# Pseudomonas brassicacearum-induced biotite weathering: Role of iron homeostasis and two siderophores

Tom Girard, Isabelle Basile-Doelsch, Sylvain Fochesato, Adrien Duvivier, Emmanuel Doelsch, Thierry Heulin, Wafa Achouak

► To cite this version:

Tom Girard, Isabelle Basile-Doelsch, Sylvain Fochesato, Adrien Duvivier, Emmanuel Doelsch, et al.. Pseudomonas brassicacearum-induced biotite weathering: Role of iron homeostasis and two siderophores. Environmental Science and Technology, 2025, 59 (16), pp.7973-7982. 10.1021/acs.est.4c07951 . hal-05183304v2

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*Pseudomonas brassicacearum*-induced biotite weathering: Role of iron homeostasis and two siderophores

Tom Girard*‡, Isabelle Basile-Doelsch‡, Sylvain Fochesato‡, Adrien Duvivier‡, Emmanuel Doelsch§α, Thierry Heulin‡, and Wafa Achouak‡

Aix Marseille Univ, CEA, CNRS, BIAM, LEMIRE, ITEM, F-13115 Saint Paul-Lez-Durance, France

Aix Marseille Univ, CNRS, IRD, INRAE, CEREGE, ITEM, F-13545 Aix-en-Provence, France

§CIRAD, UPR Recyclage et risque, F-34398 Montpellier, France

αRecyclage et Risque, University of Montpellier, CIRAD, F-34398 Montpellier, France

KEYWORDS

Pyoverdine, ornicorrugatin, iron metabolism, biotite weathering, *Pseudomonas brassicacearum*

SYNOPSIS

This study shows that bacteria capable of producing a secondary siderophore, whose production is not stopped by intracellular iron, are more effective in improving mineral weathering.
17 Soil bacteria play a crucial role in enhancing mineral weathering, thereby facilitating the release
18 of mineral structural ions into the environment. *Pseudomonas brassicacearum* NFM421 a root-
19 isolated bacterium, produces two different siderophores in the form of pyoverdine and
20 ornicorrugatin. We studied the interaction between this bacterium and biotite—a natural iron-
21 bearing phyllosilicate—to assess the factors governing siderophore-mediated biogenic weathering.

22 We demonstrated that bacterial Fe is an essential factor driving biotite weathering. Our findings
23 suggested that the lipopeptidic siderophore ornicorrugatin might be more effective than pyoverdine
24 as an iron-bearing mineral weathering agent. This secondary siderophore's production is
25 maintained even when the iron requirement of the bacteria is fulfilled. Moreover, we observed that
26 another mechanism requiring direct physical contact might enable *P. brassicacearum* to acquire
27 iron structural ions from soil minerals.

<figure><img src="image_1.png" /></figure>

28

29

30
31

# 1. INTRODUCTION

Mineral weathering—a soil mineral dissolution process that releases structural ions in the environment—is a major pedogenesis mechanism. Various abiotic factors are known to impact the mineral weathering rate in soils, and there is growing interest in the living organisms involved in this so-called biogenic weathering process¹. Among these organisms, certain bacteria have evolved to efficiently extract nutrients from soil minerals, thereby accelerating mineral weathering and ion release into the surrounding environment. Uroz et al. (2015)² proposed the term ‘mineralosphere’ in reference to the specific microenvironment surrounding soil minerals within which bacterial communities are affected by the mineral physicochemical properties. Bacteria thriving in this microenvironment are very efficient at extracting nutrients from the mineral structure. Different mechanisms can affect mineral weathering rates, such as acidification, electron transfer, bacterial motility, biofilm formation and chelation³. Complexolysis involves organic molecules that feature high chemical affinity for the mineral structural ions. Bonding of these ions leads to their release from the mineral structure and enhances the mineral weathering rate.

Iron homeostasis is crucial for bacterial metabolism and growth, as this nutrient is essential for various physiological processes such as respiration, the Krebs cycle and DNA repair⁴. Multiple studies have shown that siderophore-producing *Pseudomonas* bacteria are able to effectively extract and utilize structural Fe from different minerals (flocking asbestos waste, hematite, smectite, etc.) through siderophore production⁵⁻⁷.

*Pseudomonas* species are known for their phytobeneficial potential⁸⁻¹². Studies focused on mineral weathering bacteria frequently highlight that *Pseudomonas* spp. are efficient at mineral nutrient extraction via two main mechanisms, i.e. organic acid¹³,¹⁴ and siderophore¹⁵⁻¹⁷ production. Bacteria produce specific compounds, called siderophores, that have strong affinity for Fe (III) to
scavenge environmental Fe under Fe-deficient conditions. Fluorescent *Pseudomonas* spp. produce pyoverdine, a major siderophore, while also often producing secondary siderophores<sup>18</sup>, such as pyochelin, yersiniabactin<sup>19</sup>, corrugatin<sup>20</sup> or ornicorrugatin<sup>21</sup>. Apart from pyochelin, the impact of secondary siderophores on mineral weathering has not been thoroughly investigated.

However, free Fe can also be very toxic, leading to DNA, protein and lipid damage via the Fenton reaction, which generates hydroxyl radicals<sup>22</sup>. The intracellular Fe level is regulated through complex systems to maintain a balance that is sufficiently high without reaching levels that would be toxic for the microorganism. The ferric uptake regulator (Fur) protein has been identified as a key regulator in bacterial Fe metabolism<sup>23</sup>. In response to intracellular Fe concentration variations, Fur regulates the expression of genes encoding Fe uptake proteins, Fe-using proteins and siderophore biosynthesis, thereby optimizing Fe uptake while preventing cellular damage.

