Pheno- and Genotyping of Hopanoid Production in Acidobacteria.

Jaap S Sinninghe Damsté,Bärbel U Foesel,Laura Villanueva,W Irene C Rijpstra,Svetlana N Dedysh

doi:10.3389/fmicb.2017.00968

Abstract

Hopanoids are pentacyclic triterpenoid lipids synthesized by different bacterial groups. Methylated hopanoids were believed to be exclusively synthesized by cyanobacteria and aerobic methanotrophs until the genes encoding for the methylation at the C-2 and C-3 position (hpnP and hpnR) were found to be widespread in the bacterial domain, invalidating their use as specific biomarkers. These genes have been detected in the genome of the Acidobacterium “Ca. Koribacter versatilis,” but our knowledge of the synthesis of hopanoids and the presence of genes of their biosynthetic pathway in other member of the Acidobacteria is limited. We analyzed 38 different strains of seven Acidobacteria subdivisions (SDs 1, 3, 4, 6, 8, 10, and 23) for the presence of C30 hopenes and C30+ bacteriohopane polyols (BHPs) using the Rohmer reaction. BHPs and/or C30 hopenes were detected in all strains of SD1 and SD3 but not in SD4 (excepting Chloracidobacterium thermophilum), 6, 8, 10, and 23. This is in good agreement with the presence of genes required for hopanoid biosynthesis in the 31 available whole genomes of cultivated Acidobacteria. All genomes encode the enzymes involved in the non-mevalonate pathway ultimately leading to farnesyl diphosphate but only SD1 and 3 Acidobacteria and C. thermophilum encode all three enzymes required for the synthesis of squalene, its cyclization (shc), and addition and modification of the extended side chain (hpnG, hpnH, hpnI, hpnJ, hpnO). In almost all strains, only tetrafunctionalized BHPs were detected; three strains contained variable relative abundances (up to 45%) of pentafunctionalized BHPs. Only “Ca. K. versatilis” contained methylated hopanoids (i.e., 2,3-dimethyl bishomohopanol), although in low (<10%) amounts. These genes are not present in any other Acidobacterium, consistent with the absence of methylated BHPs in the other examined strains. These data are in agreement with the scattered occurrence of methylated BHPs in other bacterial phyla such as the Alpha-, Beta-, and Gammaproteobacteria and the Cyanobacteria, limiting their biomarker potential. Metagenomes of Acidobacteria were also examined for the presence of genes required for hopanoid biosynthesis. The complete pathway for BHP biosynthesis was evident in SD2 Acidobacteria and a group phylogenetically related to SD1 and SD3, in line with the limited occurrence of BHPs in acidobacterial cultures.

Highlights

Hopanoids are omnipresent natural products occurring in many groups of bacteria and some higher plants
Thirty-eight Acidobacteria strains distributed over seven subdivisions (SDs 1, 3, 4, 6, 8, 10, and 23) were analyzed for the presence of C30 hopenes and BHPs, and for the presence of methylated hopanoids
Since BHPs often reside in the membrane, where they act as rigidifiers (e.g., Rezanka et al, 2010), we suspected that the classical way of BHP analysis by extraction might miss a substantial fraction of BHPs in Acidobacteria

Summary

Introduction

Hopanoids are omnipresent natural products occurring in many groups of bacteria and some higher plants. The abundance of 2-methyl extended hopanes relative to their non-methylated counterparts was used to conclude that nitrogen-fixing cyanobacteria played a key role in the deposition of black shales during the early Aptian and late Cenomanian oceanic anoxic events (Kuypers et al, 2004) Such studies strongly rely on the consistency of the link between cyanobacteria and their unique ability to produce 2-methyl BHPs. Such studies strongly rely on the consistency of the link between cyanobacteria and their unique ability to produce 2-methyl BHPs This link was already somewhat questionable since non-extended 2-methyl hopanoids (i.e., 2ß-methyldiplopterol and 2ß-methyldiploptene) were reported in species falling in the group of the Alphaproteobacteria (Zundel and Rohmer, 1985; Knani et al, 1994; Vilcheze et al, 1994; Bravo et al, 2001). This revealed that the potential origins of sedimentary extended 2-methyl hopanoids are more diverse than previously thought

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Conclusion