Download Two multifunctional peptide synthetases and an 0

Printed in Great Britain
Microbiology (1996), 142,741-746
Two multifunctional peptide synthetases and
an 0-methyltransferaseare involved in the
biosynthesis of the DNA-binding antibiotic and
antitumour agent saframycin M x l from
Myxococcus xanthus
Andreas Pospiech, Jurg Bietenhader and Thomas Schupp
Author for correspondence: Andreas Pospiech. Tel: +41 61 6961746. Fax: +41 61 6966323.
Ciba-Geigy Ltd, Core Drug
Discovery Technologies,
CH-4002 Basel, Switzerland
Saframycin M x l is a DNA-binding antibiotic and antitumour agent produced by
Myxococcus xanthus. It is a heterocyclic quinone, thought to be synthesized via
the linear pepide intermediate AlaGlyTyrTyr. Analysis of 14-1kb DNA sequence
involved in saframycin production revealed genes for two large multifunctional
peptide synthetases of 1770 and 2605 amino acids, respectively, and a putative
0-methyltransferase of 220 amino acids. The three ORFs read in the same
direction and are separated by short non-translated gaps of 4 4 and 49 bp. The
peptide synthetases contain two amino-acid-activating domains each. The first
domain lacks two of the most conserved 'core' sequences, and the last domain
is followed by a putative reductase functionality, not previously seen in
peptide synthetases. Complementation tests showed that antibiotic-nonproducing mutant strains lacking one of the peptide synthetases secrete a
substrate, presumably a modified amino acid precursor, that can be used by
0-methyltransferase-def icient mutant strains to synthesize saframycin M x l
.
Keywords : Myxococcus xanthus, saframycin Mxl , antibiotic, peptide synthetase, 0methy ltransferase
INTRODUCTION
OCH,A
I
The Gram-negative myxobacteria grow in soil and have a
complex life cycle with multicellular differentiation.
Simiar to differentiating actinomycetes and bacilli, they
produce a large number of secondary metabolites
(Reichenbach e t al. , 1988). Myxococczts xantbzts produces
saframycin Mxl (Irschick e t al., l988), a heterocyclic
quinone antibiotic which has antibacterial and antitumour
activity. Other saframycins were isolated from
Streptomyces lavendztlae (Arai et al., 1977, 1980). The
saframycins bind covalently to DNA and inhibit DNA
and RNA synthesis (Ishiguro et al., 1978, 1981 ; Kishi e t
al., 1984; Lown e t al., 1982). The N-heterocyclic quinone
of saframycin Mxl (Fig. 1) is derived from two tyrosine
molecules, and the side chain is derived from glycine and
alanine (Trowitzsch-Kienast e t al., 1988).
ACH,
ACH,O
I
Previously we identified, by T n 5 mutagenesis in M.
The GenBank accession number for the sequence reported in this paper is
U24657
0002-0658 0 1996 SGM
Ala
Fig. 7. Structure o f saframycin Mxl derived from alanine,
glycine and two tyrosines. Filled triangles indicate methylations.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 20:57:33
741
A. POSPIECH, J. B I E T E N H A D E R a n d T. SCHUPP
xantbzu, genes involved in saframycin Mxl biosynthesis.
The corresponding DNA was cloned in Escbericbia coli on
overlapping cosmids. Fragments of the cloned DNA were
used to create saframycin-non-producing derivatives by
gene disruption, confirming the identity of the cloned
genes and localizing them on the cosmids. Sequencing of
part of an open reading frame revealed a putative peptide
synthetase, suggesting that saframycin Mxl may be
derived from a non-ribosomally synthesized peptide
precursor (Pospiech e t a/., 1995). Here we describe a more
detailed analysis of the saframycin Mxl biosynthetic gene
cluster, which possesses some novel features.
METHODS
Strains, plasmids and culture conditions. The bacterial strains
and plasmids used are listed in Table 1. E . coli was grown at
37 "C in Luria broth (LB) or on LB agar. Sarcina lutea was grown
at 30 "C on solid DST medium (Oxoid) or in liquid BHI
medium (BBL). M . xanthus was grown at 28 "C in S45 medium
(Pospiech e t al., 1995), or on solid S46 medium, which is S45
with 12 g agar 1-'. Antibiotics were used at the following
concentrations for E . coli and M. xanthus: 50 pg ampicillin ml-',
50 pg kanamycin ml-' and 34 pg chloramphenicol m1-l.
