Unveiling CO2 stimulon underlying the microbial community
Kenji UEDA
College of Bioresource Sciences, Nihon University
Report:In order to widely assess the influence of high CO2 atmosphere on the bacterial
cellular state, comprehensive transcriptional and protein expression analyses were carriedout. The results demonstrated that the condition remarkably affected the expression level of
20-50 genes in each organism and that each strain exhibited a different expression profile.
Short time exposure to high CO2 atmosphere not only suggested the presence of a specificgenetic regulatory system but also inferred that the condition causes cellular lesion. Isolationof multiple bacterial strains exhibiting a high-CO2-dependent colony morphotype
demonstrated the general occurrence of CO2-dependent phenomenon.
Research objectives
CO2 is an effective indicator of biological activity that probably serves as the mostgeneral quormone (a specific small molecule being an indicator of the maturation level of themicrobial community) in the natural environment. The author hypothesizes that CO2 isreceived by various microorganisms and affects their mode of action, thus constituting abasis supporting the development of the bacterial ecosystem.1-5 This study aims atunderstanding the influence of a general environmental factor on the microbial communityfrom the viewpoint of genetic regulation by comprehensively analyzing the effect of CO2 onbacterial gene expression. In particular, the study tries to elucidate the whole picture of CO2stimulon, the genetic group whose expression varies simultaneously along with the change in
CO2 level, to clarify that CO2 is the most general and effective metabolic indicator andserves as a key factor that determines the mode of microbial community structure. To date,
the argument in microbial ecology is mostly based on limited evidence from the observation
on a certain physiological property of a specific organism and the change in microbial
content, and has not yet figured out the principle fundamental to the development of themicrobial community. This study brings forth the new understanding in microbial ecology
by elucidating the role of CO2, a key factor fundamental to the microbial physiology andcommunity structuring.
Methods:
To study the cellular response to the change in CO2 concentration, expression
comparison analyses were carried out at an RNA and protein level by using bacterial cells
incubated under normal conditions (Air) and at 5% CO2 air (high-CO2). For RNA analysis,
the transcripts extracted from the cells cultivated for 24 h under the two conditions were
subjected to RNAseq and RT-PCR analyses. For protein analysis, the cells were precultured

and then incubated for 1 h under air and high- CO2 condition to study the response in a shortperiod. Proteins from intercellular and extracellular fractions were subjected to acomprehensive MS analysis. The transcriptional analysis was carried out in five model
bacterial organisms including those affiliating Bacillus. The protein analysis was done withregard to Bacillus subtilis.
In addition to the aforementioned expression analysis, new bacterial strains
exhibiting a different colony morphotype under air and high-CO2 condition were screened.
Isolates obtained mainly from water samples by cultivation under high-CO2 conditions werereplica-plated and their colony morphotypes were compared after cultivation under the twoconditions at 28°C.
The aforementioned experiments were carried out using buffered medium toprevent the pH shift due to the addition of high CO2.
Results:(1) Transcriptional analysis.
Transcriptional profiles under air and high-CO2 conditions were obtained for 6
strains that exhibited a CO2-dependent phenotype in the preliminary observation. As a resultof comparison analysis, distinctive transcriptional upregulation was observed in all strains
with regard to 20-50 genes both under air and high-CO2 conditions. The constituents of theupregulated gene group were diverged, and no common gene was identified. Any specificregulatory mechanism that commonly controls the CO2-dependent gene expression has not
yet been deduced. Meanwhile, the CO2-dependent transcription was confirmed with respect
to some of the genes of B. subtilis exhibiting a positive or negative response to high- CO2
through RT-PCR analysis. These genes included those involved in the amino acidmetabolism. The fact that the genes identified by the abovementioned RNAseq analysis
included those for primary metabolism inferred that the increase in CO2 concentration
stimulates the signal transduction connected to the metabolic control.
(2) Protein analysis.
The transcriptional analysis demonstrated the occurrence of a complex
transcriptional profile specific to each organism. Based on this result, the next trial wasperformed by focusing on a short-period cellular response at the protein level. A single batchof B. subtilis cells was divided into two fractions and incubated under air and high-CO2
conditions for 1 h, and the resultant cultures were subjected to the comprehensive protein
expression analysis using mass-spectrometry. The analysis successively detected proteins
exhibiting marked dependence on CO2, however, those included innercellular proteins suchas ribosomal proteins. This raised the possibility that the exposure to high CO2 atmospherecauses cellular lesion and leakage of innercellular contents. On the other hand, the highCO2-dependent reduction of certain proteins such as the cell wall-binding protein suggested
the occurrence of a specific repression mechanism responding to the high CO2 environment.

(3) Screening for CO2-dependent bacterial strains.
About 500 natural isolates were studied for the dependence of their colony
phenotype on high CO2. Of these, ca. 30 strains exhibited distinctive dependence. The major
phenotypes affected by the CO2 level were the production of a sticky matrix (a possible
extracellular polysaccharide), pigment production, and colony morphology. The Bacillus
strains used in the aforementioned analysis also showed a marked dependence of theirphenotype.
Conclusion:
This and the previous studies indicate that many bacteria are variously affected byhigh CO2. The influence caused by CO2 probably includes the change due to the adaptive
response of the cell, which is based on the signaling network controlling gene expression bysensing the change in CO2 concentration. On the other hand, the evidence obtained in thisstudy suggests that the change in CO2 concentration causes other events that significantly
affect the cell homeostasis such as cellular lesion and metabolic flow exchange due to the
influence on the carboxylating or decarboxylating enzymatic reactions as well as the shift ofpH. CO2 stimulon should be recognized as a global system that includes the responses tothese indirect factors affected by CO2 change. Therefore, the understanding of the whole
picture of the system should be carried out through the identification of each factor andrelating signal network.
References:
1. Ueda, K. Invisible interactions between microorganisms. In Microbes:The Foundation
Stone of the Biosphere) (Ed. C. Hurst Springer. in press.
2. Ueda, K. and Beppu, T. Syntrophic growth of Symbiobacterium thermophilum inassociation with free-living bacteria. In The Mechanistic Benefits of Microbial Symbionts.
(Ed. C. Hurst). Springer. pp. 47-65. 2016.
3. Watsuji, T. O. et al. (2014) Analysis of the tryptophanase expression in Symbiobacterium
thermophilum in a coculture with Geobacillus stearothermophilus. Appl. Microbiol.
Biotechnol. 98:10177-10186.
4. Ueda, K., Nishida, H., and Beppu, T. (2012) Dispensabilities of carbonic anhydrase inproteobacteria. Int. J. Evol. Biol. 2012:324549.
5. Nishida, H., Beppu, T., and Ueda, K. (2009) Symbiobacterium lost carbonic anhydrase in
the course of evolution. J. Mol. Evol. 68:90-96.