Isolation and characterization of peptide antibiotics LI-F04 and polymyxin B6 produced by Paenibacillus polymyxa strain JSa-9

Article abstract of DOI:10.1016/j.peptides.2011.08.004

Paenibacillus polymyxa JSa-9 had been found to produce five cyclic LI-F type antibiotics which were released into culture medium in accordance with our previous report. In this study, another three kinds of antagonistic compounds were extracted from P. polymyxa JSa-9 cell pellets and (or) spores by methanol. Using high performance liquid chromatography (HPLC) method, two antagonistic fractions were separated and collected from the methanol extract. One showed inhibition against Escherichia coli and Staphylococcus aureus, while the other was active against Aspergillus niger and S. aureus. By means of electrospray ionization mass spectroscopy (ESI-MS), infrared spectroscopy (IR), and amino acid analysis, two kinds of compounds from fraction B with molecular masses of 901 and 915 Da were characterized as the linear lipopeptide analogs of antibiotics LI-F04a and LI-F04b, respectively. Another antimicrobial substance from fraction A could be attributed to polymyxin B₆.

Full text of DOI:10.1016/j.peptides.2011.08.004

Peptides 32 (2011) 1917–1923
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Peptides
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Isolation and characterization of peptide antibiotics LI-F04 and polymyxin B6
produced by Paenibacillus polymyxa strain JSa-9
Yang Deng, Zhaoxin Lu, Hua Bi, Fengxia Lu, Chong Zhang, Xiaomei Bie∗
College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China,
1 Weigang, Nanjing 210095, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 June 2011
Received in revised form 2 August 2011
Accepted 2 August 2011
Available online 12 August 2011
Paenibacillus polymyxa JSa-9 had been found to produce five cyclic LI-F type antibiotics which were
released into culture medium in accordance with our previous report. In this study, another three kinds of
antagonistic compounds were extracted from P. polymyxa JSa-9 cell pellets and (or) spores by methanol.
Using high performance liquid chromatography (HPLC) method, two antagonistic fractions were sep-
arated and collected from the methanol extract. One showed inhibition against Escherichia coli and
Staphylococcus aureus, while the other was active against Aspergillus niger and S. aureus. By means of
electrospray ionization mass spectroscopy (ESI-MS), infrared spectroscopy (IR), and amino acid analysis,
two kinds of compounds from fraction B with molecular masses of 901 and 915 Da were characterized
as the linear lipopeptide analogs of antibiotics LI-F04a and LI-F04b, respectively. Another antimicrobial
substance from fraction A could be attributed to polymyxin B6.
Keywords:
Paenibacillus polymyxa
LI-F04
Polymyxin B6
Identification
Antimicrobial activity
