Characterizing Commas: Discovery and exploration of a novel family of
pleomorphic Rhizobiales bacteria isolated from herbivorous ants
Brianna M. Busscher and John T. Wertz, PhD, Department of Biology, Calvin College, Grand Rapids, MI 49546
Introduction
Five orders of bacteria are conserved in the guts of all herbivorous ants with bacteria in the order Rhizobiales being most closely linked to herbivory [2]. Interes:ngly, feeding studies have shown that when fed a diet of pollen, the number of Rhizobiales increases significantly in the gut of Cephalotes varians, sugges:ng these bacteria have a role in ant nutrient provisioning [3]. To beKer understand this role, we isolated several novel Rhizobiales from Cephalotes varians ants throughout the Florida Keys and characterized key metabolic characteris:cs. Interes:ngly, preliminary DNA sequencing of the Rhizobiales isolates suggested they may be members of a novel family of bacteria. Hence we also use these metabolic characteris:cs to validate their placement within a new family of bacteria, as well as name and validly publish them as such. Objectives
1.  Physiologically characterize Rhizobiales cul:vars and compare with members from the same family 2.  Place Rhizobiales within a robust phylogene:c framework 3.  Determine pec:noly:c ability of Rhizobia cul:vars General Methods
Unless otherwise specified, cells were grown at room temp on TSA or TSA+pec:n plates and in TSB liquid media. Gaseous environment alternated between 2% O2/5% CO2 (hypoxia) and 20% O2/1% CO2. Aier fixa:on in 2.5% glutaraldehyde/0.1M cacodylate solu:on, thin sec:oning and transmission electron imaging were done at the MSU Center for Advanced Microscopy.
Genotyping
CSM3390-24
JR021-5
0.90 JR021-2
CSM3382-8
0.99
CSM3370-14
Uncultured
Rhizobiales clone cv1-B-04 [KF730289]*
0.97
Uncultured Rhizobiales clone Queen-09 [KF730312]*
0.54
Uncultured Rhizobiales clone TNWA8G [KF250094]#
Candidatus “Liberibacter asiaticus” [NR_074528]
0.99
Candidatus “Liberibacter solanacearum” [NR_074494]
Candidatus “Liberibacter americanus” [EU921625]
Rhizobium aggregatum [NR_026115]
0.78
Allorhizobium undicola [Y17047]
0.86
Aureimonas altamirensis DUCC3708 [KP318455]
Neorhizobium alkalisoli [LN774523]
0.84
Rhizobiaceae
Agrobacterium rhizogenes [D14501]
0.71
Rhizobium leguminosarum [AY509900]
Ensifer adhaerens [AB681163]
0.93 0.89
Sinorhizobium meliloti JNVU TF7 [JN228090]
Shinella zoogloeoides [AB681761]
Mesorhizobium loti [KM192336]
0.97
Phyllobacteriaceae
Aminobacter aminovorans [NR_025301]
Xanthobacter autotrophicus [NR_026308]
Xanthobacteriaceae /
0.87
Azorhizobium doebereinerae [NR_041839]
Bradyrhizobiaceae
Bradyrhizobium diazoefficiens [AB909430]
0.89
Caulobacter crescentus [AF125194]
Escherichia coli [NR_114042]
40 Genera0on Time (Hrs) 35 30 25 20 15 A
B
10 5 0 0% 1% 2% 3% 4% 5% 0.3
6% Figure 5. Maximum likelihood-­‐based 16S rRNA gene phylogeny of JR021-­‐5. The phylogeny is based on 1051 shared nucleo:de posi:ons. Branch point support is indicated by the numbers at branch nodes. Other members of the division Rhizobia are given as reference species. GenBank accession numbers are shown in brackets. Scale bar = 0.3 changes per nucleo:de. Phylogene:c analysis was done using phylogeny.fr soiware. CO2 Concentra0on Figure 1. Effect of CO2 concentra:on on genera:on :me of JR021-­‐5. Since bacteria were isolated under an atmosphere of 5% CO2 (with 2% O2, 93% N2), we sought to determine if CO2 was a requirement for growth or was growth-­‐s:mula:ng. Under a star:ng headspace of 100% N2, Balch tubes were injected with pure CO2 and atmospheric air to obtain a final headspace composi:on of 0-­‐5% CO2 in 0.5% and then 1% increments. O2 was maintained at 2% in each tube. Values are expressed as mean±SD of hours and were collected from 3 replicates. 20 18 17 16 15 14 13 12 11 10 0/0 5/1 10/1 20/1 20/0 O2/CO2 Concentra0on Figure 2. To assess anaerobic, aerobic, or possible microaerophilic metabolism, we measured the effect of O2 and CO2 concentra:on on growth of JR021-­‐5. Under a star:ng headspace of 100% N2, Balch tubes were injected with pure CO2 and atmospheric air to obtain a final headspace composi:on of 0% O2 with 0% CO2 (0/0), 5% O2 with 1% CO2 (5/1), 10% O2 with 1% CO2 (10/1), 20% O2 with 1% CO2 (20/1), and 20% O2 with 0% CO2 (20/0). Values are expressed as mean±SD of hours and were collected from 3 replicates. While growth occurred at 20/0 in liquid media, JR021-­‐5 did not grow on solid media incubated on the bench top. 1 0.8 0.4 0.2 0 0/0 5/1 10/1 20/1 20/0 O2/CO2 Concentra0on Figure 3. Effect of O2 and CO2 concentra:ons on overall cell yield as determined by maximum absorbance data from the Balch tubes from Figure 2. Cell yield was tracked to assist in interpreta:on of genera:on :mes (Figure 2) calculated from the experiment. Values are expressed as mean±SD of OD and were collected from 3 replicates. D
C
D
Figure 4. Transmission electron micrographs of JR021-­‐5 (A and B) and CSM3370-­‐14 (C and D). All scale bars, 0.200 μM. Characteristic*
Rhizobiales!cultivar!
