Download Expression and purification of four different rhizobial acyl carrier

Microbiology (2000), 146, 839–849
Printed in Great Britain
Expression and purification of four different
rhizobial acyl carrier proteins
Isabel M. Lo! pez-Lara† and Otto Geiger†
Author for correspondence : Otto Geiger. Tel : j52 73 131697. Fax : j52 73 175581.
e-mail : Otto!cifn.unam.mx
Institute of Biotechnology,
Technical University of
Berlin, Seestrasse 13,
D-13353 Berlin, Germany
In rhizobia, besides the constitutive acyl carrier protein (AcpP) involved in the
biosynthesis and transfer of common fatty acids, there are at least three
specialized acyl carrier proteins (ACPs) : (1) the flavonoid-inducible nodulation
protein NodF ; (2) the RkpF protein, which is required for the biosynthesis of
rhizobial capsular polysaccharides ; and (3) AcpXL, which transfers 27hydroxyoctacosanoic acid to a sugar backbone during lipid A biosynthesis.
Whereas the nucleotide sequences encoding the three specialized ACPs are
known, only the amino acid sequence of the AcpP of Sinorhizobium meliloti
was available. In this study, using reverse genetics, the genes for the
constitutive AcpPs of S. meliloti and of Rhizobium leguminosarum were cloned
and sequenced. Previously, it had been shown that NodF and RkpF can be
overproduced in Escherichia coli using the T7 polymerase expression system.
Using the same system, the constitutive AcpPs of S. meliloti and of R.
leguminosarum , together with the specialized ACP AcpXL, were overproduced
and purified. All the known ACPs of rhizobia can be labelled in vivo during
expression in E. coli with radioactive β-alanine added to the growth medium
due to their modification with a 4’-phosphopantetheine prosthetic group. The
availability of all functionally different ACPs should help to unravel how
different fatty acids are targeted towards different biosynthetic pathways in
one organism.
Keywords : Rhizobium, acyl carrier protein, acpP, acpXL
INTRODUCTION
Biosynthesis and transfer of fatty acids is of major
importance during the formation of biological membranes, storage lipids, or certain amphiphilic signal
molecules [i.e. Nod factors in rhizobia (De! narie! et al.,
1996) or autoinducers in Gram-negative bacteria (Fuqua
et al., 1996)]. In higher animals, fatty acid biosynthesis
occurs on a multienzyme complex whereas in bacteria,
fatty acids are formed by the catalytic activity of a
number of monofunctional enzymes. In the latter
organisms, a small acidic protein carries the growing
fatty acyl chains via a thioester linkage of a 4hphosphopantetheine prosthetic group, which itself is
.................................................................................................................................................
† Present address : CIFN – UNAM, Av. Universidad s/n, Colonia Chamilpa,
Apdo postal 565-A, CP 62210, Cuernavaca, Morelos, Mexico.
Abbreviations : ACP, acyl carrier protein ; iPCR, inverse PCR.
The GenBank accession numbers for the nucleotide sequences reported in
this paper are AF159244 and AF159243 for the acpP-containing regions of
Sinorhizobium meliloti 1021 and of Rhizobium leguminosarum LPR5045,
respectively.
attached to a conserved serine residue. This protein is
called acyl carrier protein (ACP). In bacteria, ACPs are
involved not only in the synthesis of fatty acyl chains,
but also in their transfer during phospholipid, lipid A
or α-haemolysin biosynthesis (Issartel et al., 1991 ;
Jackowski et al., 1991). In Escherichia coli, all these
functions seem to be realized by a single and absolutely
essential ACP which is produced constitutively from the
acpP gene (Jackowski et al., 1991 ; Rawlings & Cronan,
1992) and which is now named AcpP.
Some bacteria have additional ACPs which are involved
in the biosynthesis of special β-ketides or fatty acids
(Bibb et al., 1989 ; Shearman et al., 1986). In rhizobial
species infecting legume plants of the Trifolieae, Vicieae,
and Galegeae tribes, one specialized ACP, the nodulation protein NodF, is involved, together with NodE, in
the synthesis of polyunsaturated fatty acids which are
host-specific substituents of nodulation (Nod) factors
(Yang et al., 1999).
In addition to AcpP and NodF, there are at least two
additional ACPs in rhizobia. The first was identified as
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the rkpF gene product at the fix23 locus in Sinorhizobium meliloti 41 (Petrovics et al., 1993 ; Reuhs,
1996 ; Epple et al., 1998). The second novel ACP is
involved in the transfer of the unusually long 27hydroxyoctacosanoic acid to a sugar backbone during
lipid A biosynthesis in Rhizobium leguminosarum and
has been named AcpXL (Brozek et al., 1996). The
overall amino acid sequence identities of these four
ACPs range from only 26 to 32 % (Brozek et al., 1996),
and a well-conserved amino acid region can only be
observed around the phosphopantetheine-binding site.
However, the three-dimensional structure of the AcpP
of E. coli has been determined (Kim & Prestegard, 1990)
and an initial characterization of the secondary structure
and the general tertiary fold of the NodF protein (Ghose
et al., 1996) demonstrates that the overall structures of
ACPs are surprisingly well conserved. In spite of the
general structural similarity between NodF and AcpP of
E. coli, AcpP cannot substitute for NodF in vivo in the
synthesis of polyunsaturated fatty acids (Ritsema et al.,
1998). Furthermore, these authors have shown that the
NodF-specific functions are encoded in the C-terminal
half of the protein. The presence in rhizobia of four
different ACPs offers an unusual opportunity for investigation of the structure–function relationship of the
ACPs.
The nodF gene of R. leguminosarum bv. viciae had been
cloned in the pET9a vector (Ritsema et al., 1994). With
this system it is possible to overexpress NodF in E. coli
to comprise 40 % of the total soluble cell protein (Ghose
et al., 1996). In an analogous way, the rkpF gene of S.
meliloti has been cloned and its protein product overproduced in E. coli (Epple et al., 1998). Overexpression
of the different rhizobial ACPs will allow detailed
structural investigations. Also, the different rhizobial
ACPs will be invaluable tools for comparative biochemical studies in which acyl-ACPs are used as
cosubstrates. In particular, the constitutive AcpP of S.
meliloti will be required to elucidate the biosynthesis of
a phosphorus-free ornithine-derived lipid recently described in this species (Geiger et al., 1999).
In the present study the acpXL gene from R. leguminosarum was amplified using the PCR technique and
cloned in an overexpression vector. Previously, only the
amino acid sequence of the constitutive ACP of S.
meliloti was known (Platt et al., 1990). Using reverse
genetics, we have cloned and sequenced the acpP gene of
S. meliloti and of R. leguminosarum. Furthermore, we
have established the methods for the overproduction
and subsequent purification of AcpXL and rhizobial
AcpPs. In vivo labelling of the ACP-overproducing E.
coli strains with β-alanine has shown that each of the
four ACPs of rhizobia can be post-translationally
modified in E. coli with a 4h-phosphopantetheine prosthetic group.
