**1. Introduction**

Iron (Fe) is an essential element for life. In microorganisms, it is used as a co-factor for enzymatic processes, such as in electron transfer during respiration and photosynthesis, nucleic acid or chlorophyll synthesis, nitrate reduction, nitrogen fixation and detoxification of oxygen radicals [1–3]. The use of Fe in bacteriochlorophyll (BChl) synthesis and the process of photophosphorylation makes it particularly important to phototrophic organisms. Aerobic anoxygenic phototrophs (AAP) are one such physiological group that uses photosynthesis in oxic conditions as an additional energy source to respiration [4]. They make up a significant proportion of many bacterial communities from a host of environments [5], and therefore likely require a substantial Fe uptake. In addition, Fe may be crucial to protect the cells from oxidative stress due to singlet oxygen formation during BChl *a* synthesis. While necessary for metabolism, biologically active Fe is typically quite sparse in nature as its soluble level is very low at soil and water surfaces [2]. In response to this limitation, both bacteria and fungi have developed siderophores to compete for the available Fe [6]. Siderophores, aptly named from the Greek root representing "iron-bearing" [7], are low weight molecules (no more than 1500–2000 Da and generally lower than 1000 Da) with a high-affinity for Fe [2,3,8–10]. The molecules are often short polypeptides with modified or D-amino acids [2,9,11]. They can also be made from dicarboxylic acids and diamine or amino alcohols, linked by amide and ester bonds. These building blocks retain some characteristics of amino acids [2]. Siderophores can be classified into two main functional groups. The first is the hydroxamate group, which involves hydroxamic acid and is produced by both fungi and bacteria. Second is the catechol group, compounds of which

**Citation:** Kuzyk, S.B.; Hughes, E.; Yurkov, V. Discovery of Siderophore and Metallophore Production in the Aerobic Anoxygenic Phototrophs. *Microorg* **2021**, *9*, 959. https://doi.org/10.3390/ microorganisms9050959

Academic Editor: Johannes F. Imhoff

Received: 22 March 2021 Accepted: 28 April 2021 Published: 29 April 2021

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contain catechol rings and are only produced by bacteria [6,9,10]. Smaller groups such as the hydroxyacids and the α-hydroxy carboxylates are only rarely used by bacteria [9,10]. Usually, siderophores are synthesized and secreted by cells under iron-deplete growth conditions [8]. Once bound to iron, the complex is taken up by the cells in a substrate specific process [2].

While the main purpose of siderophores is Fe acquisition, they may also play some additional roles. In *Pseudomonas aeruginosa*, pyoverdine controls virulence factor production [12]. *Escherichia coli* can be protected from oxidative stress by the catechol type siderophore enterobactin [13]. Pyochelin in *P. aeruginosa* has a toxic effect on eukaryotic cells, possibly aiding in the bacterium's virulence [13]. Watasemycins, sideromycins, oxachelin and fusigen have antibacterial activity, which may aid in community competition by preventing other populations from growing [13]. Additionally, siderophores have been shown to bind more than one metal [3,14], including some that have higher affinity for Cu or Zn rather than Fe [15,16]. This broad-spectrum activity has required the classification "metallophore" [17,18], which is a term used for secondary metabolites capable of binding a range of metal(loid) cations. When a metallophore would have a specific metal affinity, it would have a sub-categorical name, where siderophore is for Fe-binding, chalkophore for Cu, or zincophore for Zn, all named when discovered.

This concept, of metallophores capable of capturing multiple metal cations has implicated usefulness in toxic heavy metal tolerances. Particularly in extreme environments, where metals can be present at elevated concentrations that inhibit a variety of life [19,20]. Many metal ions can diffuse freely through the cellular membrane, which is inhibited if the metal is bound to a siderophore that is too large to move without active transport. Furthermore, membrane receptors specific to siderophore-iron complexes can differentiate between those containing substitute cations, causing the cell to reject the alternative metalcontaining siderophore. The reduction of free metal concentrations in proximity of the bacterium and decreased passive diffusion of unwanted metals into the cell will lower their overall toxic effect [3]. This could be a compelling concept, as AAP possess very high levels of resistance to toxic heavy metal(loid) oxides [21]. While internal enzymatic reduction takes place [22], the external production of siderophores may provide an additional layer of defense. Additionally, as mentioned above, the siderophores' ability to reduce reactive oxygen species could help AAP as they need protection against oxidative stress due to their aerobic production of BChl *a*.

*Chromocurvus halotolerans* EG19, is a γ-proteobacterial AAP that was isolated during the spring of 2002 from floating microbial mats within the East German Creek System, Manitoba, Canada [23]. As this is a landlocked hypersaline spring system, it likely contains highly endemic communities of microorganisms that have not been mixed with or affected by allochthonous populations [23]. EG19 forms motile, short rod or longer curved rodshaped, orange-pink bacteria. When grown with complex carbon sources, EG19 produces a brown pigmented hydrophilic compound, which is excreted into the growth medium. While a similar phenomenon had never been reported in other AAP, it was hypothesized that the compound could be a siderophore [24], as ferric bound siderophores can be visually yellow-brown or red-brown [25]. Our study confirms the identity of this extracellular product and describes it as the first siderophore discovered in an AAP. Other AAP from a vast array of environments that do not pigment growth medium were also tested for their production of siderophores, as most of these metal chelating small molecules are colourless, and synthesis is therefore possible.

