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.