Báo cáo Y học: The opgGIH and opgC genes of Rhodobacter sphaeroides form an operon that controls backbone synthesis and succinylation of osmoregulated periplasmic glucans pot

The
opgGIH
and
opgC
genes of
Rhodobacter sphaeroides
form
an operon that controls backbone synthesis and succinylation
of osmoregulated periplasmic glucans
Virginie Cogez
1
, Evgueni Gak
2
, Agnes Puskas
2
, Samuel Kaplan
2
and Jean-Pierre Bohin
1
1
Unite
´
de Glycobiologie Structurale et Fonctionnelle, CNRS UMR8576, Universite
´
des Sciences et Technologies de Lille, France;
2
Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, TX, USA
Osmoregulated periplasmic glucans (OPGs) of Rhodobacter
sphaeroides are anionic cyclic molecules that accumulate in
large amounts in the periplasmic space in response to low
osmolarity of the medium. Their anionic character is pro-

vided by the substitution of the glucosidic backbone by
succinyl residues. A wild-type strain was subject to trans-
poson mutagenesis, and putative mutant clones were
screened for changes in OPGs by thin layer chromatogra-
phy. One mutant deficient in succinyl substitution of the
OPGs was obtained and the gene inactivated in this mutant
was characterized and named opgC. opgC is located
downstream of three ORFs, opgGIH, two of which are
similar to the Escherichia coli operon, mdoGH, governing
OPG backbone synthesis. Inactivation of opgG, opgI or
opgH abolished OPG production and complementation
analysis indicated that the three genes are necessary for
backbone synthesis. In contrast, inactivation of a gene
similar to ndvB, encoding the OPG-glucosyl transferase in
Sinorhizobium meliloti, had no consequence on OPG syn-
thesis in Rhodobacter sphaeroides. Cassette insertions in
opgH had a polar effect on glucan substitution, indicating
that opgC is in the same transcription unit. Expression of
opgIHC in E. coli mdoB/mdoC and mdoH mutants allowed
the production of slightly anionic and abnormally long
linear glucans.
Keywords: periplasm; osmoregulation; cyclic glucans;
glucosyl transferase; operon.
Osmoregulated periplasmic glucans (OPGs) are found in
the periplasmic space of proteobacteria [1]. These oligosac-
charides exhibit quite different structures among various
species but they share four common characteristics: (a) a
small size, with a degree of polymerization (DP) in the range
of 5–24; (b)
D

-glucose being the only sugar unit; (c)
b-glucosidic bonds being the main type of linkages; (d) the
periplasmic concentration increasing in response to a
decrease of environmental osmolarity. Four families of
OPGs are described on the basis of structural features of the
polyglucose backbone [1]: family I, heterogeneously sized
linear and branched b-1,2;b-1,6 glucans; family II, hetero-
geneously sized cyclic b-1,2 glucans; family III, homogen-
eously sized cyclic and branched b-1,3;b-1,6 glucans;
family IV, homogeneously sized cyclic b-1,2;a-1,6 glucans.
In several bacterial species, OPGs are substituted by one or
several of a series of different residues, originating from
either the membrane phospholipids (phosphoglycerol,
phosphoethanolamine, and phosphocholine) or from inter-
mediate metabolism (acetyl, succinyl, and methylmalonyl).
Thus, depending on the bacterial strain and growth
conditions, OPGs can be found unsubstituted, neutral or
anionic.
The function of OPGs in the bacterial envelope remains
obscure. However, mutants defective in OPG synthesis have
a highly pleiotropic phenotype, indicative of an overall
alteration of their envelope properties. When some bacteria
interact with a eucaryotic host, as pathogens or symbionts,
mutants defective in backbone synthesis are partially or
completely impaired in this interaction [1]. This is the case
for mutants of Agrobacterium tumefaciens, Pseudomonas
syringae, Bradyrhizobium japonicum, P. aeruginosa, Erwinia
chrysanthemi and Brucella abortus [2–8]. One highly attenu-
ated Salmonella (enterica) typhimurium mutant resulted
from a transposon insertion in the mdoB gene known to

govern OPG substitution by phosphoglycerol in Escherichia
coli [9]. In contrast, a S. meliloti mutant impaired in OPG
substitution by phosphoglycerol effectively nodulated
alfalfa [10], but the anionic character of these OPGs was
more or less retained by an increase of succinyl substitution.
Obviously, further genetic analyses of different model
organisms are needed to understand the OPG function(s).
Rhodobacter sphaeroides is a free-living photohetero-
trophic bacterium of the alpha subdivision of the proteo-
bacteria, whose genome is composed of two distinct circular
chromosomes [11]. Genetic analysis is highly developed in
this organism, which is a model for the study of bacterial
photosynthesis [12]. R. sphaeroides produces OPGs belong-
ing to family IV. They mainly consist of a cyclic glucan
homogenous in size (DP
1
¼ 18) in which 17 glucose units
Correspondence to J P. Bohin, CNRS UMR8576, Baˆ t.C9, U.S.T.L.,
59655 Villeneuve d’Ascq Cedex, France.
Fax: + 33 3 20 43 65 55, Tel.: + 33 3 20 43 65 92,
E-mail:
Abbreviations: DP, degree of polymerization; SIS, Sistrom’s succinic
acid minimal medium; LOS, low-osmolarity medium; Amp,
ampicillin; Kan, kanamycin; Rif, rifampicin; Spc, spectinomycin;
Str, streptomycin; Tet, tetracycline; Tmp, trimethoprim; Cml,
chloramphenicol; MP, maximum-parsimony; TMS, transmembrane
segment; ACP, acyl carrier protein.
(Received 19 October 2001, revised 11 March 2002,
accepted 22 March 2002)
Eur. J. Biochem. 269, 2473–2484 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02907.x

are linked by b-1,2 linkage and one glucose unit is linked
by a-1,6 linkage. This backbone is substituted to various
degrees by two kinds of residues: O-acetyl residues (0–2 per
mol) and O-succinyl residues (1–7 per mol) that confer a
highly anionic character to these OPGs [13]. Because
R. sphaeroides shows a close relationship to those organisms
that share an interactive lifestyle with a eucaryotic host, but
itself does not, it represents a model organism in which to
study OPG synthesis.
Our initial purpose was to obtain OPG defective mutants
by screening a transposon insertion library with a thin layer
chromatographic assay [14]. A single mutant clone was
detected that produced neutral OPGs. Actually, this partic-
ular phenotype allowed the demonstration of acetyl substi-
tution of the OPGs [13]. The transposon insertion was
located just downstream of a series of genes similar to the
E. coli mdoGH operon that govern the synthesis of OPGs
belonging to family I. In this paper, we describe the
molecular and functional characterization of these genes,
opgGIH, necessary for OPG backbone synthesis and of a
new gene, opgC, necessary for OPG succinylation.
MATERIALS AND METHODS
Bacterial strains and media
The bacterial strains and plasmids used are detailed in
Table 1. R. sphaeroides strains were grown at 30 °Cin
Sistrom’s succinic acid minimal medium (SIS; [22]); anaero-
bically, illuminated at 100 WÆm
)2
. To determine OPG
production, aerobic chemoheterotrophic cultures were

grown, with shaking, in Luria–Bertani broth [23]. E. coli
strains were grown at 37 °C in Luria–Bertani. When low
osmolarity medium was required, Luria–Bertani without
NaCl or low-osmolarity medium (LOS; [15]) was used.
Solid media were obtained by adding agar (15 gÆL
)1
).
Antibiotics were added to the medium at the following
concentrations: ampicillin (Amp), 100 lgÆL
)1
; kanamycin
(Kan), 25 lgÆL
)1
;rifampicin(Rif)100lgÆL
)1
; spectino-
mycin (Spc), 50 lgÆL
)1
; streptomycin (Str), 50 lgÆL
)1
;tetra-
cycline (Tet), 1.0 lgÆL
)1
; trimethoprim (Tmp), 50 lgÆL
)1
for
R. sphaeroides and ampicillin, 50 lgÆL
)1
; chloramphenicol
(Cml), 25 lgÆL

