Introduction

Certain chemolithotrophic bacteria inhabit ore-bearing geological formations exposed to the atmosphere and obtain all of their energy for growth from the oxidation and dissolution of minerals within the ore. Energy is derived from oxidative phosphorylation coupled to respiratory electron transfer. The ability to respire aerobically on soluble ferrous ions under strongly acidic conditions is currently known to be expressed by at least 34 species in 14 genera distributed throughout the Gram-negative (Markosyan, 1972;?Huber and Stetter, 1989;?Kelly and Wood, 2000;?Hallberg et al., 2010), Gram-positive (Clark and Norris, 1996;?Norris et al., 1996;?Johnson et al., 2008,?2009;?Guo et al., 2009;?Jiang et al., 2009), and Archaea bacteria (Segerer et al., 1986;?Huber et al., 1989;?Huber and Stetter, 1991;?Karavaiko et al., 1994;?Golyshina et al., 2000,?2009). Given the genetic diversity within this collection of phenotypically related bacteria, it would not be surprising to learn that phylogenetically distinct groups of bacteria express different electron transfer biomolecules and pathways to accomplish aerobic respiration on soluble iron.

Classic reductionist studies that involve the structural and functional characterization of highly purified proteins in dilute solution have described a bewildering variety of different redox-active electron transport proteins in cell-free extracts derived from iron-grown Gram-negative (Cox and Boxer, 1978;?Hart et al., 1991;?Blake et al., 1992;?Yarzábal et al., 2002,?2004), Gram-positive (Blake et al., 1993;?Takai et al., 2001;?Dinarieva et al., 2010), and Archaea (Hettmann et al., 1998;?Dopson et al., 2005;?Auernik and Kelly, 2008) bacteria. The most promising efforts to date have focused on the iron respiratory chain of?Acidithiobacillus ferrooxidans, where an iron “respirasome” super complex has been defined that is comprised of 2?c-type cytochromes, a blue copper protein called rusticyanin, and an?aa₃-type terminal oxidase (Castelle et al., 2008). The proteins in the aerobic iron respiratory pathway of?At. ferrooxidans?do not appear to be expressed in many of the phylogenetically distinct bacteria that also respire on iron. Similarly, redox-active proteins expressed in other iron-grown bacteria do not appear to be expressed in iron-grown?At. ferrooxidans. Comparative analyses conducted using those relevant bacterial genomes where partial or complete DNA sequence data is available (Chen et al., 2005;?Ram et al., 2005;?Valdes et al., 2008;?Clum et al., 2009;?Siezen and Wilson, 2009) have not yet provided significant insight into other iron respiratory proteins or pathways. There is little information in the DNA databases to compare with because the proteins in the aerobic iron respiratory pathway of?At. ferrooxidans?do not appear to be universal among those bacteria that respire on iron. In either case, actual respiratory electron transfer in the intact organism is not directly observed. Rather, the functional properties of the intact electron transfer chain are inferred from observations on isolated molecules.

This paper introduces a new means to study respiratory electron transfer reactions?in situ?in intact bacteria under physiological conditions. The premise is that accurate UV-visible spectroscopy of electron transfer reactions among colored cytochromes can be conducted in highly turbid suspensions if the live bacteria are irradiated in an isotropic homogeneous field of incident measuring light. Under those conditions, the absorbed radiant power is independent of scattering effects (Elterman, 1970;?Fry et al., 1992;?Javorfi et al., 2006;?Hodgkinson et al., 2009). We conducted equilibrium and kinetic studies on the Fe(II)-dependent reduction and O₂-dependent oxidation of cytochromes in intact?Leptospirillum ferrooxidans?at pH 1.7. We used a commercial integrating cavity absorption meter (ICAM) where the cuvette comprised a reflecting cavity completely filled with the absorbing suspension.?L. ferrooxidans?was selected because it is only known to respire on one substrate, reduced iron (Harrison, 1984). We observed that a cytochrome with a reduced spectral peak at 579 nm is an obligatory intermediate in the aerobic iron respiratory chain of?L. ferrooxidans.

