Introduction
Bacteria can often utilize multiple energy sources; however, the
bioavailability of substrates and constraints on cellular resources
leads to myriad of substrate utilization patterns (Chubukov, Gerosa,
Kochanowski, & Sauer, 2014; Monod, 1949; Wong, Gladney, & Keasling,
1997). Complex regulation networks, influenced by substrate energies and
preculture histories, typically drive the mechanisms that lead to
diauxic growth or simultaneous substrate consumption behaviors (Narang
& Pilyugin, 2007; Okano, Hermsen, Kochanowski, & Hwa, 2020).Acidithiobacillus ferrooxidans is a key member of the microbial
consortia involved in the industrial-scale bioleaching of copper, and it
has been commonly reported to have diauxic behavior on iron and sulfur
with a preference for iron oxidation when both sources are present
(Beck, 1960; Espejo & Romero, 1987; Ponce, Moinier, Byrne, Amouric, &
Bonnefoy, 2012; Suzuki, Takeuchi, Yuthasastrakosol, & Oh, 1990; Wu et
al., 2019). However, conflicting behaviors from different strains have
been reported for these bacteria, as some of the iron- and
sulfur-oxidizing strains studied previously have been phylogenetically
reclassified (Amouric, Brochier-Armanet, Johnson, Bonnefoy, & Hallberg,
2011; Espejo, Escobar, Jedlicki, Uribe, & Badilla-Ohlbaum, 1988;
Falagán & Johnson, 2016; Ghosh & Dam, 2009; Hallberg, González-Toril,
& Johnson, 2010; Hedrich & Johnson, 2013; Kelly & Wood, 2000; Norris
et al., 2020; Ponce et al., 2012; Suzuki et al., 1990; Yarzabal,
Appia-Ayme, Ratouchniak, & Bonnefoy, 2004). Although iron oxidation is
generally assumed to precede sulfur oxidation, A. ferrooxidanscells cultivated on ferrous iron have been observed to adsorb to sulfur
particles, despite the hydrophobic nature of the surface, and this
process can be modeled through a Langmuir equation (Konishi, Takasaka,
& Asai, 1994; Xia et al., 2013). Additionally, the adsorption ofA. ferrooxidans to sulfur has been found to involve extracellular
polymeric substances (EPS) which consist of lipopolysaccharides
(Dispirito, Dugan, & Tuovinen, 1983; Gehrke, Telegdi, Thierry, & Sand,
1998). Gene expression data in A. ferrooxidans has suggested that
genes in both the iron and sulfur pathways can be transcribed and
upregulated simultaneously, even when the bacteria are cultivated on
ferrous iron (Kucera et al., 2013). The interactions between the sets of
enzymes involved in iron and sulfur metabolism are poorly understood,
other than the existence of two bc1 complexes allowing
for the iron and sulfur oxidation systems to co-exist (Brasseur et al.,
2004; Bruscella et al., 2007; Kucera, Sedo, et al., 2016; Valdes et al.,
2008). Thus, despite the critical importance of these microorganisms in
industrial metal bioleaching operations, gaps still remain in the
fundamental understanding of the chemolithotrophic behavior of these
bacteria.
The metabolic oxidation of sulfur can be challenging as it requires
interfacial interactions that require treatment from the cells. Previous
attempts to introduce surfactants to improve the contact between
acidophiles and the hydrophobic surface of sulfur yielded only small
increases in the oxidation rates of sulfur, and high concentrations of
these surfactants inhibited cell growth (Frederick, Jones, & Starkey,
1956; Knickerbocker, Nordstrom, & Southam, 2000; Peng, Liu, Nie, &
Xia, 2012). Although the production of EPS specific to sulfur and other
proteins by A. ferrooxidans is known to be necessary for the
processing of sulfur into a bioavailable form for oxidation, a method to
significantly enhance sulfur oxidation with these microorganisms has
proven to be elusive (Gehrke et al., 1998; He et al., 2011).
Specifically, the identification of the minimum components necessary for
the activation of sulfur remain unresolved (Ramirez, Guiliani,
Valenzuela, Beard, & Jerez, 2004). As such, little is known about the
consequences of the discrepancy in bioavailability between the soluble
ferrous iron and hydrophobic sulfur particles. Given that the oxidation
of sulfur involves more favorable reduction potentials for generating
reducing equivalents in the cell than using ferrous iron, we sought to
address the hypothesis that A. ferrooxidans may actually consume
sulfur preferentially to ferrous iron oxidation under certain conditions
(Bird, Bonnefoy, & Newman, 2011; Quatrini et al., 2009).
The high concentrations of iron present in acidic growth media
formulations presents a challenge for genetic studies with A.
ferrooxidans . Although kanamycin and streptomycin are unstable, they
have been used as selectable transformation markers as they provide
acceptable selective pressure in Acidithiobacillus family for
fundamental genetic work (Inaba, Banerjee, Kernan, & Banta, 2018;
Kernan et al., 2016; Peng, Yan, & Bao, 1994; Wang et al., 2012; Yu,
Liu, Wang, Li, & Lin, 2014). While the limitations and instability of
these selective pressures have been shown for sulfur medium forAcidithiobacillus caldus cultures, the lack of growth inhibition
in iron medium has not been measured in liquid cultures with A.
ferrooxidans (Wang et al., 2017). Moreover, despite the relative
stability of incompatibility group Q (IncQ) plasmids without
antibiotics, methods for plasmid maintenance in transconjugant strains
beyond replating strains onto selective solid plates have not been
explored (Liu, Borne, Ratouchniak, & Bonnefoy, 2001). Additional
development of various techniques and media to improve the ease of
genetic engineering is a goal in A. ferrooxidans to be used in
applications such as commercial biomining (Gumulya et al., 2018).
Here, we explore the effects of concentrations of iron and sulfur, cell
density, and the physical properties of sulfur on the apparent substrate
utilization preference of A. ferrooxidans ATCC 23270. We created
a simple dispersion formulation of sulfur that shortens the lag time for
sulfur consumption. By using this dispersed sulfur, we find that the
phenotypes associated with sulfur oxidation are observed more quickly
than when using untreated sulfur. Notably, we demonstrate that sulfur
oxidation can precede iron oxidation when the sulfur is in this
bioavailable form, even at low sulfur concentrations where unformulated
sulfur had little impact on ferrous iron oxidation. This formulation
mimics the activation of sulfur by cellular material and debris under
natural conditions. Furthermore, we demonstrate that this dispersed
sulfur allows maintenance of engineered strains in liquid media and that
improved growth on elemental sulfur benefits expression of GFP withinA. ferrooxidans. Thus, the application of this new bioavailable
sulfur formulation opens a new avenue for working with engineeredA. ferrooxidans and controlling iron and sulfur oxidation.