METHODS
al., 1990), which expresses the T7 polymerase under the
control of the lac promoter, was used as a host for the
overproduction of ACPs. E. coli strains were grown at 37 mC in
Luria–Bertani (LB) medium (Sambrook et al., 1989), except
for large-scale cultures for the overproduction of ACPs that
were grown in M9 minimal medium (Miller, 1972). Antibiotics
were added, when required, to obtain the following final
concentrations (µg ml-") : carbenicillin, 100 ; kanamycin, 50 ;
and chloramphenicol, 20.
DNA techniques and DNA sequence analysis. DNA manipulations were carried out using standard procedures (Sambrook
et al., 1989). Labelling of DNA probes and colorimetric
detection were carried out with the DIG-DNA labelling and
detection kit of Boehringer Mannheim. DNA sequencing was
performed using the dideoxy chain-termination method
(Sanger et al., 1977) using the T7 Sequenase 7-deaza-dGTP
sequencing kit (Amersham). Plasmids for DNA sequencing
were isolated using a QIAprep Spin Miniprep kit (Qiagen).
When required, PCR products were purified using the
QIAquick PCR purification kit (Qiagen). DNA and derived
protein sequences were analysed with the  search
program (Altschul et al., 1997). Alignments of DNAs or amino
acid sequences were done with the program  at the
Genestream network server IGH Montpellier, France (http :\\
vega.igh.cnrs.fr\bin\lalign-guess.cgi).
Cloning of the acpP gene of S. meliloti. This was was carried
out in three steps.
(1) Cloning of the first half of the acpP gene. Two degenerate
oligonucleotides were used in a PCR to amplify and subsequently clone that part of the gene encoding the aminoterminal half of the AcpP of S. meliloti. These oligonucleotides
were deduced from the amino acid sequence of different ACPs,
among them the sequence of the AcpP of S. meliloti 1021 (Platt
et al., 1990). The forward primer oACP4 was based on the
amino terminus of constitutive ACPs and the backward primer
oACP3 was based on the conserved phosphopantetheinebinding region of several ACPs (Fig. 1). Using genomic DNA
of S. meliloti 1021 as a template PCR was carried out with
degenerate primers (oACP3 and oACP4), Taq polymerase
(MBI Fermentas) and a PCR programme especially designed
for degenerate oligonucleotides. The programme started with
3 cycles consisting of 30 s at 94 mC, 30 s at 37 mC and 30 s at
72 mC, and was followed by 30 cycles with identical parameters except that the annealing temperature was 55 instead
of 37 mC. The products of the PCR reaction were separated in
a 3 % NuSieveR GTGR agarose (FMC Bioproducts) gel and the
DNA fragment migrating according to the expected size of
140 bp was excised. The piece of agarose was melted for
10 min at 65 mC and a 10-fold dilution was used as template in
a new round of PCR with degenerate oligonucleotides oACP4
and oACP3. For this second round of PCR, conditions of
amplification were 30 cycles each of 30 s at 94 mC, 30 s at 65 mC
and 20 s at 72 mC. The products were purified, digested with
NdeI and BamHI and ligated to pET9a that had been digested
with the same enzymes. Four transformants carrying pET9a
with an insert of 120–140 bp were sequenced using oligonucleotides oACP4 and oACP3. Plasmid pTB5013 harbours a
fragment with an internal core sequence of 67 nucleotides
encoding amino acids 11– 32 of AcpP of S. meliloti (Platt et al.,
1990).
Bacterial strains, plasmids and media. The strains and
(2) Amplification and cloning of inverse PCR (iPCR) products to
identify the full-length coding sequence of AcpP. Essentially the
plasmids used in this study are listed in Table 1. E. coli DH5α
was used for cloning purposes. E. coli BL21(DE3) (Studier et
iPCR method described by Triglia et al. (1988) was followed.
A pair of divergent oligonucleotide primers (oTBC16 : BamHI,
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Rhizobial acyl carrier proteins
Table 1. Bacterial strains and plasmids used
Strain/plasmid
Relevant characteristics*
Source/reference
Escherichia coli
DH5α
Host used for cloning
BL21(DE3)
OG7001
Host used for expression
panD mutant of BL21(DE3)
Bethesda Research
Laboratory
Studier et al. (1990)
Epple et al. (1998)
Rhizobia
Sinorhizobium meliloti 1021
Rhizobium leguminosarum
LPR5045
SmR mutant of SU47 wild-type
RifR, symbiosis-plasmid-cured
Meade et al. (1982)
Hooykaas et al. (1981)
CbR, cloning vector
KnR, expression vector
CmR, production of lysozyme for
repression of T7 polymerase
KnR, used for overexpression of
NodF
KnR, used for overexpression of
RkpF
KnR, used for overexpression of
AcpXL
KnR, used for overexpression of
AcpP of S. meliloti
KnR, used for overexpression of
AcpP of R. leguminosarum
KnR, used for overexpression of
AcpP of E. coli
Yanisch-Perron et al. (1985)
Studier et al. (1990)
Studier et al. (1990)
Plasmids†
pUC18
pET9a
pLysS
pMP2301
pTB1003
pTB5011
pTB5035
pTB5040
pTB5079
Ritsema et al. (1994)
Epple et al. (1998)
This work
This work
This work
This work
* SmR, RifR, CbR, KnR and CmR : streptomycin, rifampicin, carbenicillin, kanamycin and chloramphenicol resistance, respectively.
† Other plasmids constructed in this work were pCU18 derivatives carrying the fragments as shown in
Fig. 2.
HindIII 5h-AAAGGATCC GCA AGC TTC ATC GAT
GAC-3h ; and oTBC18 : BamHI, SalI 5h-AAAGGATCCGC
GTC GAC GCC AAG ATG ATC-3h ; restriction sites are
underlined and respective enzymes are given before the
sequence) were designed from the internal sequenced region
(67 bp) of the acpP gene cloned in pTB5013 (Fig. 2a) in such a
way that they annealed to the target DNA template 18 bp
apart and their 3h termini were in opposite directions. S.
meliloti 1021 genomic DNA was completely digested with
individual restriction enzymes, the DNA was cleaned using
phenol\chloroform extraction followed by ethanol precipitation, and was then used for self-circularization by ligase
treatment at low DNA concentrations (Maiti & Ghosh, 1996 ;
Triglia et al., 1988). iPCR reactions were performed with the
self-circularized DNA samples using the standard reaction
mixture, primers oTBC16 and oTBC18 and Taq polymerase.