© 2011 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Nowadays, antibiotic-resistant bacteria are continuously
emerging. Consequently, people have to develop the engineered
drugs or screen for the novel natural antibiotics from new microbial
sources for the pharmaceutical industry [22].
2.1. Microorganisms
P. polymyxa JSa-9 (CGMCC accession no: 4314; China General
Microbiology Culture Collection Center) was isolated and charac-
terized as described elsewhere [3].
All 11 indicator strains used in the study (Table 1) were supplied
by the China Committee for Culture Collection of Microorganisms.
The bacteria were incubated at 37 ◦C and maintained on LB agar for
24 h. The fungi were grown on potato dextrose agar (PDA) at 28 ◦C
for 3–7 days.
Paenibacillus polymyxa (formerly Bacillus polymyxa; reclassified
by Ash et al. [1]) is a spore-forming Gram-positive bacterium that
has been frequently isolated from rhizosphere soils. Strains of the
species are capable of suppressing plant diseases and promot-
ing plant growth [19]. P. polymyxa strains have long been known
for their ability to produce peptide antibiotics. One kind includes
polymyxins, polypeptins, gavaserin, saltavalin, and jolipeptin,
being active against both Gram-positive and Gram-negative bacte-
ria [5,7,8,17,20], and another kind named as LI-F antibiotics (LI-F03,
LI-F04, LI-F05, LI-F06, LI-F07, and LI-F08) or gatavalin and fusari-
cidins A, B, C, and D [2,9–13,15] showing inhibitory effects against
fungi and Gram-positive bacteria.
P. polymyxa strain JSa-9, which was previously isolated from
the soil and characterized in our laboratory, could produce a
high-molecular-weight glycoprotein and LI-F antibiotics with a
broad-spectrum antibacterial and antifungal activities [3,4]. In this
paper, further study is made to identify another three antimicrobial
compounds from the same strain JSa-9.
2.2. Extraction and purification of antimicrobial compounds
Strain JSa-9 was inoculated in a 250 ml flask containing 50 ml of
LB medium, and cultivated at 30 ◦C at 180 rpm for 24 h as a pre-
culture. 50 ml of pre-culture was inoculated in 500 ml of Landy
medium [14] at 30 ◦C in a rotary shaker at 150 rpm for 3 days.
After centrifugation of the culture at 12,000 × g for 20 min, the cell
pellets were resuspended in methanol (1 ml l−1 of culture), placed
at 4 ◦C overnight and then centrifuged as above. The supernatant,
herein referred to as the antimicrobial crude extract, was further
detected the antimicrobial activity by the agar-well diffusion assay
[21].
The crude methanol extract was fractioned by reversed phase
high performance liquid chromatography (RP-HPLC) using Ailent
1100 series C18 column (250 mm × 4.6 mm, USA) as previously
∗
Corresponding author. Tel.: +86 25 84396583; fax: +86 25 84396431.
E-mail address: bxm43@njau.edu.cn (X. Bie).
0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2011.08.004

