Zetaproteobacteria Pan-Genome Reveals Candidate Gene Cluster for Twisted Stalk Biosynthesis and Export

Elif Koeksoy,Clara S Chan,Timm Bayer,David Emerson,Oliver M Bezuidt

doi:10.3389/fmicb.2021.679409

Abstract

Twisted stalks are morphologically unique bacterial extracellular organo-metallic structures containing Fe(III) oxyhydroxides that are produced by microaerophilic Fe(II)-oxidizers belonging to the Betaproteobacteria and Zetaproteobacteria. Understanding the underlying genetic and physiological mechanisms of stalk formation is of great interest based on their potential as novel biogenic nanomaterials and their relevance as putative biomarkers for microbial Fe(II) oxidation on ancient Earth. Despite the recognition of these special biominerals for over 150 years, the genetic foundation for the stalk phenotype has remained unresolved. Here we present a candidate gene cluster for the biosynthesis and secretion of the stalk organic matrix that we identified with a trait-based analyses of a pan-genome comprising 16 Zetaproteobacteria isolate genomes. The “stalk formation in Zetaproteobacteria” (sfz) cluster comprises six genes (sfz1-sfz6), of which sfz1 and sfz2 were predicted with functions in exopolysaccharide synthesis, regulation, and export, sfz4 and sfz6 with functions in cell wall synthesis manipulation and carbohydrate hydrolysis, and sfz3 and sfz5 with unknown functions. The stalk-forming Betaproteobacteria Ferriphaselus R-1 and OYT-1, as well as dread-forming Zetaproteobacteria Mariprofundus aestuarium CP-5 and Mariprofundus ferrinatatus CP-8 contain distant sfz gene homologs, whereas stalk-less Zetaproteobacteria and Betaproteobacteria lack the entire gene cluster. Our pan-genome analysis further revealed a significant enrichment of clusters of orthologous groups (COGs) across all Zetaproteobacteria isolate genomes that are associated with the regulation of a switch between sessile and motile growth controlled by the intracellular signaling molecule c-di-GMP. Potential interactions between stalk-former unique transcription factor genes, sfz genes, and c-di-GMP point toward a c-di-GMP regulated surface attachment function of stalks during sessile growth.

Highlights

Twisted stalks are organo-mineral composites that were first recognized in the early nineteenth century for their distinctive ribbon-shaped morphology (Ehrenberg, 1836)
We identified the degree of genomic similarity between all Zetaproteobacteria and Betaproteobacteria genomes included in our study by calculating their average amino acid identity (AAI) using CompareM2 with default parameters of 30% sequence identity and 70% sequence alignment length
Accessory and strain-specific genes made up the majority of the Zetaproteobacteria pan-genome, whereas the core genome was relatively small with only 0.04–10.3% of the total gene count (90–30% blastp identity, respectively, Supplementary Table 1)

Summary

Introduction

Twisted stalks are organo-mineral composites that were first recognized in the early nineteenth century for their distinctive ribbon-shaped morphology (Ehrenberg, 1836). The full metabolic potential of the Zetaproteobacteria is yet to be uncovered (Field et al, 2015; Brooks and Field, 2020) and so are the evolutionary grounds for the rise of the stalk formation phenotype in two distinct Proteobacteria classes Their requirement for O2 as terminal electron acceptor limits microaerophilic Fe(II)-oxidizers to low-O2 environmental niches (typically < 50 μM) where they can outcompete the kinetics of chemical Fe(II) oxidation with ambient O2 (Rentz et al, 2007; Druschel et al, 2008; McAllister et al, 2019). The source environments of neutrophilic Fe(II)-oxidizing Betaproteobacteria were primarily reported as ferruginous groundwater discharge points (Emerson et al, 2013; Kato et al, 2015), while metagenomic data indicates their presence in marine environments such as the Arctic MidOcean Ridge, where they co-occur with Zetaproteobacteria (Vander Roost et al, 2017)

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Results

Conclusion