*Pseudomonas brassicacearum* NFM421, which was isolated from the *Arabidopsis thaliana* rhizosphere by Achouak et al. (2000)<sup>24</sup>, is able to produce both pyoverdine and a secondary lipopeptidic siderophore known as ornicorrugatin<sup>25</sup>. Under Fe-starvation conditions, active *prrF1* gene transcription in *P. brassicacearum* leads to production of the non-coding RNA (ncRNA) *prrF1*. Subsequently, *prrF1* ncRNA suppresses the translation of target messenger RNAs encoding Fe-using proteins<sup>26</sup>. Conversely, in Fe-repletion conditions, Fur represses *prrF1* RNA transcription, hence indirectly activating target messenger RNA translation and subsequent Fe-using protein production<sup>27,28</sup>. Previous studies have demonstrated that *P. brassicacearum* can remotely degrade Fe-doped imogolite nanotubes through siderophore production<sup>29</sup>. This degradation resulted in down-regulation of *prrF1* expression, indicating that the bacteria had acquired a sufficient amount of Fe through this mechanism.
Here we mainly aimed to study factors governing microbial weathering of soil minerals. We hypothesized that *P. brassicacearum* growth would be enhanced by iron acquisition from biotite particles through siderophore-mediated and -independent mechanisms, and that biotite structural elements would be released during this process. We evaluated whether pyoverdine and ornicorrugatin were efficient to different extents in this mechanism and whether the genetic regulation processes involved in their production were dissimilar. To determine the efficiency of pyoverdine and ornicorrugatin in this mechanism, as well as the genetic regulation processes governing their production, we generated mutants incapable of producing pyoverdine, ornicorrugatin, or both siderophores. We then monitored Fe homeostasis regulation and bacterial growth during the interaction between *P. brassicacearum* and biotite. Furthermore, we examined how iron genetic regulation and direct physical accessibility of the mineral influence biotite weathering.

## 2. MATERIALS & METHODS

### Bacterial strains and mutants

The bacterial strain used in this study was *P. brassicacearum* NFM421. In addition to the wild-type (WT) strain, mutants lacking the pyoverdine encoding gene (*pvdL*) (PSEBR_a1667) and the ornicorrugatin encoding gene (PSEBR_a2391) were used in this study. Details on the method used to generate the studied mutants and plasmids are provided in SI-1 and SI-2. For clarity, the mutants are hereafter referred to as *ΔpvdL* and *Δorni*, while the double mutant is referred to as *ΔpvdLΔorni*.

### Mineral

The mineral used was biotite, a phyllosilicate with the following composition³⁰: $K_{0.92}(Fe^{2+}_{1.06}Mg_{1.66})(Fe^{3+}_{0.094}Ti_{0.12}Mn_{0.046})[Si_3AlO_{10}] (OH_{1.1}F_{0.9})$. Biotite particles in the 25–50 µm size range were isolated via water sieving and subsequently sterilized through autoclaving (121°C, 15 min).
100 **Growth conditions and adjusted preculture preparation**

101 Fe-deficient casamino acid medium (CAA) with the following composition was used in the
102 experiments: 5.0 g.L⁻¹ casamino acid; 1.18 g.L⁻¹ dipotassium phosphate; 0.25 g.L⁻¹ magnesium
103 sulfate. This medium was chosen due to its low iron concentration (SI-3) and to the fact that it can
104 support robust bacterial growth and promote efficient siderophore production³¹. An isolated colony
105 was picked up and inoculated in a single use PETG culture tube containing 5 ml of CAA medium.
106 Following overnight incubation at 30°C and agitation at 150 rpm, the culture was centrifuged
107 (10,000 rpm, 10 min) and the supernatant was discarded. The resulting pellet was resuspended in
108 sterile CAA to achieve a final 600 nm optical density (OD₆₀₀) of 0.6. This suspension, hereafter
109 referred to as the 'adjusted preculture' was used as inoculum in the subsequent experiments.

110 **Intracellular iron level monitoring during the *P. brassicacearum*-biotite interaction in a 96-**
111 **well microplate assay**

112 A 96-well microplate assay was used to monitor iron homeostasis and bacterial growth during the
113 bacteria-biotite interaction. Briefly, adjusted precultures of the different mutants were inoculated
114 in 200 µl microplate wells containing either CAA alone, CAA supplemented with FeCl3 (100 µM)
115 or CAA supplemented with 0.1% (w/v) biotite. The microplates were covered and then placed in
116 a microplate reader (TECAN infinite M1000) at 28°C with shaking. Three replicates (three
117 individual wells) were performed per condition. Bacterial growth was evaluated by measuring the
118 600 nm optical density increase (OD600) every 15 min for 48 h. The TECAN Multiple Reads per
119 Well option was used to minimize interference from biotite particles during OD600 measurements.
120 A total of 16 measurements were obtained per well in a circular-like pattern and the mean of these
121 obtained values was used as the OD600. Control wells containing CAA with biotite particles (0.1%)
122 but no bacteria (CAAbiot) or CAA alone (CAA) were used to calculate the corrected OD600
(ΔOD<sub>600</sub>) for the experimental wells, as explained hereafter. Moreover, under a microscope, we
did not observe any bacterial cell aggregation, even after 48 h of growth (SI-4).