)1
; kanamycin, 50 lgÆL
)1
; tetracycline,
10 lgÆL
)1
; trimethoprim, 50 lgÆL
)1
for E. coli.
Transformation and mating
E. coli cells were made competent and transformed using
the rubidium chloride technique [24]. The broad host range
plasmids (originating from pLA2917, pRK415 or pSUP202)
were mobilized from S17-1 into R. sphaeroides strains.
Matings were performed on nitrocellulose filters laid on
Luria–Bertani plates and the exconjugants selected on Tet,
Tmp or Kan Luria–Bertani plates, or SIS Str+Spc or SIS
Kan plates.
Transposon mutagenesis
The mobilizable suicide plasmid pSUPTn5TpMCS [25,26]
was introduced into R. sphaeroides WS8bymatingat30 °C
with E. coli S17-1 and spread onto Luria–Bertani Tmp Str
plates. After a 3-day incubation, clones of R. sphaeroides
containing transposon insertions (conferring trimethoprim
resistance) were picked and arranged to construct libraries
of putative mutants.
Thin-layer chromatographic screening method
Each Tn5TpMCS mutant generated from WS8 was
screened for production of OPGs as described previously:
4 mL of an overnight culture in Luria–Bertani without

NaCl were treated to give a 15-lL extract in water [14].
Samples of 5 lL were analyzed by chromatography on
aluminium silica gel 60 plates (Merk) in ethanol/butanol/
water (5 : 5 : 4) solvent. Glucans were revealed by spray-
ing dried plates with 0.2% orcinol in 20% sulfuric acid
followed by heating at 110 °C.Thesameprocedurewas
used for rapid determination of the OPG synthesis by the
various mutants obtained from strain 2.4.1. This proce-
dure was poorly quantitative and used only to check the
presence or absence of OPGs and their anionic or neutral
character.
DNA purification, restriction and modification enzymes
and ligase
Standard procedures [27] were used for large scale plasmid
isolation and rapid analysis of recombinant plasmids.
Genomic DNA extraction was done as described by Davis
et al. [28]. Restriction endonucleases (Eurogentec or Gibco
BRL), the large (Klenow) fragment of DNA polymerase I
and T4 DNA ligase (Gibco BRL) were used according to
manufacturer’s recommendations.
In vitro
construction of plasmids
For the sequencing of a fragment of the opgC gene, a
genomic DNA fragment of the opgC1::Tn5TpMCS strain
NFB4000 was cloned in the EcoRI site of plasmid pUC19.
This restriction enzyme cut once in the Tn5TpMCS DNA
outside the gene conferring trimethoprim resistance to the
transposon. Trimethoprim-resistant clones harboring a
plasmid containing a 6-kb DNA insert were isolated on
plates containing trimethoprim and ampicillin. In this

plasmid, called pNFR2, a translational fusion occurred
fortuitously between the eighteenth codon of the a-lacZ
fragment present in the vector and the third codon of opgI,
while opgH was intact and opgC inactivated.
For complementation tests in R. sphaeroides,a4.5-kb
SalI fragment from cosmid pUI8166 [21], containing an
intact copy of opgC (opgH being truncated), was inserted
into the SalI site of pUC19 to give pNFR12. The 4.5 kb
fragment was then liberated by digesting pNFR12 with
HindIII and KpnI and inserted into the broad host rang
mobilizable vector pRK415 [18] digested with HindIII and
KpnI, to give pNFR13 (Fig. 3).
A2.2-kbEcoRV–BglII fragment from pUI8166, con-
taining an intact copy of opgH, was inserted into Litmus
28 (Amp
r
; New England Biolabs), to give pGAK115.
Then, the 2.2-kb fragment was liberated by digesting
pGAK115 with HindIII and BglII, and inserted into
pRK415 digested with HindIII and BamHI, to give
pGAK136 (Fig. 3).
A3.5SalI fragment from cosmid pUI8166, containing an
intact copy of opgGI, was inserted into the SalIsiteofpBSII
SK(+) (Amp
r
; Statagene), to give pUI2509. Then, the
2474 V. Cogez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
3.5 kb was liberated by digesting pUI2509 with HindIII and
KpnI,andinsertedintopRK415digestedwithHindIII and
KpnI, to give pGAK247 (Fig. 3).

A1.2-kbBglII–KpnI fragment from pUI2509, containing
an intact copy of opgI, was inserted into the BamHI site of
pRK415, to give pGAK135 (Fig. 3).
A1.8-kbBamHI fragment from pUI2509, containing an
intact copy of opgI, was inserted into the BamHI site of
pRK415, to give pGAK245 (opgI in the tet promoter
orientation) and pGAK246 (opgI opposite to the tet
promoter orientation; Fig. 3).
A5-kbApaI–BalI fragment from cosmid pUI8166,
containing an intact copy of opgGIH (opgC being truncated),
was blunt ended and inserted into the SmaI site of pUC19, to
give pNFR14. A 5-kb fragment was liberated by digesting
pNFR14 with HindIII and KpnI,andinsertedintopRK415
digested with HindIII and KpnI, to give pNFR21 (Fig. 3).
A2.0-kbHindIII–EcoRV fragment from pNFR14 was
inserted between the HindIII and SmaI sites of pUC19 to
give pNFR15. A 2.0-kb fragment was liberated by digesting
pNFR15 with HindIII and KpnI,andinsertedintopRK415
digested with HindIII and KpnI, to give pNFR20 (Fig. 3).
A1.8-kbStuI–SmaI fragment from pNFR12, containing
opgC, was inserted into the SmaI site of pUC19 to give
pNFR18. A 1.8-kb fragment was liberated by digesting
pNFR18 with KpnIandSmaI,andinsertedintothe
expression vector pYZ4 to give pNFR25 (Fig. 3).
A3-kbEcoRI fragment from pNF14, containing opgIH,
was inserted into the EcoRI site of pUC19 in the same
orientation as a-lacZ, to give pNFR35 (Fig. 3). A 2.2-kb
HindIII–BglII from pNFR35 and a 1.2-kb BglII–EcoRI
from pNFR25 were ligated and inserted into the site created
by digestion with HindIII and EcoRI of pUC19, to give

pNFR37 (Fig. 3). This plasmid is similar to pNFR2 except
that opgC is now intact.
A4-kbEcoRI–HindIII fragment from pNFR2 was blunt
ended and inserted into the SmaI site of pYZ4, to give
pNFR30.
Table 1. Bacterial strains and plasmids used in this study.
Strain or plasmid Relevant genotype Source or reference
Escherichia coli
S17-1 thi pro hsdR
–
hsdM
+
recA RP4 plasmid integrated Tc::Mu-Km::Tn7 [15]
DH5a F
–
recA1 endA1 gyrA96 thi-1 hsdR17 glnV44 relA1 DlacU169 k (/80 dlacZDM15) Lab stock
NFB216 D(lac-pro) ara mdoH200::Tn10 pyrC46 rpsL thi (/80 dlacZDM15) [16]
NFB702 D(lac-pro) ara mdoG202::neo pyrC46 rpsL thi (/80 dlacZDM15) [16]
NFB1933 his pgi::Mu D(zwf-edd)1 eda-1 rpsL mdoB214::Tn10 mdoC1::Tn5 [14]
NFB1100 pNF309/NFB216 [4]
NFB4234 pNFR30/NFB216 This work
NFB4245 pNFR37/NFB1933 This work
Rhodobacter sphaeroides
WS8 Wild type, Str
r
strain Laboratory stock
NFB4000 WS8 opgC1::Tn5TpMCS This work
2.4.1 Wild type Laboratory stock
AP5 2.4.1 opgG5::W (Str
r

