Leucine-Responsive Regulatory Protein in Acetic Acid Bacteria Is Stable and Functions at a Wide Range of Intracellular pH Levels

• Published in: Journal of bacteriology Vol. 203; no. 18; p. e0016221
• Main Authors: Ishii, Yuri, Shige, Yuki, Akasaka, Naoki, Trinugraha, Afi Candra, Higashikubo, Haruka, Fukuda, Wakao, Fujiwara, Shinsuke
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 20.08.2021
• Subjects: Acetic acid; Acetic Acid - metabolism; Acetic acid bacteria; Acetobacteraceae - genetics; Acetobacteraceae - growth & development; Acid production; Acidification; Acids; Affinity; Bacteria; Bacteriology; Binding; Cell culture; Circular dichroism; Deoxyribonucleic acid; Dichroism; DNA; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; E coli; Electrophoretic mobility; Escherichia coli - genetics; Fluorescence; Gene Expression Regulation, Bacterial; Green fluorescent protein; Green Fluorescent Proteins - metabolism; Hydrogen-Ion Concentration; Intracellular; Leucine; Leucine-Responsive Regulatory Protein - chemistry; Leucine-Responsive Regulatory Protein - genetics; Leucine-Responsive Regulatory Protein - metabolism; pH effects; Protein Binding; Proteins; Research Article; Spectroscopy; Transcription factors; acetic acid bacteria; Komagataeibacter europaeus; leucine-responsive regulatory protein; pHluorin; cytoplasmic acidification
• ISSN: 0021-9193; 1098-5530
• DOI: 10.1128/JB.00162-21

Acetic acid bacteria grow while producing acetic acid, resulting in acidification of the culture. Limited reports elucidate the effect of changes in intracellular pH on transcriptional factors. In the present study, the intracellular pH of Komagataeibacter europaeus was monitored with a pH-sensitive green fluorescent protein, showing that the intracellular pH decreased from 6.3 to 4.7 accompanied by acetic acid production during cell growth. The leucine-responsive regulatory protein of ( Lrp) was used as a model to examine pH-dependent effects, and its properties were compared with those of the Escherichia coli ortholog ( Lrp) at different pH levels. The DNA-binding activities of Lrp and Lrp with the target DNA ( and ) were examined by gel mobility shift assays under various pH conditions. Lrp showed the highest affinity with the target at pH 8.0 ( [dissociation constant], 0.7 μM), decreasing to a minimum of 3.4 μM at pH 4.0. Conversely, Lrp did not show significant differences in binding affinity between pH 4 and 7 ( , 1.0 to 1.5 μM), and the highest affinity was at pH 5.0 ( , 1.0 μM). Circular dichroism spectroscopy revealed that the α-helical content of Lrp was the highest at pH 5.0 (49%) and was almost unchanged while being maintained at >45% over a range of pH levels examined, while that of Lrp decreased from its maximum (49% at pH 7.0) to its minimum (36% at pH 4.0). These data indicate that Lrp is stable and functions over a wide range of intracellular pH levels. Lrp is a highly conserved transcriptional regulator found in bacteria and archaea and regulates transcriptions of various genes. The intracellular pH of acetic acid bacteria (AAB) changes accompanied by acetic acid production during cell growth. The Lrp of AAB ( Lrp) was structurally stable over a wide range of pH and maintained DNA-binding activity even at low pH compared with Lrp from E. coli living in a neutral environment. An experiment showed DNA-binding activity of Lrp to the target varied with changes in pH. In AAB, change of the intracellular pH during a cell growth would be an important trigger in controlling the activity of Lrp .