Salt Stress-Induced Loss of Iron Oxidoreduction Activities and Reacquisition of That Phenotype Depend on rus Operon Transcription in Acidithiobacillus ferridurans

Abstract

The type strain of the mineral-oxidizing acidophilic bacterium was grown in liquid medium containing elevated concentrations of sodium chloride with hydrogen as electron donor. While it became more tolerant to chloride, after about 1 year, the salt-stressed acidophile was found to have lost its ability to oxidize iron, though not sulfur or hydrogen. Detailed molecular examination revealed that this was due to an insertion sequence, IS, which belongs to the IS subgroup of the IS family, having been inserted downstream of the two promoters PI and PII of the operon (which codes for the iron oxidation pathway in this acidophile), thereby preventing its transcription. The ability to oxidize iron was regained on protracted incubation of the culture inoculated onto salt-free solid medium containing ferrous iron and incubated under hydrogen. Two revertant strains were obtained. In one, the insertion sequence IS had been excised, leaving an 11-bp signature, while in the other an ∼2,500-bp insertion sequence (belonging to the IS family) was detected in the downstream inverted repeat of IS The transcriptional start site of the operon in the second revertant strain was downstream of the two ISs, due to the creation of a new "hybrid" promoter. The loss and subsequent regaining of the ability of to reduce ferric iron were concurrent with those observed for ferrous iron oxidation, suggesting that these two traits are closely linked in this acidophile. Iron-oxidizing acidophilic bacteria have primary roles in the oxidative dissolution of sulfide minerals, a process that underpins commercial mineral-processing biotechnologies ("biomining"). Most of these prokaryotes have relatively low tolerance to chloride, which limits their activities when only saline or brackish waters are available. The study showed that it was possible to adapt a typical iron-oxidizing acidophile to grow in the presence of salt concentrations similar to those in seawater, but in so doing they lost their ability to oxidize iron, though not sulfur or hydrogen. The bacterium regained its capacity for oxidizing iron when the salt stress was removed but simultaneously reverted to tolerating lower concentrations of salt. These results suggest that the bacteria that have the main roles in biomining operations could survive but become ineffective in cases where saline or brackish waters are used for irrigation.

Influence of mobile genetic elements and insertion sequences in long- and short-term adaptive processes of Acidithiobacillus ferrooxidans strains

“…To evaluate the persistence and/or mobility of iMGEs and eMGEs in A. ferrooxidans strains under different growth conditions, we chose sublineage 2A strain CCM 4253 as a test case. In iron-oxidizing acidithiobacilli, changes in growth and stress conditions have proved to increase the transposition of IS elements at new sites, particularly in genes involved in iron oxidoreduction 36 – 38 . Thus, we performed genome resequencing of long-term iron- and sulfur-adapted cultures ( Experiment 1 , 20 generations).…”

“…Furthermore, the stress induced by elevated sodium chloride concentration caused ISAfd1 to be inserted downstream of the two promoters PI and PII of the rus operon (which encodes the iron oxidation pathway), thereby preventing its transcription. The ability to oxidize iron was restored after prolonged cultivation in the absence of sodium chloride, and two revertant strains were obtained 38 . Given the scarcity of genetic mutants of the taxon, we analyzed this mutation in further detail.…”

“… 19 . However, their activity has only been demonstrated for some IS elements, including the 1.2-kb ISAfe1 (formerly IST1 or ISTfe1, from the ISL3 family 34 ) found in A. ferrooxidans and the 1.3-kb ISAfd1 (from the IS701 family) found in A. ferridurans , both if which have been associated with a loss of the ability to oxidize/reduce iron 35 – 38 . Thus, the reversible transposition of mobile genetic elements may be responsible for phenotypic switching in these acidophilic iron/sulfur oxidizers.…”

A Model of Aerobic and Anaerobic Metabolism of Hydrogen in the Extremophile Acidithiobacillus ferrooxidans

“…The loss of the ability to anaerobically reduce Fe 3+ was also observed in At. ferridurans, which was caused by salt stress-induced insertional inactivation of the rus operon (Bonnefoy et al, 2018). This evidence pointed to the essential role of some gene(s) encoded by the rus operon in the mechanism of anaerobic respiratory Fe 3+ reduction in iron-oxidizing acidithiobacilli.…”

Effect of Sodium Chloride on Pyrite Bioleaching and Initial Attachment by Sulfobacillus thermosulfidooxidans

“…These reduced forms of sulfur are substrates for S. thermosulfidooxidans as it can oxidize not only iron but also sulfur compounds (Karavaiko et al, 2005). Several studies have reported that iron oxidation is more sensitive than sulfur oxidation to inhibitory effects by chloride and salt (Harahuc et al, 2000;Bonnefoy et al, 2018). Therefore, exposure to chloride possibly affects the preference for substrate utilization of S. thermosulfidooxidans, so that sulfur compounds may be preferred over Fe 2+ .…”