Control of Novel Energy Production by Anaerobic Respiration
Using Citrate via a Citrate-Responsive Two-Component SystemTomohiro SHIMADA
Department of Agricultural Chemistry, School of Agriculture, Meiji
University
Research objective
The aim of this study was to understand the mechanisms of citrate utilization andresponse in Escherichia coli, including the genomic transcriptional regulation of the citrateresponsive two-component regulatory system, CitAB, and its physiological role. E. coli strain
K-12 cannot grow on citrate as a single carbon source, but can use it as a substrate for a typeof metabolism known as “citrate fermentation,” which utilizes part of the tricarboxylic acidcycle in a reductive direction to consume reduction forces produced from metabolism, such as
glycolysis under anaerobic conditions. This type of metabolism has been reported to betranscriptionally regulated by the two-component system, CitAB (CitA is the histidine kinaseand CitB is the response regulator), which responds to extracellular citrate under anaerobicconditions. However, the role of CitAB in regulating the whole genome is unknown. Therefore,
we aimed to identify the genomic transcriptional regulatory network of CitB in E. coli anddetermine its physiological role.
Methods
Transcriptional regulatory networks form a hierarchical structure. Therefore, tounderstand the essential regulatory mechanisms of transcriptional control, it is necessary to
identify direct regulatory targets that are distinct from indirect effects. In addition, the presenceof other factors in the cell causes competitive inhibition of the genome, and the tested
transcription factors are not always expressed or activated in vivo. In this study, we
comprehensively identified the genomic binding regions of CitB using a novel GenomicSELEX (gSELEX) method. gSELEX is an in vitro method in which purified transcription
factors are mixed with genomic DNA fragments, and the DNA sequences of the complexes
formed are analyzed. The gSELEX method enables the comprehensive identification of directbinding sequences in the genome1). This method has the advantage that direct genomic target
sequences can be obtained, even for transcription factors whose expression or activation
conditions are unknown, and it is not affected by the presence of other competitive factors inthe cell. In this study, CitB target sequences obtained using the gSELEX method were analyzed
using tiling arrays. The binding activity of the CitB target was confirmed using gel shift assays.

To validate the regulation of the newly identified CitB target genes, wild-type and citABdeficient E. coli strains were cultured in glucose-containing medium under anaerobic
conditions, and mRNA levels with and without citrate (20 mM) addition were compared using
reverse transcription-quantitative polymerase chain reaction. Next, citrate and glucose
concentrations in the culture medium were measured over time to observe the effect of citrateon glucose consumption. ATPase activity was measured to verify that energy production wasregulated by CitAB. Furthermore, the growth-stimulating effect of citrate was verified using
strains deficient in genes associated with citrate metabolism and anaerobic respiration. Insummary, we determined the mechanism of anaerobic respiration using citrate and CitAB.
Results
The gSELEX method successfully identified approximately 30 CitB-binding regionsin the E. coli genome (Fig. 1). All previously reported CitB target genes were identified.
Several novel target genes involved in carbon metabolism and respiration were also identified.
These genes were selected as the targets for this study. The ability of CitB to bind to thesenovel targets was verified using a gel shift assay, and it was confirmed that CitB specificallybound to all targets.
To further examine the effect of CitAB on transcriptional regulation, the expression levelsof the novel target genes were compared in wild-type and citAB-deficient E. coli strains inglucose minimal medium under anaerobic conditions, with or without the addition of citrate.
It was found that the mRNA levels of all target genes increased more than 2-fold in the wildtype strain upon the addition of citrate.

Cell growth was monitored under the same conditions as above. The addition ofcitrate enhanced the growth of the wild-type strain, but no effect was observed in the citABdeficient strain. When the concentrations of citrate, succinate, and glucose in the medium
under these conditions were measured over time, accelerated succinate production and glucose
consumption were observed with the consumption of citrate in the wild-type strain, but not in
the citAB-deficient strain. These results demonstrated a citrate- and citAB-dependent increasein the growth rate by promoting glucose consumption. Furthermore, as the transcription ofgenes encoding ATP synthase was activated by CitAB, ATP synthase activity was measured
under the same conditions. The results indicated that ATP synthase activity is dependent on
both citrate and CitAB. ATP production by ATP synthases requires a proton concentration
gradient. Therefore, the growth-promoting effect of citrate was observed in strains deficient in
respiration-related genes and was abolished in strains deficient in fumarate reductase andNADH:quinone oxidoreductase.
Conclusion

The results of this study suggest that the role of the citrate-responsive two-component
system CitAB regulatory network in E. coli is not only the consumption of reducing power(NADH) by “citrate fermentation” under anaerobic conditions, but also the activation ofglycolysis by activating pyruvate dehydrogenase (AceEF-Lpd) and ATP synthase(AtpABCDEFGHI) to produce ATP using a proton concentration gradient (Fig. 2).

References
1) Shimada, T., Ogasawara, H. and Ishihama, A. (2018) Genomic SELEX screening ofregulatory targets of Escherichia coli transcription factors. Methods Mol. Biol. 1837:49-69.