Microbial iron management mechanisms in extremely acidic environments: comparative genomics evidence for diversity and versatility

Abstract

Background: Iron is an essential nutrient but can be toxic at high intracellular concentrations and organisms have evolved tightly regulated mechanisms for iron uptake and homeostasis. Information on iron management mechanisms is available for organisms living at circumneutral pH. However, very little is known about how acidophilic bacteria, especially those used for industrial copper bioleaching, cope with environmental iron loads that can be 1018 times the concentration found in pH neutral environments. This study was motivated by the need to fill this lacuna in knowledge. An understanding of how microorganisms thrive in acidic ecosystems with high iron loads requires a comprehensive investigation of the strategies to acquire iron and to coordinate this acquisition with utilization, storage and oxidation of iron through metal responsive regulation. In silico prediction of iron management genes and Fur regulation was carried out for three Acidithiobacilli: Acidithiobacillus ferrooxidans (iron and sulfur oxidizer) A. thiooxidans and A. caldus (sulfur oxidizers) that can live between pH 1 and pH 5 and for three strict iron oxidizers of the Leptospirillum genus that live at pH 1 or below.

Results: Acidithiobacilli have predicted FeoB-like Fe(II) and Nramp-like Fe(II)-Mn(II) transporters. They also have 14 different TonB dependent ferri-siderophore transporters of diverse siderophore affinity, although they do not produce classical siderophores. Instead they have predicted novel mechanisms for dicitrate synthesis and possibly also for phosphate-chelation mediated iron uptake. It is hypothesized that the unexpectedly large number and diversity of Fe(III)-uptake systems confers versatility to this group of acidophiles, especially in higher pH environments (pH 4-5) where soluble iron may not be abundant. In contrast, Leptospirilla have only a FtrI-Fet3P-like permease and three TonB dependent ferri-dicitrate siderophore systems. This paucity of iron uptake systems could reflect their obligatory occupation of extremely low pH environments where high concentrations of soluble iron may always be available and were oxidized sulfur species might not compromise iron speciation dynamics. Presence of bacterioferritin in the Acidithiobacilli, polyphosphate accumulation functions and variants of FieF-like diffusion facilitators in both Acidithiobacilli and Leptospirilla, indicate that they may remove or store iron under conditions of variable availability. In addition, the Fe(II)-oxidizing capacity of both A. ferrooxidans and Leptospirilla could itself be a way to evade iron stress imposed by readily available Fe(II) ions at low pH. Fur regulatory sites have been predicted for a number of gene clusters including iron related and non-iron related functions in both the Acidithiobacilli and Leptospirilla, laying the foundation for the future discovery of iron regulated and iron-phosphate coordinated regulatory control circuits.

Conclusion: In silico analyses of the genomes of acidophilic bacteria are beginning to tease apart the mechanisms that mediate iron uptake and homeostasis in low pH environments. Initial models pinpoint significant differences in abundance and diversity of iron management mechanisms between Leptospirilla and Acidithiobacilli, and begin to reveal how these two groups respond to iron cycling and iron fluctuations in naturally acidic environments and in industrial operations. Niche partitions and ecological successions between acidophilic microorganisms may be partially explained by these observed differences. Models derived from these analyses pave the way for improved hypothesis testing and well directed experimental investigation. In addition, aspects of these models should challenge investigators to evaluate alternative iron management strategies in non-acidophilic model organisms.

Figure 1

Predicted ferrous iron transporter gene organization and function in Acidithiobacilli and Leptospirilla. A) FeoPABC system, B) NRAMP-family transporter MntH with the DNA sequences of their predicted Fur boxes, C) EfeU-MCO system, D) Model for ferrous iron transport. ■: predicted Fur box. AT: A. thiooxidans; AF: A. ferrooxidans; AC: A. caldus; LIIa: Leptospirillum sp. Group II UBA; LIIb: Leptospirillum sp. Group II 5-way GC; LIII: Leptospirillum sp. Group III 5-way GC.

