Mechanism of growth delay induced in Escherichia coli by near ultraviolet radiation.

Abstract

Continuously growing cultures of E. coli B/r were irradiated with a fluence of broad-band near-ultraviolet radiation (315-405 nm) sufficient to cause extensive growth delay and complete cessation of net RNA synthesis. Chloramphenicol treatment was found to stimulate resumption of RNA synthesis, similar to that observed with chloramphenicol treatment after amino-acid starvation. E. coli strains in which amino-acid starvation does not result in cessation of RNA synthesis ("relaxed" or ret strains) show no cessation of growth and only a slight effect on the rate of growth or of RNA synthesis. These findings show that such near-UV fluences do not inactivate the RNA synthetic machinery but affect the regulation of RNA synthesis, in a manner similar to that produced by amino-acid starvation. Such regulation is believed to be mediated through alterations in concentration of guanosine tetraphosphate (ppGpp), and our estimations of ppGpp after near-UV irradiation are consistent with such an interpretation. These data, combined with earlier published data, strongly suggest that the mechanism of near-UV-induced growth delay in E. coil involves partial inactivation of certain tRNA species, which is interpreted by the cell in a manner similar to that of amino-acid starvation, causing a rise in ppGpp levels, a shut-off of net RNA synthesis, and the induction of a growth delay.Near-ultraviolet radiation (near-UV; 300-380 nm) is present in sunlight, but is absorbed only marginally, if at all, by proteins and nucleic acids. Yet the well-known deleterious effects of far-ultraviolet radiation (far-UV; below 300 nm), such as killing, induction of mutation, and inhibition of growth, are found to occur in bacteria throughout the near-UV region (1, 2).Of these effects, the one induced by the lowest fluences of near UV is the inhibition of growth*. Bacterial studies have shown that growth delay has a narrow action spectrum, peaking at 340 nm. Earlier suggestions that quinones might be the chromophores and oxidative phosphorylation the cellular target (1) have not been supported by recent work showing that coenzyme Q is resistant to near-UV radiation in vivo (3) and that ATP synthesis shows a threshold response (B. Lakchaura, T. Fossum, and J. Jagger, J. Bacteriol., 125, in press). In addition, it is unlikely that DNA is the target for near-UV-induced growth inhibition (4).Inhibition of growth clearly must involve inhibition of macromolecular synthesis. Swenson et al. (5) He has also shown that the action spectrum for induction of this inhibition of RNA synthesis is similar to the action spectrum for induction of growth delay in E. coli, and that the growth delay action spectrum fits very closely to the absorption spectrum of tRNAs that contain the unusual nucleoside 4-thiouridine (4Srd). It is known that near-UV irradiation in vitro of such tRNAs can induce an adduct between the 4Srd in the 8-position and a cytidine in the 13-position of the tRNA, and that this lowers the rate at which the tRNA accepts amino acids (7). The f...

Biosynthesis of 4-Thiouridine in tRNA in the Methanogenic Archaeon Methanococcus maripaludis

“…These cross-linked tRNAs are poor substrates for aminoacylation (6,8), leading to the accumulation of uncharged tRNAs (9). This effect mimics amino acid starvation and triggers the stringent response through the synthesis of ppGpp and ApppGpp, which inhibit cell growth and induce the expression of specific proteins that enhance cell survival after the stress (2,4,7,10).…”

The Role of the Cysteine Residues of ThiI in the Generation of 4-Thiouridine in tRNA

“…The s 4 U undergoes a photoactivated 2 ϩ 2 cycloaddition with cytidine 13 when the tRNA is exposed to light of a wavelength similar to the 334 nm absorbance maximum of s 4 U (13-15). The resulting cross-linked tRNA are poor aminoacylation substrates (16), and the accumulation of uncharged tRNA arrests growth by triggering the stringent response (17,18). Lipsett and co-workers (19,20) investigated the enzymology of s 4 U biosynthesis in Escherichia coli and reported that the overall reaction utilized cysteine as the sulfur donor and required ATP as a substrate.…”

Photoreactive Bicyclic Amino Acids as Substrates for Mutant Escherichia coli Phenylalanyl-tRNA Synthetases

“…A potential caveat of using UVA irradiation ( , ϳ335 nm) involves the formation of photo adducts between thiouridine and cytidine at positions 8 and 13 in some tRNAs of E. coli (18), causing an amino acid starvation response. Once the cells are removed from UV exposure, however, the effects are reversed, and they recover by synthesis of new functional tRNAs (19).…”