella viridisRE OperuvianusRickettsiella GCA_964417555.1Aquirickettsiella gammari Rickettsiella grylliRickettsiella isopodorumRE Litargus connexusRickettsiella GCA 965662625.1Rickettsiella GCA 964341305.1Rickettsiella GCA 049797115.1RE Miltochrista miniataRE Aleochara curtulaRickettsiella GCA 037439325.1 RE Xylota segnisRE Rhagonycha lignosa Piscirickettsia salmonis Berkiella GCA_947478905.1Berkiella aquaeBerkiella GCA_963976875.1Berkiella cookevillensis CLE Aamericanum C904CLE Rmicroplus CLERMCLE RappendiculatusCLE RturanicusCLE Rsanguineus CRS CATL adelaidensisL septentrionalisL londiniensisL oakridgensisL nagasakiensisL yabuuchiaeL impletisoliL erythraL taurinensisL rubrilucensL quinlivaniiL birminghamensisL busanensisL beliardensisL lansingensisL jordanisL cardiacaL brunensisL hackeliaeL jamestowniensis DSM 19215L clemsonensisL feeleiiL donaldsoniiL fairfieldensisL massiliensisL micdadeiL maceacherniiL nautarumL drozanskii LLAP 1L israelensisL waltersiiL pneumophilaL fallonii LLAP 10L antarcticaL shakespearei DSM 23087L worsleiensisL quateirensisL moravicaL saoudiensisL rowbothamiiL lyticaL gratianaL sainthelensiL longbeachae NSW150L santicrucisL cincinnatiensisF dumoffii Tex KLL steeleiL steigerwaltiiL cherrii DSM 19213L qingyiiF gormaniiL wadsworthiiL parisiensisL tucsonensisL anisa Legionella adelaidensis Legionella septentrionalis Legionella londiniensis Legionella oakridgensis Legionella nagasakiensis Legionella yabuuchiae Legionella impletisoli Legionella erythra Legionella taurinensis Legionella rubrilucens Legionella quinlivanii Legionella birminghamensis Legionella busanensis Legionella beliardensis Legionella lansingensis Legionella jordanis Legionella cardiaca Legionella brunensis Legionella hackeliae Legionella jamestowniensi Legionella clemsonensis Legionella feeleii Legionella donaldsonii Legionella fairfieldensis Legionella massiliensis Legionella micdadei Legionella maceachernii Legionella nautarum Legionella drozanskii Legionella israelensis Legionella waltersii Legionella pneumophila Legionella fallonii Legionella antarctica Legionella shakespearei Legionella worsleiensis Legionella quateirensis Legionella moravica Legionella saoudiensis Legionella rowbothamii Legionella lytica Legionella gratiana Legionella sainthelensi Legionella longbeachae Legionella santicrucis Legionella cincinnatiensis Legionella dumoffii Legionella steelei Legionella steigerwaltii Legionella cherrii Legionella qingyii Legionella gormanii Legionella wadsworthii Legionella parisiensis Legionella tucsonensis Legionella anisa Paracoxiella cheracis CE Amblyomma sculptum CE Amblyomma americanum CE Dermacentor nuttalli CE Dermacentor silvarum CE Dermacentor marginatus CE Haemaphysalis qinghaiensis CE Haemaphysalis japonica CE Rhipicephalus turanicus CE Rhipicephalus sanguineus CE Rhipicephalus appendiculatus CE Rhipicephalus microplus CE Haemaphysalis longicornis CE Amblyomma nuttalli CE Ornithodoros amblus CE Ornithodoros maritimus CE Ornithodoros peruvianus Coxiella burnetii X X X Mrp X Functional Absent Pseudogenized .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 16. Changes in sugar catabolism, glycolytic control, and respiratory metabolism. (A) Summary of gene gain events that have affected sugar utilization in the lineage leading to C. burnetii. (B) Sugar and sugar alcohol catabolism pathways in C. burnetii. AldT: aldohexose dehydrogenase; DHAP: dihydroxyacetone phosphate; eda: 2-dehydro-3-deoxyphosphogluconate aldolase; F-1,6-diP: Fructose-1,6-bisphosphatase; F6P: Fructose-6-phosphatase; G3P: glyceraldehyde 3-phosphate; KDG: 2-keto-3-deoxygluconate; KdgK: 2-dehydro-3- deoxygluconokinase; PFK: ATP-dependent phosphofructokinase; PFP: inorganic pyrophosphate- phosphofructokinase; UQ: ubiquinone; UQH2: ubiquinol; UxuA: mannonate dehydratase; X5P: xylulose 5-phosphate; XI: xylose isomerase; XK: xylulokinase PFPPFK F-1,6-diP F6P PPi Pi AT P ADP ADP PEP Glucose G3P DHAP non-oxidative pentose phosphate pathway GlpK GlpD Pyruvate Glycerol Glycerol-3P XI XK Xylose Xylulose X5PUQ UQH2 edaAldT UxuA KdgKMannose Mannonate KDG Coxiella bu rnetii str. RSA493 Co xiella bu rne tii str. Dugwa y Legionella CEs of soft ticks Paracoxiella CEs of hard ticks Rickettsiella Aquicella Mannose catabolism gain Xylose catabolism gain A B Ubiquinol-based respiratory chain usage PFK1 glycolysis regulation gain .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint Figure 17. Distribution of cytochrome genes in Legionellales. bo3: cytochrome bo3 ubiquinol oxidase; bd: cytochrome bd ubiquinol oxidase; caa3: cytochrome caa3 oxidase; bc1: cytochrome bc1 complex; Ccm: cytochrome c maturation machinery. bo₃ bd caa₃ Ccm Ricke tt siell a Paracoxiell a Legionell a Coxiella_burneti i CE_of_hard_tick s Aquicell a CE_o f_so ft _tick s Berkiell a Aquicella Paracoxiella Legionella Coxiella burnetii CEs of Hard Ticks Rickettsiella CEs of Soft Ticks Berkiella X X bc1 X Functional Pseudogenized Absent .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 15, 2026. ; https://doi.org/10.64898/2026.05.14.724635doi: bioRxiv preprint