After a preheating step (95 mC, 30 min), conditions for
amplification were 50 cycles each consisting of 45 s at 94 mC,
45 s at 65 mC and 150 s at 72 mC. A specific fragment of 1n5 kb
from the KpnI digest was purified on an agarose gel. After
digestion of the purifed fragment with KpnI and HindIII, the
DNA sequences downstream of the acpP gene were cloned in
pUC18 that had been digested with the same two enzymes
(Fig. 2a ; plasmid pTB5021). After digestion of the 1n5 kb iPCR
product with SalI, a SalI fragment of 214 bp carrying sequences
upstream of the acpP gene was cloned in pUC18 that had been
digested with SalI (Fig. 2a ; plasmid pTB5030).
(3) Cloning of the acpP gene of S. meliloti in the overexpression
plasmid pET9a. When the complete DNA sequence encoding the
AcpP of S. meliloti had been determined, the ORF was
amplified from genomic DNA of S. meliloti 1021 using
homologous primers oTBC19 (NdeI 5h-AGGAATA
CAT ATG AGC GAT ATC GCA GAA CG-3h) and oTBC20
(BamHI 5h-AAAGGATCC TCA GGC CTG GGC TTT
CTC-3h), together with cloned Pfu DNA polymerase
(Stratagene). The PCR product was digested with NdeI
and BamHI and ligated with pET9a also digested with
these enzymes. After transformation in DH5α, plasmids with
an insert of the correct size were selected and sequenced. They
all showed the same DNA sequence and pTB5035 was chosen
for further studies.
Cloning of the acpP gene of R. leguminosarum. The same
specific primers (oTBC19 and oTBC20) used for amplification
of the acpP gene of S. meliloti were used for the amplification
and subsequent cloning in pET9a of the gene encoding the
constitutive AcpP of R. leguminosarum LPR5045. Identical
DNA sequences were obtained from several inserts derived
from independent PCR amplifications. By this method the
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(a)
N-terminal amino acid sequences of AcpPs
AcpP of E. coli
AcpP of S. meliloti
Degenerate primer oACP4
(b)
Conserved amino acid sequences of diverse ACPs
AcpP of E. coli
NodF of R. leguminosarum bv. viciae
AcpP of S. meliloti
ACP gra of S. violaceoruber
ACP tcm of S. glaucescens
Degenerate primer oACP3
.................................................................................................................................................................................................................................................................................................................
Fig. 1. Design of two degenerate oligonucleotide primers for cloning and sequencing part of the acpP gene of S. meliloti.
(a) Nucleotide sequence of the N-terminal degenerate primer oACP4 with a NdeI restriction site. (b) Nucleotide sequence
of the degenerate primer oACP3 with a BamHI restriction site. Conserved amino acids of the phosphopantetheinebinding motif of ACPs are boxed. Sequence data were obtained from the following references from top to bottom :
Rawlings & Cronan (1992), Shearman et al. (1986), Platt et al. (1990), Sherman et al. (1989), and Bibb et al. (1989).
Specialized ACPs gra from Streptomyces violaceoruber and tcm from Streptomyces glaucescens are involved in granaticin
and tetracenomycin biosynthesis, respectively.
core DNA region encoding the AcpP of R. leguminosarum
was obtained.
To determine the DNA sequence flanking this internal core
region, a pair of divergent primers for iPCR were selected.
One of the primers used was oTBC18, which anneals 100 % in
the last 13 nucleotides of the 3h end to the DNA of R.
leguminosarum and adds a SalI site which is not present in the
DNA of R. leguminosarum. A new primer specific for DNA
sequences of acpP of R. leguminosarum was designed,
oTBC25 (BamHI, 5h-AAAGGATCCTTTCGAA GAA GAA
TTC GGC-3h), which annealed 80 bp apart from oTBC18 and
the 3h terminus of which was in the opposite direction.
Genomic DNA of R. leguminosarum LPR5045 was digested,
religated and used in iPCR reactions with primers oTBC18
and oTBC25 as described for genomic DNA of S. meliloti. A
specific product of 1n2 kb was obtained with a SalI digest.
Making use of the restriction sites introduced by the primers,
the sequences upstream of the acpP were cloned as a SalI
fragment (pTB5062) and those downstream of the acpP gene
as a BamHI\SalI fragment (pTB5065) (Fig. 2b). Sequencing of
the iPCR fragments revealed that the oligonucleotide oTBC19
was identical to corresponding DNA sequences of the acpP of
R. leguminosarum. The oligonucleotide oTBC20 differed only
in one base pair (A at position 225 instead of G) from
corresponding DNA sequences of the acpP of R. leguminosarum. As this change is in the third position of the respective
codon it encodes K in both cases and does not lead to any
difference in the amino acid sequence of AcpP of R. leguminosarum. Therefore, oTBC19 and oTBC20 were used to amplify
acpP of R. leguminosarum and after cloning in pET9a the
plasmid obtained (pTB5040) was chosen for overproduction
of AcpP of R. leguminosarum.
Cloning of the acpXL gene of R. leguminosarum and of the
acpP gene of E. coli in pET9a. Based on the DNA sequence of
the acpXL gene (Brozek et al., 1996 ; GenBank accession no.
AF081835), a pair of homologous primers was designed for
the PCR amplification. In the forward primer oTBC12 (5hGGAATACATATG ACC GCT ACA TTC GAT AAA G-3h),
the GTG starting codon occurring in R. leguminosarum
(Brozek et al., 1996) was changed to ATG and a NdeI
restriction site was added. The backward primer oTBC13 (5hAAAGGATCC TCA GGC CTT GGC CGC-3h) adds a BamHI
restriction site. As template, genomic DNA of R. leguminosarum LPR5045 was used. Design of the primers for amplification of the acpP gene of E. coli was based on the DNA
sequence of the gene (Rawlings & Cronan, 1992) (GenBank
accession no. M84991), the forward primer being oTBC30
(NdeI, 5h-AGGAATACAT ATG AGC ACT ATC GAA GAA
CG-3h) and the backward primer oTBC31 (BamHI, 5hAAAGGATCC TTA CGC CTG GTG GCC GTT G-3h). As
template, genomic DNA of E. coli DH5α was used. After
purification of the amplified products, they were digested with
NdeI and BamHI and ligated to pET9a that had been
previously digested with the same two enzymes. After transformation of DH5α, plasmids with inserts of the correct size
were selected and sequenced. Plasmid pTB5011 carries a DNA
insert of identical sequence as that published for acpXL by
Brozek et al. (1996) except that the starting GTG codon is
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(a)
(1) deg PCR
(2) iPCR
(3) Pfu PCR
(b)
(1) Pfu PCR
(2) iPCR
.................................................................................................................................................................................................................................................................................................................