, Spc
r
) This work
EG7 2.4.1 opgH7::kan, opposite to gene orientation This work
EG13 2.4.1 opgI13::kan, gene orientation This work
EG18 2.4.1 opgH18::kan, gene orientation This work
EG131 2.4.1 opgI131::kan, opposite to gene orientation This work
EG238 2.4.1 ndvB238::W (Str
r
, Spc
r
) This work
Plasmids
pHP45W Source of the W interposon (Str
r
, Spc
r
) [17]
pRK415 Tet
r
, broad host range mobilizable vector [18]
pUC19 Amp
r
, cloning vector [19]
pUC4K Source of the kan cassette (Kan
r
) Pharmacia
pYZ4 Kan
r
, cloning vector [20]

pUI8166 pLA2917-derived cosmid clone from R. sphaeroides 2.4.1
T
library [21]
pNF309 Kan
r
, pYZ4 carrying mdoH
+
[4]
pGAK135 Tet
r
, pRK415 carrying opgI
+
This work
pGAK136 Tet
r
, pRK415 carrying opgH
+
This work
pGAK245 Tet
r
, pRK415 carrying opgI
+
(in the tet promoter orientation) This work
pGAK246 Tet
r
, pRK415 carrying opgI
+
(opposite of the tet promoter orientation) This work
pGAK247 Tet
r

, pRK415 carrying opgGI
+
This work
pNFR13 Tet
r
, pRK415 carrying opgC
+
This work
pNFR20 Tet
r
, pRK415 carrying opgG
+
This work
pNFR21 Tet
r
, pRK415 carrying opgGIH
+
This work
pNFR25 Kan
r
, pYZ4 carrying opgC
+
This work
pNFR30 Kan
r
, pYZ4 carrying opgIH
+
This work
pNFR35 Amp
r

, pUC19 carrying opgIH
+
This work
pNFR37 Amp
r
, pUC19 carrying opgIHC
+
This work
Ó FEBS 2002 The opgGIHC operon of R. sphaeroides (Eur. J. Biochem. 269) 2475
Construction of mutants
A2.6-kbEcoRI fragment from pUI2509 was inserted into a
pBS II SK(+) derivative in which the BamHI was filled in,
to give pUI2511. The W interposon was liberated by
digesting pHP45W with BamHI and inserted in the unique
BamHI of pUI2511. The construct, containing opgG
disrupted 244 bp from its predicted start codon, was
subcloned as a 4.6-kb EcoRI fragment into the EcoRI site
of the broad host rang mobilizable vector pSUP202 [15] to
give pUI2513. pUI2513 was mobilized from S17-1 into
R. sphaeroides 2.4.1. Exconjugants were selected on SIS
Str+Spc plates and the structure of the Tet-sensitive clones
was confirmed by Southern hybridization [29].
A1.3-kbEco47III fragment was deleted from pUI2509
to give pGAK112. Then, a 2.1-kb SalI fragment from
pGAK112 was inserted into the SalI site of pUC19 to give
pGAK114. A kan cassette was liberated as a 1.2-kb SalI
fragment from pUC4K (Pharmacia), and inserted (in both
orientations) into the unique XhoI site of pGAK114. The
two resulting constructs, containing opgI disrupted 19 bp
from its predicted start codon, were subcloned as 3.8 kb

AatII-Eco47III fragments between the ScaIandAatII sites
of the broad host range mobilizable vector pSUP202D
(M. Gomelsky, Department of Microbiology and Molecu-
lar Genetics, University of Texas Health Center, Houston,
Texas, USA)
2
to give pGAK118 (kan and opgI in the same
orientation) and pGAK119 (kan and opgI in opposite
orientations). The two plasmids were mobilized into
R. sphaeroides 2.4.1. Exconjugants were selected on SIS
Kan plates and the structure of the Tet-sensitive clones was
confirmed by Southern hybridization.
The 1.2 kb SalI fragment, containing the kan cassette
described above, was inserted (in both orientations) into the
unique SalI site of pGAK115. The two resulting constructs,
containing opgH disrupted 609 bp from its predicted start
codon, were subcloned as 3.5 EcoRV–SnaBI fragments into
the ScaI site of pSUP202D to give pGAK120 (kan and
opgH in the same orientation) and pGAK121 (kan and
opgH in opposite orientations). The two plasmids were
mobilized into R. sphaeroides 2.4.1. Exconjugants were
selected on SIS Kan plates and the structure of the Tet
sensitive clones was confirmed by Southern hybridization.
A9.5-kbSacI–KpnI cosmid DNA fragment mapping
to contig 12 of the R. sphaeroides 2.4.1 genome (http://
mmg.uth.tmc.edu/sphaeroides/) was inserted into Litmus 28
to give pGAK227. This DNA sequence contains a portion
of the ndvB gene beginning 569 nucleotides downstream of
the purported ndvB start codon. The W interposon was
liberated by digesting pHP45W with SmaI and combined

with the 8.7 kb MscI fragment of pGAK227 to give
pGAK232. A 7.9-kb EcoRI fragment from pGAK232 was
subcloned into pSUP202 (pGAK238) and mobilized into
R. sphaeroides 2.4.1. Exconjugants were selected on SIS
Str+Spc plates and the structure of the Tet-sensitive clones
was confirmed by Southern hybridization.
DNA sequencing
Small scale DNA sequencing was carried out with the
Sequenase version 2.0 kit (USB corporation) except
for the Tn5 insertion point where the oligonucleotide
5¢-CATGGAAGTCAGATCCTGG-3¢ (Eurogentec), cor-
responding to the end of both IS50 delineating Tn5 was
used as a primer.
The opgGIHC DNA sequence was determined by primer
walking and performed at the DNA Core Facility of the
Department of Microbiology and Molecular Genetics
(University of Texas Health Science Center, Houston,
Texas, USA), on an ABI 377 automatic DNA sequencer
using the Big Dye terminator sequencing kit (Perkin-Elmer,
Applied Biosystem Division). Gibco BRL and Integrated
DNA Technology synthesized custom primers. Assembly
and analysis of the DNA sequences were performed using
the DNA Strider (Institut de Recherche Fondamentale,
Commisariat a
`
l’Energie Atomique, Paris, France),
PHRED
AND PHRAP
(CodonCode Corporation) and
GCG

(Genetics
Computer Group, Wisconsin Package, Madison, Wiscon-
sin, USA) softwares. The opgGIHC nucleotide sequence has
been deposited in GenBank under accession no. AF016298.
The DNA sequences and deduced amino-acid seq-
uences were analyzed by using computer programs and
sequence data made freely available from Infobiogen
( and from ERGO (http://wit.
integratedgenomics.com/IGwit/CGI/).
A preliminary alignment of the full-length sequences of
MdoH homologues was generated by
CLUSTAL W
,using
default gap penalties. The
CLUSTAL W
alignment was then
refined by manually deleting N- and C-terminal noncon-
served sequences. However, the P1 domain [30], which is
almost absent in a series of MdoH homologues, was
considered as phylogenetically relevant and included (in
MdoH, 637 out of the 847 amino acids were thus
considered). Phylogenetic trees were constructed by using
maximum-parsimony (MP) and neighbor-joining methods.
The MP analyses used the program
PROTPARS
implemented
in
PHYLIP
(Phylogeny Inference Package, Joe Felsenstein,
Department of Genetics at the University of Washington).