1918
Y. Deng et al. / Peptides 32 (2011) 1917–1923
Table 1
Fourier transform infrared (FTIR) spectrophotometer (Thermo Fis-
cher Scientific, Waltham, MA, USA). To determine the amino acid
composition, the sample was hydrolyzed with 6 mol l−1 HCl at
110 ◦C for 24 h, and was analyzed by a Hitach L-8800 amino acid
analyzer (Japan).
Minimum inhibitory concentrations (MIC) for the HPLC-purified fractions from
Paenibacilus polymyxa strain JSa-9 and polymyxin B.
Indicator strain
MIC (␮g ml−1
)
Fraction A
Fraction B
Polymyxin B
Gram-positive bacteria
2.5. Minimum inhibitory concentrations
Staphylococcus aureus AS1.2465
Micrococcus luteus AS1.191
Bacillus cereus AS1.1846
Paenibacillus polymyxa JSa-9
Gram-negative bacteria
>128
128
128
4
16
16
>128
64
128
>128
Both P. polymyxa JSa-9 peaks with antimicrobial activity from
HPLC purification of 1 l Landy media culture were combined. For
analysis of the P. polymyxa JSa-9 antimicrobials and polymyxin
B (Amresco, US), minimum inhibitory concentration (MIC) deter-
minations were performed according to the National Committee
for Clinical Laboratory Standards (NCCLS) (NCCLS 2003). The com-
mercial polymyxin B sulfate was used as a control. The final
concentrations of the antibiotic samples in the wells ranged from
0.25 to 128 ␮g ml−1. MIC analysis was performed in triplicate and
results did not vary.
>128
>128
Escherichia coli AS1.487
8
8
16
64
>128
>128
2
4
4
Pseudomonas fluorescens AS1.1802
Pseudomonas aeruginosa AS1.2620
Fungal
Aspergillus niger AS3.350
Aspergillus oryzae AS3.951
Penicillium notatum AS3.4356
Penicillium expansum AS3.4042
Rhizopus stolonifer AS 3.2336
128
>128
>128
>128
>128
8
16
32
32
32
64
128
>128
>128
32
MIC was performed in triplicate, and identical values were obtained each time.
3. Results
described [4]. Elution was monitored by UV detection at 210 nm,
and all isolatable fractions were collected manually for the antimi-
crobial activity assay by the method described earlier. The fractions
with antimicrobial activity were vacuum evaporated to dryness.
3.1. Extraction and purification of antimicrobial compounds
P. polymyxa JSa-9 cultures were separated into cell pellets and
supernatant by centrifugation, and the cell pellets was extracted
using methanol. The inhibition assay indicated that the cell extract
exhibited antagonistic effects on all 11 pathogenic microorganisms
tested, especially Staphylococcus aureus AS1.2465, Escherichia coli
AS1.487 and Aspergillus niger AS3.350, whereas no activity was
observed against the producer strain (P. polymyxa JSa-9) (Fig. 1).
Besides, the methanol (solvent only) used as a control had no
activity against all the indicators (data not shown). This implies
that the antimicrobial substance is tightly associated with bacte-
rial cells or cell wall-bound polymer. Then the methanol extract of
cell pellets was further purified through HPLC method, two major
fractions were obtained in the elution time period of 25–28 min
(Fig. 2). Of the two fractions in the chromatogram, the earlier
2.3. Mass spectroscopy
The active HPLC fractions (fractions A and B) were respectively
analyzed by electrospray ionization/collision-induced dissociation
mass spectrometry (ESI/CID-MS) using the method as previously
described [4]. Data acquisition was in the positive mode.
2.4. Infrared spectroscopy and amino acid analysis
The infrared spectrum was measured as a potassium bromide
pellet. Four scans of the sample were taken using a Mattson
Fig. 1. Inhibition of Staphylococcus aureus AS1.2465 (a), Micrococcus luteus AS1.191 (b), Bacillus cereus AS1.1846 (c), Paenibacillus polymyxa JSa-9 (d), Escherichia coli AS1.487
(e), Pseudomonas fluorescens AS1.1802 (f), Pseudomonas aeruginosa AS1.2620 (g), Aspergillus niger AS3.350 (h), Aspergillus oryzae AS3.951 (i), Penicillium notatum AS3.4356 (j),
Penicillium expansum AS3.4042 (k) and Rhizopus stolonifer AS 3.2336 (l) by the antimicrobial crude extract of P. polymyxa strain JSa-9 cells. (1–3): 60 ␮l of the antimicrobial
crude extract.

