Iron Homeostasis in Bacillus subtilis Requires Siderophore Production and Biofilm Formation.

Iron (Fe) is the most important metal in biology. Despite its abundance, Fe is mostly present as a ferric form in soils, strongly limiting its bioavailability. To overcome the challenge of Fe acquisition, many microorganisms produce siderophores to retrieve Fe from natural sources. Another ubiquitous feature of bacteria in natural environments is biofilm formation. Previous studies showed that external Fe strongly influenced biofilm formation in several bacteria, suggesting that this microenvironment plays a mechanistic role in micronutrient acquisition for bacteria. Here, we applied a complementary set of analytical methods and deletion mutants to evaluate the role of biofilm formation, siderophore production, and their interaction in Fe homeostasis in Bacillus subtilis We observed that Fe homeostasis, i.e., active growth at a constant intracellular Fe concentration, requires both siderophore production and biofilm formation. Also, we report that in B. subtilis, both biofilm formation and siderophore production are required to achieve active Fe acquisition from the medium and to sustain normal growth. Furthermore, we provide evidence that the formation of biofilm slightly enhances the kinetics of Fe complexation by catechol siderophores and markedly improves siderophore use efficiency. These results provide new perspectives on the mechanism underlying siderophore-based acquisition of Fe in biofilm-forming bacteria.IMPORTANCE Iron acquisition is of fundamental importance for microorganisms, since this metal is generally poorly bioavailable under natural conditions. In the environment, most bacteria are found tightly packed within multicellular communities named biofilms. Here, using the soil Gram-positive bacterium Bacillus subtilis, we show that biofilm formation and the production of siderophores, i.e., small molecules specifically binding metals, are both essential to ensure Fe uptake from the medium and maintain cellular Fe homeostasis. The biofilm matrix appears to play an important role favoring the efficient usage of siderophores. Taken together, our results demonstrate a close link between biofilm formation and iron acquisition in B. subtilis, allowing a better comprehension of how bacteria can cope with metal limitation under environmental conditions.

Anthropogenic deposition of heavy metals and phosphorus may reduce biological N2 fixation in boreal forest mosses.

A study was undertaken to test the effects of molybdenum (Mo) and phosphorus (P) amendments on biological nitrogen (N) fixation (BNF) by boreal forest moss-associated cyanobacteria. Feather moss (Pleurozium schreberi) samples were collected on five sites, on two dates and at different roadside distances (0-100m) corresponding to an assumed gradient of reactive N deposition. Potential BNF of Mo and P amended moss samples was measured using the acetylene reduction assay. Total N, P and heavy metal concentrations of mosses collected at 0 and 100m from roadsides were also measured. Likewise, the needles from Norway spruce trees (Picea abies) at different roadside distances were collected in late summer and analyzed for total N, P and heavy metals. There was a significant increase in BNF with roadside distance on 7-of-10 individual Site×Date combinations. We found no clear evidence of an N gradient across roadside distances. Elemental analyses of feather moss and Norway spruce needle tissues suggested decreasing deposition of heavy metals (Mo-Co-Cr-Ni-V-Pb-Ag-Cu) as well as P with increasing distance from the roadside. The effects of Mo and P amendments on BNF were infrequent and inconsistent across roadside distances and across sites. One particular site, however, displayed greater concentrations of heavy metals near the roadside, as well as a steeper P fertility gradient with roadside distance, than the other sites. Here, BNF increased with roadside distance only when moss samples were amended with P. Also at this site, BNF across all roadside distances was higher when mosses were amended with both Mo and P, suggesting a co-limitation of these two nutrients in controlling BNF. In summary, our study showed a potential for car emissions to increase heavy metals and P along roadsides and underscored the putative roles of these anthropogenic pollutants on BNF in northern latitudes.

Molybdenum and phosphorus limitation of moss-associated nitrogen fixation in boreal ecosystems.

Biological nitrogen fixation (BNF) performed by moss-associated cyanobacteria is one of the main sources of new nitrogen (N) input in pristine, high-latitude ecosystems. Yet, the nutrients that limit BNF remain elusive. Here, we tested whether this important ecosystem function is limited by the availability of molybdenum (Mo), phosphorus (P), or both. BNF in dominant mosses was measured with the acetylene reduction assay (ARA) at different time intervals following Mo and P additions, in both laboratory microcosms with mosses from a boreal spruce forest and field plots in subarctic tundra. We further used a 15 N2 tracer technique to assess the ARA to N2 fixation conversion ratios at our subarctic site. BNF was up to four-fold higher shortly after the addition of Mo, in both the laboratory and field experiments. A similar positive response to Mo was found in moss colonizing cyanobacterial biomass. As the growing season progressed, nitrogenase activity became progressively more P limited. The ARA : 15 N2 ratios increased with increasing Mo additions. These findings show that N2 fixation activity as well as cyanobacterial biomass in dominant feather mosses from boreal forests and subarctic tundra are limited by Mo availability.

Identification of the hydroxamate siderophore ferricrocin in Cladosporium cladosporioides.

The hydroxamate siderophore ferricrocin was identified in Cladosporium cladosporioides growth medium by solid phase extraction and ultra high pressure liquid chromatography coupled to a time of flight mass spectrometer (UHPLC/QTOF-MS). Both desferricrocin and ferricrocin were detected in the extracellular medium assisted by high resolution mass spectrometry. This is the first identification of a hydroxamate siderophore in Cladosporium cladosporioides. This finding emphasizes the common meaning of ferricrocin in fungi.

Molybdenum and phosphorus interact to constrain asymbiotic nitrogen fixation in tropical forests.

Biological di-nitrogen fixation (N(2)) is the dominant natural source of new nitrogen to land ecosystems. Phosphorus (P) is thought to limit N(2) fixation in many tropical soils, yet both molybdenum (Mo) and P are crucial for the nitrogenase reaction (which catalyzes N(2) conversion to ammonia) and cell growth. We have limited understanding of how and when fixation is constrained by these nutrients in nature. Here we show in tropical forests of lowland Panama that the limiting element on asymbiotic N(2) fixation shifts along a broad landscape gradient in soil P, where Mo limits fixation in P-rich soils while Mo and P co-limit in P-poor soils. In no circumstance did P alone limit fixation. We provide and experimentally test a mechanism that explains how Mo and P can interact to constrain asymbiotic N(2) fixation. Fixation is uniformly favored in surface organic soil horizons--a niche characterized by exceedingly low levels of available Mo relative to P. We show that soil organic matter acts to reduce molybdate over phosphate bioavailability, which, in turn, promotes Mo limitation in sites where P is sufficient. Our findings show that asymbiotic N(2) fixation is constrained by the relative availability and dynamics of Mo and P in soils. This conceptual framework can explain shifts in limitation status across broad landscape gradients in soil fertility and implies that fixation depends on Mo and P in ways that are more complex than previously thought.