125 - Wells supplemented with 0.1% (w/v) biotite: ΔOD<sub>600</sub> = OD<sub>600</sub>(measured) – OD<sub>600</sub>(CAA<sub>biot</sub>)

126 - Wells without biotite: ΔOD<sub>600</sub> = OD<sub>600</sub>(measured) – OD<sub>600</sub>(CAA)

127 The Growthcurver R package³² was used to obtain the fastest generation time in the exponential
128 phase (T<sub>gen</sub>) and the maximum population capacity (K) per condition.

129 The intracellular iron levels of bacteria during their interaction with biotite were also monitored
130 using a fluorescence-based approach. NFM421 WT, ΔpvdL, Δorni and ΔpvdLΔorni were
131 transformed with a plasmid (pOT1-d2EGFP) containing a transcriptional fusion of prrF1-d2EGFP
132 (destabilized enhanced green fluorescent protein). d2EGFP expression was controlled in these
133 transformed strains by the prrF1 gene promotor. The d2EGFP fluorescence level was thus directly
134 linked to the prrF1 gene expression, thereby enabling the monitoring of intracellular iron levels
135 according to the fluorescence intensity²⁸,²⁹. d2EGFP fluorescence (λ<sub>excitation</sub>: 473 nm; λ<sub>emission</sub>: 510
136 nm) was measured in the microplate reader. The d2EGFP fluorescence intensity (FI) was
137 normalized by ΔOD<sub>600</sub> (FI/ΔOD<sub>600</sub>) to accurately reflect the cellular Fe state and facilitate inter-
138 strain comparisons. The detailed protocol and explanations regarding the transcriptional fusion
139 approaches used are provided in SI-5 and SI-6.

140 P. brassicacearum culture with or without physical accessibility to biotite

141 50 µl of adjusted precultures of the WT strain or Δ*pvdLΔorni* harboring the *prrF1* promoter –
142 d2EGFP transcriptional fusion were used to inoculate PETG flasks containing the following:

143 - 100 ml of CAA (control)

144 - 100 ml of biotite supplemented (0.1% w/v) CAA (bacteria in contact with biotite)
145 - 100 ml of CAA + a dialysis membrane (50 kDa pore diameter) containing biotite (0.1%
146 relative to the total liquid volume of the flask) (bacteria physically separated from biotite)

147 Non-inoculated controls of biotite-supplemented CAA or CAA without biotite were included.
148 Periodic sampling was conducted to measure OD600 and d2EGFP FI using a microplate reader
149 (TECAN infinite M1000).

150 **Biotite weathering during bacterial growth**

151 10 µl of adjusted precultures of the different strains were placed in glass tubes containing 10 ml
152 of CAA supplemented or not with 0.05% (w/v) biotite particles. The glass tubes were then
153 incubated at 30°C with agitation at 150 rpm for 7 days. Following incubation, the cultures were
154 centrifuged (10,000 rpm, 10 min) and filtered through a 0.22 µm pore diameter membrane
155 (cellulose acetate). Non-inoculated controls (CAA + biotite without bacteria) were included. Four
156 replicates were performed per condition (see SI-7 for the protocol chart). The glass tubes were
157 thoroughly washed with HCl and rinsed three times with miliQ H₂O before being autoclaved and
158 used for the experiments.

159 **Biotite weathering with bacterial supernatants**

160 50 µl of adjusted precultures of different strains were placed in triplicate PETG flasks, each
161 containing 50 ml of CAA medium. Following a 48-h incubation at 30°C with agitation at 150 rpm,
162 the cultures were centrifuged (10,000 rpm, 10 min) and the supernatants were filtered through a
163 0.22 µm pore diameter membrane (cellulose acetate). The triplicate filtered supernatants were
164 pooled before measuring the relative pyoverdine concentration and relative total siderophore
165 production (see SI-8 and SI-9 for pyoverdine and total siderophore measurement protocols).
166 Subsequently, 25 ml of the pooled supernatant was placed in glass flasks, with or without 0.05%
167 (w/v) sterile biotite particles; five replicates were performed per condition. Five glass flasks
containing biotite particles in sterile CAA medium were used as control. After 7 days of incubation at 30°C with agitation at 150 rpm, the supernatant was collected through centrifugation (10,000 rpm, 10 min) and filtration using a 0.22 µm pore diameter cellulose acetate membrane (see SI-10 for the protocol chart). The pooled supernatant pH was measured with a pH-meter (Five Easy – Mettler Toledo).

pvdL and orni genes expression monitoring during P. brassicacearum-biotite interaction in a 96-well microplate

A 96-well microplate assay was used to monitor *pvdL* and *orni* gene expression in the WT strain during bacteria-biotite interactions and at increasing FeCl₃ concentrations. 10 µl of an adjusted WT strain preculture were inoculated in 200 µl microplate wells containing 190 µl of either CAA alone, CAA supplemented with increasing FeCl₃ concentrations (0.1, 0.5, 1, 5, 10 or 100 µM) or CAA supplemented with 0.1% (w/v) biotite. The microplates were covered and then placed in a microplate reader (TECAN infinite M1000) at 28°C with shaking. Three replicates (three individual wells) were performed per condition. Bacterial growth was assessed by measuring the OD₆₀₀ every 15 min for 48 h. Wells containing CAA with biotite particles (0.1%) but no bacteria (CAA_biot), or CAA alone (CAA), were used to calculate the corrected OD₆₀₀ (ΔOD₆₀₀) of the experimental wells according to the procedure outlined in the intracellular iron level monitoring experiment.

