1. Ants play an integral role in terrestrial
ecosystems. Yet, surprisingly, little is known
about their behavior, feeding habits, and
source of nutrition. Bacterial symbionts are
known to play a key role in the diversification
and ecological adaptation of numerous
organisms, including ants. Previous studies
have shown that “turtle ants” from the tribe
Cephalotini possess an abundance of
symbiotic bacteria in their gut [1], and that
members from the phyla Verrucomicrobia,
Bacteroidetes, and Proteobacteria are
consistently detected in the gut despite
variations in diet, population, and
geographical location [2].
Thus, it is clear that isolating, identifying, and
understanding the physiological
characteristics of these core bacteria will help
to elucidate the mechanisms by which
bacterial symbionts interact with Cephalotes
and thereby drive their ecological adaptation
and evolution.
Previously, we successfully isolated two novel
bacteria belonging to Verrucomicrobia from
ant guts. Here, we present preliminary
results on isolates CV41 and CAG34,
cultivated from C. varians and C. rohweri,
respectively
Physiological Characterization of Bacteria Belonging to a Novel Genus of
Verrucomicrobia from the Guts of Cephalotes Ants
Jonathan Y. Lin and John T. Wertz, PhD, Department of Biology, Calvin College, Grand Rapids, MI 49546
Introduction
Objectives
Growth Experiments
Microscopy, Phenotyping, and
Genotyping Conclusions
References and
Acknowledgements
1. Physiologically characterize and determine
the roles of the isolates in ant guts.
2. Systematically categorize and properly name
the isolates.
1. 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.
2. 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.
Growth was possible under an atmosphere
consisting of 0.5-20% O2 and up to 5% CO2 for
CV41 and CAG34. However, CV41 could not
grow in the absence of CO2, making it a
capnophile. This suggests that the bacteria
grow in atmospheric levels of O2 but higher
than atmospheric levels of CO2 in their gut
environment.
Optimum growth for CV41 and CAG34 was
observed between pH 6.9-7.7 and 0.5-1.5%
NaCl (w/v). Both isolates grew optimally at 37
oC. While this growth temperature is higher
than expected, it is possible that the bacteria
have adapted to the warm tropical habitat of
the ants.
Most cultivated Verrucomicrobia solely use
sugars as substrates. Here, our isolates use
sugars as well as organic acids, suggesting that
both are important in the gut. CV41 and CAG34
have slightly different substrate preferences,
perhaps reflective of long-term co-speciation
and dietary preferences of the ants.
From comparisons of their 16S rRNA gene
sequences, CV41 and CAG34 are 98% similar,
suggesting, along with physiological
differences, they are distinct species. Both
isolates share a 93% similarity with their closest
cultivated neighbor Opitutus terrae, suggesting
that CV41 and CAG34 form a novel genus.
Future work will include determining new
genus and species names for the isolates,
obtaining data on their membrane fatty acids,
and developing eco-physiological models for
these bacteria, particularly in relationship to
nitrogen reducing mechanisms in Cephalotes
guts .
General Methods
Figure 4. Transmission electron micrographs of CV41(A) and CAG34 (B).
Scale bars, .200 μM (A and B); Gram-stain light micrographs of CV41(C)
and CAG34 (D). Scale bars, 500 μM (C and D).
Figure 1. Effect of CO2 concentration on generation times of CV41 and
CAG34. Under a starting headspace of 100% N2, Balch tubes were
injected with pure CO2 and atmospheric air to obtain a final headspace
composition of 0-5% CO2 in 0.5% increments. O2 was maintained at 2%
in each tube. Values are expressed as mean±SD of hours and are
obtained from 3 replicates.
Figure 3. Effect of O2 and CO2 concentration on growth rates of CV41 and
CAG34. Under a starting headspace of 100% N2, Balch tubes were
injected with pure CO2 and atmospheric air to obtain a final headspace
composition of 0% O2 with 0% CO2 (0/0), 0% O2 with 1% CO2 (0/1), 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 are obtained
from 6 and 3 replicates for CV41 and CAG34, respectively.
