Brighter near-infrared (NIR) fluorescent proteins (FPs) are required for multicolor microscopy and deep-tissue imaging. Here, we present structural and biochemical analyses of three monomeric, spectrally distinct phytochrome-based NIR FPs, termed miRFPs. The miRFPs are closely related and differ by only a few amino acids, which define their molecular brightness, brightness in mammalian cells, and spectral properties. We have identified the residues responsible for the spectral red-shift, revealed a new chromophore bound simultaneously to two cysteine residues in the PAS and GAF domains in blue-shifted NIR FPs, and uncovered the importance of amino acid residues in the N-terminus of NIR FPs for their molecular and cellular brightness. The novel chromophore covalently links the N-terminus of NIR FPs with their C-terminal GAF domain, forming a topologically closed knot in the structure, and also contributes to the increased brightness. Based on our studies, we suggest a strategy to develop spectrally distinct NIR FPs with enhanced brightness.

Endolithic microbial communities survive nutrient and energy deficient conditions while contributing to the weathering of their mineral substrate. This study examined the mineral composition and microbial communities of fully serpentinized weathered rock from 0.1 to 6.5 m depth at a site within the Khalilovsky massif, Orenburg Region, Southern Ural Mountains, Russia. The mineral composition includes a major content of serpentinite family (mostly consisting of lizardite and chrysotile), magnesium hydrocarbonates (hydromagnesite with lesser amounts of hydrotalcite and pyroaurite) concentrated in the upper layers, and clay minerals. We found that the deep-seated weathered serpentinites are chrysotile-type minerals, while the middle and surface serpentinites mostly consist of lizardite and chrysotile types. Microbial community analysis, based on 16S rRNA gene sequencing, showed a similar diversity of phyla throughout the depth profile. The dominant bacterial phyla were the Actinobacteria (of which unclassified genera in the orders Acidimicrobiales and Actinomycetales were most numerous), Chloroflexi (dominated by an uncultured P2-11E order) and the Proteobacteria (predominantly class Betaproteobacteria). Densities of several groups of bacteria were negatively correlated with depth. Occurrence of the orders Actinomycetales, Gaiellales, Solirubrobacterales, Rhizobiales and Burkholderiales were positively correlated with depth. Our findings show that endolithic microbial communities of the Khalilovsky massif have similar diversity to those of serpentine soils and rocks, but are substantially different from those of the aqueous environments of actively serpentinizing systems.