Y. Deng et al. / Peptides 32 (2011) 1917–1923
1919
Fig. 2. C18 RP-HPLC analysis of the antimicrobial crude extract of P. polymyxa JSa-9 cells (HPLC type: Agilent 1100 (USA), chromatography C18 RP-column, 250 mm × 4.6 mm;
detected at 210 nm). Mass spectrometry analysis showed that the fraction A had a molecular weight of 1219.72 Da (M+H)+ while fraction B had molecular weights of 901.55
and 915.51 Da (M+H)+.
fraction A with retention time at 25.8 min exhibited inhibitory
activity against E. coli and S. aureus, but there was no activity against
A. niger, while the later fraction B at retention time of approxi-
mately 27 min was only active against S. aureus and A. niger (data
not shown). Therefore, these two fractions were separately col-
lected using the same HPLC program, and prepared for structural
identification.
in m/z 915.51 were revealed. They were found to be structurally
related to the members of the LI-F family: LI-F04a and LI-F04b by
comparing their partial amino acid sequences with those from the
literature [12,13]. Fig. 4a shows the CID spectrum of m/z 901.55,
in which a product ion at m/z 256.31 as well as a product ion at
646.19 (the difference between the molecular ion of m/z 901.55
and the m/z 256.31 fragment) were observed. The ion at m/z
256.31 was interpreted to represent the side chain (15-guanidino-
3-hydroxypentadecanoic acid, GHPD) that was easily fragmented
from the peptide, and m/z 646.19 comprised the remaining struc-
ture of the peptide. The fragment ion at m/z 830.44 was inferred
as the molecular ion with loss of terminal moieties of the side
chain. Product ions and fragmentation patterns similar to those
described above were also observed in MS/MS analysis of m/z
915.51 (Fig. 4b). These results were consistent with those obtained
by previous MS studies of ring-opened LI-F04 peptides which were
prepared using alkaline hydrolysis [12]. Furthermore, amino acid
analysis of the mixture from fraction B showed the presence of
Thr, Ala, Val, Asp/Asn, and Glu/Gln in a molar ratio of 4:2:4:1:1,
which verified the tentative amino acid sequences from MS results.
The two components shared identical the fatty acid residue and
amino acid sequences and differed by a molecular mass 18 Da dif-
ference in molecular weight with LI-F04a and LI-F04b peptides,
suggesting that they were probably linear LI-F04a and LI-F04b,
respectively.
3.2. Identification of antimicrobial compounds
ESI-MS analysis of fraction A from the final HPLC purification
revealed a molecular weight of m/z 1219.72 (Fig. 2), which was
suspected to be polymyxin B6 (1218 Da) [16]. To confirm its struc-
ture, tandem mass spectrometry (MS/MS) was employed on the
[M+H]+ ion (m/z 1219.72). The result given in Fig. 3a exhibited
that the sequence of the antimicrobial peptide could be attributed
to Dab–Thr–Dab–Dab–Thr–Dab–Dab, in which Dab represent 2,4-
diaminobutyric acid. The product ion m/z 257.62 corresponding
to fatty acyl–Dab+ indicated that the elemental composition of
the fatty acyl completely matched C9H17O2 of polymyxin B6,
While another ion at m/z 261.58 might be well in agreement with
Phe–Leu/Ile in the ring (Fig. 3b). These fragmentation patterns were
similar to that obtained by previous MS studies [6]. In addition,
amino acid analysis suggested that the compound had the same
amino acid composition as polymyxin B6: Dab, Thr, Phe, and Leu/Ile
in a molar ratio of nearly 6:2:1:1.
The kinds of functional groups and chemical bonds present in
fraction B was then analyzed with FTIR spectroscopy. The infrared
spectrum indicated that the mixture contains peptide bonds. Char-
acteristic bands at 1550 and 1665 cm−1, corresponding to amide II
and amide I, respectively (C O, C N stretching), and at 3390 cm−1
(N H stretching of proteins) were observed (Fig. 5). Addition-
ally, the spectrum revealed bands with recorded wave numbers of
2853–2925 cm−1, characteristic of aliphatic C–H stretching of fatty
Data from ESI-MS analysis showed that [M+H]+ ion peaks were
901.55 and 915.51 corresponding to the retention time of 27 min
(Fig. 2). In order to characterize their primary structures, the molec-
ular ions of m/z 901.55 and 915.51 were chosen as precursor ions for
further CID analysis. As shown in the MS/MS spectra (Fig. 4a and b),
a series of fragment ions were observed and the tentative sequences
Val–Val–Thr–Asn–Ala in m/z 901.55 and Val–Val–Thr–Gln/Lys–Ala