The
PHYLIP
programs
SEQBOOT
,
PROTPARS
,and
CONSENSE
were used sequentially to generate an MP tree that was
replicated in 100 bootstraps; on this basis bootstrap
confidence levels were determined.
Analysis of OPGs from
R. sphaeroides
Cultures (100–500 mL) of R. sphaeroides were grown
overnight in Luria–Bertani without NaCl. After 20 min
centrifugation at 10 000 g, OPGs were extracted by 70%
ethanol from the cell pellets. The extracts were concentrated
by rotary evaporation, and lipids and proteins were then
removed by the addition of a mixture of chloroform and
methanol (2 : 1). The aqueous phase, containing OPGs, was
chromatographied on a Biogel P4 column (Bio-Rad). The
column (1.5 cm in diameter, 68 cm in height) was equili-
brated with acetic acid 0.5% and eluted at a rate of
15 mLÆh
)1
in the same buffer. Fractions (1.5 mL) contain-
ing OPGs were pooled, concentrated by rotary evaporation,
desalted on a Biogel P2 column (Bio-Rad), and fractions
containing OPGs were pooled and lyophilized. Sugar
content was determined colorimetrically by using the

anthrone-sulfuric acid reagent procedure [27].
Analysis of OPGs from
E. coli
Strain NFB1933 and its derivatives were grown in LOS
medium (5 mL) supplemented with 0.24 m
MD
-[U-
14
C]glu-
2476 V. Cogez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
cose (125 MBqÆmmol
)1
). OPGs were extracted by the char-
coal adsorption procedure [15]. Pyridine extract obtained by
this procedure was chromatographied on a Biogel P4
column (Bio-Rad) and then on DEAE-Sephacel column
(Pharmacia).
Determination of neutral and anionic characteristics
of OPGs
OPGs were desalted on a PD10 column (Pharmacia) and
equilibrated with a Tris/HCl 10 m
M
pH 7.4 buffer. OPG-
containing fractions were pooled and chromatographied on
a DEAE-Sephacel (Pharmacia) column (1.5 cm in diameter,
38 cm in height) equilibrated with Tris/HCl 10 m
M
pH 7.4
and eluted with the same buffer containing increasing
concentrations of NaCl ranging from 0 to 0.2

M
by steps of
0.05
M
. A volume of 60 mL was used for each NaCl
concentration and the volume of each collected fraction was
4mL.
Matrix-assisted laser desorption-ionization (MALDI)
mass spectrometry (MS)
To remove all substituents, 250 lg glucose equivalent of
lyophilized OPGs from E. coli were dissolved in 100 lL
of fluorohydric acid (HF) and left for 60 h at 4 °C.
OPGs were then neutralized by the addition of seven
volumes of saturated lithium hydroxide (LiOH) solution.
The LiF precipitate was separated by centrifugation and
washed several times. The different supernatants were
pooled and neutralized with AG 50 W-X8 (H
+
form,
Bio-Rad), and then desalted on a Biogel P2 column (Bio-
Rad). Fractions containing OPGs were pooled and
lyophilized.
For removal of the succinyl and acetyl substituents,
OPGs from R. sphaeroides were de-esterified in 0.1
M
KOH
at 37 °C for 1 h. After neutralization with AG 50 W-X8
(H + form, Bio-Rad), the samples were desalted on a Bio-
Gel P-2 column.
The matrix used for carbohydrate analysis was 3-amino-

quinolin (10 gÆL
)1
in water; [13]). Lyophilized oligosac-
charides samples were redissolved in doubly distilled water
andthendilutedwithanappropriatevolumeofthematrix
solution (1 : 5, v/v). One microliter of the resulting solution
was deposited onto a stainless steel target, and the solvent
was evaporated under gentle stream of warm air.
The experiments were carried out on a VISION 2000
(Finnigan MAT) time-of-flight mass spectrometer, as pre-
viously described [13].
RESULTS
Isolation of a mutant with a OPG altered phenotype
A random Tn5TpMCS mutagenesis was performed in
R. sphaeroides WS8 and glucans extracted from 436
random Tn5TpMCS insertion mutants were analyzed by
thin layer chromatography to find a clone (Fig. 1, lane 2)
whose OPGs showed a slower migration when compared to
the wild type (Fig. 1, lane 1). An isolated clone was called
NFB4000.
Mild alkali treatment of OPGs removes substituents
attached to the glucan backbone by O-ester linkages. As
expected, treated wild-type OPGs (Fig. 1, lane 3) exhibited
a reduced migration similar but not identical to that of
mutant OPGs (Fig. 1, lane 2). To observe identical
migration between the two types of OPGs, mild alkali
treatment of the mutant OPGs was necessary (Fig. 1, lane
4). The growth rates and growth yields of the two strains
grown in Luria–Bertani without NaCl were identical.
Accurate measurements showed that their OPG levels

were also identical (22 ± 2 lg of glucose equivalent per
mg of cell protein), indicating that the observed change
was not the consequence of a reduction in glucose
backbone synthesis.
OPGs isolated from the mutant are neutral
OPGs produced by the mutant and wild-type strains were
further analyzed by DEAE-Sephacel chromatography,
which allows for the separation of subfractions of glucan
by their anionic character. OPGs extracted from WS8 were
separated into five main subfractions eluted at increasing
NaCl concentration higher than 100 m
M
, showing their
highly anionic character (Fig. 2). OPGs produced by wild-
type R. sphaeroides are essentially homogeneous in size with
a degree of polymerization of 18 glucose residues [13]. These
are substituted to various degrees with succinyl residues that
Fig. 1. Thin layer chromatographic analysis of OPGs extracted from
WS8 (wild type, lanes 1 and 3) or NFB4000 (opgC1::Tn5TpMCS, lanes
2 and 4). Extracts (see Materials and methods) were applied directly to
thin-layer chromatography plates (lanes 1 and 2) or first subjected to
mild alkali treatment to remove substituents attached by O-ester
linkages (lanes 3 and 4). Arrows on the left side indicate the position of
three different levels of OPG substitution.
Ó FEBS 2002 The opgGIHC operon of R. sphaeroides (Eur. J. Biochem. 269) 2477
are negatively charged at pH 7.4, and acetyl residues that
are neutral. Thus, one could expect that each subfraction
separated by DEAE-Sephacel corresponded to an increas-
ing number of succinyl residues. The second, third and
fourth subfractions were collected separately, desalted and

analyzed by MALDI-mass spectrometry. This analysis
revealed that these subfractions were still heterogeneous
with different degrees of substitution by succinyl residues
(data not shown). Thus, we must consider that each
subfraction corresponded to a different charge-to-mass
ratio due to various levels of substitution by succinyl and
acetyl residues.
OPGs extracted from NFB4000 were not adsorbed on
the DEAE-Sephacel column, showing their neutral charac-
ter (Fig. 2). An accurate structural analysis revealed that the
OPGs from NFB4000 lacked succinyl residues but remained
substituted by acetyl residues [13]. The gene interrupted by
the Tn5TpMCS insertion was called opgC,byanalogywith
the mdoC gene that governs OPG succinylation in E. coli
[14].
opgC
lies downstream from a locus similar
to
mdoGH
of
E. coli
The opgC1::Tn5TpMCS mutation was cloned into the
pUC19 vector from genomic DNA using the trimethoprim
resistance conferred by the transposon as a selection.
Several clones were obtained that contained plasmids with
the same 6 kb insert in one or the other orientation. The
DNA sequence of this 6 kb insert was determined using a
primer from within the IS50 DNA (see Materials and
methods). This sequence was compared with the avail-
able sequence data from R. sphaeroides 2.4.1 (http://