Fig. 2. Steps for the cloning of the acpP genes of rhizobia in pET9a. (a) The three steps followed for the cloning and
sequencing of the acpP of S. meliloti 1021 : (1) PCR with degenerate primers, (2) iPCR using specific primers derived from
the region cloned with the degenerate primers, and (3) PCR amplification with Pfu polymerase and specific primers. (b)
The two steps followed for the cloning and sequencing of the acpP of R. leguminosarum LPR5045 : (1) PCR amplification
with the specific primers devised for acpP of S. meliloti, and (2) iPCR to check the sequences at the borders of acpP of R.
leguminosarum. B, BamHI ; H, HindIII ; K, KpnI ; N, NdeI ; S, SalI. At the bottom of both (a) and (b) schemes of the
sequenced regions are shown. RBS denotes putative ribosome-binding sites, a T above two arrowheads denotes a
putative transcription terminator and inverted arrows in (a) denote the palindromic area downstream of acpP of S.
meliloti. The area with homology to RIME1 in (a) is marked with a dashed line.
changed to ATG. Plasmid pTB5079 carries a DNA insert of
identical sequence to the one previously published for acpP of
E. coli by Rawlings & Cronan (1992).
Overproduction of ACPs in E. coli. E. coli strain BL21(DE3)
(Studier et al., 1990) was individually transformed with the
selected plasmid for overproduction of the respective ACP. In
the case of the AcpPs, which are known to be toxic to E. coli
when cloned in a high-copy-number plasmid, E. coli strain
BL21(DE3) carrying the plasmid pLysS was used as host. The
presence of pLysS represses the basal activity of the T7
polymerase.
Small-scale overproduction was used for establishing the best
conditions for each of the ACPs produced. Cultures of 30 ml
in LB medium containing the required antibiotics were grown
at 37 mC to an OD of approximately 0n4, then IPTG was
'#!
added to a final concentration of 0n1 mM and cells were
harvested by centrifugation at different time points after
induction. The cell pellet was resuspended in 1n5 ml of 50 mM
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Tris\HCl, 0n1 M KCl, pH 6n8 and kept frozen at k20 mC.
After thawing, the cells were broken by a single passage
through a French press at 20 000 p.s.i. (138 MPa). Broken cell
suspensions were centrifuged for 5 min in an Eppendorf
centrifuge at 15 000 r.p.m. and the supernatants were analysed
for the presence of the overproduced protein by 20 % native
PAGE (Jackowski & Rock, 1983).
For large-scale overproduction of ACPs, the strains were
grown at 37 mC in 2 l flasks containing 500 ml M9 minimal
medium supplemented with the appropriate antibiotics. At an
OD of 0n4, IPTG was added to a final concentration of
'#! and cells were harvested by centrifugation 4 h later.
0n1 mM
The cell pellets (1 vol.) were resuspended in 10 vols of either
50 mM Tris\HCl, 0n1 M KCl, pH 6n8 in the case of AcpXL
and E. coli AcpP, or of 50 mM MOPS\KOH, 6 mM MgSO ,
%
1 mM DTT, pH 7n4 in the case of rhizobial AcpPs and kept
frozen at k20 mC. After thawing, the cells were broken by
passing the suspension twice through a French press at 20 000
p.s.i. The lysates were centrifuged at 7000 g for 20 min at 4 mC
and the supernatants were used as cell-free extracts.
In vivo labelling of rhizobial ACPs with β-alanine. In vivo
labelling of ACPs with β-alanine, the biosynthetic precursor of
4h-phosphopantetheine, was carried out essentially as described by Epple et al. (1998). A β-alanine auxotroph, strain
OG7001 (Epple et al., 1998), was transformed with pLysS and
the new strain OG7001(pLysS) was individually transformed
with the different plasmids for overproduction of ACPs or
with pET9a. The presence of the pLysS plasmid facilitates the
lysis of the cell pellets.
Purification of ACPs. NodF from R. leguminosarum (Ghose et
al., 1996) and RkpF from S. meliloti (Epple et al., 1998) were
purified as described before with the exception that 2-propanol
was removed by rotatory evaporation, as described below,
instead of by dialysis.
The procedure described below was applied to extracts
obtained from cells of 2 l cultures. For the purification of
AcpXL, cold 2-propanol was added dropwise to a cell-free
extract of the E. coli strain overproducing the ACP to a final
concentration of 60 % (v\v). After 60 min at 4 mC, the
precipitate was removed by centrifugation at 7000 g at 4 mC
for 20 min. The supernatant was concentrated by rotatory
evaporation to remove the 2-propanol and then applied to a
30 ml column of DEAE-52 cellulose (Whatman), which had
been equilibrated with 50 mM Tris\HCl pH 6n8, 1 mM
CHAPS. The column was washed with 90 ml buffer A (10 mM
Bistris\HCl pH 6, 1 mM CHAPS). Elution was performed
with a linear gradient from 0 to 1 M NaCl in buffer A in a total
volume of 160 ml. Fractions of 3 ml were collected and
analysed by PAGE.
For the purification of AcpPs of rhizobia, cold 2-propanol was
added dropwise to the cell-free extracts to a final concentration
of 20 %. After 60 min at 4 mC, the precipitate was removed by
centrifugation at 7000 g at 4 mC for 20 min. The supernatant
was applied to a 30 ml DEAE-52 cellulose column equilibrated
with buffer A containing 1 mM DTT. Washing and elution of
the column were as described for the chromatography of
AcpXL with the exception that all buffers contained 1 mM
DTT. Fractions containing AcpP were pooled and after
dialysis against 5 mM Tris\HCl pH 6n8 at 4 mC overnight, the
volume was reduced by a sixth by rotatory evaporation.
Because the rhizobial ACPs run distinctly ahead of the E. coli
AcpP in the non-denaturing PAGE (Platt et al., 1990 ; Figs 3
and 4), the concentrated samples carrying the overproduced
AcpP of rhizobia were further purified by a preparative native
PAGE (1n5 mm thick gels) in order to obtain separation from
the E. coli AcpP. The unstained gel band containing the
rhizobial AcpP (after identification by parallel staining) was
sliced into small pieces and the AcpP protein was electroeluted
with a Schleicher & Schuell Biotrap electroseparation system.
Electroelution was carried out for 7 h at 200 V.
AcpP of E. coli was purified from the overproducing strain
using the same protocol as described for the purification of
AcpXL.
RESULTS
Cloning of the acpP gene of S. meliloti
The DNA sequences of the genes encoding the three
specialized ACPs described in rhizobia, NodF, RkpF
and AcpXL, are known. However, only the amino acid
sequence of the constitutive AcpP of S. meliloti has been
reported (Platt et al., 1990). Our aim was to overproduce
and purify the four functionally different ACPs of
rhizobia using the T7 polymerase expression system
(Studier et al., 1990).
Initially, we tried to amplify the whole gene encoding
AcpP by PCR using degenerate oligonucleotides that
were based on the amino acid sequence of S. meliloti.