*pvdL* and *orni* expression levels were monitored using another fluorescence-based approach. The WT strain was transformed with a plasmid containing a transcriptional fusion of *pvdL*-mCherry or *orni*-mCherry. Consequently, mCherry expression was controlled by a *pvdL* or *orni* gene promotor. The mCherry fluorescence level was thus directly linked to the pyoverdine or ornicorrugatin production levels. mCherry FI (λ<sub>excitation</sub>: 587 nm; λ<sub>emission</sub>: 610 nm) was measured
every 15 min in the microplate reader and normalized to ΔOD₆₀₀ to accurately reflect the cellular
pyoverdine or ornicorrugatin production level in the tested conditions. 20 µg.ml-1 of kanamycine
were added to maintain the mCherry transcriptional fusion plasmids in the strains.

ICP-OES analysis

Inorganic ions concentrations were measured using an Agilent 5800 VDV ICP-OES
spectrometer. All supernatants were acidified with 2.5% HNO₃ (v/v) prior to measurement.
Calibration curves were plotted using the CCS-4 CCS-5 & CCS-6 ICP standards. Quality control
was done by measuring the EU-L, ES-H (SCP Science) and SLRS-6 certified reference materials
in each run. The detection limits were 0.18 µM for Fe, 0.26 µM for Al and 0.6 µM for Si. The
concentrations presented here correspond to the concentration increase calculated by subtracting
the measured concentrations of a non-inoculated control from those of the samples.

Statistical analysis

The null hypothesis was systematically rejected in the Kruskal-Wallis tests, thus indicating that
at least one of the tested conditions (WT, Δ*pvdL*, Δ*orni*, or Δ*pvdLΔorni*) gave significantly
different concentrations. Conover-Iman pairwise multiple comparisons (with *p*-value adjustment
according to Benjamini-Hochberg) were conducted to analyze differences between each tested
condition. Conditions showing significant differences according to this test (*p* < 0.05) are indicated
by different letters in the figures.

The same statistical analysis was used to compare the T<sub>gen</sub> and K growth parameters obtained
with the Growthcurver R package.

3. RESULTS AND DISCUSSION

Iron homeostasis during *P. brassicacearum* growth with biotite
A microplate assay was conducted to monitor Fe homeostasis and assess the roles of pyoverdine and ornicorrugatin in Fe uptake from biotite. Figures 1A and B illustrate the OD600 increase ($ΔOD600$ – representing the bacterial growth) and the associated growth parameters. Figure 1C illustrates the F.I/$ΔOD600$ ratio, i.e. reflecting the cellular expression of *prrF1*, thereby enabling the monitoring of Fe deficiency at the cellular level. The WT growth rate was higher in the presence of biotite, which served as an iron source, thereby promoting bacterial growth. However, the $ΔpvdL$ mutant, which only produces ornicorrugatin, had the shortest generation time compared to the WT and other mutants, regardless of biotite presence, hence indicating its superior fitness. In contrast, the $Δorni$ mutant had the longest generation time, despite producing pyoverdine. These findings suggest that ornicorrugatin plays a crucial role in bacterial fitness, a topic that would warrant further investigation (Fig. 1A and B). Notably, in the absence of biotite, the F.I/$ΔOD600$ rate continued to increase for at least 24 h for all strains, and no significant decrease was noted during the 48-h culture period (Fig. 1C).
<figure><img src="image_2.png" /><figcaption>Figure 1. Analysis of bacterial growth and *prrF1* ncRNA expression in response to biotite exposure.</figcaption></figure>

A – OD<sub>600</sub> increase (ΔOD<sub>600</sub>) of the different strains; B – Growth parameters of the different strains with or without biotite. T<sub>gen</sub>: generation time (h<sup>-1</sup>), K: maximum population capacity. Different letters indicate significant differences (P < 0.05) based on the Conover-iman test; C – FI/ΔOD<sub>600</sub> ratio (*prrF1* expression level) of the different strains during growth in CAA with or without biotite particles (0.1%). CAA medium + biotite particles: solid lines, CAA medium: dashed lines. Strain colors: WT: purple, Δ*pvdL*: red, Δ*orni*: blue, Δ*pvdLΔorni*: grey. Vertical lines represent the standard deviations of the replicates at each time point. If the lines are smaller than the curve, they may not be visible.

In the presence of biotite, all strains showed a significantly higher maximum population capacity (K) than when biotite was absent (Fig. 1A and B). However, the Δ*pvdLΔorni* double mutant
showed a less marked K increase compared to the WT strain and Δ*pvdL* mutant and it reached maximum ΔOD600 later (18 h for the WT strain and Δ*pvdL* mutant and 21 h for the Δ*pvdLΔorni* double mutant). Although the Δ*orni* mutant had a slower Tgen than the other strains, its growth was also stimulated in the presence of biotite (Fig. 1A and B). The expression of *prrF1* ncRNA, as indicated by the FI/ΔOD600 ratio, ranged from ≈10,000 (Δ*orni*) to ≈20,000 units (WT) in the absence of biotite, whereas in its presence, this ratio initially increased for all strains, with different maximum values (Fig. 1C). The WT strain and Δ*pvdL* mutant reached maximum FI/ΔOD600 values of 5,955 and 6,792 units, respectively, while the Δ*pvdLΔorni* double mutant peaked at 9,647 units. The expression of *prrF1* ncRNA began to rapidly decline—indicating sufficient intracellular iron levels—after 20 h of growth for the WT strain and Δ*pvdL* mutant, and 24 h for the Δ*pvdLΔorni* double mutant. The Δ*orni* strain reached its FI/ΔOD600 peak after 24 h of growth, yet it had a lower peak value (4,705 units) than the other strains and its fluorescence ratio subsequently decreased (Fig. 1C).

