Hopanoids in marine cyanobacteria: probing their phylogenetic distribution and biological role

Sáenz, J. P.; Waterbury, J. B.; Eglinton, T. I.; Summons, R. E. (2012) Geobiology, 10(4), 311–319. DOI: 10.1111/j.1472-4669.2012.00318.x

Summary

We surveyed 16 axenic marine cyanobacterial cultures for Bacteriohopanepolyols (BHPs) and investigated hopanoid regulation in [[crocosphaera-watsonii|Crocosphaera watsonii]]. In pure culture, BHPs were detected only in species capable of nitrogen fixation — C. watsonii, Trichodesmium erythraeum, and four strains of Cyanothece — implicating hopanoids as potential markers for diazotrophy in the oceans. The most abundant marine cyanobacteria (Prochlorococcus, Synechococcus) do not produce hopanoids. C. watsonii has high membrane hopanoid content (~5–10% of TLE by mass), but BHP abundance did not vary significantly under nitrogen-limiting versus nitrogen-replete conditions, suggesting that hopanoids are not directly linked to nitrogen fixation physiology. We hypothesized that high hopanoid content in C. watsonii may reduce membrane permeability to lipophilic antimicrobial compounds in the environment, though this remains untested.

Key Findings

• Of 16 axenic marine cyanobacterial strains screened, only 6 produced detectable BHPs — all 6 are capable of nitrogen fixation (C. watsonii, T. erythraeum, 4 Cyanothece strains). No BHPs were detected in Synechococcus, Prochlorococcus, Dermocapsa, or Microcoleus.
• C. watsonii produces BHT-CE as its dominant hopanoid (~99% of total BHP) with trace BHT (~1%). BHT-CE abundance was estimated at 5–10% of TLE by mass.
• BHP cellular abundance in C. watsonii did not vary significantly between nitrogen-replete (SN), nitrogen-free (SO), and ammonium-amended (SNH4) growth conditions, nor between light and dark sampling points — arguing against a direct role in nitrogen fixation.
• Marine cyanobacterial enrichment cultures exhibited higher BHP structural diversity (up to 5 compounds) than pure cultures, indicating uncharacterized marine bacteria contribute diverse BHP structures.
• 2-Methylhopanoids were absent from all marine cyanobacterial pure cultures surveyed, creating a paradox for the interpretation of 2-methylhopanes as cyanobacterial biomarkers in ancient marine sediments.
• The absence of cyanobacterial squalene-hopene cyclase (sqhC) genes from the Global Ocean Sampling (GOS) database may reflect a size bias: GOS screened cells <0.8 μm, while hopanoid-producing C. watsonii and Trichodesmium are >1 μm.

Methods

• Pure cultures obtained from John Waterbury (WHOI), Sallie Chisholm (MIT), and Pasteur Culture Collection. Enrichment cultures prepared from marine samples in Bermuda, Cape Verde, and Falmouth, MA.
• Lipid extraction by modified Bligh and Dyer method; BHP analysis by HPLC-APCI/MSⁿ on a Finnigan Surveyor LC / LTQ-MS.
• Quantification via external standard curve of acetylated BHT with PD internal standard. C. watsonii diel experiments performed in triplicate batch cultures sampled during mid-light and mid-dark periods.

Significance

This paper was the most comprehensive survey of hopanoids in marine cyanobacteria to its date. The exclusive association of BHP production with diazotrophic marine cyanobacteria raised the question of whether hopanoids serve an ecological role related to the physiological demands of nitrogen fixation (e.g., oxygen protection). The lack of nitrogen-dependent regulation, however, led us toward an alternative hypothesis — that hopanoids function as a general permeability barrier — which was later extended in the biophysical work on lipid ordering.