Insights into Systems for Iron-Sulfur Cluster Biosynthesis in Acidophilic Microorganisms

Abstract

Fe-S clusters are versatile and essential cofactors that participate in multiple and fundamental biological processes. In Escherichia coli, the biogenesis of these cofactors requires either the housekeeping Isc pathway, or the stress-induced Suf pathway which plays a general role under conditions of oxidative stress or iron limitation. In the present work, the Fe-S cluster assembly Isc and Suf systems of acidophilic Bacteria and Archaea, which thrive in highly oxidative environments, were studied. This analysis revealed that acidophilic microorganisms have a complete set of genes encoding for a single system (either Suf or Isc). In acidophilic Proteobacteria and Nitrospirae, a complete set of isc genes (iscRSUAX-hscBA-fdx), but not genes coding for the Suf system, was detected. The activity of the Isc system was studied in Leptospirillum sp. CF-1 (Nitrospirae). RT-PCR experiments showed that eight candidate genes were co-transcribed and conform the isc operon in this strain. Additionally, RT-qPCR assays showed that the expression of the iscS gene was significantly up-regulated in cells exposed to oxidative stress imposed by 260 mM Fe₂(SO₄)₃ for 1 h or iron starvation for 3 h. The activity of cysteine desulfurase (IscS) in CF-1 cell extracts was also up-regulated under such conditions. Thus, the Isc system from Leptospirillum sp. CF-1 seems to play an active role in stressful environments. These results contribute to a better understanding of the distribution and role of Fe-S cluster protein biogenesis systems in organisms that thrive in extreme environmental conditions.

Keywords: Fe-S cluster; Isc; Leptospirillum; Suf; acidophiles; iron starvation; oxidative stress.

Fig. 1. Organization of suf and isc gene clusters in different microorganisms.

Genes are represented by arrows orientated according to the strand location. “H”: hypothetical gene, “fdx”: ferredoxin and “fdr”: ferredoxin reductase. Asterisk (*) indicates neutrophilic microorganisms.

Fig. 2. Phylogenetic analysis of SufS and IscS from extremophilic and neutrophilic bacteria.

The evolutionary history was inferred by the Maximum Likelihood algorithm and the JTT matrix-based model using amino acidic sequences of SufS and IscS. The analysis was performed using the MEGA-X program.

Fig. 3. Genetic organization of the isc gene cluster in Leptospirillum sp. CF-1.

(A) Schematic map of the isc operon. The Rho-independent-like structure between icsX and hyp2 was predicted with the RNAold finding terminator tool. The primers designed to amplify each intergenic region are indicated below the operon (1-18). (B) RT-PCR amplification of intergenic regions using cDNA, RNA (without addition of reverse transcriptase) as negative control, and genomic DNA (gDNA) as positive control of amplification. The primer pairs used for amplification are indicated above each panel and the expected amplicon sizes are shown below each panel. St – molecular weight standards.

Fig. 4. Relative mRNA levels of cysteine desulfurase iscS in Leptospirillum sp. CF-1.

Relative expression was evaluated in cells exposed to (A) 260 mM Fe₂(SO₄)₃ for 0.5 and 1 h, and (B) decreasing FeSO₄ concentrations (33 and 0 mM) for 3 h. The data represent the average of 3 independent experiments (bars indicate standard deviation). Statistical analysis was carried out by the ANOVA test.

Fig. 5. Cysteine desulfurase activity in Leptospirillum sp. CF-1.

Cysteine desulfurase activity in whole protein extracts from cells exposed to (A) 260 mM Fe₂(SO₄)₃ for 0.5 and 1 h, and (B) decreasing FeSO₄ concentrations (33 and 0 mM) for 3 h. The data represent the average of 3 independent experiments (bars indicate the value range). Statistical analysis was carried out by the ANOVA test.