DNA manipulations. Standard genetic techniques for in vitro
DNA manipulations and cloning were used (Sambrook e t al.,
1989).
PCR amplification. A 519 bp fragment internal to ORF C was
amplified using the primers 5'-ATGAATTCACGTCGAATTGACACAGTCC and 5'-ATGAATTCACAGCGTGTTGTCGAGGATG 3' (the non-homologous tails containing EcoRI
sites are underlined), and pAP93/8 (Pospiech e t al., 1995) as
template. Reaction buffer and Tag polymerase from Boehringer
Mannheim were used. Thirty cycles of 30 s at 96 "C, 1 min at
55 "C, 1 min at 72 "C were performed in a Hybaid thermocycler
(MWG-Biotech).
Gene disruption of M. xanthus DM504/15. pAP107-10 was
introduced into M . xanthus DM504/15 by conjugation from E.
coli. An overnight culture of E. coli S17-1 containing pAP10710, grown under kanamycin and ampicillin selection, was
diluted 100-fold with LB medium without antibiotics and
incubated for a further 6 h. Subsequently 1 x lo1' cells of this E.
coli culture and 5 x lo8 cells of M. xanthzis DM504/15 (grown at
28 OC to a cell density of 2 x lo8 ml-' and preincubated for
10 min at 45 "C) were mixed, sedimented (SOOOg, 3 min),
resuspended in 150 pl S45 medium and spotted onto S46 agar
without antibiotics. After overnight incubation at 28 "C, cells
were scraped off the agar and resuspended in 1 ml S45 medium.
Portions (100 pl) were plated onto S46 plates containing 50 pg
kanamycin ml-' and incubated for 6 d at 28 "C. Kanamycinresistant transconjugants, containing plasmids integrated into
the genome via homologous recombination, were selected.
Biological assay (biotest) for saframycin M x l production. M.
xanthus DM504/15 and its derivatives were grown for 5 d at
28 "C on S46 agar (20 ml per plate) without antibiotics. Agar
plugs (3 mm in diameter) with single colonies were placed onto
DST agar freshly overlaid with 10 ml 45 "C DST agar containing 100 pl of an overnight culture of Sarcina lutea. After
overnight incubation at 30 "C, the inhibition zones of S. lutea
were measured. This biological assay detected b 0.5 ng saframycin Mxl. Colonies of strain DM504/15 produce 10-25 ng
saframycin Mxl and inhibition zones of 10-14 mm diameter;
non-producing mutants did not inhibit the growth of S.htea.
Complementation test. M. xanthus mutant strains were grown
for 3 d at 28 "C in S45 medium without antibiotics. Subse-
Table 1. Bacterial strains and plasmids
Strain or
plasmid
E. coli
XL1-Blue
S17-1
M y xococcus
xanthus
DM504/15
APlO9-2
AP100/1-1
AP100/6-1
Sarcina lutea
ATCC 9341
Plasmids
PAP107-10
pCIBl32*
pBluescript I1 SK'
Characteristicst
Source or reference
recA 1 endA 1 g y r A 9 6 tbi- 1 bsdR 17 supE44 r e l A 1 lac [F'
proA+B+ facIq AfacZM15 Tn 10 (Tc')]
recA pro tbi bsdR chr :: RP4-2
Stratagene Cloning Systems
Saframycin Mxl production strain
DM504/15, safC: :pAP107-10, Saf Km'
DM504/15, s a p ::pAP98-1, Saf Km'
DM504/15, s a f A : :pAP98-17, Saf- Km'
Pospiech e t al. (1995)
This study
Pospiech et al. (1995)
Pospiech e t al. (1995)
Saframycin Mxl sensitive
American Type Culture Collection
0.5 kbp internal PCR amplified fragment of sajC cloned
in the EcoRI site of pCIB132
Amp' Cm' Km' oriT (RK2)
Amp'
This study
Simon e t al. (1986, 1983)
J. M. Ligon, pers. comm.$
Stratagene Cloning Systems
* Derivative of pSUP2021 (Simon et al., 1986) constructed by reversal of the 5 kb Not1 fragment, followed by removal of the resulting 3 kb
BamHI fragment, leaving a unique BamHI cloning site.
t Saf+'-, saframycin productionlnon-production.
$ J. M. Ligon; Ciba-Geigy Corp., PO Box 12257, Research Triangle Park, NC 27709-2257, USA.