These degenerate primers did not give amplification of
the desired product in any of the various conditions
assayed (data not shown). We then decided to amplify
part of the acpP gene using a pair of degenerate
oligonucleotides, the sequences of which were derived
from the alignment of different ACPs (Fig. 1) as
described in the Methods section. Amplification from
genomic DNA of S. meliloti 1021 with oACP4 and
oACP3 gave several unspecific products and a minor
amount of a DNA fragment of the expected size of
140 bp (data not shown). This fragment was used for a
second round of PCR and the fragment of the appropriate size was cloned in pET9a. Sequencing analysis
of DNA from four independent clones showed that two
of them, each with a DNA insert of 123 bp, had an
internal region of 67 bp that differed in only one base
pair. Because the two primers for PCR were degenerate,
the nucleotide sequences of the two insert DNAs were
slightly different in the region where the primers
annealed. For plasmid pTB5013, the deduced 22 amino
acid sequence corresponding to the 67 bp core region
was that of amino acids 11– 32 of the AcpP of S. meliloti
(Platt et al., 1990).
iPCR with the two gene-specific primers oTBC16 and
oTBC18 gave a specific product from the KpnI digest of
1n5 kb. To further confirm that the iPCR product was
specific, a Southern DNA hybridization was carried out
using as a probe the DNA insert of pTB5013. The probe
hybridized strongly with the iPCR product and genomic
DNA of S. meliloti digested with KpnI gave a specific
band at 1n5 kb, in agreement with the size of the iPCR
product (data not shown).
The nucleotide sequences flanking the 67 bp core acpP
region were obtained after sequencing of two fragments
of the iPCR product of the KpnI digest (Fig. 2a). The
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Rhizobial acyl carrier proteins
homologous primers oTBC19 and oTBC20 were then
used to amplify the complete acpP gene from genomic
DNA of S. meliloti. Three independent acpP inserts
cloned in pET9a were sequenced and they all showed an
identical DNA sequence for acpP. The deduced amino
acid sequence is identical to that determined by protein
sequencing (Platt et al., 1990). Plasmid pTB5035 was
chosen for further studies.
Cloning of the acpP gene of R. leguminosarum
The amino acid sequence of the acpP of R.
leguminosarum was not known. We tried the same pair
of specific primers, oTBC19 and oTBC20, designed for
the amplification of the acpP of S. meliloti in a PCR
reaction with genomic DNA of R. leguminosarum
LPR5045. A unique and specific fragment of the expected
size was obtained and subsequently cloned in pET9a.
The deduced amino acid sequence differed in only three
positions from that of the AcpP of S. meliloti ; these were
(S. meliloti to R. leguminosarum) : E 21 D, S 24 V and G
26 S. The DNA sequences of the regions flanking the
core region of acpP of R. leguminosarum were obtained
using iPCR (Fig. 2b and Methods). Plasmid pTB5040
was chosen for the overproduction of AcpP of R.
leguminosarum.
Analysis of the nucleotide sequences of acpP genes
of rhizobia
The complete nucleotide sequences encoding AcpPs of
S. meliloti 1021 and of R. leguminosarum LPR5045
together with the surrounding regions were obtained.
The high level of similarity between the DNA sequences
encoding S. meliloti AcpP and R. leguminosarum AcpP
(86n5 %) does not extend into the surrounding areas.
Using the  program, we identified a partial ORF
downstream of acpP of R. leguminosarum with homology to FabF (3-oxoacyl-ACP synthase II) from
different organisms. The highest similarity is to FabF
from E. coli (56 % identity and 71 % similarity in an
overlap of 39 amino acids). In E. coli the fabF gene is
also located directly downstream of acpP (Rawlings &
Cronan, 1992). By comparison with the sequences of R.
leguminosarum, we identified downstream of acpP of S.
meliloti a partial ORF encoding the sinorhizobial FabF
(Fig. 2a). However, whilst in R. leguminosarum the
acpP–fabF intergenic region is 92 nucleotides, in S.
meliloti the intergenic region is much larger (305
nucleotides) (Fig. 2). Downstream of both rhizobial
acpP genes the typical structure of a rho-factor-independent transcription terminator was identified (Fig.
2). The same kind of transcription terminator structures
have been found downstream of the acpP genes of E. coli
and of Azospirillum brasilense (Rawlings & Cronan,
1992 ; Maiti & Ghosh, 1996). Further analysis of the
intergenic region of S. meliloti revealed a complex
secondary structure with several inverted repeats (shown
by inverted arrows in Fig. 2a). A  search with the
acpP–fabF intergenic region of S. meliloti revealed the
presence of a homologue of the mosaic sequence RIME
1 (Fig. 2a). RIME are Rhizobium-specific intergenic
mosaic elements characterized by two large palindromes
(Østera/ s et al., 1995). RIME 1 has been found in different
rhizobial species while RIME 2 seems to be restricted to
S. meliloti (Østera/ s et al., 1995). In the acpP–fabF
intergenenic region of S. meliloti, upstream of RIME 1
there is another mosaic element consisting of two
palindromes, only one of which shows homology to
RIME 2. Putative RBS were identified for acpP of S.
meliloti, acpP of R. leguminosarum and fabF of R.
leguminosarum (Fig. 2). No significant similarity was
observed between the DNA sequences upstream of the
acpP genes of S. meliloti and of R. leguminosarum.
Optimization of overproduction of ACPs
We have amplified and cloned in pET9a the genes
encoding AcpXL of R. leguminosarum (Brozek et al.,
1996) and for AcpP of E. coli (Rawlings & Cronan,
1992) using homologous primers (see Methods). AcpP of
E. coli is the best studied ACP and therefore it was used
in our studies as a control.
The growth of strain BL21(DE3) was not affected when
carrying the plasmid for overproduction of AcpXL,
pTB5011. However, when carrying the plasmids expressing the constitutive AcpPs of rhizobia, pTB5035 or
pTB5040, the growth rate of host strains was reduced.
This result was not unexpected since the full-length acpP
of Gram-negative bacteria cloned in a high-copynumber vector is unstable in the E. coli host (Rawlings
& Cronan, 1992 ; Maiti & Ghosh, 1996). Although
plasmids pTB5035 and pTB5040 could be maintained in
BL21(DE3), the overproduction of encoded AcpPs was
very poor (data not shown). Good overproduction of
AcpPs was obtained only if in addition the plasmid
pLysS was present (Fig. 3a, lanes 2 and 3). A similar
effect has been described for the acpP gene of E. coli (Hill
et al., 1995). Therefore, for overproduction of the three
AcpPs, BL21(DE3) carrying pLysS was always used as
the host.
Using small cultures of 30 ml, the best conditions for the
overproduction of each ACP were established. In all
cases maximal protein overproduction occurred between 3 and 4 h after induction with IPTG (data not
shown). The best yield was obtained when cultures were
induced at an OD of 0n4–0n5. Induction with concentrations of IPTG higher than 0n1 mM did not give any
significant increase in the protein yield (data not shown).
