Identification of Genetic Variants via Bacterial Respiration Gas Analysis

Naoki Koga; Takuro Hosomi; Martijn Zwama; Chaiyanut Jirayupat; Takeshi Yanagida; Kunihiko Nishino; Seiji Yamasaki

doi:10.3389/fmicb.2020.581571

Identification of Genetic Variants via Bacterial Respiration Gas Analysis

Front Microbiol. 2020 Nov 16:11:581571. doi: 10.3389/fmicb.2020.581571. eCollection 2020.

Authors

Naoki Koga¹, Takuro Hosomi², Martijn Zwama³, Chaiyanut Jirayupat², Takeshi Yanagida^{2

4}, Kunihiko Nishino^{1

3}, Seiji Yamasaki^{1

3

5}

Affiliations

¹ School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
² Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.
³ Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan.
⁴ Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka, Japan.
⁵ Institute for Advanced Co-Creation Studies, Osaka University, Osaka, Japan.

Abstract

Indole is a signal molecule derived from the conversion of tryptophan, and it is present in bacterial respiratory gas. Besides influencing bacterial growth, indole exhibits effects on human health, including a positive effect on inflammation and protection against pathogens. However, a high fecal indole concentration (FIC) can suggest an unbalanced gut flora or the presence of certain pathogens. To analyze the indole produced by bacteria, its collection and detection is required. Traditional methods usually require centrifugation of liquid bacterial culture medium and subsequent extraction of indole from the medium or partial purification of indole from fecal samples (e.g., by distillation or extraction). In this study, we demonstrate the possibility of identifying gas contents directly from bacteria, and we distinguish the difference in species and their genetics without the need to centrifuge or extract. Using an absorbent sheet placed above a liquid culture, we were able to collect gas content directly from bacteria. Gas chromatography-mass spectrometry (GC-MS) was used for the analysis. The GC-MS results showed a clear peak attributed to indole for wild-type Escherichia coli cells (MG1655 and MC4100 strains), whereas the indole peak was absent in the chromatograms of cells where proteins, part of the indole production pathway from tryptophan (TnaA and TnaB), were not expressed (by using tnaAB-deleted cells). The indole observed was measured to be present in a low nmol-range. This method can distinguish whether the bacterial genome contains the tnaAB gene or not and can be used to collect gas compounds from bacterial cultures quickly and easily. This method is useful for other goals and future research, such as for measurements in restrooms, for food-handling facilities, and for various applications in medical settings.

Keywords: Escherichia coli; bacteria; gas; gas chromatography-mass spectrometry; indole; tnaAB.