742
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 20:57:33
Saframycin Mxl biosynthesis in M . xanthtrs
quently they were mixed (5 pl from each strain), spotted onto
S46 agar and incubated for a further 2 d at 28 "C. Agar plugs
(3 mm in diameter) were then placed onto DST agar freshly
overlaid with 10 ml molten (45 "C) DST agar and 100 p1 of an
overnight culture of S. lutea. After overnight incubation at
30 "C, the inhibition zones of S.Iutea were measured.
HPLC analysis of saframycin Mxl production. S45 medium
(20 ml) was inoculated with 1 ml cells from a -70 "C stock in
20 % (v/v) glycerol and incubated with shaking for 3 d at 28 "C.
Then a second preculture in 20 ml S45 medium was inoculated
with 5 YOof the first culture and incubated with shaking for 3 d
at 28 "C, after which 1.5 ml of the second culture was transferred
to a 200 ml Erlenmeyer flask containing 50 ml Saf3 medium
[containing, per litre: 3 g casein peptone, 32 g dextrin A-332,
2 g MgSO, .7H,O, 0.5 g CaC1,. 2H,O, 4 g sodium citrate, 50 g
absorber resin XAD 1180 (Rohm and Hass), pH 7.21. After 3 d
shaking at 28 "C, 5 ml 5 % (w/v) casitone was added. After
additional incubation for 3 d at 28 "C the absorber resin XAD
1180 was harvested on a polyester sieve (Satorius, B 420-47-N)
and eluted with 20 ml 90% (v/v) 2-propanol by shaking for
30 min in 50 ml Falcon tubes at room temperature. The 2propanol eluate was centifuged for 5 min and the clear
supernatant was analysed by HPLC for its saframycin Mxl
content. For reverse-phase HPLC analysis, the silica-based
stationary phase Lichrospher 60 A RP-18 (5 pm; Merck) was
used in a 12.5 cm column. A potassium phosphate buffer
(2.5 mM, pH 3) acetonitrile gradient was the mobile phase.
Saframycin Mxl was detected by absorption at 260 nm. Yields
for strain DM504/15 were between 18 and 24 mg (1 culture)-'.
The detection limit was 3 mg 1-'.
DNA sequencing. Double-stranded D N A fragments were
cloned into pBluescript I1 SI<' and both strands were examined
by DNA cycle sequencing with dye-labelled terminators (Sanger
et al., 1977) and an Applied Biosystems automated sequencer
(model 373A). Universal primer and oligonucleotide primers
designed according to the newly obtained D N A sequences were
used. The standard cycling conditions were 25 cycles of 96 "C
for 20 s and 60 "C for 240 s. All reactions were performed in a
Perkin-Elmer GeneAmp 9600 PCR system using the AmpliTaq
DNA polymerase from Perkin-Elmer.
Computer analysis of DNA and protein sequences. The D N A
sequences were assembled and analysed using software version
1.20 from Applied Biosystems and the UWGCG program
package version 8.0 (Devereux e t al., 1984).
RESULTS AND DISCUSSION
Sequence of three ORFs involved in saframycin M x l
biosynthesis
The sequence of saframycin biosynthesis genes was
extended in both directions to cover 14.1 kb of DNA with
an overall G C content of 71 mol YO,typical for myxobacteria. Three ORFs with a codon usage typical for highG + C genes were identified using the program
CODONPREFERENCE (Fig. 2). The three ORFs read in the
same direction and are separated by short, presumably
untranslated, regions of 44 and 49 bp (Fig. 2). Each ORF
is preceded by potential ribosome-binding sites upstream
of the ATG start codons (Table 2). Promoter sequences
are not evident from the sequence; no consensus
promotor sequence has yet been established for
M~yxococcz~s.
Two very large ORFs encode the putative
peptide synthetases SafA (2605 amino acids, M,285745)
+
kb
-
AP 100/1-1
1
B
BE
E
4%
AP10016-1
p
&
,
safB
B
B
@
.
APx9-2
p
B
&
,
,
6
@
B
B
+@& +@
safA
safC
Fig. 2. Organization of the saframycin Mxl biosynthesis gene
cluster of M. xanthus DM504/15. Open boxes in ORF A (safA)
and ORF B (safB) show the amino-acid-activating domains.
