α-fur, an antisense RNA gene to fur in the extreme acidophile Acidithiobacillus ferrooxidans

Abstract

A large non-coding RNA, termed a-Fur, of~1000 nt has been detected in the extreme acidophile Acidithiobacillus ferrooxidans encoded on the antisense strand to the iron-responsive master regulator fur (ferric uptake regulator) gene. A promoter for a-fur was predicted bioinformatically and validated using gene fusion experiments. The promoter is situated within the coding region and in the same sense as proB, potentially encoding a glutamate 5-kinase. The 39 termination site of the a-fur transcript was determined by 39 rapid amplification of cDNA ends to lie 7 nt downstream of the start of transcription of fur. Thus, a-fur is antisense to the complete coding region of fur, including its predicted ribosome-binding site. The genetic context of a-fur is conserved in several members of the genus Acidithiobacillus but not in all acidophiles, indicating that it is monophyletic but not niche specific. It is hypothesized that a-Fur regulates the cellular level of Fur. This is the fourth example of an antisense RNA to fur, although it is the first in an extreme acidophile, and underscores the growing importance of cis-encoded non-coding RNAs as potential regulators involved in the microbial iron-responsive stimulon. INTRODUCTIONIron is an important micronutrient for micro-organisms because it is used as a cofactor for a large number of enzymes and electron transport proteins (Williams, 2012). However, it can also catalyse the Fenton reaction, generating hydroxyl radicals ( ? OH) that can damage many biological components, including DNA and proteins (Imlay, 2008;Cornelis et al., 2011). It is found at levels of~10 218 M (Andrews et al., 2003;Williams, 2012) in neutral, aerobic environments, which is far below the minimum requirement for bacterial growth (10 27 M) (Andrews et al., 2003). Consequently, micro-organisms have evolved complex systems for the uptake of iron, and have developed tight intracellular homeostatic controls to balance iron uptake and storage to avoid possible oxidative stress (Imlay, 2008;Cornelis et al., 2011). In many bacteria, this regulation is achieved by the action of the Fur (ferric uptake regulator) protein.In iron-rich conditions, Fur binds Fe 2+ (Mills & Marletta, 2005) and functions as a transcriptional repressor by binding to Fur boxes near promoter sequences blocking the entry of RNA polymerase, thus inhibiting initiation of RNA transcription. Genes regulated in this manner include those involved in the biosynthesis of siderophores and iron uptake (Friedman & O'Brian, 2003; Quatrini et al., 2007;Gao et al., 2008;Butcher et al., 2012), genes encoding small RNAs such as fsrA and ryhB (Smaldone et al., 2012;Massé & Gottesman, 2002;Salvail & Massé, 2012), and many other genes involved in iron homeostasis (reviewed by Salvail & Massé, 2012). Under iron-limiting conditions, Fur no longer binds Fe 2+ , reducing its affinity for Fur boxes and thus allowing transcription to proceed (Baichoo et al., 2002;Ochsner et al., 2002;Butcher et al., 2012). In the case of the siderophore and iron-uptake genes, t...

Physiological roles of antisense RNAs in prokaryotes

“…Under oxidative stress, mimicked by hydrogen peroxide or methyl viologen, a high expression of α-fur, which was associated with an activation of fur transcription, lead to a global increase of total Fur protein in M. aeruginosa PCC 7806 [36] in order to prevent hydroxyl radical formation. An antisense to a fur mRNA was also found in the extreme acidophile Acidithiobacillus ferrooxidans [37], an organism which lives in iron-rich conditions and which uses iron as an energy source. During iron depletion and accumulation of sulfur, both the sense and the antisense of fur were induced, possibly due to opening of a secondary structure on the RBS of the fur transcript, which allows efficient translation.…”

The world of asRNAs in Gram-negative and Gram-positive bacteria

“…PCC 7120, Synechocystis sp. PCC 6803, Microcystis aeruginosa and in the proteobacteria Acidithiobacillus ferrooxidans [33][34][35][36][37]. This asRNA named α-fur is encoded in the 3′ untranslated region (UTR) of the fur gene and is involved in the repression of fur expression by an unknown mechanism.…”

“…Strikingly, the α-fur promoters, located downstream of their respective fur genes, are not conserved and are likely to have emerged independently. Therefore, this suggests a strong evolutionary pressure toward asRNA-mediated regulation of Fur expression; possibly due to the ability of asRNAs to modulate their function according to intracellular Fe 2+ concentrations in iron-limited environments such as ocean for cyanobacteria and V. anguillarum or saturated in iron in the case of A. ferrooxidans, which is found in acidic water outflow from metal mines [37].…”