Comparative genomic analysis of nickel homeostasis in cable bacteria.

BACKGROUND: Cable bacteria are filamentous members of the Desulfobulbaceae family that are capable of performing centimetre­scale electron transport in marine and freshwater sediments. This long­distance electron transport is mediated by a network of parallel conductive fibres embedded in the cell envelope. This fibre network efficiently transports electrical currents along the entire length of the centimetre­long filament. Recent analyses show that these fibres consist of metalloproteins that harbour a novel nickel­containing cofactor, which indicates that cable bacteria have evolved a unique form of biological electron transport. This nickel­dependent conduction mechanism suggests that cable bacteria are strongly dependent on nickel as a biosynthetic resource. Here, we performed a comprehensive comparative genomic analysis of the genes linked to nickel homeostasis. We compared the genome­encoded adaptation to nickel of cable bacteria to related members of the Desulfobulbaceae family and other members of the Desulfobulbales order. RESULTS: Presently, four closed genomes are available for the monophyletic cable bacteria clade that consists of the genera Candidatus Electrothrix and Candidatus Electronema. To increase the phylogenomic coverage, we additionally generated two closed genomes of cable bacteria: Candidatus Electrothrix gigas strain HY10­6 and Candidatus Electrothrix antwerpensis strain GW3­4, which are the first closed genomes of their respective species. Nickel homeostasis genes were identified in a database of 38 cable bacteria genomes (including 6 closed genomes). Gene prevalence was compared to 19 genomes of related strains, residing within the Desulfobulbales order but outside of the cable bacteria clade, revealing several genome­encoded adaptations to nickel homeostasis in cable bacteria. Phylogenetic analysis indicates that nickel importers, nickel­binding enzymes and nickel chaperones of cable bacteria are affiliated to organisms outside the Desulfobulbaceae family, with several proteins showing affiliation to organisms outside of the Desulfobacterota phylum. Conspicuously, cable bacteria encode a unique periplasmic nickel export protein RcnA, which possesses a putative cytoplasmic histidine­rich loop that has been largely expanded compared to RcnA homologs in other organisms. CONCLUSION: Cable bacteria genomes show a clear genetic adaptation for nickel utilization when compared to closely related genera. This fully aligns with the nickel­dependent conduction mechanism that is uniquely found in cable bacteria.

Multi-wavelength Raman microscopy of nickel-based electron transport in cable bacteria.

Cable bacteria embed a network of conductive protein fibers in their cell envelope that efficiently guides electron transport over distances spanning up to several centimeters. This form of long-distance electron transport is unique in biology and is mediated by a metalloprotein with a sulfur-coordinated nickel (Ni) cofactor. However, the molecular structure of this cofactor remains presently unknown. Here, we applied multi-wavelength Raman microscopy to identify cell compounds linked to the unique cable bacterium physiology, combined with stable isotope labeling, and orientation-dependent and ultralow-frequency Raman microscopy to gain insight into the structure and organization of this novel Ni-cofactor. Raman spectra of native cable bacterium filaments reveal vibrational modes originating from cytochromes, polyphosphate granules, proteins, as well as the Ni-cofactor. After selective extraction of the conductive fiber network from the cell envelope, the Raman spectrum becomes simpler, and primarily retains vibrational modes associated with the Ni-cofactor. These Ni-cofactor modes exhibit intense Raman scattering as well as a strong orientation-dependent response. The signal intensity is particularly elevated when the polarization of incident laser light is parallel to the direction of the conductive fibers. This orientation dependence allows to selectively identify the modes that are associated with the Ni-cofactor. We identified 13 such modes, some of which display strong Raman signals across the entire range of applied wavelengths (405-1,064 nm). Assignment of vibrational modes, supported by stable isotope labeling, suggest that the structure of the Ni-cofactor shares a resemblance with that of nickel bis(1,2-dithiolene) complexes. Overall, our results indicate that cable bacteria have evolved a unique cofactor structure that does not resemble any of the known Ni-cofactors in biology.