Exact amino acid positions of domains are indicated by the
numbers above. The black bars indicate DNA fragments whose
integration into the DM504/15 genome abolishes saframycin
M x l production. Filled triangles indicate the positions of Tn5
insertions. The complete sequence is available under GenBank
accession number U24657. Nucleotide positions of the start and
stop codons: safA, 5491-13305; safB, 138-5447; safC,
13354-14013. E, EcoRl; B, BamHI.
and SafB (1770 amino acids, M, 192495) and the
downstream ORF encodes a putative O-methyltransferase, SafC (220 amino acids, M,24673).
Putative peptide synthetases SafA and SafB
Both SafA and SafB contain two domains of approximately 520-600 amino acids resembling amino-acidactivating domains of known peptide synthetases. Highly
conserved 'core' sequences, defined by Stachelhaus &
Marahiel (1995), are clearly recognizable in all four
domains (Fig. 3).
The domain SafBl is unusual in that it lacks core sequence
5 (ATP-binding ; Pavela-Vrancic e t al., 1994a, b ; Tohika
e t al., 1993), has a poorly conserved core sequence 1
(unknown function) and has an unusually large spacing
between the core sequences 1, 2 and 3 (Fig. 3). The core
sequences 2, 3 (ATP-binding ; Gocht & Marahiel, 1994),
4 (ATPase motif; Gocht & Marahiel, 1994) and 6 (4'phosphopantetheine binding, D'Souza e t al., 1993;
Schlumbohm e t al., 1991 ; Vollenbroich e t al., 1993; Stein
e t a/., 1994) are, however, well conserved. Safl3l also
contains a spacer motif, HHIVVDFW (unknown function; aa 838-845), downstream of core sequence 6. Spacer
motifs are present at the ends of amino-acid-activating
domains of the modules initiating peptide synthesis of
gramicidin (GrsA) and tyrocidin (TycA) (Hori e t al.,
1989; Stachelhaus e t al., 1995; Weckermann e t al., 1988).
Therefore, SafB1, the first domain in the sequence,
probably activates alanine, the first amino acid in the
putative saframycin precursor peptide. The poor conservation or lack of core sequences 1 and 5 is unusual for
peptide synthetases, but the related adenylate-forming
enzymes also lack strict conservation of these sequences
(Marahiel, 1992).
Safl32, SafAl and SafA2 contain all six core sequences. A
spacer motif is located upstream of SafAl (aa 165-172),
the normal position for peptide synthetases not initiating
a peptide chain (Stachelhaus e t al., 1995). Downstream of
SafA2, without an intervening spacer sequence, are 370
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 20:57:33
743
'
A. P O S P I E C H , J. B I E T E N H A D E R a n d T. S C H U P P
Table 2. Putative Shine-Dalgarno sequences
* Numbers
ORF
SD
5aP
safA
safC
GAGG
GGAGAAGA
GGAAGA
Spacer
-10-5-5-
Start codon*
Stop codon*
ATG (138)
ATG (5491)
ATG (13354)
TAG (5450)
TGA (13308)
TAG (14016)
in parentheses indicate the first and last nucleotides shown.
core
sequences
LKAQQA
3
SOTTQ
Function
unknown
ATP binding
1
3
4
OELC IGO
TQD
5
RIEXEIB
ATPase
motif
ATP
binding
ATP binding
6
LOOHS
4'phosphopantetheine
binding
SafBl
97
SafB2 1247
SafAl 559
SafA2 1668
TycA
98
GrsA
111
GrsB-A 540
SrfAl 532
Figrn3. Conserved protein sequences in the domains SafB1, SafB2, SafAl and SafA2, and their comparison with
presumably homologous sequences from other organisms. GrsA, gramicidin S synthethase I of Bacillus brevis (Hori e t a/.,
1989); GrsB-A, gramicidin S synthethase II of B. brevis (Turgay e t a/., 1992); TycA, phenylalanine-activating enzyme in
tyrocidine biosynthesis (Weckermann et a/., 1988); SrfA, surfactin synthetase (Cosmina e t a/., 1993). Perfectly conserved
amino acids are printed in bold. The numbers on the left give the positions of the first amino acids shown; numbers
between the amino acids indicate the distances between these sequences. The core sequences were defined by
Stachelhaus & Marahiel (1995).