mmg.uth.tmc.edu/sphaeroides/) and found 99% identical
with an ORF present in the vicinity of cerRI, a locus present
on chromosome I and governing the synthesis of an
acylhomoserine lactone signal [21]. Previous sequencing of
cosmid pUI8166 [21] revealed the presence upstream from
opgC of three ORFs, two of which (named opgG and opgH)
are very similar to the genes mdoG and mdoH of E. coli
(Fig. 3). In this organism, mdoG and mdoH form an operon
under osmotic control that governs the synthesis of linear
OPGs [16]. When pUI8166 was introduced into the mutant
strain NFB4000, restoration of the anionic character of
the OPGs was observed by thin layer chromatography
(Table 1). The opgC gene was further subcloned as a 4.5-kb
SalI fragment in plasmid pNFR13 that still complements
the NFB4000 defect.
Analysis of nucleotide sequence of opgGIHC
of
R. sphaeroides
The first ORF, opgG, encodes a 540-amino-acid polypep-
tide. OpgG starts with an AUG and no alternative initiation
codon (GUG or UUG) is found in its vicinity. Analysis of
the first 70 amino acids with the
SIGNALP
program (http://
www.cbs.dtu.dk/services/SignalP/) allowed the prediction
of a 38-amino-acid signal peptide and of a 502-amino-acid
mature protein. This protein is 40% identical and 58%
similar to the mature MdoG protein.
The second ORF was named opgI because it appeared to
be necessary to OPG backbone synthesis (see below). It

overlaps the opgG stop codon (TGATG) and is predicted to
encode a 66-amino-acid polypeptide. No similarity was
detected between OpgI and sequences available in the
databases, except with an ORF conserved in the opgGIHC
locus of R. capsulatus. This locus is 64% identical to its
R. sphaeroides counterpart over 5070 nucleotides. Thus, this
observation strengthens that hypothesis that the existence of
opgI is not the result of a sequencing error.
The third ORF, opgH, starts five nucleotides after the
opgI stop codon and encodes a 595-amino-acid polypeptide.
Thus, OpgH is shorter than MdoH (847 amino acids).
MdoH consists of three large cytoplasmic domains separ-
ated by eight transmembrane segments (TMS); the topology
is N-terminal, two TMS, central, six TMS, C-terminal
[30]. For both proteins, the
TOPPRED
2 program (http://
bioweb.pasteur.fr/seqanal/interfaces/toppred.html) predic-
ted seven TMS while the eighth segment was demonstrated
experimentally for MdoH [30]. Thus, OpgH exhibits the
same organization as MdoH with the major difference that
the N-terminal domain is almost absent and the C-terminal
domain is much shorter. Finally, within the conserved
regions, OpgH and MdoH are 40% identical and 70%
similar.
Tn5TpMCS was found to be inserted 747 bp down-
stream from the putative start codon of the fourth ORF,
opgC, encoding a putative 399-amino-acid polypeptide.
OpgG and OpgH present a high degree of sequence
similarities to MdoG and MdoH of E. coli. In contrast,

Fig. 2. DEAE-Sephacel anion exchange column chromatography pro-
files of OPGs from strains WS8 (Top) and NFB4000 (Bottom). 1200
(WS8) and 400 (NFB400) lg of glucose equivalent were loaded on the
column. Ionic strength was increased by steps of 0.05
M
NaCl at the
fractions indicated by the arrows. Fractions (4 mL) were collected and
sugar content was determined colorimetrically (see Materials and
methods). The concentration of each fraction is indicated as percent of
the total fractions.
2478 V. Cogez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
while OpgC and MdoC have similar sizes (399 and 385
amino acids, respectively), they do not show any significant
sequence similarities. However, OpgC and MdoC exhibit
stretches of hydrophobic amino acids over their entire
length. For MdoC [14], 10 TMS have been predicted by the
TOPPRED
2 program. For OpgC, the same program allowed
the prediction of 11 TMS.
Two inverted repeats of 12 bp (CGAAGGCACCCTC
ACGGGTGCCCTTGG) followed by TCGTTT are found
144 nucleotides downstream from the stop codon of opgC.
This could be an intrinsic transcription terminator as no
sizeable ORF starts before this point.
OpgG, OpgI, and OpgH are necessary to OPG backbone
synthesis, but not NdvB
A locus similar to ndvB, the gene governing OPG synthesis
in S. meliloti, has been partially described previously on
chromosome II [31]. Thus, the question was to determine
whether ndvB,oropgGIH, or both are necessary for OPG

synthesis in R. sphaeroides. Therefore, each of the four
genes were inactivated separately in R. sphaeroides 2.4.1 and
the resulting strains subjected to OPG analysis (see Mate-
rials and methods).
None of the mutants exhibited any particular phenotype
on plate when compared to the wild-type strain 2.4.1.
Growths of the various strains were compared in four
different liquid media (SIS, Luria–Bertani, Luria–Bertani
without NaCl, and LOS; see Materials and methods).
No differences were observed in the growth rates and the
growth yields of these cultures.
When OPGs were extracted from the ndvB mutant and
analyzed by thin layer chromatography, spots correspond-
ing to anionic OPGs were observed. These OPGs were
further purified, deesterified, and analyzed by MALDI-MS.
The resulting spectra (Fig. 4) revealed the presence of
one quasimolecular ion with the calculated mass for an
[M + Na]
+
ion based on an unsubstituted 18-member
cyclic glucan. The glucan produced seems to be mostly
homogeneous in size, and only minor species corresponding
to cyclic glucans composed of 16, 17, 19, 21, 22, 23, and 24
glucose residues are also present (Fig. 4). These data
allowed two major conclusions: (a) NdvB is not necessary
for OPG synthesis in R. sphaeroides; (b) OPG structures are
identical amongst various strains of R. sphaeroides as
identical spectra were obtained for OPGs extracted from
strains 2.4.1 and WS8 [13]. Similar results were previously
observed for different strains of X. campestris [13].

When extracts from the opgG, opgI,oropgH mutants
were analyzed by thin layer chromatography, no spots
corresponding to OPGs could be detected. These results
Fig. 3. Restriction map of a 8-kb fragment
present in cosmid pUI8166 and its derivatives.
Arrows indicated ORFs of opgGIHC.
Horizontal bars indicate the structure of the
various inserts of the relevant plasmids.
Fig. 4. Positive-ion MALDI mass spectra of OPGs extracted from the
R. sphaeroides 2.4.1 derivative EG238 (ndvB). Mass assignments are
based on an external calibration. The number on the top of each peak
refers to the degree of polymerization of glucose residues.
Ó FEBS 2002 The opgGIHC operon of R. sphaeroides (Eur. J. Biochem. 269) 2479
were confirmed by Biogel P4 chromatography of extracts
obtained from 100-mL cultures. Complementation analysis
were performed by introducing various plasmids into each
of the mutants tested (Table 2).
The opgG mutation was complemented with a plasmid
containing only the putative promoter and opgG.TheW
insertion had no polar effect on downstream gene expres-
sion. One can suggest the presence of a secondary promoter
inside the opgG ORF as previously observed in E. coli and
E. chrysanthemi [7].
The opgI mutations behaved differently according to the
orientation of the kan cassette. When the cassette was in the
same orientation as the opg genes, the opgI mutation could
be complemented by a plasmid containing only opgI and the
putative secondary promoter. When the plasmid contained
a shorter sequence upstream of opgI, complementation was
not possible. This allowed a more accurate localization of