Figure 2

Abundance and diversity of predicted ferric iron siderophore transporters in Acidithiobacilli and Leptospirilla. The Ven diagram shows species-specific and shared TonB dependent outer membrane receptors. Color coding indicates predicted siderophore specificity. Red: dicitrate, Green: linear catecholate, Blue: cyclic catecholate, Purple: hydroxamate.

Figure 3

Genomic context for ferric iron transport candidate genes. Predicted functions of the genes are listed in Additional file 2 according to gene cluster number. AT: A. thiooxidans. AF: A. ferrooxidans. AC: A. caldus. LIIa: Leptospirillum sp. Group II UBA. LIIb: Leptospirillum sp. Group II 5-way GC. LIII: Leptospirillum sp. Group III 5-way GC.

Figure 4

Examples of predicted novel metabolic functions grouped together with iron uptake functions in putative co-regulated gene clusters (operons).

Figure 5

Predicted novel citrate synthesis-efflux system and Fe(III)-dicitrate uptake system in A. ferrooxidans and A. thiooxidans. Inset: Predicted conserved gene cluster coding for a dicitrate TonB-dependent receptor (FecA1), a dicarboxylate efflux pump (MarC), a malate dehydrogenase (Mdh), an ACT domain carrying protein (Act), TonBExbBD biopolymer transport system, and a GNAT acetyltransferase (Gnat). Colors in the membrane model correspond to genes in the gene context scheme.

Figure 6

Model for phosphate/phosphonate associated Fe(III) uptake in Acidithiobacilli and Leptospirilla. (A) Partially conserved gene cluster in all three Acidithiobacilli coding for a FecA3 TonB-dependent receptor, the biopolymer transport system ExbBDTonB, a surface layer protein (SLP) and an alkaline phosphatase (PhoD). (B) Partially conserved gene cluster in all three Leptospirilla coding for orthologous TonB-dependent receptors (FecA9), the biopolymer transport system ExbBDTonB, a phosphate activated transcriptional regulator (PhoB) and a surface layer protein (SLP). Colors in the membrane model correspond to genes in the gene context scheme. AT: A. thiooxidans. AF: A. ferrooxidans. AC: A. caldus. LIIa: Leptospirillum sp. Group II UBA. LIIb: Leptospirillum sp. Group II 5-way GC. LIII: Leptospirillum sp. Group III 5-way GC.

Figure 7

Nitrogenase dedicated ferric iron and molybdate transport in A. ferrooxidans. A. Genomic context and gene organization of the predicted bifunctional Fe and Mo transport operon. B. Model for A. ferrooxidans dedicated metal import for nitrogenase function. Colors in the membrane model correspond to genes in the gene context scheme. ■: Fur box. Violet: Genes encoding nitrogenases. Orange : genes encoding bifunctional metal transporters. AT: A. thiooxidans. AF: A. ferrooxidans. AC: A. caldus.

Figure 8

Sequence alignment of bacterioferritins. Rhodopseudomonas palustris (RPA) NP_948938, Bradyrhizobium japonicum (BJA) NP_773320, Rhodobacter sphareoides (RSP) YP_351589, Chromobacterium violaceum (CVI) NP_903069, E. coli (ECO) NP_417795, A. caldus ACA, A. thiooxidans ATH and A. ferrooxidans AFE. The binuclear metallic center is indicated in blue (Glu-18 Glu-51 His-54 Glu-94 Glu-127 His-130) and the heme ligand in red (Met-52).

Figure 9

Fur family transcriptional regulators and their genomic context in A) Acidithiobacilli, B) Leptospirilla. Color coding indicates orthology and bars linking genes through genomes indicate percentage of amino acid sequence similarity. AT: A. thiooxidans. AF: A. ferrooxidans. AC: A. caldus. LIIa: Leptospirillum sp. Group II UBA. LIIb: Leptospirillum sp. Group II 5-way GC. LIII: Leptospirillum sp. Group III 5-way GC.

Figure 10

Diversity of alternative iron acquisition modules and putative regulatory connections in acidophiles. Light blue: ferrous iron uptake module, Violet: Ferric-dicitrate uptake module, Orange: Ferric-siderophore uptake module, Grey: Metalophosphate/phosphonate uptake module, Orange arrows: Regulatory connections.