SafA3 (2216)
HetM
(130)
E q A 2 (3051)
Lys2
(967)
ANYLAPSSLLTGATGYLGAAFLLEQLLKRTRAT
PVTQPKKVFLTGGTGFLGAFLIRELLQQTQADV
YAVPGGTILVTGGTAGLGAEVARWLAGRGAEHL
EGKTTINVFVTGVTGFLGSYILADLLGRSPKNY
Fig. 4. Alignment of a putative NAD(P)H-bindingsite of SafA3
with those from other proteins. HetM, putative polyketide
synthetase from Anabaena sp. (Black & Wolk, 1994); EryAZ, pketoreductase domain of the polyketide synthetase involved in
erythromycin biosynthesis of Saccharopolyspora erythraea
(Donadio & Katz, 1992); Lys2, a-aminoadipate reductase of
Saccharomyces cerevisiae (Morris & links-Robinson, 1991). The
numbers in parentheses give the positions of the first amino
acids shown. Perfectly conserved amino acids are shown in
bold.
amino acids similar to the ketoreductase function of
HetM, a putative polyketide synthase of Anabaenaf sp.
(44% identity; Black & Wolk, 1994) and Lys2 of
SaccharomJyces cereuisiae, which reduces a-aminoadipate to
a-aminoadipic-d-semialdehyde using N ADPH (33 YO
identity ; Morris & Jinks-Robertson, 1991). SafA2, HetM
and Lys2 contain an NAD(P)H binding motif (Fig. 4)
characteristic for diverse reductive enzymes. SafA2 is the
first peptide synthetase amino-acid-activating domain
with a putative reductase function but interestingly, Lys2
contains all six peptide synthetase core sequences in the
correct order upstream of the NAD(P)H-binding motif,
but with a different spacing (Morris & Jinks-Robertson,
1991).
744
SafC
MIHHVELTQSVLQYIRDSSVRDNTlILRDLREETSKLP.LRTMQ1PPEQGQ4 9
MdmC
VADQTTLSPALLDYARSVALREDGLLRELIiDMTAQLPGGRAMQIMPEEAQ50
. . ... . I . . . : / : I I . . : l : : : : l l : [ : : I . . I I
I . I ) ) II::I
S a f C LLSLLVRLI
STLCAALALPADGRVIACDLSEEWVSI 9 9
MdmC
STLCKARALPAGGRIVTCDISDKWPGI 1 0 0
III I I IIll:I l::-ll:l:.l-:l
FLGLLIRL
S a f C ARRYWQRAGVADRIEVRLGDAHHSLEALVGSEHR
:
.:IIIIII.:
~
:
:
: . I~
..[
: . :~
:
~
~
MdmC GAPFWQRAGVDGLIDLRIGDAARTLAELRERDG
S a f C FYY
SGKVADPSWGDPETDSLRRINAKLLT 1 9 9
MdmC HYY
FFGRVADPA.ADDPDTVAVRTLNDLLRD 1 9 9
II
S a f C DERVDLSMLPIADGLTLARKR 220
IIIII:.:I.:III:IIII:I
MdmC DERVDIALLTVADGITLARRR 220
Fig. 5. Alignment of SafC and the 0-methyltransferase MdmC
of Streptomyces mycarofaciens (Hara & Hutchinson, 1992).
Shadowed regions indicate conserved sequence motifs in Sadenosylmethionine-dependent methyltransferases.
The putative 0-methyltransferase SafC
SafC shows 51 Yo identity over its entire length to the 0methyltransferase MdmC of the midecamycin producer
Streptomyes mycarofaciens (Fig. 5) (Hara & Hutchinson,
1992) and 39.5 YO identity to the caffeoyl-CoA-3-0methyltransferase (cCoAMT) of Petrosilinum crispum
(Schmitt e t al., 1991). Three highly conserved motifs
of S-adenosylmethionine-dependentmethyltransferases
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 20:57:33
Saframycin Mxl biosynthesis in M . xanthtcs
Motif I
SafC
cCoAMT
EryG
MdmC
63 TLEVGVFTG
77 TMEIGVYTG
85 VLDVGFGLG
64 VLEIGTFTG
Motif I1
134
149
149
135
Motif I11
GTFDLAFI 158
GTFDFVFV 173
ETFDRVTS 176
GAFDLVFV 159
LVRPGGLIIL
LVKIGGLIGY
VLKPGGVLAI
LVRPGGLVAI
Fig, 6. Conserved sequence motifs I, II and Ill in Sadenosylmethionine-dependent methyltransferases (Kagan &
Clarke, 1994). cCoAMT, caffeoyl-CoA-3-O-methyitransferase o f
Petrosilinum crispum (Schmitt et a/., 1991); EryG, erythromycin
biosynthesis O-methyltransferase o f S. erythraea (Weber et a/.,
1 989); M d mC, midecamycin-O-methy Itra nsferase of Streptomyces mycarofaciens (Hara & Hutchinson, 1992).
