Osmotic response in Leptospirillum ferriphilum isolated from an industrial copper bioleaching environment to sulfate

Abstract

Iron and sulfur-oxidizing microorganisms play important roles in several natural and industrial processes. Leptospirillum (L.) ferriphilum, is an iron-oxidizing microorganism with a remarkable adaptability to thrive in extreme acidic environments, including heap bioleaching processes, acid mine drainage (AMD) and natural acidic water. A strain of L. ferriphilum (IESL25) was isolated from an industrial bioleaching process in northern Chile. This strain was challenged to grow at increasing concentrations of sulfate in order to assess changes in protein expression profiles, cells shape and to determine potential compatible solute molecules. The results unveiled changes in three proteins: succinyl CoA (SCoA) synthetase, isocitrate dehydrogenase (IDH) and aspartate semialdehyde dehydrogenase (ASD); which were notably overexpressed when the strain grew at elevated concentrations of sulfate. ASD plays a pivotal role in the synthesis of the compatible solute ectoine, which was identified along with hydroxyectoine by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The relationship between IDH, SCoA, and ectoine production could be due to the TCA cycle, in which both enzymes produce metabolites that can be utilized as precursors or intermediates in the biosynthesis of ectoine. In addition, distinct filamentous cellular morphology in L. ferriphilum IESL25 was observed when growing under sulfate stress conditions. This study highlights a new insight into the possible cellular responses of L. ferriphilum under the presence of high sulfate levels, commonly found in bioleaching of sulfide minerals or AMD environments.

Keywords: Lesptospirillum ferriphilum; bioleaching; compatible solutes; osmotic strength; sulfate.

Figure 1

Autotrophic iron oxidized colonies growing onto overlay plates obtained from Minera Escondida copper bioleaching plant (A). Bacterial colonies of L. ferriphilum IESL 25 (B).

Figure 2

Growth curves of L. ferriphilum IESL 25 at different sulfate concentrations.

Figure 3

Microphotographs obtained by scanning electron and contrast phase microscopy of L. ferriphilum IESL25 cultures grown at different sulfate concentrations, A,B,E grown at 8 g/L and C,D,F at 80 g/L of sulfate (red arrows indicate cells in contrast phase microphotographs).

Figure 4

MALDI-TOF spectra using the stainless-steel matrix with TiO₂⁺ CsI molecular weight peaks of 150–300 Da (A) and the NALDI⁺ CsI matrix with molecular weight peaks of 140–250 (B). The upper spectra represent the condition of 80 g/L of sulfate, the central spectra correspond to 8 g/L of sulfate (control), and the lower spectra correspond to the control of the matrix or blank. The mass/charge (m/z) peaks indicated with the red arrow coincide with the adducts (a chemical compound that arises from the direct combination of two chemical species) Hydroxyectoine-H⁺ (159.026), Hydroxyectoine-Cs⁺ (290.95) and Ectoine-H⁺ (143.25).

Figure 5

Spots in 2-DE gels in the pH range 5-8NL grown under the control condition of 8 g/L of sulfate and with 80 g/L of sulfate. Spots 1 to 4 represent the proteins differentially expressed and identified by LC-MS/MS.