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. 2012 May;78(9):3156-65.
doi: 10.1128/AEM.07782-11. Epub 2012 Feb 24.

Isolation of a Paenibacillus sp. strain and structural elucidation of its broad-spectrum lipopeptide antibiotic

Affiliations

Isolation of a Paenibacillus sp. strain and structural elucidation of its broad-spectrum lipopeptide antibiotic

Yaoqi Guo et al. Appl Environ Microbiol. 2012 May.

Abstract

This research was initiated to search for novel antimicrobial compounds produced by food or environmental microorganisms. A new bacterial strain, designated OSY-SE, which produces a unique and potent antimicrobial agent was isolated from soil. The isolate was identified as a Paenibacillus sp. through cultural, biochemical, and genetic analyses. An antimicrobial compound was extracted from Paenibacillus OSY-SE with acetonitrile and purified using liquid chromatography. After analyses by mass spectrometry (MS) and nuclear magnetic resonance (NMR), the antimicrobial compound was determined to be a cyclic lipopeptide consisting of a C(15) fatty acyl (FA) chain and 13 amino acids. The deduced sequence is FA-Orn-Val-Thr-Orn-Ser-Val-Lys-Ser-Ile-Pro-Val-Lys-Ile. The carboxyl-terminal Ile is connected to Thr by ester linkage. The new compound, designated paenibacterin, showed antagonistic activities against most Gram-positive and Gram-negative bacteria tested, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium. Paenibacterin is resistant to trypsin, lipase, α-glucosidase, and lysozyme. Its antimicrobial activity was lost after digestion by pronase and polymyxin acylase. Paenibacterin is readily soluble in water and fairly stable to exposure to heat and a wide range of pH values. The new isolate and its antimicrobial agent are being investigated for usefulness in food and medical applications.

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Figures

Fig 1
Fig 1
Scanning electron microscope examination of Paenibacillus OSY-SE cells.
Fig 2
Fig 2
MALDI-TOF MS analysis of paenibacterin and its linear form produced by alkaline hydrolysis. (A) Spectrogram of paenibacterin (m/z 1605.14) and its three homologues (m/z 1591.12, 1619.17, and 1633.21). (B) Spectrogram of linearized paenibacterin (m/z 1622.97); the ion at m/z 1644.96 corresponds to a sodium adduct.
Fig 3
Fig 3
NMR analysis of the peptidyl fragment in the amide region. (A) 2D 1H-15N HSQC recorded for the sample in H2O, showing the 12 main-chain NH amide cross-peaks and a cluster of folded peaks (f) attributable to an Arg, Lys, or Orn side chain NH3+ group; (B) 2D 1H NOESY of the same sample, showing the amide region cross-peaks, with assignments.
Fig 4
Fig 4
Elucidation of amino acid sequence and linkage of paenibacterin by HMBC. (A) 2D 1H-13C HSQC recorded for the sample dissolved in CD3OD, showing the 13C resonances in the region between 45 and 75 ppm. The CHα assignments of the 13 amino acids, together with Thr3 CH2β, Ser5 CH2β, Ser8 CH2β, and Pro10 CH2δ assignments, are labeled to assist the analysis of cross-peaks in panel B. The unlabeled methyl group at 3.30/49.1 ppm (1H/13C) is attributed to the residual solvent methanol. (B) 2D 1H-13C HMBC acquired for the same sample, showing the connectives associated with Hα protons. Sequential assignment is traced according to intraresidue Hα(i)-C′(i) and C′(i − 1)-Hα(i) (asterisk) multiple-bond J-coupling connectivities. The stretch starts from the connectivity of fatty acid carbonyl carbon (fat) to Orn1 Hα and ends with Lys12 C′ to Ile13 Hα. Also noted by the broken lines are the long-range J couplings of Thr3 Hβ-Thr3 C′ and Thr3 Hβ-Ile13 C′. The latter is the strong evidence for a cyclic peptide with an ester bond formed between the Thr3 hydroxyl group and the Ile13 C-terminal carboxylic group. It is important to note that the tilted and spit HMBC cross peaks are due to 1H-1H coupling (J modulation) (15).
Fig 5
Fig 5
Tertiary structure of the peptide moiety of paenibacterin calculated from NMR constraints in aqueous solution. The five bulky aliphatic side chains (V2, V6, I9, V11, and I13) are labeled.
Fig 6
Fig 6
Fragmentation of b and y ion series of linearized paenibacterin, examined by MS/MS.
Fig 7
Fig 7
1D 13C NMR spectrum, revealing the iso- and anteiso- fatty acyl chains.
Fig 8
Fig 8
Molecular structure of paenibacterin; R = CH3(CH2)13 (normal chain), CH3CH(CH3)(CH2)11 (iso- chain), or CH3CH2CH(CH3)(CH2)10 (anteiso- chain).

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