(Kagan & Clarke, 1994) are also present in SafC (Fig. 6).
Due to its high degree of similarity to S-adenosyldependent O-methyltransferases, SafC can be grouped in
a class of enzymes catalysing O-methylations of small
molecules (Kagan & Clarke, 1994) and may be responsible
for one, two or all three O-methylation reactions necessary
for the biosynthesis of saframycin Mxl (Fig. 1).
Targeted disruption of the genes encoding SafA, SafB
and SafC
Four amino-acid-activating domains, reductive and
methylase functions are consistent with the structure of
saframycin Mxl. Further confirmation for the involvement of SafA, Sam and SafC in saframycin biosynthesis
was obtained from gene disruption experiments. Three
fragments, internal to the three ORFs and indicated in
Fig. 2, were cloned into the mobilizable vector pCIB132
and introduced by conjugation from E. coli S17-1 into M.
xantbtcs DM504/15, the saframycin producer (Pospiech e t
a/., 1995). The pCIB132 derivatives cannot replicate
autonomously in M. xantbus, but they can integrate into
the chromosome by homologous recombination to give
kanamycin-resistant transcon jugants at a frequency of
about 1O r ' per recipient cell. Saframycin production,
determined using the biotest and HPLC analysis, was
abolished in all tested transconjugants (number of transconjugants in biotest : 10 for s a f A , 36 for s a p , 17 for safC;
number of transcon jugants in HPLC analysis : 3 for s a f A ,
3 for s a p , 6 for safC). These results are consistent with,
but do not prove, involvement of the three safgenes in
antibiotic production.
Cross-feeding experiments
The above s a f A , s a p and safC mutant strains were used
for cross-feeding studies designed to detect the existence
of diffusible saframycin Mxl precursors. Mixed culture of
the s a f A and s a p mutant strains, presumably lacking
peptide synthetase activity, did not result in antibiotic
production. Mixed growth of either of these strains with
the safC mutant strain resulted in a 4 mm inhibition zone,
indicating a low but significant level of saframycin biosynthesis. The DM504/15 control gave an inhibition zone
of 12 mm. When the safC mutant strain was grown near
the s a f A or s a p mutant strain, antibiotic appeared near
the former, indicating that the safC strain converted a
precursor secreted by the peptide-synthetase-deficient
strain. Therefore, the peptide synthetases probably use
methylated amino acid derivatives either as natural
substrates or as alternative substrates.
Conclusions
The two putative peptide synthetases SafB and SafA (Fig.
2) involved in saframycin Mxl biosynthesis contain two
amino-acid-activating domains each, consistent with the
hypothetical linear tetrapeptide structure of the saframycin precursor. The first domain, S a m l , probably
responsible for activating the amino-terminal alanine,
lacks the usually well-conserved peptide synthase core
sequence 1, and the last domain, SafA3, has attached a
putative reductase function, possibly involved in the
modification of the carboxy-terminal tyrosine. Both of
these domains represent new types of amino-acidactivating domains.
The targeted gene disruptions, together with the crossfeeding experiments, proved the involvement of the three
sequenced ORFs in saframycin biosynthesis. The crossfeeding experiment using strains growing close together
rather than mixed indicated that the putative 0methyltransfrerase encoded by safC presumably modifies
tyrosine before it becomes a substrate for the peptide
synthetase. This, of course, has implications for the
possible use of the saframycin peptide synthetase modules
for the construction of recombinant peptide synthetases
for the synthesis of new metabolites as pioneered by
Stachelhaus e t a/. (1995).
ACKNOWLEDGEMENTS
We thank J. Heim for supporting the project, and K. Hess and
H. B. Jenny for performing HPLC analysis.
REFERENCES
Arai, T., Takahashi, K. & Kubo, A. (1977). New antibiotics,
saframycins A, B, C, D, and E. J Antibiot 30, 1015-1018.
Arai,T., Takahashi, K., Ishiguro, K. & Mikami, Y. (1980). Antitumor
antibiotics, saframycin A and C. Gann 71, 790-796.
Black, T. A. & Wolk, C. P. (1994). Analysis of a Het- mutation in
Anabaena sp. strain PCC7120 implicates a secondary metabolite in
the regulation of heterocyst spacing. J Bacterioll76, 2282-2292.