Table 1. Phenotypic characteristics of isolates CV41 and CAG34 compared
with other cultivated members of phylum Verrucomicrobia.
aDiplosphaera colitermitum BAA2264T, bOpitutus terrae DSM11246T,
cPuniceicoccus vermicola IMCC1545T, and dRubritalea marina
DSM177716T. +, utilized; -, not utilized; ND, not determined. Substrate
usage and enzyme activity were determined using BIOLOG microplates
and API 20NE strips.
Figure 2. Effect of O2 concentration on generation times of CV41 and
CAG34. Under a starting headspace of 100% N2, Balch tubes were
injected with atmospheric air and pure CO2 to obtain a final headspace
composition of 0, 0.5, 1, 2, 4, 8, 12, 16, or 20% O2. CO2 was maintained
at 1% in each tube. Values are expressed as mean±SD of hours and are
obtained from 3 replicates.
0
10
20
30
40
50
60
70
0.0% 1.0% 2.0% 3.0% 4.0% 5.0% 6.0%
GenerationTime(Hrs)
CO2 Concentration
CV41
CAG34
0
10
20
30
40
50
60
0.0% 5.0% 10.0% 15.0% 20.0%
GenerationTime(Hrs)
O2 Concentration
CV41
CAG34
0/0 0/1 10/1 20/1 20/0
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
GrowthRate(Generations/Hour)
CAG34
CV41
Unless otherwise specified, Cells were maintained on
TSA plates and in TSB liquid medium under 2% O2 and
5% CO2 at room temperature. After fixation in 2.5%
glutaraldehyde/0.1 M. cacodylate solution, thin
sectioning and transmission electron imaging was
done at the MSU Center for Advanced Microscopy.
Characteristic CV41 CAG34 D. colitermituma O. terraeb P. vermicolac R. marinad
Isolation Source Ant gut Ant gut Termite gut Paddy soil Clamworm gut Sea sponge
Cell Shape Coccus Coccus Diplococcus Coccus Coccus Coccus or
Rod
Cell Diameter (μm) 0.5-0.6 0.5-0.6 0.5-0.6 0.4-0.6 0.6-1.0 0.5-1.1
Color Colorless Colorless Colorless Colorless Pale Red Red
Anaerobic Growth - - + + + -
Catalase - - - - - -
Oxidase + - - - - +
Motility ND ND - + - -
Temp. range (oC) 23-37 23-37 15-35 10-37 8-37 8-30
pH range 6.9-7.3 6.9-7.7 5.5-7.5 5.5-9.0 5.5-9.5 6.8-8.2
NaCl range (%) 0.5-1.5 0.5-1.5 1.5 3.0 1.0-7.5 ND
G+C content (% mol) 60.5 60.7 60.5 73.7 52.1 50.9
Arabinose - - - + + -
Cellobiose + - + - - +
Galactose + - + + + +
Glucose + + + + + +
Lactose + - ND + + +
Mannitol + + - + + +
Mannose + + - + + +
Melibiose - + ND + ND +
A B
C D
Figure 5. Maximum likelihood-based 16S rRNA gene phylogeny of CAG34
and CV41. The phylogeny is based on 1,091 shared nucleotide positions.
* = 16S rRNA gene clones from C. rohweri guts. # = 16S rRNA gene clones
from C. varians guts. Branch point support is indicated by the numbers
at branch nodes. Other members of the division Verrucomicrobia are
given as reference species. Genbank accession numbers are shown in
brackets. Scale bar = 0.2 changes per nucleotide. Phylogenetic analysis
was done using phylogeny.fr software.
Lori Keen; Alicia Withrow (MSU); Calvin College Science Division and
Harvey Rozema Student Research Fellowship