82NISR RESEARCH GRANT
2004 Research Grant

Studies on the Growth Characteristics of Unculturable
Environmental Microorganisms Using a Syntrophic Bacterium, Symbiobacterium thermophilum, as a Model
Kenji UEDA
College of Bioresource Sciences, Nihon University
Research aims
The commensal relationship between microorganisms is
not well studied because of the difficulties in its manipulation techniques, in contrast to the great progress for the microbial symbiosis with animals and plants. This study aims
at elucidating the basic molecular mechanisms that underlie
the microbe-microbe interaction. Especially, it focuses andcarries out precise analysis on the physiological characteristics of Symbiobacterium thermophilum. S. thermophilum isa unique microorganism, which does not propagate when it
is isolated but normally grows when it is cultured with athermophilic Bacillus strain S.1–3) To identify the factor(s)that support the growth of this organism should provide insight into the molecular basis for the microbial commensalism.
Methods
The strains used were S. thermophilum IAM14863,3) Escherichia coli yadF  mutant and its parental strain4) andBacillus subtilis ATCC6633. E. coli and B. subtilis weregrown under the standard culture conditions, and S. thermophilum was cultivated as described previously.2,5) In thecultures for the air-phase CO2 concentration measurement,
an airtight 0.5 L Erlenmeyer flask with two side arms wasused. For culture media, LB medium (containing 1% Tryptone, 0.5% yeast extract, and 0.5% NaCl) was used as abase medium. The amount of each constituent was changed
when needed. For the measurement of cellular concentration, the specific quantitative PCR technique was used forS. thermophilum,5) and the colony-counting method wasused for others.
Results
The precise analysis on the culture conditions that induce
mono-growth of S. thermophilum revealed that this organism performs marked proliferation when CO2-containing
N2 gas was introduced in the culture medium. Since the organism did not grow when CO2-free N2 gas was introduced,
we concluded that CO2 is the factor that induces the monogrowth of this organism. By using the closed cultivation
system, the threshold CO2 concentration for the initiation ofproliferation of S. thermophilum was determined to be200 ppm.
Recently, similar phenomena as above were reported as
to the phenotypes of E. coli and Ralstonia eutropha mutants for carbonic anhydrase (CA).6,7) CA is a widely-occurring enzyme that mediates interconversion between CO2and HCO3. The deficiency of the CA gene in E. coli andR. eutropha causes lethal effect under normal culture condition but does not affect the growth under CO2-introduced
culture condition. Based on this, we performed cocultivation a CA mutant (yadF ) of E. coli and B. subtilis, andfound that the mutant performed marked growth even under
the normal culture condition. The threshold CO2 concentration for the initiation of the CA mutant was determined tobe 7,500 ppm.
The genome sequencing of S. thermophilum8) revealed
that there is no CA gene in the genome of this organism.
The evidence strongly suggests that as observed with the E.
coli CA mutant, S. thermophilum requires CO2 due to thelack of this enzyme, and it is compensated for by the commensalism with another microorganism.
In order to demonstrate the presence of CO2-dependent
microorganism like S. thermophilum, we screened for microorganisms that grow only under the CO2-introduced culture condition. As a result, about 60 bacterial strains weresuccessfully isolated. The 16S rDNA-based phylogenetic
analysis revealed that they contain several strains that are
taxonomically very unique.
Conclusion
1) The most critical factor for the initiation of proliferation of S. thermophilum is CO2.
2) S. thermophilum can proliferate by utilizing the CO2
generated by other organisms.
3) The candidate for the genetic background that causes
the CO2 requirement is the deficiency of carbonic anhydrase.
4) CO2-dependent microorganisms are probably predominant in the natural environment.
5) CO2-dependent microorganisms may contain unknown
strains, since they can not be isolated under the normal culture condition although they grow under commensal cultivation.

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