1920
Y. Deng et al. / Peptides 32 (2011) 1917–1923
Fig. 3. (a) CID MS/MS analysis of a precursor ion at m/z 1219.72. m/z values of diagnostic product ions of the first series and the second series are noted with and without
asterisk, respectively. The first fragmentation series begins when the terminal fatty acid moiety (3-hydroxy-6-methyloctanoic acid) and 2,4-diaminobutyric acid (Dab) are
lost, resulting in m/z 963.31 ion; fragmentation then continues sequentially. The second series begins with fragmentation on the cyclic peptide portion of the molecule with
loss of Dab–Phe–Leu, followed by sequential fragmentation. Mass spectrometry was done in positive ion mode. (b) Two proposed mechanisms of ESI/CID fragmentation for
polymyxin B6.

Y. Deng et al. / Peptides 32 (2011) 1917–1923
1921
acids (Fig. 5). However, comparison of the results with the report
of Kurusu and Ohba [13] revealed that there was not a absorption
band at about 1750 cm−1 to show the presence of a lactone ring in
the two compounds. These data illustrated that fraction B appeared
to be a mixture of two linear peptides. From the above analysis,
the two antimicrobial compounds were identified as the linear
lipopeptide analogs of antibiotic LI-F04a and LI-F04b, respectively
(Fig. 4c).
In this paper, three antibiotics were isolated from P. polymyxa
JSa-9 cells, and purified by HPLC. Mass spectrum and amino acid
composition of the peptide present in fraction A was identical
to that of polymyxin B6. Polymyxin B6 is a cyclic polypep-
tide which was isolated and characterized for the first time by
Orwa et al. [16] It has the same composition as polymyxin B1
except that the fatty acid moiety is 3-hydroxy-6-methyloctanoic
acid.
Initial MS analysis of fraction B indicated the presence of two
molecules which had a 14 Da difference in molecular weight each
other. On the basis of further structural elucidation, they differed
only in a single amino acid residue (Asn/Gln) at position 2 thus giv-
ing two masses of 901 and 915 Da (Fig. 4c). The molecular weights of
these two compounds were respectively 18 Da more than those of
LI-F04a and LI-F04b, however their amino acid sequences and lipid
moieties were identical. A reasonable explanation for this is that
the two compounds are ring-opened peptides of LI-F04. Analysis of
FTIR spectrum confirmed this assumption. LI-Fs are peptide antibi-
otics that have been isolated from Paenibacillus sp., which have a
ring structure composed of six amino acids in addition to GHPD
[13]. It was found that the linear peptides of LI-F04 produced by
JSa-9 strongly inhibit a wide range of plant pathogenic fungi such
as A. niger and Gram-positive bacteria such as S. aureus in this work.
Because of the excellent activity, these antimicrobial compounds
produced by P. polymyxa JSa-9 exhibit a potential application in
biological controls and pharmaceutics.
3.3. Assay for antimicrobial activity
The MICs of HPLC-purified fractions A and B were measured
using microdilution methods against a number of fungi, Gram-
positive and Gram-negative bacteria (Table 1). Of the several
indicator organisms, fraction A only exhibited strong antagonis-
tic activity toward Gram-negative bacteria, which was similar to
polymyxin B. However, fraction B had low MIC values against
the Gram-positive bacteria and fungi tested, especially S. aureus
AS1.2465 and A. niger AS3.350 showing relatively high sensitivity
with MICs of 4 and 8 ␮g ml−1, respectively.
4. Discussion
Tandem mass spectrometry has been used extensively for elu-
cidating primary structures of peptides and proteins, and requires
samples in only small quantities. The infrared spectroscopy and
amino acid analysis can offer additional information about the
structural features of the peptides.
Rautenbach et al. [18] reported that the structure and stability
of a linear analog of iturins by ESI/CID-MS. To our knowledge, we
Fig. 4. CID MS/MS analysis when using m/z 901.55 (a) and 915.51 (b) as precursor ion, respectively. Mass spectrometry was done in positive ion mode. (c) Primary structures
of the LI-F04a and LI-F04b from Paenibacillus sp. and the proposed molecular structures of these two antimicrobials (m/z 901.55 and 915.51) from P. polymyxa JSa-9 cells.
GHPD indicates 15-guanidino-3-hydroxypentadecanoic acid.

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Y. Deng et al. / Peptides 32 (2011) 1917–1923
.
Fig. 4. (Continued ).
firstly identify structure-linear LI-F peptide from a natural bacterial
isolate. Besides, P. polymyxa strain JSa-9 showing inhibitory effects
on many pathogens in vitro had been found to produce more than
one antagonistic metabolites in the cell-free culture supernatant
according to our previous reports [3,4]. Up to now, P. polymyxa JSa-9
has been to found to produce polymyxin B6 and a series of LI-F type
antibiotics including five cyclic and two linear LI-Fs, a phenomenon
that has never been reported earlier.

Y. Deng et al. / Peptides 32 (2011) 1917–1923
1923
Fig. 5. Fourier transform infrared (FTIR) spectroscopy of the mixture of m/z 901.55 and 915.51.
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