Role of alarmone (p)ppGpp in the regulation of indole formation depending on glucose content in Escherichia coli

 Signaling molecules such as indole (product of tryptophan catabolism) and (p)ppGpp (stringent response regulator) are involved in regulation of physiological processes in bacterial cells aimed to adapt to antibiotics and stresses. However, question of existence of relationship between the stringent response and indole signaling requires more detailed investigation.
The aim. To study effect of stringent response regulator (p)ppGpp on indole production in Escherichia coli depending on glucose content.
Materials and methods. In this work, we studied the dynamics of indole accumulation in batch cultures of parent E. coli BW25141 ((p)ppGpp+ strain) and deletion mutant BW25141∆relA∆spoT ((p)ppGpp0 strain) in glucose-mineral tryptophan-free M9 medium, as well as with 2 mM tryptophan addition. In order to study effect of starvation stress on bacterial cell ability to synthesize indole, we used a model of growth limitation by carbon substrate at two glucose concentrations, 0.1 % and 0.4 %.
Results. We have shown here that (p)ppGpp absence in E. coli cells reduces their ability to produce indole in the tryptophan-free medium and significantly slows down the rate of its accumulation in the tryptophan-containing one. Low glucose concentration (0.1 %) leads to decrease in indole production by (p)ppGpp+ cells in the tryptophan-free medium. The presence of indole synthesis precursor, tryptophan, in growth medium, on the contrary, increases the production of indole at lower glucose concentration in both (p)ppGpp+ and (p)ppGpp0 strains demonstrating direct dependence of delay time for onset of indole formation on glucose content, which is more pronounced in the culture of deletion mutant unable of synthesizing (p) ppGpp. The data obtained can be interpreted as result of complex regulatory effect of catabolic repression and the stringent response caused by alarmone (p)ppGpp action on expression level of tnaCAB operon responsible for indole biosynthesis.

1. Kim J, Park W. Indole: A signaling molecule or a mere metabolic byproduct that alters bacterial physiology at a high concentration? J Microbiol. 2015; 53(7): 421-428. doi: 10.1007/s12275-015-5273-3

2. Vega N, Allison K, Khalil A, Collins J. Signaling-mediated bacterial persister formation. Nat Chem Biol. 2012; 8(5): 431-433. doi: 10.1038/nchembio.915

3. Potrykus K, Cashel M. (p)ppGpp: Still magical?Annu Rev Microbiol. 2008; 62: 35-51. doi: 10.1146/annurev.micro.62.081307.162903

4. Korch SB, Henderson TA, Hill TM. Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis. Mol Microbiol. 2003; 50(4): 1199-1213. doi: 10.1046/j.1365-2958.2003.03779.x

5. Hauryliuk V, Atkinson GC, Murakami KS, Tenson T, Gerdes K. Recent functional insights into the role of (p)ppGpp in bacterial physiology. Nat Rev Microbiol. 2015; 13(5): 298-309. doi: 10.1038/nrmicro3448

6. Wood TK, Song S. Forming and waking dormant cells: The ppGpp ribosome dimerization persister model. Biofilm. 2020; 2: 100018. doi: 10.1016/j.bioflm.2019.100018

7. Atkinson GC, Tenson T, Hauryliuk V. The RelA/SpoT Homolog (RSH) superfamily: Distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life. PLoS One. 2011; 6(8): e23479. doi: 10.1371/journal.pone.0023479

8. Srivatsan A, Wang J. Control of bacterial transcription, translation and replication by (p)ppGpp. Curr Opin Microbiol. 2008; 11(2): 100-105. doi: 10.1016/j.mib.2008.02.001

9. Liu S, Wu N, Zhang S, Yuan Y, Zhang W, Zhang Y. Variable persister gene interactions with (p)ppGpp for persister formation in Escherichia coli. Front Microbiol. 2017; 8: 1795. doi: 10.3389/fmicb.2017.01795

10. Zarkan A, Liu J, Matuszewska M, Gaimster H, Summers DK. Local and universal action: The paradoxes of indole signalling in bacteria. Trends Microbiol. 2020; 28(7): 566-577. doi: 10.1016/j.tim.2020.02.007

11. Sanchez-Vazquez P, Dewey CN, Kitten N, Ross W, Gourse RL. Genome-wide effects on Escherichia coli transcription from ppGpp binding to its two sites on RNA polymerase. Proc Natl Acad Sci USA. 2019; 116(17): 8310-8319. doi: 10.1073/pnas.1819682116

12. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000; 97(12): 6640-6645. doi: 10.1073/pnas.120163297

13. Kim D, Sitepu IR, Hashidokoa Y. Induction of biofilm formation in the betaproteobacterium Burkholderia unamae CK43B exposed to exogenous indole and gallic acid. Appl Environ Microbiol. 2013; 79(16): 4845-4852. doi: 10.1128/AEM.01209-13

14. Han TH, Lee JH, Cho MH, Wood TK, Lee J. Environmental factors affecting indole production in Escherichia coli. Res Microbiol. 2011; 162(2): 108-116. doi: 10.1016/j.resmic.2010.11.005

15. Hu M, Zhang C, Mu Y, Shen Q, Feng Y. Indole affects biofilm formation in bacteria. Indian J Microbiol. 2010; 50(4): 362-368. doi: 10.1007/s12088-011-0142-1

16. Isaacs HJr, Chao D, Yanofsky C, Saier MHJr. Mechanism of catabolite repression of tryptophanase synthesis in Escherichia coli. Microbiology. 1994; 140(8): 2125-2134. doi: 10.1099/13500872-140-8-2125

17. Stewart V, Yanofsky C. Evidence for transcription antitermination control of tryptophanase operon expression in Escherichia coli K-12. J Bacteriol. 1985; 164(2): 731-740. doi: 10.1128/jb.164.2.731-740.1985

18. Amato SM, Orman MA, Brynildsen MP. Metabolic control of persister formation in Escherichia coli. Mol Cell. 2013; 50(4): 475-487. doi: 10.1016/j.molcel.2013.04.002