These results demonstrated that *P. brassicacearum* NFM421 could effectively acquire Fe from biotite through pyoverdine or ornicorrugatin production. This was in line with previous research findings on *Caballeronia mineralivorans* PML1(12), which can also achieve siderophore-mediated Fe-extraction from minerals³³,³⁴. The observed enhanced growth in the presence of biotite underscores the bacteria's ability to utilize the structural elements of biotite, including Fe and potentially other ions such as Mg³⁵. The enhanced growth provided by biotite appeared to be less marked for the Δ*pvdLΔorni* mutant than for the WT strain, highlighting the importance of siderophores for nutrient acquisition.

Nonetheless, the Δ*pvdLΔorni* double mutant ultimately obtained sufficient Fe from the mineral, suggesting the involvement of other mechanisms in addition to siderophore production in Fe
scavenging from biotite. Fluorescent *Pseudomonas* strains are known to produce gluconic and α-ketogluconic acid, which could potentially dissolve biotite through complexation and medium acidification³⁶. However, this mechanism may not have been involved under our experimental conditions, as it would require glucose as carbon source. Otherwise, another mechanism involving direct physical contact might facilitate Fe scavenging from biotite, as suggested by Kuhn et al. (2013)³⁷ in their study on the enzymatic reduction of montmorillonite Fe(III) by *P. mendocina*.

In conclusion, *P. brassicacearum* successfully extracted Fe from biotite through both siderophore-dependent and -independent mechanisms, thereby fulfilling its metabolic needs (allowing Fe-using protein production) and enhancing its growth.

## Biotite physical accessibility impacts iron homeostasis

We sought to confirm whether another mechanism requiring physical accessibility would enable *P. brassicacearum* to extract Fe from biotite by monitoring bacterial Fe homeostasis of the WT strain and Δ*pvdLΔorni* double mutant in the presence of biotite particles that were either physically accessible (suspended, S) or not (encased in a dialysis membrane, DM). In the absence of biotite, *prrF1* ncRNA expression in both Δ*pvdLΔorni* double mutant and WT strain increased, reaching an initial peak at around 24 h of growth (Fig. 2). Subsequently, the fluorescence slightly decreased until 48 h, followed by a gradual increase until reaching a plateau at 112 h. This pattern indicated a low intracellular Fe level. When biotite was added as suspended particles in the growth medium (S), the fluorescence rapidly decreased after 16 and 24 h of growth for the WT strain and Δ*pvdLΔorni* mutant, respectively (Fig. 2). The fluorescence levels were higher for the double mutant (Δ*pvdLΔorni*) than for the WT strain, suggesting that although both strains could extract Fe from biotite, the lack of siderophores limited the capacity of the Δ*pvdLΔorni* double mutant to scavenge it.
282 When biotite was present in a dialysis membrane (i.e. not physically accessible, DM), the extent
283 of the WT strain fluorescence decrease was very similar to that noted when biotite was free, but
284 with a delay due to the time required for the siderophores to diffuse within the dialysis bag and
285 subsequently return to the bacterial cells. In contrast, the fluorescence level of the double mutant
286 ($\Delta pvdL\Delta orni$) did not get as low as it did when biotite was suspended in the medium (≈ 30,000 vs
287 10,000 units). These results suggest that the inability to produce pyoverdine and ornicorrugatin
288 restrained the $\Delta pvdL\Delta orni$ double mutant from scavenging biotite Fe when it was not physically
289 accessible.

290 This was consistent with the findings of Parrello et al. (2016)¹⁷, who identified a similar
291 unknown contact mechanism enabling *Pseudomonas aeruginosa* PAO1 to acquire Fe from
292 nontronites when direct physical contact was possible. This was also in line with previous findings
293 showing that the ability to adhere and colonize specific mineral surfaces is essential to enable the
294 bacteria to access environmental Fe¹⁴,³⁸. Our results confirmed that, apart from siderophore-
295 mediated Fe uptake, *P. brassicacearum* can perform direct Fe uptake when it is in physical contact
296 with biotite. In this context, siderophore production appears to be an effective strategy to maximize
297 the chances of scavenging Fe from minerals in the environment that may not be directly accessible
298 to the cells. Moreover, this Fe extraction may increase the mineral weathering rate.

299
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Impact of the physical accessibility on iron homeostasis. FI/ΔDO₆₀₀ ratio (*prrF1* expression level) of *P. brassicacearum* WT (**A**) and Δ*pvdLΔorni* (**B**) strains during growth in CAA with: **0.05% suspended (S) biotite particles**: solid lines, **CAA without biotite (no biot)**: dotted lines, or **CAA with 0.05% dialysis membrane (DM)-encased biotite particles**: dashed lines. **Strain colors**: WT: purple, Δ*pvdLΔorni*: grey. Error bars represent standard deviation.</figcaption></figure>

Mineral weathering ability of *P. brassicacearum*
The biotite weathering process was assessed by quantifying aluminum (Al), iron (Fe), and silicon (Si) release. Fe, Al and Si concentrations were measured in the culture supernatants after 7 days of culturing the mutants and the WT strain in presence of biotite particles (Fig. 3).

