Iron is not everything: unexpected complex metabolic responses between iron-cycling microorganisms.

Coexistence of microaerophilic Fe(II)-oxidizers and anaerobic Fe(III)-reducers in environments with fluctuating redox conditions is a prime example of mutualism, in which both partners benefit from the sustained Fe-pool. Consequently, the Fe-cycling machineries (i.e., metal-reducing or -oxidizing pathways) should be most affected during co-cultivation. However, contrasting growth requirements impeded systematic elucidation of their interactions. To disentangle underlying interaction mechanisms, we established a suboxic co-culture system of Sideroxydans sp. CL21 and Shewanella oneidensis. We showed that addition of the partner's cell-free supernatant enhanced both growth and Fe(II)-oxidizing or Fe(III)-reducing activity of each partner. Metabolites of the exometabolome of Sideroxydans sp. CL21 are generally upregulated if stimulated with the partner´s spent medium, while S. oneidensis exhibits a mixed metabolic response in accordance with a balanced response to the partner. Surprisingly, RNA-seq analysis revealed genes involved in Fe-cycling were not differentially expressed during co-cultivation. Instead, the most differentially upregulated genes included those encoding for biopolymer production, lipoprotein transport, putrescine biosynthesis, and amino acid degradation suggesting a regulated inter-species biofilm formation. Furthermore, the upregulation of hydrogenases in Sideroxydans sp. CL21 points to competition for H2 as electron donor. Our findings reveal that a complex metabolic and transcriptomic response, but not accelerated formation of Fe-end products, drive interactions of Fe-cycling microorganisms.