at 158.8 ppm, and the three additional nitrogens predicted
by HRFABMS (Table 1) to be present in the final structure.
genized with the genome priming system (GPS, New
England Biolabs) and then retransformed into E. coli.
Mutagenized cosmids that no longer conferred the pro-
duction of antibiosis to E. coli were recovered, and the
DNA flanking each transposon insertion was sequenced
using transposon specific primers. Both eDNA clones were
found to contain single open reading frames (ORF) that
appeared to confer the production of antibacterial activity
to E. coli. Neither the proposed long-chain N-acyltryptophan
synthase from CSL1 (NasW-NAS tryptophan) nor the
proposed long-chain N-acylarginine synthase from CSL11
(NasR-NAS arginine) showed any significant sequence
identity (>20%) to sequences from cultured bacteria that
have been deposited in GenBank or the long-chain N-
acyltyrosine synthases (NasY-NAS tyrosine) found in other
eDNA clones.15
Table 1. High-Resolution FABMS Data for the Major N-Acyl
Amino Acids Present in Ethyl Acetate Extracts of E. coli
Transformed with NASs from CSL1 (NasW), CSL11 (NasR),
and CSL12 (NasY)
(m/z) [M + H]+
eDNA clone
compounda
molecular
formula
calcd
found
CSL1
Trp-C16:1
Trp-C16
Trp-C18:1
CSL11
C
C
C
27H40N2O3
27H42N2O3
29H44N2O3
441.3117
443.3274
469.3430
441.3119
443.3276
469.3430
Arg-C14:1
Arg-C14
Arg-C15
Arg-C16:1
Arg-C16
CSL12
C20H38N4O3
C20H43N4O3
C21H42N4O3
C22H42N4O3
C22H44N4O3
383.3022
385.3179
399.3335
411.3335
413.3492
383.3020
385.3177
399.3334
411.3335
413.3494
To confirm that the two proposed NASs were neces-
sary and sufficient to confer the production of long-chain
N-acyl amino acids to E. coli, each ORF was subcloned
as a GST fusion protein, and these constructs were then
assayed for the ability to confer antibiosis to E. coli.
NasW and NasR were amplified from the cloned eDNA
using the polymerase chain reaction and cloned as GST
fusion proteins in pGEX3X (Pharmacia Biotech) to give
pNasWGST and pNasRGST, respectively.16 E. coli cultures
transformed with pNasWGST and pNasRGST were grown
at 37 °C to an OD600 of 0.7 at which point the temperature
was reduced to 20 °C and the cultures induced with 10 µM
IPTG. After an additional 18 h at 20 °C, the entire culture
was extracted with ethyl acetate. FABMS, bioautography,
and NMR analysis of these extracts confirmed that the
predicted long-chain N-acyl amino acid synthases NasR and
NasW are necessary and sufficient to confer the production
of long-chain N-acylarginines and N-acyltryptophans to E.
coli, respectively.
Tyr-C14:1
Tyr-C14
Tyr-C16:1
Tyr-C16
Tyr-C18:1
C
C
C
C
C
23H35NO4
23H37NO4
25H39NO4
25H41NO4
27H43NO4
390.2644
392.2801
418.2957
420.3114
446.3270
390.2645
392.2802
418.2956
420.3114
446.3271
a Compounds are listed as amino acid-fatty acid side chain.
Although the presence of the long-chain fatty acid side
chain was obvious from the methyl triplet and the large
methylene envelope seen in the 1H spectrum, the individual
fatty acids present in each extract could not be resolved by
NMR. The major long-chain N-acyl metabolites present in
each extract were therefore purified by reversed-phase HPLC
from the active mixture (acetonitrile and water with 0.1%
triethylamine, gradient, Supelco ODP50) and then character-
ized using high-resolution FABMS (Table 1). On the basis
of the molecular formulas predicted by HRFABMS, CSL1
produces long-chain N-acyltryptophans modified with both
saturated and monounsaturated fatty acids that range from
16 to 18 carbons in length, and CSL11 produces long-chain
N-acylarginines modified with both saturated and monoun-
saturated fatty acids that range from 14 to 16 carbons in
length. Synthetic samples of N-palmitoleoyl-L-tryptophan and
N-palmitoleoyl-L-arginine that were produced from the
appropriate L-amino acids and the long-chain fatty acid
chloride13 are spectroscopically identical to the natural
samples and, as seen with the natural samples, antibacterially
active.14
Long-chain N-acyl amino acid-producing clones have been
found in every eDNA library that we have examined to date.
The biosynthesis of long-chain N-acyl amino acids appears
to be a common phenomenon among uncultured bacteria
whose DNA can be readily expressed in E. coli. The
frequency with which this family of compounds is found
(14) In disc diffusion assays against Staphylococus aureus, 50 µg of
Trp-C16:1 produced an 8 mm clear (14 mm hazy) zone of growth, and 5
µg of the ampicillin control produced a 14 mm clear zone of growth
inhibition. Arg-C16:1 is only weakly active as an antibiotic against S. aureus.
Growth inhibition against S. aureus was only observed when the compound
was directly applied to the surface of a bacterial lawn. N-Palmiteoyl-L-
tryptophan [a]25D +10.8 (c 0.79, methanol). N-Palmiteoyl-L-arginine +23.6
(c 0.50, methanol/CH2Cl2 (1:1)).
(15) Sequence data for NasW and NasR have been deposited in GenBank
under accession numbers AY214919 and AY214920, respectively.
(16) NasW and NasR were amplified from the cloned eDNA using the
polymerase chain reaction and the following primer pairs: NasW 5’-
CGTGGGATCCCCATGTTGATGGGCGATGAAGGGCA-3’ and 5’-GCT-
CAATTGGCGGGATTGCTTGGCTTTGAAGCTGA-3’ (BamH I and Mfe
I), NasR 5’-GGAGGGGATCCTCATGCAGCCAGAGATCTTCGCGC-3’
and 5’-ATCGAATTCCTGGTCTCAGTCGCTCACATCTC-3’ (BamH I and
EcoR I). The gel-purified PCR products were digested with the appropriate
restriction endonucleases (as indicated above) and ligated into BamH I-
and EcoR I-digested pGEX-3X vector to give plasmids pNasWGST and
pNasRGST.
To identify the genes responsible for the biosynthesis of
each family of N-acyl amino acids, the eDNA cosmids
isolated from CSL1 and CSL11 were transposon muta-
(13) A 2-fold molar excess of each amino acid was stirred with 40 mg
of palmitoleoyl chloride in 2 mL of 1:1 CH2Cl2/DMF with 100 µL of
pyridine. After 48 h, the reactions were diluted with 10 mL of 1 N HCl
and then extracted two times with 10 mL of CH2Cl2. The CH2Cl2 was
removed under vacuum, and the synthetic long-chain N-acyl amino acids
were then purified using silica gel chromatography (CHCl3/MeOH + 0.1%
HOAc, step gradient).
Org. Lett., Vol. 7, No. 17, 2005
3615