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Gram-negative bacterial 3-hydroxy fatty acids (3-OH-FAs) 20 biomarkers are widespread in a variety of environments including both marine 21 and terrestrial sediments (including speleothems). In this study we analysed 22 the hydroxylated membrane lipids of 26 soil samples from an altitudinal 23 transect of Shennongjia Mountain (Mt.) in central China to study the 24 environmental factors controlling the relative distribution of 3-OH-FAs. Our 25 results show that both the ratio of the summed iso and anteiso to the total 26 amount of normal 3-OH-FAs (RIAN), and the ratio of summed iso and anteiso 27 to the total amount of all 3-OH-FAs (Branched Index) were primarily related to 28 the pH of soil (R 2 = 0.70 and 0.70, respectively). Additionally, the anteiso to 29 normal 3-hydroxy fatty acids ratio of the C15 and C17 homologues (RAN15 and 30 RAN17) shows a significant negative correlation with mean annual air 31 temperature (MAAT) (R 2 =0.51 and 0.48, respectively). When comparing the 3-32 OH-FA based indices with established glycerol dialkyl glycerol tetraether 33 (GDGT) based indices from the same soil samples, the RIAN and Branched 34 Index show strong linear correlations with the cyclisation ratio of branched 35 tetraethers (CBT) (R 2 = 0.77 and 0.74, respectively), and the RAN15 and RAN17 36 show negative correlations with the MBT/CBT-MAAT (MBT, methylation index 37 of branched tetraethers) (R 2 = 0.61 and 0.36, respectively). Our new field-based 38 correlations demonstrate the physiological response of Gram-negative bacterial 39 cell membranes to the external environment and suggest that 3-hydroxy fatty 40 3 acids can be applied in palaeoenvironmental studies to estimate past MAAT 41 and soil pH.

A three-stage linear gradient strategy using reverse-phase high-performance liquid chromatography (HPLC) was optimized for rapid, high-quality, and simultaneous purification of the lipopeptide isoforms of iturin, fengycin, and surfactin, which may differ in composition by only a single amino acid and/or the fatty acid residue. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was applied to detect the lipopeptides harvested from each reversed-phase HPLC peak. Amino acid analysis based on phenyl isothiocyanate derivatization was further used for confirmation of the amino acid species and molar ratio in a certain HPLC fraction. By this MALDI-TOF-MS/MS coupled with amino acid analysis, it was revealed that iturin at m/z 1,043 consists of a circular Asn-Tyr-Asn-Gln-Pro-Asn-Ser peptide and C14 β-OH fatty acid. Surfactin homologs from Bacillus subtilis THY-7 at m/z 1,030, 1,044, 1,058, and 1,072 possess a circular Glu-Leu-Leu-Val-Asp-Leu-Leu peptide and the β-OH fatty acid with a different length (C13-C16). Fengycin species at m/z 1,463 and 1,477 are homologs possessing the circular peptide Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Ile linked to a C16 or C17 γ-OH fatty acid, whereas fengycin at m/z 1,505 contains a Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile sequence with a Val instead of Ala at position 6. The method developed in this work provided an efficient approach for characterization of diverse lipopeptide isoforms from the iturin, fengycin, and surfactin families.