Discussion
Combining FISH and colonization experiments, we revealed the
colonization specificity of Apibacter and its distribution in the
bee gut. Comparative genomic analyses of 30 genomes from theFlavobacteriaceae family, including 14 newly sequencedApibacter genomes from this study and publicly available genomes
for the outgroups, we characterized gene signatures underlying lifestyle
transition and adaptation to bee gut symbionts.
FISH visualization indicates that Apibacter coinhabit withSnodgrassella and colonize the epithelium of the bee gut. As core
members of gut bacteria in A. cerana , both Apibacter andSnodgrasella are microaerophilic, sharing nutritional sources
(Zheng, Powell, Steele, Dietrich, & Moran, 2017). We showed thatApibacter isolated from A. cerana were able to colonizeA. mellifera , although at a significantly lower colonization
rate. These results suggest that host incompatibility is probably not
the constraining factor responsible for the rarity of Apibacterin A. mellifera . However, it is not yet possible to examine
inter-host competition between Apibacter isolated from different
honeybee species, because isolates from A. mellifera were not
available to us.
Comparative genomic analysis revealed key gene functions potentially
associated with the adaptation to bee gut niche. In a typical symbiotic
system, benefits provision was considered crucial for the establishment
of a mutualistic relationship (EWALD, 1987; Sachs et al., 2013). Our
findings reveal that Apibacter are indeed providing beneficial
traits to the host. For example, genes involved in amino acid
biosynthesis are preserved in Apibacter spp., at a background of
overall genome reduction, which echoes those previously reported in
other bee gut symbionts (Kwong et al., 2014). Furthermore, theApibacter group retained the mannose catabolic gene, which was
responsible for monosaccharide detoxification in the honeybee therefore
broadening food choice for the host (Zheng et al., 2016).
Polysaccharides utilization is a prominent property carried by bee gut
symbionts including Gilliamella , Bifidobacterium andLactobacillus (Bonilla-Rosso & Engel, 2018; Engel, Martinson, &
Moran, 2012; Kešnerová et al., 2017). However, relevant genes are
substantially lost in the Apibacter group. Interestingly, the
core bacterial species Snodgrasella that coinhabit withApibacter at A. cerana gut epithelium also lack the
capacity to utilize polysaccharides (Kwong et al., 2014). We speculate
that polysaccharides might be limited in the niche that they share.
The gut lumen is mainly anaerobic, where the dominant symbiotic
anaerobes inhabit. However, oxygen can diffuse from the intestinal
epithelium cells and create a microaerobic environment for facultative
anaerobes (He et al., 1999; Zheng, Powell, et al., 2017). A previous
study found both cytochrome bd and cbb3 in
the Apibacter genome, which were presumably involved in
microaerobic respiration (Kwong et al., 2018). In the present work, we
identified additional anaerobic respiration NAR operon that was
conserved within the Apibacter group and in the coinhabitingSnodgrassella , but absent from the other four core bee gut
bacteria species. These observations suggest that the NAR pathway might
be important for the microbiome to colonize intestinal epithelium. Such
respiratory flexibility might enable Apibacter to survive altered
oxygen tensions. This finding is congruent with the observation in mouseE. coli , where they require both microaerobic and anaerobic
respirations for successful colonization (Jones et al., 2007). A further
study proved that the NAR pathway played a key role in E. colicolonization of the mouse gut, because the NarG mutant showed
colonization deficiency for both commensal bacteria and pathogenicE. coli (Jones et al., 2011). These results are in line with the
observation that nitrate reduction could facilitate the growth of gut
microaerobic bacteria at low oxygen conditions (Tiso & Schechter,
2015). Therefore, we conclude that the NAR operon is an important
genetic signature for Apibacter adaptation to the bee gut.
Genes that are shared between the LCAs of Clade C and Clade E but absent
from the Apibacter group, contain functions either deleterious to
the mutualistic relationship with the host, or redundant for the
symbiotic lifestyle. Histidine biosynthesis is one of the most energy
consuming processes for bacteria, such that the degradation of histidine
as carbon and nitrogen sources is strictly regulated (Bender, 2012). The
histidine catabolism is limited in bee gut environments, as oxygen is
required for the activation of the Hut operon (Goldberg & Hanau, 1980).
Considering that the bee gut is mostly
anoxic,
the Hut pathway is highly likely to be malfunctioning inApibacter and is susceptive to be lost. In addition, histidine
degradation is important for pathogens to recognize eukaryotic hosts and
to activate virulence factors (Zhang et al., 2014).
In conclusion, combining molecular and colonization experiments, for the
first time, we visualized and quantified the distribution ofApibacter spp. inside the bee gut, and proved thatApibacter isolates of A. cerana could survive in A.
mellifera . Genomic comparisons with relatives living on other
lifestyles revealed that host beneficial traits and respiration nitrate
reduction (NAR pathway) were key functions for adaptation to the bee gut
environment.