#### **2. Materials and Methods**

#### *2.1. Bacterial Strains and Growth Conditions*

For this study, 101 strains of AAP originating from an assortment of environments, as well as phylogenetically diverse throughout numerous proteobacterial clades were selected from Dr. Vladimir Yurkov's vast collection. A complete list of chosen strains, original source of isolation, relatedness to type species, and 16S rRNA partial gene sequence accession numbers are listed in Tables A1 and A2.

Freshwater AAP were cultivated on rich organic (RO) medium as described [26], with one minor modification. Bactopeptone was reduced to 0.5 g/L and casamino acids were supplemented at 0.5 g/L, which provided a larger variety of complex nutrients. Marine AAP requiring salt were propagated on RO medium described above supplemented with 2% NaCl. AAP originally isolated from the East German Creek System, Manitoba, Canada, were grown using medium A (MA) [23]. Those isolated from biological soil crusts of Sandy Lands Forest and Spruce Woods National Park were cultured on Biological Soil Crust Medium A or B (BSCA or BSCB) [27]. Strains from meromictic Mahoney Lake in British Columbia, Canada were grown on N1 medium [28].

In addition to bacterial isolates formerly described elsewhere, some of those chosen for siderophore testing had not been previously published. AAP isolated from the meromictic Blue Lake in British Columbia, Canada, were cultured using a Blue Lake medium (BLM) containing (g/L): MgSO4, 0.5; NH4Cl, 0.3; KH2PO4, 0.3; KCl, 0.3; CaCl2, 0.05; NaCl, 12.0; NaHCO3, 0.5; Na-acetate, 1.0; malic acid, 1.0; yeas<sup>t</sup> extract, 1.0; bactopeptone, 0.5; with vitamins and trace elements solutions, 2.0 mL each; autoclaved at pH 5.9 and then adjusted after autoclaving to pH 7.5 with 0.5 N NaOH.

Furthermore, several AAP had been recovered from the freshwaters of Lake Winnipeg whilst the habitat was under study [29,30]. Specifically, in the spring of 2017, strain AJ72 was collected at Grand Beach from littoral water, AM19, AM27 and AM91 were from littoral sediment of Victoria Beach. During that summer, BA23 and BE100 were isolated at limnetic sites S1 and S5, respectively, while BL67 and BK61 originated within the littoral water and sediment of Grand Beach and Victoria Beach, respectively. Fall samples of Grand Beach sediment contained CK155 and CK182, while Victoria Beach littoral waters revealed isolate CL63. All were purified on the slightly modified freshwater RO medium as described.

#### *2.2. Iron Chelating Chromeazurol S Assay*

Every chosen strain was grown on 2% agar plates containing their specific growth medium supplemented with the dye chromeazurol S (CAS), which turns blue when bound to Fe and reverts to yellow/orange being released [31]. To make media, 60.5 mg CAS were dissolved in 50 mL ddH2O, then mixed with 10 mL of an iron solution containing 1 mM FeCl3 and 10 mM HCl. HDTMA (72.9 mg) was dissolved in 40 mL ddH2O prior to mixing with the CAS/iron solution, bringing the total volume to 100 mL. Separately, 900 mL of each growth medium (MA, RO, RO 2% NaCl, BSCA, or BLM) were prepared without the addition of iron, but with the correct amount of components for 1 L, as that would be the final volume of medium after combining with CAS/iron solution. Media were then autoclaved at pH 5.9, separate from the CAS mixture. After autoclaving, each medium was adjusted to pH 6.8 and the CAS solution was added. Agar plates would turn blue when solidified. If measurements of ingredients were not exact or added in an incorrect order, CAS would precipitate out of solution and plates with pH higher than 6.8 would appear green instead of blue. CAS plates were heavily inoculated with each strain, and streaked to achieve isolated colonies after 5 days of growth at 28 ◦C in the dark.

#### *2.3. Variant Cation Chromeazurol S Assay*

Due to the principle chemistry behind the siderophore assay [31], which used negatively charged CAS dye that would weakly bind the cation Fe3+, we decided to replace Fe3+ with different metal(loid) cations, including Mg2+, V3+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Se4+ and Te2+. Microbial growth with an aura of colour change in medium from blue to yellow hue would signify that microbially produced siderophore could bind the substituted cation, causing the released dye to revert to its yellow colour. These variant cation CAS plates were successfully created as explained in the previous section, cured to diverse shades of blue dependent on the metal(loid), and were screened for metallophore production identically

as in the Fe-chelation CAS assay. All cations were purchased from Sigma Aldrich, USA as chloride salts, which were soluble once acidified as described.