Cosmina, P., Rodriguez, F., de Ferre, F., Grandi, G., Perego, M.,
Venema, G. & van Sinderen, D. (1993). Sequence and analysis of
the genetic locus responsible for surfactin synthesis in Bacillus
subtih. Mol Microbial 8, 821-831.
Devereux, J., Haeberli, P. & Smithies, 0. (1984). A comprehensive
set of sequence analysis programs for the VAX. Nucleic Acids Res
12, 387-395.
Donadio, 5. & Katz, L. (1992). Organization of the enzymatic
domains in the multifunctional polyketide synthase involved in
erythromycin formation in Saccharopobspora eytbraea. Gene 111,
51-60.
D'Souza, C., Nakanno, M. M., Corbell, N. & Zuber, P. (1993).
Aminoacylation site mutations in amino acid-activating domains of
surfactin synthetase : effects on surfactin production and competence development in Bacillus subtilis. J Bacterioll75, 3502-351 0.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 20:57:33
745
A. P O S P I E C H , J. B I E T E N H A D E R a n d T. S C H U P P
Gocht, M. & Marahiel, M. A. (1994). Analysis of core sequences in
the D-Phe activating domain of the multifunctional peptide
synthetase TycA by site-directed mutagenesis. J Bacteriof 176,
2654-2662.
Hara, 0. & Hutchinson, C. R. (1992). A macrolide 3-0acyltransferase gene from the midecamycin-producing species
Sfreptomyces mycarofaciens. J Bacteriof 174, 5141-5144.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). MofecztfarCloning,
a Laborat09 Manual. Cold Spring Harbor, N Y : Cold Spring Harbor
Laboratory.
Sanger, F., Nicklen, 5. & Coulson, A. R. (1977). DNA sequencing
with chain terminating inhibitors. Proc Natf Acad Sci US2474,
5463-5467.
Schlumbohm, W., Stein, T., Ullrich, C., Vater, J., Krause, M.,
Marahiel, M. A,, Kruft, V. & Wittmann-Liebhold, B. (1991). An
active serine is involved in covalent substrate amino acid binding at
each reaction center of gramicidin S synthetase. J Biof Chem 266,
Hori, K., Yamamoto, Y., Minetoki, T., Kurotsu, T., Kanda, M.,
Miura, S., Okamura, K., Furujama, 1. & Saito, Y. (1989). Molecular
cloning and nucleotide sequence of the gramicidin S synthetase 1
gene. J Biochem 106, 639-645.
23135-23141.
Irschick, H., Trowitzsch-Kienast, W., Gerth, K., Hefle, G. &
Reichenbach, H. (1988). Saframycin Mxl, a new natural saframycin
isolated from a myxobacterium. J Antibiot 41, 993-998.
Schmitt, D., Pakusch, A. E. & Matern, U. (1991). Molecular cloning,
induction, and taxonomic distribution of caffeoyl-CoA-3-0methyltransferase, an enzyme involved in disease resistance. J Biof
Ishiguro, K., Sakiyama, S., Takahashi, K. & Arai, K. (1978). Mode
of action of saframycin A, a novel heterocyclic quinone antibiotic.
Inhibition of RNA synthesis in vivo and in vitro. Biochemistcy 17,
2545 2550.
Ishiguro, K., Takahashi, S., Yazawa, K., Sakiyama, S. & Arai, K.
(1981). Binding of saframycin A, a heterocyclic quinone anti-tumor
antibiotic to DNA as revealed by the use of the antibiotic labeled
with [“C]-tyrosine or [14C]-cyanide.J Biof Chem 256, 2162-2167.
Kagan, R. M. & Clarke, 5. (1994). Widespread occurrence of three
sequence motifs in diverse S-adenosylmethione-dependent methyltransferases suggests a common structure for these enzymes. Arch
Biochem Biopbyys 310, 41 7-427.
Kishi, K., Yazawa, K., Takahashi, K., Maeda, A. & Arai, K. (1984).
Structure-activity relationships of saframycins. J.Antibiot 37,
847-852.
Kleinkauf, H. & von Dt)hren, H. (1990). Nonribosomal biosynthesis
of peptide antibiotics. Eztr J Biochem 192, 1-15.
Lown, 1. W. , Joshua, A. V. & Lee, 1. 5. (1982). Molecular
mechanisms of binding and single-strand scission of desoxyribonucleic acid by the antitumor antibiotics saframycin A and C.
Biochemisty 21, 419-428.