this promoter between the BamHI and BglII sites of opgG.
When the cassette was in the opposite orientation, comple-
mentation was not observed, indicating a polar effect on
opgH expression.
opgC
is cotranscribed with
opgH
The putative start codon of opgC is located five nucleotides
before the stop codon of opgH, which strongly suggests
cotranscription. Actually, when opgH was disrupted by a
cassette in the same orientation (EG18) the mutation could
be complemented with only opgH and the OPGs produced
had an anionic character. When the cassette was in the
opposite orientation (EG7), the OPGs produced were
neutral. When cosmid pUI8166 was introduced into the
EG7 and EG18 strains, both synthesis and anionic substi-
tution of the OPGs were restored whatever the cassette
orientation. Therefore, we concluded that opgC and opgH
are cotranscribed.
Phylogenetic analysis of MdoH homologues
The E. coli mdoH gene product shows structural features
of a glycosyltransferase belonging to family 2 [1]. With
the growing number of complete genomes sequenced,
phylogenetic analyses of MdoH homologues is now pos-
sible. Figure 5 shows a phylogenetic tree obtained by the
maximum-parsimony method (the neighbor-joining method
gave similar results). Two cellulase synthase from cyano-
bacteria can be considered as an out-group and indicate the
possible location of the root. No similarities were found
between the MdoH and the NdvB homologues. A very

similar tree was observed for the MdoG homologues, but
with no out-group (data not shown). One may wonder if a
correlation exists between the phylogenetic position of a
particular OPG-glucosyltransferase and the structure of the
OPGs produced. At the present time, we have only partial
information. However, the OPGs produced by X campestris
and R. sphaeroides belong to the same family and the
corresponding OPG-glucosyltransferases appear related
(Fig. 5).
In our current working model [1], the OPG-glucosyl-
transferase H is assisted by the MdoG-like protein, possibly
a transglycosidase. Depending on the proteins considered,
the glucan produced would be a linear b,1-2 with b,1-6
branches as in E. coli, or cyclic b,1-2 with an a,1-6 closure
like in R. sphaeroides. If this hypothesis is true, it may be
possible to obtain b,1-2 polymerization of glucose residues
in E. coli when expressing the opgH of R. sphaeroides.
opgIH of
R. sphaeroides
can complement a mdoH
mutation in
E. coli
Several difficulties emerged when trying to express
R. sphaeroides genes in E. coli. We have known that
R. sphaeroides genes are not readily expressed in E. coli,
Table 2. OPG production in various opg mutants of R. sphaeroides 2.4.1.
Strain
Chromosomal
mutation Plasmid
OPG

synthesis
OPG
character
AP5 opgG5::W – – Absent
AP5 opgG5::W pNFR20 + Anionic
EG13 opgI13::kan – – Absent
EG13 opgI13::kan pGAK135 – Absent
EG13 opgI13::kan PGAK245 + Anionic
EG13 opgI13::kan PGAK246 + Anionic
EG131 opgI131::kan – – Absent
EG131 opgI131::kan pGAK135 – Absent
EG131 opgI131::kan pGAK245 – Absent
EG131 opgI131::kan pGAK246 – Absent
EG7 opgH7::kan – – Absent
EG7 opgH7::kan pGAK136 + Neutral
EG7 opgH7::kan pNFR21 + Neutral
EG7 opgH7::kan pUI8166 + Anionic
EG18 opgH18::kan – – Absent
EG18 opgH18::kan pGAK136 + Anionic
EG18 opgH18::kan pNFR21 + Anionic
EG18 opgH18::kan pUI8166 + Anionic
NFB4000 opgC1::Tn5– + Neutral
NFB4000 opgC1::Tn5 pUI8166 + Anionic
NFB4000 opgC1::Tn5 pNFR13 + Anionic
2480 V. Cogez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
even in an in vitro system [32]. Moreover, the first gene of
this locus encodes a periplasmic protein translated with an
uncommonly long signal-peptide that may not be recog-
nized by the E. coli secretory machinery. Actually, the
introduction of cosmid pUI8166 in either mdoG or mdoH

mutants of E. coli failedtorestoreanyOPGsynthesis
(data not shown). Placing the opgGIH genes downstream of
the lac promoter in pUC19 (pNFR14) was also unsuc-
cessful. However, when pNFR2 was introduced into a
mdoH200::Tn10 strain, material corresponding to the OPGs
was detected by thin layer chromatography. Sequencing of
the plasmid revealed that a translational fusion should have
fortuitously occurred between the eighteenth codon of the
a-lacZ fragment present in the vector and the third codon of
opgI, thus allowing the expression of the downstream opgH
gene. This was confirmed by the construction of pNFR30
where an opgIH-containing fragment with cohesive ends
was blunt ended using the Klenow fragment of DNA
polymerase I (see Materials and methods). Among plasmids
presenting the correct orientation of insert with respect to
the lac promoter, some were able to complement a mdoH
mutation, others not.
Plasmids pNFR30 and pNF309 where opgIH (Fig. 3)
and mdoH [4], respectively, are governed by the lac
promoter in the same vector, were introduced in the same
mdoH mutant strain. OPGs were extracted and analyzed by
gel filtration chromatography. The amount of OPGs was
threefold lower with pNFR30 than with pNF309. As OPG
synthesisisincreasedbyafactorof1.5whenmdoH
+
is
present on a multicopy plasmid like pNF309 [30], the level
observed with the R. sphaeroides genes was considered to
be the result of an efficient complementation. The OPGs
produced by the two strains were treated to remove all

substituents and then subjected to a MALDI-MS analysis.
With pNF309, the spectra were characteristic of linear-
branched glucans found in E. coli, with a DP of five to 14
glucose residues, the three principal species containing six,
seven, and eight glucose residues (Fig. 6A). It should be
noted that the presence of pNF309 induced a slight
increased of the maximal DP normally observed [33]. With
pNFR30, the spectra were very similar but the maximal DP
was at least of 18 glucose units and the principal species
contained five, six, seven, eight, and nine glucose residues
(Fig. 6B).
opgC of
R. sphaeroides
transfers succinyl residues
to OPGs in
E. coli
As shown above, very similar proteins are implicated in the
synthesis of quite different glucosidic backbones, but
Fig. 6. Positive-ion MALDI mass spectra of OPGs extracted from
E. coli strains NFB1100 (pmdoH
+
/mdoH, panel A) or from NFB4234
(popgI
+
H
+
/mdoH, panel B). Mass assignments are based on an
external calibration.
Fig. 5. Unrooted phylogenetic tree for MdoH homologues prepared using the maximum parsimony method as described in Materials and methods.
Numbers on forks are bootstrap confidence levels. Cell. synth., cellulose synthase. Full species names are as follows: Caulobacter crescentus;

Nitrosomonas europeae; Pseudomonas fluorescens; Ralstonia eutropha; Rhodopseudomonas palustris; Shewanella putrefaciens; Vibrio cholerae;
Xanthomonas citri; Xylella almond; Xylella oleander; Xylella fastidiosa; Yersinia pseudotuberculosis. All sequence data are from ERGO with the
exception of X. citri (E. G. M. Lemos, School of Agronomy and Vetenary Sciences, Campus of Jaboticabal, Sao Paulo State University, Brazil,
personal communication)
5
.
Ó FEBS 2002 The opgGIHC operon of R. sphaeroides (Eur. J. Biochem. 269) 2481
membrane proteins, with no sequence similarities, are
probably involved in the transfer of succinyl residues
through the cell membrane to OPGs present in the
periplasmic space [14]. The open question was whether the
R. sphaeroides genes can be expressed in E. coli,andwhat
properties they confer in this context.
The highly anionic character of OPGs synthesized by
E. coli is due to the presence of phosphoglycerol and
succinyl residues. Two genes mdoB and mdoC govern these
two kind of substitution and a mdoB mdoC double mutant
produce neutral OPGs [14]. To test the transfer ability of
OpgC in a heterologous context, pNFR37 (Fig. 3) was
introduced in strain NFB1933. We had previously observed
that pNFR12, which contains only opgC, was ineffective.
Plasmid pNFR37 is expected to express opgI as a transla-
tional fusion with a-lacZ and opgH, and opgC which
overlaps opgH. The specifically labeled OPGs produced in
the presence of pNFR37 were purified and analyzed by
DEAE-Sephacel chromatography. Under these conditions,
OPGs synthesized by the recipient strain are totally neutral
and are not retained by the column [14]. After introduction
of the plasmid, 20% of the radioactivity, corresponding to
anionic glucans, were retained by the column and eluted