Materials and Methods

Cell Culture

Leptospirillum ferrooxidans?DSMZ 2705 was cultured autotrophically on soluble ferrous ions at 30°C in the medium described elsewhere (Tuovinen and Kelly, 1973), adjusted to pH 1.5 and amended with 44 g/l of FeSO₄·7H₂O. Cells grown to stationary phase were harvested by centrifugation, washed three times with 0.02 M H₂SO₄, pH 1.7, and resuspended in sufficient 0.02 M H₂SO₄?to achieve a stock suspension of 1.5 × 10¹⁰?cells/ml. The stock suspension was stored at 4°C for up to 2 weeks while spectroscopic experiments were conducted on aliquots of the cells. Previous stock suspensions of this organism have been stored in dilute sulfuric acid at 4°C for over 6 weeks before changes in the bacterium’s energy metabolism could be detected.

Quantification of Bacteria

Absolute numbers of?L. ferrooxidans?cells were determined by electrical impedance measurements in a Multisizer 4 particle counter (Beckman Coulter, Inc., Brea, CA, USA) fitted with a 30-μm aperture. The instrument was programmed to siphon 50 μl of sample that contained Isoton II as the electrolyte. The current applied across the aperture was 600 μA. Voltage pulses attendant with impedance changes as particles passed through the aperture were monitored with an instrument gain of four.

Relative numbers of?L. ferrooxidans?cells were determined by photon correlation scattering spectroscopy with a DelsaNano C particle size analyzer, also from Beckman Coulter, Inc. Cell densities were adjusted to 8.3 × 10⁶?cells/ml in 0.02 M sulfuric acid to give an attenuator obscuration of 47%. Determination of the relative numbers of light scattering species as a function of particle diameter was accomplished by the time domain method with operating and analysis software provided by Beckman Coulter, Inc.

Absorbance Measurements with Cell Suspensions

Absorbance measurements on intact cells in suspension were conducted in an Olis CLARiTY 1000 A spectrophotometer (On Line Instrument Systems, Inc., Bogart, GA, USA) that employed a novel ICAM. In a typical experiment, identical 4.2 ml solutions that contained ferrous sulfate in 0.02 M sulfuric acid, pH 1.7, were added to both the sample and reference observation cavities of the spectrophotometer. After recording a stable baseline from 350 to 650 nm, 140 μl were withdrawn from the sample cavity and replaced with 140 μl of the stock cell suspension of?L. ferrooxidans. Apparent absorbance spectra (typically 6.2 s^?1) were then collected until any visible absorbance changes had ceased. Raw apparent absorbance values were converted to absorbance values per cm using Fry’s method (Fry et al., 2010) as described in the text.

Results

The Redox State of Electron Transfer Proteins Can be Monitored?in situ?in Intact Bacteria under Physiological Conditions

The principal features of the novel CLARiTY spectrophotometer used to conduct absorbance measurements in turbid solutions are included in the schematic diagram shown in Figure?1. The sample and reference observation cells of this dual beam spectrophotometer were each comprised of a 4.2-ml spherical quartz cuvette fused with a 6-mm ID quartz tube. Each quartz chamber was surrounded by a tightly packed proprietary white powder that served to maximize diffuse reflectance of light on the exterior walls of the spherical flask. The apertures in the reflecting sphere through which the measuring light entered and the transmitted/scattered light exited to the photomultiplier tube were positioned at a 90° angle such that the light had to undergo many reflections and cell transversals before it was quantified using the photomultiplier tube. A white Teflon plug with a 6-mm OD was inserted into the quartz tube to minimize the loss of light out of the neck. A 1.0-cm white stir bar was included in the sample chamber to facilitate sample mixing and suspension of any particulate matter.