In vivo labelling of rhizobial ACPs with radioactive βalanine
ACPs need to be substituted with their prosthetic group
4h-phosphopantetheine in order to be functional in fatty
acid\β-ketide synthesis or transfer. During biosynthesis
of CoA, β-alanine is a building block and can be used as
a marker to demonstrate whether CoA derivatives,
like 4h-phosphopantetheine, are covalently linked to
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I . M . L O! P E Z -L A R A a n d O . G E I G E R
(a)
1
1
2
3
4
5
6
2
3
4
5
6
7
RkpF
RkpF
Apo
Holo
Apo
Holo
AcpPEc
Apo
Holo
AcpPEc
Apo
Holo
.................................................................................................................................................
(b)
1
2
4
5
6
7
Fig. 4. Purification of ACPs. Conformationally sensitive 20 %
native PAGE gel showing purified ACPs AcpP of E. coli (lane 1),
AcpP of S. meliloti (lane 2), AcpP of R. leguminosarum (lane 3),
NodF of R. leguminosarum (lane 4), RkpF of S. meliloti (lane 5),
and AcpXL of R. leguminosarum (lane 6). In each well about 5
µg of protein was loaded and after electrophoresis proteins
were visualized with Coomassie blue. In lanes 1 and 5 apo- and
holo-forms have different mobilities.
suggests that, for all ACPs tested, addition of the 4hphosphopantetheine prosthetic group to apo-ACPs can
be performed by an enzyme activity present in E. coli,
presumably by holo-ACP synthase (AcpS) (Lambalot &
Walsh, 1995).
Purification of rhizobial ACPs
AcpPEc
.................................................................................................................................................
Fig. 3. In vivo overproduction of ACPs. (a) Optimization of the
overproduction of ACPs. Analysis of cell-free extracts from E.
coli strains overproducing the different ACPs was performed by
conformationally sensitive 20 % native PAGE and proteins were
visualized with Coomassie blue. In each lane the equivalent of
100 µl of culture was loaded. In lanes 1 and 5 apo- and holoforms have different mobilities. (b) Autoradiogram of cell
extracts of E. coli OG7001(pLysS) overproducing the different
ACPs after labelling with radioactive β-alanine. In (a) and (b)
the overproduced ACPs are : AcpP of E. coli (lane 1), AcpP of S.
meliloti (lane 2), AcpP of R. leguminosarum (lane 3), NodF of R.
leguminosarum (lane 4), RkpF of S. meliloti (lane 5), AcpXL of R.
leguminosarum (lane 6), and none (E. coli strain carrying pET9a)
(lane 7).
proteins. We have checked for the in vivo labelling with
β-alanine of ACPs overproduced in E. coli. Cell-free
extracts of OG7001(pLysS) carrying each of the overproducing plasmids or the empty vector pET9a that had
been grown in the presence of $H-labelled β-alanine
were analysed by PAGE and subsequent autoradiography showed the labelling of proteins with similar
mobility to the overproduced ACPs (Fig. 3). This result
A simple purification procedure allowed us to purify
the NodF protein to homogeneity in two steps (a 2propanol precipitation followed by chromatography on
DEAE-cellulose) (Ghose et al., 1996). NodF purified in
this way could be used for NMR investigations (Ghose
et al., 1996). In the case of E. coli AcpP and AcpXL of R.
leguminosarum, we have established similar procedures
(see Methods) that yield the respective homogeneous
proteins (Fig. 4, lanes 1 and 6, respectively). For
obtaining homogeneous rhizobial AcpPs, a third step of
purification consisting of preparative PAGE was
required in order to separate them from the AcpP of the
E. coli strain used as host for the overproduction.
Samples of purified AcpP of S. meliloti and of AcpP of
R. leguminosarum are shown in Fig. 4, lanes 2 and 3,
respectively. As previously described (Platt et al., 1990),
the AcpPs of rhizobia run in this system distinctly ahead
of the E. coli AcpP. Also, both rhizobial AcpPs purified
from the overproducing E. coli strains migrate as doublet
bands in a manner similar to that found for the AcpP
isolated from S. meliloti (Platt et al., 1990).
DISCUSSION
We have sequenced the genes for the constitutive AcpPs
of S. meliloti and of R. leguminosarum, as well as the
areas surrounding these genes. In constrast to the high
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Rhizobial acyl carrier proteins
level of similarity observed within the coding regions
(acpP and part of fabF), the intergenic regions are quite
different in length and do not show any significant DNA
similarity. Whilst in R. leguminosarum two specialized
ACPs were known, AcpXL and NodF (Brozek et al.,
1996 ; Shearman et al., 1986), here we report another
ACP from this organism which probably is the one
responsible for the biosynthesis and transfer of common
fatty acids.
Previous reports (Ghose et al., 1996 ; Epple et al., 1998),
together with the data presented here, allow for the
overproduction and purification of the four different
ACPs described in rhizobia. Since AcpP of E. coli is the
best studied ACP, we have also cloned and expressed
this AcpP in an analogous way. The availability of the
four different rhizobial ACPs, each with a different
biological function, should help to unravel how the
different fatty acids are directed towards different
biosynthetic pathways in one organism. Of special
interest will be the study of the distinct ACP substrate
specificities of NodA proteins. The nodulation protein
NodA seems to be involved in the central step of the
biosynthetic pathway leading to lipo-chitin oligosaccharide signal molecules in rhizobia (Atkinson et al.,
1994 ; Ro$ hrig et al., 1994). It is thought that NodA
transfers an acyl residue from an ACP to a Ndeacetylated chitooligosaccharide. Different rhizobia
seem to have different preferences as to the nature of the
fatty acyl residue they transfer to the chitooligosaccharide backbone (Debelle! et al., 1996 ; Ritsema et
al., 1996). NodAs from some rhizobia seem to transfer
preferentially the nodFE-dependent fatty acids (R.
leguminosarum and S. meliloti) or 1ω-hydroxylated
fatty acids (S. meliloti). These fatty acids are known to
be synthesized and carried on the specialized ACPs
NodF and AcpXL, respectively. The selectivity of the
NodA proteins can be explained either by a specific
recognition of the respective fatty acids or, more likely,
by a specific protein–protein interaction with the
specialized ACP carrying such a fatty acid. It has been
shown that acyl-AcpP from E. coli (but not free fatty
acids) can function as an acyl donor in NodA-directed
fatty acid transfer (Ritsema et al., 1997), suggesting that
NodA might interact with ACP for the transfer of the
fatty acid. A more detailed study of NodA specificity
will now be possible using the functionally different
ACPs of rhizobia.