into two subfractions by increasing the ionic strength
(Fig. 7).
DISCUSSION
This paper describes the isolation of an R. sphaeroides
mutant defective in OPG succinylation. No OPG-defective
mutants (our initial purpose) were obtained, as was the case
while screening potential mutants (several thousands in
total) in S. meliloti [10], E. coli [14], and E. chrysanthemi
(V. Cogez, & J P. Bohin, unpublished data). The reason for
this fact remains unknown. The gene inactivated in the
mutant, opgC, was then isolated and characterized. OpgC is
predicted to be a highly hydrophobic protein, most
probably inserted into the cell membrane. As we have
previously postulated for MdoC, OpgC should transfer
succinyl residues, probably provided by the succinyl-CoA
pool, through the membrane, to the OPGs accumulating in
the periplasmic space [14]. The opgC gene is located
downstream of opgGIH, three genes necessary for OPG
backbone synthesis. opgC overlaps opgH and a polar
mutation in opgH prevent opgC expression. Thus, opgC
forms an operon with opgGIH.
When expressed together, OpgI and OpgH can comple-
ment a mdoH mutation in E. coli. That means that proteins
that normally catalyze the synthesis in R. sphaeroides of
b-1,2; a-1,6 cyclic glucans comprised of 18 glucose residues
can catalyze the synthesis in E. coli of b-1,2; b-1,6 linear
glucans comprised of varying numbers (five to 18) of
glucose residues. The small ORF encoded by opgI was
found necessary for OPG synthesis in R. sphaeroides.In
E. coli, opgI is most probably necessary too, but we cannot

exclude that only translational coupling is necessary for
opgH expression. OpgH is shorter than MdoH, and OpgI
appears to correspond to the N-terminal domain of MdoH
[30], a domain poorly conserved among the different
homologues found in the proteobacteria. One should notice
that another small protein, the acyl carrier protein (ACP),
participates in OPG synthesis in E. coli [33]. ACP from
E. coli (or closely related species) functions in an unknown
way that does not require the presence of the phospho-
pantetheine prosthetic group. ACP from R. sphaeroides was
shown to be inactive in the in vitro glucosyltransferase
reaction both as an activator or an inhibitor [34]. Thus,
OpgI could play in R. sphaeroides, a role similar to that of
ACP in E. coli.
MdoH and OpgH have typical motifs found in glycosyl
transferases and one can imagine that both proteins catalyze
the polymerization of long chains of b-1,2 glucose residues.
We have previously postulated that this kind of protein,
embedded in the membrane by a number of TMSs, could be
directly involved in the translocation of the nascent glucan
chains to the periplasmic face of the membrane [30].
Therefore, other proteins such as MdoG or OpgG may
rearrange this backbone to add branches (MdoG) or make
a cyclic molecule (OpgG). As mutants of this second protein
do not accumulate any glucan molecules (this work; [4]), the
periplasmic and the membrane-bound proteins must inter-
act in a very coordinate manner during the process. Thus,
the abnormal control of the degree of polymerization of the
OPG synthesized when the R. sphaeroides opgIH genes were
expressed, would be the result of a partially defective

interaction between MdoG and OpgH. Until now, we had
not obtained the expression of OpgG in E. coli.This
experiment will be important in order to determine to what
extent this protein determines the structural differences
between the OPGs produced by the two different bacterial
species.
When expressed in E. coli, together with OpgI and
OpgH, OpgC can transfer charged residues to the OPGs.
Together with the loss of succinyl transfer in the opgC
mutant, this is a confirmation that OpgC is an OPG-
succinyltransferase. However, this activity remained at a
low level in E. coli, especially if we consider the highly
anionic character of the R. sphaeroides OPGs. Two hypo-
theses, not mutually exclusive, can be formulated to explain
this observation: one regarding the protein organization in
the membrane and one regarding the substrate specificity.
As already mentioned above, OpgC and MdoC are two
functional homologues that do not share significant amino-
acid sequence similarity. In E. coli, succinylation occurs
Fig. 7. DEAE-Sephacel anion exchange column chromatography pro-
files of [U-
14
C] glucose labeled OPGs from strain NFB4245
(popgI
+
H
+
C
+
/mdoC m doB). Ionic strength was increased by steps of

0.05
M
NaCl at fractions indicated by the arrows. Fractions (4 mL)
were collected and radioactivity was determined on aliquots.
2482 V. Cogez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
very early in the OPG biosynthetic process, most probably
while nascent polyglucose chains are still attached to, or in
the close vicinity of, the backbone synthetic enzymes
(Y. Lequette & J P. Bohin, unpublished data). Primary
substitution by phosphoglycerol residues is catalyzed by
another membrane bound protein, while secondary sub-
stitution is determined by a periplasmic enzyme [33]. There
is no information on the protein(s) that catalyze the
phosphoethanolamine transfer. Thus, at least three or four
membrane proteins could stably interact with each other
and with several periplasmic proteins, and form a complex
OPG synthetic machinery. The same complex machinery is
expected in R. sphaeroides whereOPGsarethetargetsof
succinyl and acetyl transfers. If this is true, one can envisage
that heterologous proteins cannot interact correctly, and as
a consequence, that their activities are lowered.
OpgC expression was obtained together with OpgI and
OpgH in a mdoH
+
background, and the OPG backbones
produced were highly heterogeneous in size, and probably
in the number and position of the branches as in E. chry-
santhemi [35].AsOPGsproducedinR. sphaeroides do not
possess branches, the succinyl transfer should occur on
glucose residues attached to other residues only by b-1,2

linkages, before or after cyclization of the molecules.
Therefore, only a small subfraction of the OPGs produced
in E. coli could be recognized as substrates for the
R. sphaeroides OPG-succinyl transferase.
ACKNOWLEDGEMENTS
We thank Je
´
roˆ me Lemoine for the recording of MALDI-MS spectra.
This research was supported by grants from the Centre National de la
Recherche Scientifique (UMR8576) and the Ministe
`
re de l’Education
nationale, de la Recherche et de la Technologie to J. P. B., and grant
GM-15590 to S. K.
REFERENCES
1. Bohin, J P. (2000) Osmoregulated periplasmic glucans in proteo-
bacteria. FEMS Microbiol. Lett. 186, 11–19.
2. Bahgwat, A.A., Mitho
¨
fer, A., Pfeffer, P.E., Kraus, C., Spickers,
N., Hotchkiss, A., Ebel, J. & Keister, D.L. (1999) Further studies
of the role of cyclic b-glucans in symbiosis. An ndvC mutant of
Bradyrhizobium japonicum synthesizes cyclodecakis-(1–3)-b-gluc-
osyl. Plant Physiol. 119, 1057–1064.
3. Breedveld, M.W. & Miller, K.J. (1994) Cyclic b-glucans of the
familly Rhizobiaceae. Microbiol. Rev. 58, 145–161.
4. Loubens, I., Debarbieux, L., Bohin, A., Lacroix, J M. & Bohin,
J P. (1993) Homology between a genetic locus (mdoA)involvedin
the osmoregulated biosynthesis of periplasmic glucans in Escher-
ichia coli and a genetic locus (hrpM) controlling the pathogenicity