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Biotite weathering during bacterial growth. Changes in iron, aluminum and silicon concentrations measured in the culture supernatant after 7 days of culturing the different *P. brassicacearum* strains in biotite-amended CAA medium (0.05% w/v). WT: purple Δ*pvdL*: red Δ*orni*: blue Δ*pvdLΔorni*: grey. The data indicate the mean ± standard deviation of three replicates. Different letters indicate significant differences (P < 0.05) based on the Conover-Iman test.</figcaption></figure>

All strains were able to dissolve Fe from biotite, yet the total amount of Fe extracted from biotite could not be determined because the intracellular Fe content of the bacterial cells was not measured, since the cells and remaining biotite particles were removed by filtration. Given that Al and Si are not major bacterial metabolism components, their dissolved concentrations could be considered as relevant indicators of biotite weathering. The Δ*pvdL* mutant dissolved more Al than the WT strain, whereas both strains dissolved comparable amounts of Si. The Δ*orni* mutant dissolved significantly more Al than the Δ*pvdLΔorni* double mutant, but substantially less Al and Si than both the WT strain and Δ*pvdL* mutant (Fig. 3). Siderophores preferentially bind Fe but they
can chelate other metals like Al³⁹, which could explain the observed dissolution of Al.

Azotobactin—a pyoverdine-like siderophore—forms complexes with molybdate and vanadate
more quickly than with Fe⁴⁰, which highlights that siderophores can strongly interact with other
elements. Our results suggest that, compared to pyoverdine, ornicorrugatin potentially has higher
affinity for Al or that there is higher ornicorrugatin production. It is well established that
pyoverdine production is inhibited once the bacterium's Fe requirements are met⁴¹, whereas
ornicorrugatin regulation has yet to be elucidated. Kimura et al. (2021)⁴² showed that the
siderophore produced by *Pseudomonas* sp. dissolves Fe, Al, Si and Mg from the broken edges of
biotite particles. Our results confirmed that pyoverdine and ornicorrugatin could similarly release
structural inorganic ions from biotite through complexolysis. Differential regulation of pyoverdine
and ornicorrugatin production during biotite exposure could lead to the production of unequal
amounts of these siderophores, thereby impacting their weathering rate.

### Biotite weathering by *P. brassicacearum* supernatants

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Siderophore production assessment. A – Relative pyoverdine concentration and B – relative total siderophore production in the pooled 48 h culture supernatants of the different *P. brassicacearum* strains in iron-deficient CAA medium. WT: purple ΔpvdL: red Δorni: blue ΔpvdLΔorni: grey.</figcaption></figure>

To evaluate the weathering potential of *P. brassicacearum* in total Fe deficiency, we performed
an experiment in which the bacteria were grown under constant Fe starvation conditions (without
any soluble Fe or biotite particles). We harvested culture supernatants containing the bacterial
342 metabolites and exposed biotite particles to supernatants of the WT strain and Δ*orni*, Δ*pvdL*
343 Δ*pvdLΔorni* mutants.

344 The pyoverdine fluorescence measured in the culture supernatants revealed that the WT strain
345 produced the highest amount of pyoverdine, followed by the Δ*orni* mutant (Fig. 4A). The
346 Δ*pvdLΔorni* and Δ*pvdL* mutants produced < 1% of the pyoverdine produced by the WT strain. The
347 relative total siderophore production in the supernatant was also higher for the WT strain (Fig.
348 4B). The Δ*orni* and Δ*pvdL* mutants siderophore production was 62% and 40% that of the WT
349 strain, respectively. The bacterial supernatant pH increased similarly for all strains, i.e. from 7.3
350 (the sterile CAA pH) to between 8.6 and 8.7 (see SI-11).

<figure><img src="image_6.png" /></figure>

351 **Figure 5.** Biotite weathering by *P. brassicacearum* supernatants. Increased iron, aluminum and silicon
352 concentrations measured in the supernatant after 7 days of incubation with biotite particles (0.05% w/v).
353 Strain colors: **WT**: purple Δ*pvdL*: red Δ*orni*: blue Δ*pvdLΔorni*: grey. The data indicate the mean ± standard
354 deviation for five replicates. Different letters indicate significant differences (P < 0.025) according to the
355 Conover-Iman test.