Post-translational modification with 4h-phosphopantetheine is required to render ACPs functionally active in
fatty acid biosynthesis. The in vivo labelling experiment
with β-alanine indicates that the post-translational
modification of adding the prosthetic group to the apoform of the different rhizobial ACPs can be performed
by an enzyme activity present in E. coli. However, the in
vivo substitution of the overexpressed ACPs is in no way
complete (Fig. 3a, lanes 1 and 5). Purified holo-ACP
synthase (AcpS) of E. coli (Lambalot & Walsh, 1995) is
able to transfer in vitro 4h-phosphopantetheine to the
specialized rhizobial ACPs NodF (Ritsema et al., 1998)
and RkpF (Epple et al., 1998). To quantify the in vitro
reaction between apo-ACPs and AcpS, we are presently
determining the kinetic constants (KM, kcat) for each one
of the rhizobial ACPs as substrates.
To use ACPs in biochemical studies, the holo-ACPs
should be thioesterified to particular fatty acyl residues
(or modified fatty acyl residues). We are presently
carrying out different studies in order to establish a
feasible and (if possible) general method for the
acylation of all the ACPs described here. A chemical
method for acylation of ACPs has been described
(Cronan & Klages, 1981), but preferably, simpler
enzymic methods should be used. Acyl-ACP synthetase
(Aas) of E. coli is the enzyme that can acylate E. coli
AcpP (Ray & Cronan, 1976). AcpP from S. meliloti
functions as effective substrate for E. coli Aas (Platt et
al., 1990), whilst NodF is not a good substrate for the E.
coli Aas (Ritsema et al., 1998). Therefore, it seems that
E. coli acyl-ACP synthetase has a substrate specificity
that is narrower than that of E. coli AcpS and can only
be used for acylation of constitutive ACPs. The Vibrio
harveyi acyl-ACP synthetase seems to have a broader
specificity than its E. coli counterpart (Fice et al., 1993 ;
Shen et al., 1992). It will be interesting to see if the V.
harveyi enzyme can acylate the specialized ACPs of
rhizobia.
Another enzymic approach might be to use E. coli AcpS
for loading altered phosphopantetheine moieties, from
CoA analogues, directly to the apo-ACP. Such a method
has been successful in the preparation of acylated
derivatives of Streptomyces ACPs (Gehring et al., 1997).
ACKNOWLEDGEMENTS
We thank Viola Ro$ hrs for excellent technical assistance.
I. M. L. L. was the recipient of a long-term EMBO fellowship.
This work was supported by grant Ge556\3-1 from the
Deutsche Forschungsgemeinschaft.
REFERENCES
Altschul, S. F., Madden, T. L., Scha$ ffer, A. A., Zhang, J., Zhang, Z.,
Miller, W. & Lipman, D. J. (1997). Gapped  and - : a
new generation of protein database search programs. Nucleic
Acids Res 25, 3389–3402.
Atkinson, E. M., Palcic, M. M., Hindsgaul, O. & Long, S. R. (1994).
Biosynthesis of Rhizobium meliloti lipooligosaccharide Nod
factors : NodA is required for an N-acyltransferase activity. Proc
Natl Acad Sci USA 91, 8418–8422.
Bibb, M. J., Biro, S., Motamedi, H., Collins, J. F. & Hutchinson,
C. R. (1989). Analysis of the nucleotide sequence of the Strepto-
myces glaucescens tcmI genes provides key information about the
enzymology of polyketide antibiotic biosynthesis. EMBO J 8,
2727–2736.
Brozek, K. A., Carlson, R. W. & Raetz, C. H. R. (1996). A special
acyl carrier protein for transferring long hydroxylated fatty acids
to lipid A in Rhizobium. J Biol Chem 271, 32126–32136.
Cronan, J. E. & Klages, A. L. (1981). Chemical synthesis of acyl
thioester of acyl carrier protein with native structure. Proc Natl
Acad Sci USA 78, 5440–5444.
Debelle! , F., Plazanet, C., Roche, P., Pujol, C., Savagnac, A.,
847
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 02:21:32
I . M . L O! P E Z -L A R A a n d O . G E I G E R
Rosenberg, C., Prome! , J. C. & De! narie! , J. (1996). The NodA
F. M. (1982). Physical and genetic characterization of symbiotic
proteins of Rhizobium meliloti and Rhizobium tropici specify the
N-acylation of Nod factors by different fatty acids. Mol Microbiol
22, 303–314.
De! narie! , J., Debelle! , F. & Prome! , J. C. (1996). Rhizobium lipochitooligosaccharide nodulation factors : signaling molecules
mediating recognition and morphogenesis. Annu Rev Biochem
65, 503–535.
and auxotrophic mutants of Rhizobium meliloti induced by
transposon Tn5 mutagenesis. J Bacteriol 149, 114–122.
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold
Spring Harbor, NY : Cold Spring Harbor Laboratory.
Østera/ s, M., Stanley, J. & Finan, T. M. (1995). Identification of
Rhizobium-specific intergenic mosaic elements within an essential
two-component regulatory system of Rhizobium species. J
Bacteriol 177, 5485–5494.
Epple, G., van der Drift, K. M. G. M., Thomas-Oates, J. E. &
Geiger, O. (1998). Characterization of a novel acyl carrier protein,
RkpF, encoded by an operon involved in capsular polysaccharide
biosynthesis in Sinorhizobium meliloti. J Bacteriol 180,
4950–4954.
Fice, D., Shen, Z. & Byers, D. (1993). Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio
harveyi. J Bacteriol 175, 1865–1870.
Fuqua, C., Winans, S. C. & Greenberg, E. (1996). Census and
consensus in bacterial ecosystems : the LuxR-LuxI family of
quorum sensing transcriptional regulators. Annu Rev Microbiol
50, 727–751.
Gehring, A. M., Lambalot, R. H., Vogel, K. W., Drueckhammer,
D. G. & Walsh, C. T. (1997). Ability of Streptomyces spp. acyl
carrier proteins and coenzyme A analogs to serve as substrates in
vitro for E. coli holo-ACP synthase. Chem Biol 4, 17–24.
Geiger, O., Ro$ hrs, V., Weissenmayer, B., Finan, T. M. & ThomasOates, J. E. (1999). The regulator gene phoB mediates phosphate
stress-controlled synthesis of the membrane lipid diacylglycerylN,N,N-trimethylhomoserine in Rhizobium (Sinorhizobium)
meliloti. Mol Microbiol 32, 63–73.
Ghose, R., Geiger, O. & Prestegard, J. H. (1996). NMR investigations of the structural properties of the nodulation protein,
NodF, from Rhizobium leguminosarum and its homology with
Escherichia coli acyl carrier protein. FEBS Lett 388, 66–72.