of Pseudomonas syringae. Mol. Microbiol. 10, 329–340.
5. Mukhopadhyay, P., Williams, J. & Mills, D. (1988) Molecular
analysis of a pathogenicity locus in Pseudomonas syringae pv.
Syringae. J. Bacteriol. 170, 5479–5488.
6. Mahajan-Miklos,S.,Tan,M W.,Rahme,L.G.&Ausubel,F.M.
(1999) Molecular mechanisms of bacterial virulence elucidated
using a Pseudomonas aeruginosa-Caenorhabditis elegans patho-
genesis model. Cell 96, 47–56.
7. Page, F., Altabe, S., Hugouvieux-Cotte-Pattat, N., Lacroix, J M.,
Robert-Baudouy, J. & Bohin, J P. (2001) Osmoregulated peri-
plasmic glucan synthesis is required for Erwinia chrysanthemi
pathogenicity. J. Bacteriol. 183, 3134–3141.
8. Briones, G., In
˜
on de Iannino, N., Roset, M., A.Vigliocco, Paulo,
P.S. & Ugalde, R.A. (2001) Brucella abortus cyclic b-1,2-glucan
mutants have reduced virulence in mice and are defective in intra-
cellular replication in HeLa cells. Infect. Immun. 69, 4528–4535.
9. Valentine, P.J., Devore, B.P. & Heffron, F. (1998) Identification of
three highly attenuated Salmonella typhimurium mutants that are
more immunogenic and protective in mice than a prototypical
aroA mutant. Infect. Immun. 66, 3378–3383.
10. Breedveld, M.W., Hadley, J.A. & Miller, K.J. (1995) A novel
cyclic b-1,2-glucan mutant of Rhizobium meliloti. J. Bacteriol. 177,
6346–6351.
11. Suwanto, A. & Kaplan, S. (1989) Physical and genetic mapping of
the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique
circular chromosomes. J. Bacteriol. 171, 5850–5859.
12. Oh, J I. & Kaplan, S. (2001) Generalized approach to the
regulation and integration of gene expression. Mol. Microbiol.

39, 1116–1123.
13. Talaga, P., Cogez, V., Wieruszeski, J M., Stahl, B., Lemoine, J.,
Lippens, G. & Bohin, J P. (2002)
4
Osmoregulated periplasmic
glucans of the free-living photosynthetic bacterium Rhodobacter
sphaeroides. Eur. J. Biochem. 269, 2464–2472.
14.Lacroix,J M.,Lanfroy,E.,Cogez,V.,Lequette,Y.,Bohin,
A. & Bohin, J P. (1999) The mdoC gene of Escherichia coli
encodes a membrane protein that is required for succinylation
of osmoregulated periplasmic glucans. J. Bacteriol. 181, 3626–
3631.
15. Simon, R., Priefer, U. & Pu
¨
hler, A. (1983) A broad-host-range
mobilization system for in vivo genetic engineering: transposon
mutagenesis in Gram-negative bacteria. Bio/Technol. 1, 37–45.
16. Lacroix, J M., Loubens, I., Tempeˆ te, M., Menichi, B. & Bohin,
J P. (1991) The mdoA locus of Escherichia coli consists of an
operon under osmotic control. Mol. Microbiol. 5, 1745–1753.
17. Prentki, P. & Hirsch, H.M. (1984) In vitro insertional mutagenesis
with a selectable DNA fragment. Gene 29, 303–313.
18. Keen, N.T., Tamaki, S., Kobayashi, D. & Trollinger, D. (1988)
Improved broad-host-range plasmids for DNA cloning in Gram-
negative bacteria. Gene 70, 191–197.
19. 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.
20. Broome-Smith, J.K. & Spratt, B.G. (1986) A vector for the con-
struction of translational fusions to TEM b-lactamase and the

analysis of protein export signals and membrane protein topology.
Gene 49, 341–349.
21. Puskas, A., Greenberg, E.P., Kaplan, S. & Schaefer, A.L. (1997) A
quorum-sensing system in the free-living photosynthetic bacterium
Rhodobacter sphaeroides. J. Bacteriol. 179, 7530–7537.
22. Cohen-Bazire, G., Sistrom, W.R. & Stanier, R.Y. (1956) Kinetic
studies of pigment synthesis by non-sulfur purple bacteria. J. Cell.
Comp. Physiol. 49, 25–68.
23. Miller, J.H. (1992) A Short Course in Bacterial Genetics. A
Laboratory Manual and Handbook for Escherichia coli and Related
Bacteria. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
24. Hanahan, D. (1983) Studies on transformation of Escherichia coli
with plasmids. J. Mol. Biol. 166, 557–580.
25. Choudhary, M., Mackenzie, R.C., Nereng, K., Sodergren, E.,
Weinstock, M.G. & Kaplan, S. (1994) Multiple chromosomes in
bacteria: structure and function of chromosome II of Rhodobacter
sphaeroides. J. Bacteriol. 176, 7694–7702.
26. Sasakawa, C. & Yoshikawa, M. (1987) A series of Tn5 variants
with various drug-resistance markers and suicide vector for
transposon mutagenesis. Gene 56, 283–288.
27. Silhavy, T.J., Berman, M.L. & Enquist, L.W. (1984) Experiments
with Gene Fusions. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.
28. Davis, R.W., Botstein, D. & Roth, J.R. (1980) Manual for Genetic
Engineering. Advanced bacterial genetics. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY.
Ó FEBS 2002 The opgGIHC operon of R. sphaeroides (Eur. J. Biochem. 269) 2483
29. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular
Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY.
30. Debarbieux, L., Bohin, A. & Bohin, J P. (1997) Topological
analysis of the membrane-bound glucosyltransferase, MdoH,
required for osmoregulated periplasmic glucan synthesis in
Escherichia coli. J. Bacteriol. 179, 6692–6698.
31. Choudhary, M., Mackenzie, R.C., Nereng, K., Sodergren, E.,
Weinstock, M.G. & Kaplan, S. (1997) Low-resolution sequencing
of Rhodobacter sphaeroides 2.4.1
T
: chromosome II is a true chro-
mosome. Microbiology 143, 3085–3099.
32. Chory, J. & Kaplan, S. (1982) The in vitro transcription-transla-
tion of DNA and RNA templates by extracts of Rhodopseudo-
monas sphaeroides. Optimization and comparison of template
specificity with Escherichia coli extracts and in vivo synthesis.
J. Biol. Chem. 257, 15110–15121.
33. Kennedy, E.P. (1996) Membrane derived oligosaccharides (peri-
plasmic beta-
D
-glucans) of Escherichia coli.InEscherichia Coli and
Salmonella. Cellular and Molecular Biology, 2nd edn. (Neidhardt,
F.C.Curtiss,R., IIIIngraham, J.L.Lin, E.C.C.Low, K.B.Magana-
sik, B.Reznikoff, W.S.Riley, M.Schaechter, M. & Umbarger,
H.E., eds), pp. 1064–1074, American Society for Microbiology,
Washington, DC.
34. Tang, L., Weissborn, A.C. & Kennedy, E.P. (1997) Domains of
Escherichia coli acyl carrier protein important for membrane-
derived-oligosaccharide biosynthesis. J. Bacteriol. 179, 3697–
3705.
35. Cogez, V., Talaga, P., Lemoine, J. & Bohin, J P. (2001) Osmo-

regulated periplasmic glucans of Erwinia chrysanthemi. J. Bacteriol.
183, 3127–3133.
2484 V. Cogez et al. (Eur. J. Biochem. 269) Ó FEBS 2002