Physiological Characterization of Bacteria Belonging to a Novel Genus of Verrucomicrobia

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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

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

Bacteroidetes

, and

Verrucomicrobia

Proteobacteria

are

, consistently detected in the gut despite variations in diet, geographical location [2].

population, and

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 ant guts.

Verrucomicrobia

from

Here, we present preliminary results on cultivated from respectively isolates CV41

C. varians

and and CAG34,

C. rohweri

,

Objectives

1. Physiologically characterize and determine the roles of the isolates in ant guts.

2. Systematically categorize and properly name the isolates.

General Methods

Unless otherwise specified, Cells were maintained on

TSA plates and in TSB liquid medium under 2% O

2

and

5% CO

2

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

.

Growth Experiments

Microscopy, Phenotyping, and

Genotyping

70

60

50

40

30

20

10

0

0.0%

CV41

CAG34

1.0% 2.0% 3.0%

CO

2

Concentration

4.0% 5.0% 6.0%

Figure 1

. Effect of CO

2 concentration on generation times of CV41 and

CAG34. Under a starting headspace of 100% N

2

, Balch tubes were injected with pure CO

2 and atmospheric air to obtain a final headspace composition of 0-5% CO

2 in each tube.

in 0.5% increments. O

2 was maintained at 2%

Values are expressed as mean

±

SD of hours and are obtained from 3 replicates.

A B

60

50

20

10

40

30

0

0.0%

CV41

CAG34

5.0% 10.0%

O

2

Concentration

15.0% 20.0%

Figure 2

. Effect of O

2 concentration on generation times of CV41 and

CAG34. Under a starting headspace of 100% N

2

, Balch tubes were injected with atmospheric air and pure CO

2 to obtain a final headspace composition of 0, 0.5, 1, 2, 4, 8, 12, 16, or 20% O

2

. CO

2 was maintained at 1% in each tube. Values are expressed as mean

±

SD of hours and are obtained from 3 replicates.

0.16

0.14

0.12

0.10

0.08

0.06

0.04

CAG34

CV41

0.02

0.00

0/0

0/1

10/1

20/1

20/0

Figure 3

. Effect of O

2 and CO

2 concentration on growth rates of CV41 and

CAG34. Under a starting headspace of 100% N

2

, Balch tubes were injected with pure CO

2 composition of 0% O

2 and atmospheric air to obtain a final headspace with 0% CO

2

(0/0), 0% O

2 with 1% CO

2

(0/1), 10%

O

2

CO

2 with 1% CO

2

(10/1), 20% O

2 with 1% CO

2

(20/1), and 20% O

2 with 0%

(20/0). Values are expressed as mean

±

SD of hours and are obtained from 6 and 3 replicates for CV41 and CAG34, respectively.

C D

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).

Characteristic

CV41 CAG34

D. colitermitum

a

O. terrae

b

P. vermicola

c

R. marina d

Isolation Source

Ant gut Ant gut Termite gut Paddy soil Clamworm gut Sea sponge

Cell Shape

Coccus Coccus Diplococcus Coccus Coccus Coccus or

Rod

0.5-1.1

Cell Diameter (

μ m)

Color

Anaerobic Growth

Catalase

Oxidase

Motility

Temp. range ( o

C) pH range

NaCl range (%)

G+C content (% mol)

0.5-0.6

Colorless

-

-

+

ND

23-37

6.9-7.3

0.5-1.5

60.5

0.5-0.6

Colorless

-

-

-

ND

23-37

6.9-7.7

0.5-1.5

60.7

0.5-0.6

Colorless

+

-

-

-

15-35

5.5-7.5

1.5

60.5

0.4-0.6

Colorless

+

-

-

+

10-37

5.5-9.0

3.0

73.7

0.6-1.0

Pale Red

+

-

-

-

8-37

5.5-9.5

1.0-7.5

52.1

Red

-

-

+

-

8-30

6.8-8.2

ND

50.9

Arabinose

Cellobiose

Galactose

Glucose

Lactose

Mannitol

Mannose

+

+

+

+

+

-

+

-

+

+

-

+

-

-

ND

-

-

+

+

-

+

+

+

+

+

+

+

-

+

+

+

+

+

+

-

+

+

+

+

+

-

+

Melibiose

+ ND + ND +

Table 1

. Phenotypic characteristics of isolates CV41 and CAG34 compared with other cultivated

a

Diplosphaera colitermitum

members

BAA2264

T

, of b phylum

Verrucomicrobia

Opitutus terrae

DSM11246

T

c

Puniceicoccus vermicola

IMCC1545

T

, and d

Rubritalea marina

DSM177716

T

. +, utilized; -, not utilized; ND, not determined. Substrate

,

.

usage and enzyme activity were determined using BIOLOG microplates and API 20NE strips.

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.

Conclusions

Growth was possible under an atmosphere consisting of 0.5-20% O

2 and up to 5% CO

2 for

CV41 and CAG34. However, CV41 could not grow in the absence of CO

2

, making it a capnophile. This suggests that the bacteria grow in atmospheric levels of O

2 than atmospheric levels of CO environment.

2 but higher in their gut

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 o

C. 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 guts .

Cephalotes

References and

Acknowledgements

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.

Lori Keen; Alicia Withrow (MSU); Calvin College Science Division and

Harvey Rozema Student Research Fellowship

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