JR021/5!
Rhizobiumleguminosarum!
Isolation!source!
ant!gut!
soil!
pleomorphic!
bacillus!
Peritrichous!flagella!
0!
2/6!
Arabinose!
/!
+!
Fructose!
+!
+!
Galactose!
+!
+!
Glucose!
+!
+!
Glycerol!
/!
+!
Inositol!
/!
+!
Lactose!
/!
+!
Maltose!
+!
+!
Mannitol!
/!
+!
Mannose!
+!
+!
Melibiose!
/!
+!
Raffinose!
+!
+!
Rhamnose!
/!
+!
Sorbitol!
/!
+!
Sucrose!
+!
+!
Alanine!
/!
+!
Histidine!
+!
+!
Proline!
/!
+!
Urea!
/!
+!
Erythritol!
/!
+!
pH!range!
5/6.5!
5/9!
!
0.6 -­‐0.2 C
Cell!shape!
19 Genera0on Time (Hrs) Microscopy and Phenotyping
0.86
Cell Yield Ants are one of the most abundant, diverse, and ecologically important insects on the planet. Of par:cular interest to us are herbivorous ants, specifically within the genus Cephalotes (turtle ants). Interes:ngly, the evolu:on of herbivory in ants led to extensive diversifica:on [1]. This is a somewhat surprising occurrence, as an herbivorous diet tends not to provide enough of certain necessary nutrients, like amino acids and nitrogen, for an organism to survive. Hence, we hypothesize that in the case of the Cephalotes ants, bacterial provisioning of nutrients allows an herbivorous lifestyle, and is a significant factor in ant diversifica:on, allowing the ant to move into new niches and habitats where specia:on can occur. Growth Experiments
Table 1. Substrate u:liza:on profile of isolate JR21-­‐5, compared with the well-­‐characterized Rhizobium leguminosarum. The substrate u:liza:on profile of JR21-­‐5 clearly dis:nguishes it from other members of the Rhizobiaceae, gives a glimpse into its in situ environment and metabolic role as primarily a mono-­‐ and disaccharide degrader. However, it can u:lize other non-­‐sugar compounds such as L-­‐lac:c acid, citric acid, D-­‐
glucuronic acid, L-­‐malic acid, α-­‐hydroxybutyric acid, α-­‐keto-­‐glutaric acid, glucuronamide, pyruvic acid methyl ester, and esculin ferric citrate, which suggests nutri:onal versa:lity, a possible benefit to the ant should it need to switch diets. Conclusions
The morphology, substrate u:liza:on, and DNA sequence of JR021-­‐5 and its rela:ves place these bacteria well within a new family of the Rhizobiales order. A name has not yet been determined, but the genus will likely translate to the La:n form of “bent rod” and the species, likewise, to “esculin-­‐swallowing.” The CO2 growth data suggest that 3% CO2 yields significantly faster growth (p<0.05). Growth rates at 0% O2 and 20% O2 are not significantly different, sugges:ng a faculta:vely aerobic lifestyle that may allow JR021-­‐5 to thrive in the varying condi:ons of the ant gut as the ant develops from larvae to adult. Growth at 0% O2 suggests a need for further studies focused on the ability of JR021-­‐5 to perform anaerobic respira:on. Cell yield data is not clear on whether a greater O2 concentra:on facilitates more abundant growth. Further studies are needed.
The use of sugars by JR021-­‐5 implies one func:on in the ant gut is to degrade sugar or sugar-­‐polymers. Inves:ga:on with pec:nase assays into the ability of Rhizobiales to degrade pec:n is ongoing. JR021-­‐5’s use of esculin may indicate an ability to degrade plant toxin, which would be beneficial for both the bacterium and the ant host. This study lays the groundwork for the comparison of Rhizobiales isolated from Cephalotes varians (such as JR021-­‐5) with Rhizobiales isolated from other Cephalotes species. These comparisons may shed light on metabolic differences that could have been important factors in ant specia:on. References and
Acknowledgements
1.  Hansen AK, Moran NA (2014) The impact of microbial symbionts on host plant
utilization by herbivorous insects. Mol. Ecol. 23:1473-1496.
2.  Russell JA, et al. (2009) Bacterial gut symbionts are tightly linked with the
evolution of herbivory in ants. Proc. Natl. Acad. Sci. U.S.A. 106: 21236 –21241.
3.  Hu Y, Lukasik P, Moreau CS, Russell JA (2014) Correlates of gut community
composition across an ant species (Cephalotes varians) elucidate causes and
consequences of symbiotic variability. Mol. Ecol. 23:1284-1300.
We sincerely thank Lori Keen; Alicia Withrow (MSU); Calvin College Science Division and the Na:onal Science Founda:on (Grant ID: DEB-­‐1442156)