Abstract
Absorbance spectra were collected on 12 different live microorganisms, representing six phyla, as they respired aerobically on soluble iron at pH 1.5. A novel integrating cavity absorption meter was employed that permitted accurate absorbance measurements in turbid suspensions that scattered light. Illumination of each microorganism yielded a characteristic spectrum of electrochemically reduced colored prosthetic groups. A total of six different patterns of reduced-minus-oxidized difference spectra were observed. Three different spectra were obtained with members of the Gram-negative eubacteria. Acidithiobacillus, representing Proteobacteria, yielded a spectrum in which cytochromes a and c and a blue copper protein were all prominent. Acidihalobacter, also representing the Proteobacteria, yielded a spectrum in which both cytochrome b and a long-wavelength cytochrome a were clearly visible. Two species of Leptospirillum, representing the Nitrospirae, both yielded spectra that were dominated by a cytochrome with a reduced peak at 579 nm. Sulfobacillus and Alicyclobacillus, representing the Gram-positive Firmicutes, both yielded spectra dominated by a-type cytochromes. Acidimicrobium and Ferrimicrobium, representing the Gram-positive Actinobacteria, also yielded spectra dominated by a-type cytochromes. Acidiplasma and Ferroplasma, representing the Euryarchaeota, both yielded spectra dominated by a ba3-type of cytochrome. Metallosphaera and Sulfolobus, representing the Crenarchaeota, both yielded spectra dominated by the same novel cytochrome as that observed in the Nitrospirae and a new, heretofore unrecognized redox-active prosthetic group with a reduced peak at around 485 nm. These observations are consistent with the hypothesis that individual acidophilic microorganisms that respire aerobically on iron utilize one of at least six different types of electron transfer pathways that are characterized by different redox-active prosthetic groups. In situ absorbance spectroscopy is shown to be a useful complement to existing means of investigating the details of energy conservation in intact microorganisms under physiological conditions.
Highlights
The capacity to respire aerobically on soluble ferrous ions under strongly acidic conditions is currently thought to be expressed in 42 species distributed among 19 genera in six phyla (Bonnefoy and Holmes, 2012; Johnson et al, 2012)
The reduced peak at 565 nm in Figure 1 was clearly in the spectral region normally attributed to the reduced peaks of b-type cytochromes, a feature that was absent in the spectrum obtained using intact At. ferrooxidans
There was no evidence of a broad trough of negative absorbance in the spectrum in Figure 1 that one could attribute to the iron-dependent reduction of a Integrating Cavity Absorption Meter
Summary
The capacity to respire aerobically on soluble ferrous ions under strongly acidic conditions (pH < 3) is currently thought to be expressed in 42 species distributed among 19 genera in six phyla (Bonnefoy and Holmes, 2012; Johnson et al, 2012). A variety of different electron transfer proteins and redox-active prosthetic groups have been posited by many laboratories to participate in aerobic respiration on iron These reports include studies using purified proteins and protein complexes (Cox and Boxer, 1978; Kai et al, 1992; Blake and Shute, 1997; Castelle et al, 2008; Singer et al, 2008; Bandeiras et al, 2009), spectroscopic studies on cell-free extracts (Hart et al, 1991; Blake et al, 1993; Yarzabal et al, 2002; Brasseur et al, 2004; Kappler et al, 2005; Bathe and Norris, 2007; Dinarieva et al, 2010), inventories of putative respiratory proteins deduced from whole-cell genomic sequencing activities (reviewed in Auernik and Kelly, 2008; Kozubal et al, 2011; Ilbert and Bonnefoy, 2013), and lists of likely respiratory proteins identified in transcriptomic (Auernik and Kelly, 2008; Quatrini et al, 2009) and proteomic (Dopson et al, 2005; Ram et al, 2005; Bouchal et al, 2006; Valenzuela et al, 2006) studies of cells cultured in the presence of soluble iron. When the electron-accepting capability of the soluble molecular oxygen (4 × 236 μM at 30◦C) exceeded that of the electron-donating capacity of the soluble ferrous ions (≤500 μM), the spectrum of each ironreduced organism returned to that of the resting air-oxidized organism
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