Marahiel, M. A. (1992). Muitidomain enzymes involved in peptide
synthesis. FEBS Lett 307, 40-43.
Morris, M. E. & Jinks-Robertson, S. (1991). Nucleotide sequence of
the LYS2 gene of Saccharomyces cerevisiae : homology to Bacillus (brevis
tyrocidine synthetase 1. Gene 98, 141-145.
Pavela-Vrancic, M., Pfeifer, E., van Liempt, H., Schafer, H.-J.,. von
Ddhren, H. & Kleinkauf, H. (1994a). ATP-binding in peptide
synthetases : determination of contact sites of the adenine moiety by
photoaffinity labeling of tyrocidine synthetase I with 2azidoadenosine triphos phate. Biochemist9 33, 6276-6283.
Pavela-Vrancic, M., Pfeifer, E., SchrMer, W., von Dahren, H. &
Kleinkauf, H. (1994b). Identification of the ATP-binding site in
tyrocidine synthetase I by selective modification with fluorescein 5’isothiocyanate. J Biof Chem 169, 14962-14966.
Pospiech, A,, Cluzel, B., Bietenhader, 1. & Schupp,T. (1995). A. new
~ ~ x o c o c c zxanthw
ts
gene cluster for the biosynthesis of the antibiotic
saframycin Mxl encoding a peptide synthetase. hficrobiology 141,
1793-1 803.
Reichenbach, H., Gerth, K., Irschik, H., Kunze, B. & Ht)fle, G.
(1988). Myxobacteria: a source of new antibiotics. Trends Biotwhnol
6, 115-121.
746
Chem 266, 17416-17423.
Simon, R., O’Connell, M., Labes, M. & PUhler, A. (1986). Plasmid
vectors for the genetic analysis and manipulation of rhizobia and
other Gram-negative bacteria. Methods EnZymof 118, 643-659.
Simon, R., Priefer, U. & PUhler, A. (1983). A broad host range
mobilization system for in vivo genetic engineering : transposon
mutagenesis in gram negative bacteria. Bio/ Technology 1, 784-791.
Stachelhaus, T. & Marahiel, M. A. (1995). Modular structure of
genes encoding multifunctional peptide synthetases required for
non-ribosomal peptide synthesis. F E M S Microbiol Lett 125, 3-14.
Stachelhaus, T., Schneider, A. & Marahiel, M. A. (1995). Rational
design of novel peptide antbiotics by targeted replacement of
bacterial and fungal domains. Science 269, 69-72.
Stein, T., Vater, J., Kruft, V., Wittmann-Liebold, B., Franke, P.,
Panico, M., Dowell, R. M. & Morris, H. R. (1994). Detection of 4’phosphopantetheine at the thioester binding site for L-valine of
gramicidin S synthetase 2. FEBS Lett 340, 39-44.
Tohika, K., Hori, K., Kurotzu, T., Kanda, M. & Saito, Y. (1993).
Effect of single base substitutions at glycine-870 codon of
gramicidin S synthetase 2 gene on proline activation. J Biochem 114,
522-527.
Trowitzsch-Kienast, W., Irschick, H., Reichenbach, H., Wray, V. &
Ht)fle, G. (1988). Isolierung und Strukturaufklarung der Saframycine Mxl und Mx2, neue antitumor-aktive Antibiotika aus
Myxococcw xanthtls. Liebigs A n n Chem 475-481.
Turgay, K., Krause, M. & Marahiel, M. A. (1992). Four homologous
domains in the primary structure of GrsB are related to domains in
a superfamily of adenylate-forming enzymes. Mof Microbiol 6,
529-546.
Vollenbroich, D., Kluge, B., D’Souza, C., Zuber, P. & Vater, 1.
(1993). Analysis of a mutant amino acid-activating domain of
surfactin synthetase bearing a serine-to-alanine substitution at the
site of carboxylthioester formation. FEBS Lett 325, 22&224.
Weber, J. M., Schoner, B. & Losick, R. (1989). Identification of a
gene required for the terminal step in erythromycin A biosynthesis
in Saccharopobspora eythraea. Gene 75, 235-241.
Weckermann, R. W., FUrbaB, R. & Marahiel, M. A. (1988).
Complete nucleotide sequence of the &A gene coding the
tyrocidine I synthetase from Bacillzis brevis. Ntlcleic Acids Res 16,
11841.
Received 6 December 1995; accepted 23 January 1996.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 20:57:33