356 We noted a more marked release of Fe, Al and Si from biotite when exposed to supernatants of
357 cultures from the different strains (Fig. 5) compared to experiments with living bacteria (Fig. 3).
358 The supernatants produced under Fe-deficient conditions exhibited high siderophore content.
While we observed a doubling of the Fe concentration (3.4 µM in Fig. 5 compared to 1.5 µM in Fig. 3 for WT), Al release from the biotite structure had almost quadrupled (18 µM in Fig. 5 compared to 3.5 µM in Fig. 3, for WT) and, notably, Si release had increased by more than tenfold (360 µM in Fig. 5 compared to 28 µM in Fig. 3, for WT). This higher dissolution of Si compared to Fe and Si may be attributed to the high pH of the supernatants (~9), which was favorable for Si dissolution. The WT strain supernatant exhibited the highest effectiveness in weathering biotite whereas the Δ*pvdLΔorni* supernatant showed the lowest effectiveness (Fig. 5). The limited weathering activity observed in the supernatant of the Δ*pvdLΔorni* mutant suggests that, in our experimental conditions, no other metabolites with significant weathering activity—such as organic acids—were produced in appreciable quantities. The Δ*orni* supernatant was slightly less effective in dissolving Fe but significantly less in dissolving Al and Si compared to the WT. Conversely, the Δ*pvdL* supernatant dissolved significantly less Fe and less than half of the Al and Si compared to that of the WT strain. These results highlight the efficiency of both siderophores in the mineral weathering process. Interestingly, the Δ*pvdL* supernatant was less effective overall than the Δ*orni* supernatant in weathering biotite. This was in accordance with lower ornicorrugatin production (Fig. 4B) and the fact that pyoverdine has a higher affinity for Fe than secondary siderophores⁴³. However, this contrasted with the previously presented results, where Δ*pvdL* was more effective at weathering biotite than Δ*orni* when growing in the presence of biotite particles (Fig. 3). This suggests that constant Fe starvation during supernatant production led the bacteria to produce as many siderophores as possible, resulting in higher biotite dissolution (Fig. 5). In the previous experiment (Fig. 3), the bacteria could obtain Fe from the mineral, leading to genetic regulation of siderophore production, potentially hindering pyoverdine production faster than ornicorrugatin production. Mendonca et al. (2020)¹⁶ found that *Pseudomonas putida* KT2440
supernatants increased goethite, magnetite and hematite weathering rates only when the strain was grown in Fe-deficient medium, thereby highlighting the impact of Fe availability on bacterial mineral weathering. Moreover, Dumas et al. (2013)⁴⁴ found that *P. aeruginosa* alternated between producing pyoverdine and pyochelin depending on the environmental conditions, e.g. Fe availability, temperature and pH. This phenomenon may also explain why ornicorrugatin and pyoverdine alternately dominated the weathering process in the presence of iron from biotite or in the absence of iron during bacterial growth.

## 389 Effects of Fe availability and biotite on siderophore gene expression

We compared the effects of biotite at a range of FeCl₃ concentrations on siderophore gene expression through transcriptional fusion of *pvdL* and *orni* gene promoters with the m-Cherry fluorescent protein. Fe³⁺ exhibits low water solubility and readily precipitates. However, dissolved soil organic matter can form stable complexes with Fe³⁺, thereby inhibiting its precipitation⁴⁵. Moreover, Fe-siderophore complexes demonstrate greater stability compared to Fe-organic matter complexes, enabling siderophores to efficiently sequester Fe for microbial utilization⁴⁶. Similarly, dissolved organic molecules present in CAA—mainly amino acids derived from casein hydrolysis— would likely to complex with Fe, hence enhancing its solubility and bioavailability for *P. brassicacearum* via siderophore-mediated mechanisms.

Analyses conducted in Fe-deficient medium showed high *pvdL* and *orni* gene expression (Fig. 6). The expression of both genes was gradually inhibited in the presence of increasing Fe concentrations. Interestingly, the addition of 0.1% biotite led to *pvdL* and *orni* gene expression corresponding to that obtained with a 1 to 5 µM Fe concentration. However, biotite addition led to lower *pvdL* expression (FI/ΔOD₆₀₀=675 units) compared to that of *orni* genes (FI/ΔOD₆₀₀=1350 units) (Fig. 6). *pvdL* expression halted in the presence of 5 µM or more FeCl₃ while 100 µM FeCl₃
was needed to lower *orni* expression. There was a marked increase in the expression of siderophore-coding genes after 23 h of growth because the ΔOD₆₀₀ (representing bacterial growth) had reached its maximum and plateaued. In contrast, the expression of siderophore-coding genes, as measured by FI/ΔOD₆₀₀, continued to rise, since the bacteria remained unsaturated with iron in the presence of biotite and iron concentrations < 5 µM. The dissimilar effects of Fe on *pvdL* and *orni* gene expression were probably due to the different regulation pattern of these genes. These results could explain the stronger effect of ornicorrugatin on biotite weathering, which was likely not due to higher weathering efficiency, but rather to sustained production of this siderophore over time, even when the bacterial cells had acquired sufficient Fe for their growth (Fig. 3).

<figure><img src="image_7.png" /><figcaption>**Figure 6.** Iron and biotite effects on *pvdL* and *orni* gene expression in *P. brassicacearum*. mCherry FI/ΔOD₆₀₀ (gene expression level) of WT transcriptional fusion with the (A) *pvdL* or (B) *orni* promoter.</figcaption></figure>
FeCl₃ was added to CAA medium to a final concentration of: 0 µM, 0.1 µM, 0.5 µM, 1 µM, 5 µM, 10 µM or 100 µM. The addition of 0.1% (w/v) biotite (Biot) was also tested. Vertical lines represent the standard deviations of the three replicates at each time point. If the lines are smaller than the curve, they may not be visible. Different letters indicate significant differences (P < 0.05) based on the Conover-Iman test (for the last measurement after 48 h).