Hill, R. B., MacKenzie, K. R., Flanagan, J. M., Cronan, J. E. &
Prestegard, J. H. (1995). Overexpression, purification, and
characterization of Escherichia coli acyl carrier protein and two
mutant proteins. Protein Expr Purif 6, 394–400.
Hooykaas, P. J. J., van Brussel, A. N. N., den Dulk-Raas, H., van
Slogteren, G. M. S. & Schilperoort, R. A. (1981). Sym plasmid of
Rhizobium trifolii expressed in different rhizobial species and
Agrobacterium tumefaciens. Nature 291, 351–353.
Issartel, J.-P., Koronakis, V. & Hughes, C. (1991). Activation of
Escherichia coli prehaemolysin to the mature toxin by acyl carrier
protein-dependent fatty acylation. Nature 351, 759–761.
Jackowski, S. & Rock, C. O. (1983). Ratio of active to inactive
forms of acyl carrier protein in Escherichia coli. J Biol Chem 258,
15186–15191.
Jackowski, S., Cronan, J. E., Jr & Rock, C. O. (1991). Lipid
metabolism in procaryotes. In Biochemistry of Lipids, Lipoproteins and Membranes, pp. 43–85. Edited by D. E. Vance & J.
Vance. Amsterdam : Elsevier.
Kim, Y. & Prestegard, J. H. (1990). Refinement of the NMR
structures for acyl carrier protein with scalar coupling data.
Proteins 8, 377–385.
Lambalot, R. H. & Walsh, C. T. (1995). Cloning, overproduction,
and characterization of the Escherichia coli holo-acyl carrier
protein synthase. J Biol Chem 270, 24658–24661.
Maiti, M. K. & Ghosh, S. (1996). Acyl carrier protein of Azospirillum brasilense : properties of the purified protein and
sequencing of the corresponding gene, acpP. Microbiology 142,
2097–2103.
Meade, H. M., Long, S. R., Ruvkun, G. B., Brown, S. E. & Ausubel,
Petrovics, G., Putnoky, P., Reuhs, B., Kim, J., Thorp, T. A., Noel,
K. D., Carlson, R. W. & Kondorosi, A. (1993). The presence of a
novel type of surface polysaccharide in Rhizobium meliloti requires a new fatty acid synthase-like gene cluster involved in
symbiotic nodule development. Mol Microbiol 8, 1083–1094.
Platt, M. W., Miller, K. J., Lane, W. S. & Kennedy, E. P. (1990).
Isolation and characterization of the constitutive acyl carrier
protein from Rhizobium meliloti. J Bacteriol 172, 5440–5444.
Rawlings, M. & Cronan, J. E. (1992). The gene encoding
Escherichia coli acyl carrier protein lies within a cluster of fatty
acid biosynthetic genes. J Biol Chem 267, 5751–5754.
Ray, T. K. & Cronan, J. E. (1976). Activation of long chain fatty
acyl carrier protein : demonstration of a new enzyme, acyl-acyl
carrier protein synthetase, in Escherichia coli. Proc Natl Acad Sci
USA 73, 4374–4378.
Reuhs, B. L. (1996). Acidic capsular polysaccharides (K antigens)
of Rhizobium. In Biology of Plant–Microbe Interactions, pp.
331–336. Edited by G. Stacey, B. Mullin & P. M. Gresshoff. St
Paul, MN : International Society for Molecular Plant–Microbe
Interactions.
Ritsema, T., Geiger, O., van Dillewijn, P., Lugtenberg, B. J. J. &
Spaink, H. P. (1994). Serine residue 45 of nodulation protein
NodF from Rhizobium leguminosarum bv. viciae is essential for
its biological function. J Bacteriol 176, 7740–7743.
Ritsema, T., Wijfjes, A. H. M., Lugtenberg, B. J. J. & Spaink, H. P.
(1996). Rhizobium nodulation protein NodA is a host-specific
determinant of the transfer of fatty acids in Nod factor
biosynthesis. Mol Gen Genet 251, 44–51.
Ritsema, T., Lugtenberg, B. J. J. & Spaink, H. P. (1997). Acyl-acyl
carrier protein is a donor of fatty acids in the NodA-dependent
step in biosynthesis of lipochitin oligosaccharides by rhizobia. J
Bacteriol 179, 4053–4055.
Ritsema, T., Gehring, A. M., Stuitje, A. R. & 7 other authors
(1998). Functional analysis of an interspecies chimera of acyl
carrier proteins indicates a specialized domain for protein
recognition. Mol Gen Genet 257, 641–648.
Ro$ hrig, H., Schmidt, J., Wieneke, U., Kondorosi, E., Barlier, I.,
Schell, J. & John, M. (1994). Biosynthesis of lipooligosaccharide
nodulation factors : Rhizobium NodA protein is involved in Nacylation of the chitooligosaccharide backbone. Proc Natl Acad
Sci USA 91, 3122–3126.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular
Cloning : a Laboratory Manual, 2nd edn. Cold Spring Harbor,
NY : Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing
with chain-terminating inhibitors. Proc Natl Acad Sci USA 74,
5463–5467.
Shearman, C. A., Rossen, L., Johnston, A. W. B. & Downie, J. A.
(1986). The Rhizobium leguminosarum nodulation gene nodF
encodes a polypeptide similar to acyl carrier protein and is
regulated by nodD plus a factor in pea root exudate. EMBO J 5,
647–652.
Shen, Z., Fice, D. & Byers, D. M. (1992). Preparation of fatty-
848
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 02:21:32
Rhizobial acyl carrier proteins
acylated derivatives of acyl carrier protein using Vibrio harveyi
acyl-ACP synthetase. Anal Biochem 204, 34–39.
Sherman, D. H., Malpartida, F., Bibb, M. J., Kieser, H. M., Bibb,
M. J. & Hopwood, D. A. (1989). Structure and deduced function
of the granaticin-producing polyketide synthase gene cluster of
Streptomyces violaceoruber Tu22. EMBO J 8, 2717–2725.
Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W.
(1990). Use of T7 RNA polymerase to direct expression of cloned
Yang, G.-P., Debelle! , F., Savagnac, A. & 9 other authors (1999).
Structure of the Mesorhizobium huakuii and Rhizobium galegae
Nod factors : a cluster of phylogenetically related legumes are
nodulated by rhizobia producing Nod factors with α,β-unsaturated N-acyl substitutions. Mol Microbiol 34, 227–237.
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13
phage cloning vectors and host strains : nucleotide sequences of
the M13mp18 and pUC19 vectors. Gene 33, 103–119.
genes. Methods Enzymol 185, 60–89.
Triglia, T., Peterson, M. G. & Kemp, D. J. (1988). A procedure for
.................................................................................................................................................
in vitro amplification of DNA segments that lie outside the
boundaries of known sequences. Nucleic Acids Res 16, 8186.
Received 23 September 1999 ; revised 4 January 2000 ; accepted
13 January 2000.
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