Consumption of N5, N10-methylenetetrahydrofolate in Thermus thermophilus under nutrient-poor condition

Abstract

TrmFO catalyzes the formation of 5-methyluridine at position 54 in tRNA and uses N5, N10-methylenetetrahydrofolate (CH2THF) as the methyl group donor. We found that the trmFO gene-disruptant strain of Thermus thermophilus, an extremely thermophilic eubacterium, can grow faster than the wild-type strain in the synthetic medium at 70°C (optimal growth temperature). Nucleoside analysis revealed that the majority of modifications were appropriately introduced into tRNA, showing that the limited nutrients are preferentially consumed in the tRNA modification systems. CH2THF is consumed not only for tRNA methylation by TrmFO but also for dTMP synthesis by ThyX and methionine synthesis by multiple steps including MetF reaction. In vivo experiment revealed that methylene group derived from serine was rapidly incorporated into DNA in the absence of TrmFO. Furthermore, the addition of thymidine to the medium accelerated growth speed of the wild-type strain. Moreover, in vitro experiments showed that TrmFO interfered with ThyX through consumption of CH2THF. Addition of methionine to the medium accelerated growth speed of wild-type strain and the activity of TrmFO was disturbed by MetF. Thus, the consumption of CH2THF by TrmFO has a negative effect on dTMP and methionine syntheses and results in the slow growth under a nutrient-poor condition.

Regulatory Factors for tRNA Modifications in Extreme- Thermophilic Bacterium Thermus thermophilus

“…Biosynthesis of m 5 s 2 U54 is accomplished in two steps, methylation of the C5 atom and the 2-thiolation. Although these steps occur independently at U54 in tRNA (Shigi et al, 2006a; Yamagami et al, 2016), the m 5 U54 modification in tRNA is almost fully formed in living cells even when T. thermophilus is cultured under nutrient-poor conditions (Yamagami et al, 2018). To date, therefore, an s 2 U54 modification has not been observed in tRNA from the T. thermophilus wild-type strain.…”

Transfer RNA Modification Enzymes from Thermophiles and Their Modified Nucleosides in tRNA

“…Among tRNA modification enzymes, both Fe-S cluster proteins [34,130,134,142,150,173,196,236,363] for sulfur-transfer, reduction of base and/or radical S-adenosyl-l-methionine (SAM) reaction, and enzymes with catalytic cysteine residues [141,210,364,365,366], seem to be easily changed under oxidative stress. In some cases, the substrate (e.g., electron donors and folate derivatives [126,221,227,367]) may be unstable under aerobic conditions at high temperatures. Similarly, several modified nucleosides such as D and s 4 U may be labile under oxidative stress at high temperatures.…”

“…To test this idea, the extent of modifications in tRNA from T. thermophilus cells cultured in a nutrient-poor condition was investigated [227]. Contrary to expectation, the extent of the modification of all methylated nucleosides analyzed was normal, which demonstrates that the limited nutrients were preferentially consumed in the tRNA modification systems [227]. Thus, the findings indicated the importance of tRNA modifications for the survival of T. thermophilus .…”

Unique Features and Anti-microbial Targeting of Folate- and Flavin-Dependent Methyltransferases Required for Accurate Maintenance of Genetic Information

“…Under these conditions, induced by the relatively low growth temperature, deletion of trmFO leads to defects in protein synthesis, thus influencing the cell metabolism at a global level. Moreover, the consumption of CH 2 H 4 THF by TrmFO limits the rate of dTMP synthesis by ThyX, thus resulting in slow growth under nutrient-poor conditions ( Yamagami et al, 2018 ). This implies that (ribo) thymidylate-forming enzymes may influence differentially the folate cycles in actively dividing cells, as we have previously proposed ( Leduc et al, 2007 ).…”