Ice-Binding Proteins Associated with an Antarctic Cyanobacterium, Nostoc sp. HG1.

Haruyuki Atomi,Marco Mangiagalli,Michael G. Janech,James A. Raymond

doi:10.1128/aem.02499-20

Abstract

Ice-binding proteins (IBPs) have been identified in numerous polar algae and bacteria, but so far not in any cyanobacteria, despite the abundance of cyanobacteria in polar regions. We previously reported strong IBP activity associated with an Antarctic Nostoc species. In this study, to identify the proteins responsible, as well as elucidate their origin, we sequenced the DNA of an environmental sample of this species, designated Nostoc sp. HG1, and its bacterial community and attempted to identify IBPs by looking for known IBPs in the metagenome and by looking for novel IBPs by tandem mass spectrometry (MS/MS) proteomics analyses of ice affinity-purified proteins. The metagenome contained over 116 DUF3494-type IBP genes, the most common type of IBP identified so far. One of the IBPs could be confidently assigned to Nostoc, while the others could be attributed to diverse bacteria, which, surprisingly, accounted for the great majority of the metagenome. Recombinant Nostoc IBPs (nIBPs) had strong ice-structuring activities, and their circular dichroism spectra were consistent with the secondary structure of a DUF3494-type IBP. nIBP is unusual in that it is the only IBP identified so far to have a PEP (amino acid motif) C-terminal signal, a signal that has been associated with anchoring to the outer cell membrane. These results suggest that the observed IBP activity of Nostoc sp. HG1 was due to a combination of endogenous and exogenous IBPs. Amino acid and nucleotide sequence analyses of nIBP raise the possibility that it was acquired from a planctomycete.IMPORTANCE The horizontal transfer of genes encoding ice-binding proteins (IBPs), proteins that confer freeze-thaw tolerance, has allowed many microorganisms to expand their ranges into polar regions. One group of microorganisms for which nothing is known about its IBPs is cyanobacteria. In this study, we identified a cyanobacterial IBP and showed that it was likely acquired from another bacterium, probably a planctomycete. We also showed that a consortium of IBP-producing bacteria living with the Nostoc contribute to its IBP activity.

Highlights

Ice-binding proteins (IBPs) have been identified in numerous polar algae and bacteria, but so far not in any cyanobacteria, despite the abundance of cyanobacteria in polar regions
Unlike the antifreeze proteins (AFPs) of fish and insects that are present in milligram-per-milliliter concentrations and that lower the freezing point [3], microorganismal ice-binding proteins (IBPs) are present at the microgram-per-milliliter level and have a negligible effect on the freezing point
A search of the Nostoc metagenome for bacterial 16S rRNA sequences yielded 35,791 reads with E values of Ͻ1eϪ30 that could be assigned to 359 genera representing 458 named bacterial species

Summary

Introduction

Ice-binding proteins (IBPs) have been identified in numerous polar algae and bacteria, but so far not in any cyanobacteria, despite the abundance of cyanobacteria in polar regions. NIBP is unusual in that it is the only IBP identified so far to have a PEP (amino acid motif) C-terminal signal, a signal that has been associated with anchoring to the outer cell membrane These results suggest that the observed IBP activity of Nostoc sp. Unlike the antifreeze proteins (AFPs) of fish and insects that are present in milligram-per-milliliter concentrations and that lower the freezing point [3], microorganismal IBPs are present at the microgram-per-milliliter level and have a negligible effect on the freezing point They have other effects on ice that are clearly important to microorganisms. To identify the source of activity, we sequenced the genomic DNA of an IBP-active sample of Nostoc and its associated microorganisms and attempted to identify IBPs by looking for known IBPs in the metagenome and by tandem mass spectrometry (MS/MS) proteomics analysis of ice-affinity purified proteins. Many other DUF3494-type IBPs from a consortium of surface bacteria appear to contribute to its IBP activity

Methods

Results

Conclusion