## Environmental Implications

Siderophores naturally occur in soils47,48 and various siderophores can enhance mineral weathering rates through Fe and Al chelation49-51. Our results confirmed that while a single siderophore (pyoverdine) was sufficient to fulfill the iron requirements of *P. brassicacearum*, this bacterium also produced a secondary siderophore (ornicorrugatin). These siderophores are able to extract iron even when it is tightly bound within crystalline structures, such as minerals, as demonstrated in the presence of biotite (Fig. 7). Interestingly, when this bacterial strain was grown in the presence of biotite particles, ornicorrugatin production seemed to have a more significant impact on the weathering process than pyoverdine, even though the bacteria had fulfilled their Fe requirements. Our findings suggest that the regulation of ornicorrugatin expression is likely independent of Fur (Fig. 7). It has been established that pyochelin produced by *P. aeruginosa* PAO1 as secondary siderophore is less effective than pyoverdine in enhancing smectite or nontronites weathering rates7,17. Our results highlighted the potential significance of secondary siderophores in natural processes such as mineral weathering. Ornicorrugatin, alongside corrugatin and histicorrugatin, are part of a specific set of lipopeptidic secondary siderophores. These siderophores have an octanoic fatty acid chain linked to the main peptidic sequence, yet scant information is available regarding their specific physiological roles. Elucidating the ornicorrugatin biosynthesis pathway, its regulation and physicochemical characteristics could enhance insight into the environmental impacts of these lipopeptidic siderophores.
<figure><img src="image_8.png" /><figcaption>**Figure 7.** Scheme illustrating the factors governing biotite weathering and iron homeostasis during P. brassicacearum-biotite interactions. The Fur regulator is inactive when intracellular Fe levels are low (1). *prrF1* ncRNA production inhibits the synthesis of Fe-using proteins (2). The bacterium actively produces and secretes pyoverdine and ornicorrugatin (3). Pyoverdine and ornicorrugatin chelate Fe (and eventually aluminum) through complexolysis, releasing inorganic ions from the biotite structure (4). Fe-</figcaption></figure>
445 siderophore complexes are taken up by the bacterial cells (5). Biotite-derived structural Fe is taken up by 446 the bacterial cell through an unknown mechanism requiring physical contact (6). High intracellular Fe levels 447 activate Fur (7) and enhance bacterial growth (8). Activated Fur downregulates *prrF1* expression, indirectly 448 activating Fe-using protein production (9). *PvdL* expression is promptly stopped by Fe-activated Fur (10). 449 Intracellular Fe levels also lower *Orni* expression (11). Pyoverdine production stops, while ornicorrugatin 450 continues to be produced, thereby contributing to the ongoing biotite weathering (12).

451 Sheng et al. (2024)⁵² recently showed the impact of iron-response related genes *rirA* and *irr* on 452 biotite weathering, thereby highlighting the link between iron metabolism and mineral weathering. 453 Further investigations on *P. brassicacearum* NFM421-induced weathering with different carbon 454 sources, culture media and minerals could be of great interest to help understand the factors 455 governing the mineral weathering ability of this strain.

456 Minerals release their structural elements into the environment during interactions with bacteria. 457 Soil microorganisms or surrounding plant roots can take up some of these elements as mineral 458 nutrients⁵³,⁵⁴. Weathering of phyllosilicate minerals like biotite releases inorganic elements such 459 as Fe, Al and Si into soils.

460 Biotic weathering of phyllosilicate minerals such as biotite releases essential inorganic elements 461 like Fe, Al, and Si into soils. This mineral-microbe interaction is of considerable interest for 462 developing sustainable mineral fertilizers to enhance crop growth⁵⁵. Silicon (Si) is particularly 463 valuable as it promotes plant growth and increases resilience against various abiotic stresses⁵⁶. A 464 recent study by Yang et al. (2024)⁵⁷ revealed that the silicon supply in paddy soils across major 465 rice-producing regions is insufficient, suggesting that Si fertilizers could boost rice yields and 466 enhance carbon sequestration in phytoliths. In contrast, aluminum (Al), particularly in its Al³⁺ 467 form, is toxic to plants as it inhibits root growth, plant development and photosynthesis⁵⁸. 468 However, Al toxicity primarily occurs in acidic soils with a pH < 5, i.e. a level at which Al³⁺
becomes soluble⁵⁹. Interestingly, the simultaneous dissolution of Si and Al from biotite may help mitigate Al toxicity. Silicon has been shown to alleviate Al toxicity in acidic soils through various mechanisms⁶⁰,⁶¹.

These elements can interact with soil organic molecules to form organomineral coprecipitates that are found in natural soils³⁰,⁶²⁻⁶⁴. These coprecipitates are recalcitrant to biodegradation, resulting in long-term soil organic carbon stabilization. Future research could focus on exploring whether siderophore-mediated mineral weathering in soils contributes to organomineral coprecipitate formation in nature.

*P. brassicacearum* NFM421 is known to efficiently colonize plant roots⁶⁵ and produce antifungal compounds that protect the plant from pathogens⁶⁶. The inorganic ions released through its weathering activity likely have an impact on plant metabolism and contribute to the soil's geochemical cycling. Leveraging mineral-weathering microorganisms like *P. brassicacearum* could be a promising strategy to enhance plant nutrition and growth, while promoting carbon sequestration through the formation of organomineral coprecipitates in soils⁶²,⁶³.

## 483 ASSOCIATED CONTENT

Primers and plasmids used for the mutations, additional experimental details, material and methods.

## 486 AUTHOR INFORMATION

### 487 Corresponding Author

*E-mail: tom.girard.sp21@gmail.com

### 489 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

## ACKNOWLEDGMENT

This work received support from the French government under the France 2030 Investment Plan, as part of the *Initiative d'Excellence d'Aix-Marseille Université* - A*MIDEX* (AMX-19-IET-012).

## ABBREVIATIONS

**WT**, wild-type; **Fur**, ferric uptake regulator; **ncRNA**, non-coding RNA; **Fe**, iron; **Al**, aluminum; **Si**, silicon; **CAA**, casamino acid medium; **OD600**, 600 nm optical density; **d2EGFP**, destabilized enhanced green fluorescent protein; **FI**, fluorescence intensity

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