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

doi:10.1046/j.1432-1033.2002.02907.x

Eur. J. Biochem. 269, 2473–2484 (2002) (cid:1) FEBS 2002

The opgGIHand opgC genes of Rhodobactersphaeroides form an operon that controls backbone synthesis and succinylation of osmoregulated periplasmic glucans

Virginie Cogez1, Evgueni Gak2, Agnes Puskas2, Samuel Kaplan2 and Jean-Pierre Bohin1 1Unite´ de Glycobiologie Structurale et Fonctionnelle, CNRS UMR8576, Universite´ des Sciences et Technologies de Lille, France; 2Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, TX, USA

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.

cyclic glucans; Keywords: periplasm; osmoregulation; glucosyl transferase; operon. 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

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.

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 a-1,6 glucans. In several bacterial species, OPGs are substituted by one or several of a series of different residues, originating from (phosphoglycerol, either the membrane phospholipids

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: Jean-Pierre.Bohin@univ-lille1.fr 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)

1 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 ¼ 18) in which 17 glucose units homogenous in size (DP

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resistance) were picked and arranged to construct libraries of putative mutants.

Thin-layer chromatographic screening method

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.

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 (cid:2)C. The same procedure was 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.

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.

DNA purification, restriction and modification enzymes and ligase

M A T E R I A L S A N D M E T H O D S

Bacterial strains and media

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.

Invitroconstruction of plasmids

The bacterial strains and plasmids used are detailed in Table 1. R. sphaeroides strains were grown at 30 (cid:2)C in Sistrom’s succinic acid minimal medium (SIS; [22]); anaero- illuminated at 100 WÆm)2. To determine OPG bically, production, aerobic chemoheterotrophic cultures were grown, with shaking, in Luria–Bertani broth [23]. E. coli strains were grown at 37 (cid:2)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) 100 lgÆL)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. 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.

Transformation and mating

For complementation tests in R. sphaeroides, a 4.5-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).

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

A 2.2-kb EcoRV–BglII fragment from pUI8166, con- taining an intact copy of opgH, was inserted into Litmus 28 (Ampr; 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).

The mobilizable suicide plasmid pSUPTn5TpMCS [25,26] was introduced into R. sphaeroides WS8 by mating at 30 (cid:2)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 A 3.5 SalI fragment from cosmid pUI8166, containing an intact copy of opgGI, was inserted into the SalI site of pBS II SK(+) (Ampr; Statagene), to give pUI2509. Then, the

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Table 1. Bacterial strains and plasmids used in this study.

Strain or plasmid

Relevant genotype

Source or reference

Escherichia coli

S17-1 DH5a NFB216 NFB702 NFB1933 NFB1100 NFB4234 NFB4245

thi pro hsdR– hsdM+ recA RP4 plasmid integrated Tc::Mu-Km::Tn7 F– recA1 endA1 gyrA96 thi-1 hsdR17 glnV44 relA1 DlacU169 k (/80 dlacZDM15) D(lac-pro) ara mdoH200::Tn10 pyrC46 rpsL thi (/80 dlacZDM15) D(lac-pro) ara mdoG202::neo pyrC46 rpsL thi (/80 dlacZDM15) his pgi::Mu D(zwf-edd)1 eda-1 rpsL mdoB214::Tn10 mdoC1::Tn5 pNF309/NFB216 pNFR30/NFB216 pNFR37/NFB1933

[15] Lab stock [16] [16] [14] [4] This work This work

Rhodobacter sphaeroides

Laboratory stock This work Laboratory stock This work This work This work This work This work This work

Wild type, Strr strain WS8 opgC1::Tn5TpMCS Wild type 2.4.1 opgG5::W (Strr, Spcr) 2.4.1 opgH7::kan, opposite to gene orientation 2.4.1 opgI13::kan, gene orientation 2.4.1 opgH18::kan, gene orientation 2.4.1 opgI131::kan, opposite to gene orientation 2.4.1 ndvB238::W (Strr, Spcr)

WS8 NFB4000 2.4.1 AP5 EG7 EG13 EG18 EG131 EG238 Plasmids

pHP45W pRK415 pUC19 pUC4K pYZ4 pUI8166 pNF309 pGAK135 pGAK136 pGAK245 pGAK246 pGAK247 pNFR13 pNFR20 pNFR21 pNFR25 pNFR30 pNFR35 pNFR37

Source of the W interposon (Strr, Spcr) Tetr, broad host range mobilizable vector Ampr, cloning vector Source of the kan cassette (Kanr) Kanr, cloning vector pLA2917-derived cosmid clone from R. sphaeroides 2.4.1T library Kanr, pYZ4 carrying mdoH+ Tetr, pRK415 carrying opgI+ Tetr, pRK415 carrying opgH+ Tetr, pRK415 carrying opgI+ (in the tet promoter orientation) Tetr, pRK415 carrying opgI+ (opposite of the tet promoter orientation) Tetr, pRK415 carrying opgGI+ Tetr, pRK415 carrying opgC+ Tetr, pRK415 carrying opgG+ Tetr, pRK415 carrying opgGIH+ Kanr, pYZ4 carrying opgC+ Kanr, pYZ4 carrying opgIH+ Ampr, pUC19 carrying opgIH+ Ampr, pUC19 carrying opgIHC+

[17] [18] [19] Pharmacia [20] [21] [4] This work This work This work This work This work This work This work This work This work This work This work This work

3.5 kb was liberated by digesting pUI2509 with HindIII and KpnI, and inserted into pRK415 digested with HindIII and KpnI, to give pGAK247 (Fig. 3).

A 1.2-kb BglII–KpnI fragment from pUI2509, containing an intact copy of opgI, was inserted into the BamHI site of pRK415, to give pGAK135 (Fig. 3).

give pNFR15. A 2.0-kb fragment was liberated by digesting pNFR15 with HindIII and KpnI, and inserted into pRK415 digested with HindIII and KpnI, to give pNFR20 (Fig. 3). A 1.8-kb StuI–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 KpnI and SmaI, and inserted into the expression vector pYZ4 to give pNFR25 (Fig. 3).

A 1.8-kb BamHI 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).

A 3-kb EcoRI 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. A 5-kb ApaI–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, and inserted into pRK415 digested with HindIII and KpnI, to give pNFR21 (Fig. 3).

A 4-kb EcoRI–HindIII fragment from pNFR2 was blunt ended and inserted into the SmaI site of pYZ4, to give pNFR30. A 2.0-kb HindIII–EcoRV fragment from pNFR14 was inserted between the HindIII and SmaI sites of pUC19 to

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Construction of mutants responding to the end of both IS50 delineating Tn5 was used as a primer.

A 2.6-kb EcoRI 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].

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 (http://www.infobiogen.fr/) and from ERGO (http://wit. integratedgenomics.com/IGwit/CGI/).

2

A 1.3-kb Eco47III 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 ScaI and AatII sites of the broad host range mobilizable vector pSUP202D (M. Gomelsky, Department of Microbiology and Molecu- lar Genetics, University of Texas Health Center, Houston, to give pGAK118 (kan and opgI in the same Texas, USA) 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.

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 the 847 amino acids were thus MdoH, 637 out of 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

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. A 9.5-kb SacI–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.

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]. DNA sequencing

Analysis of OPGs from E.coli

Strain NFB1933 and its derivatives were grown in LOS medium (5 mL) supplemented with 0.24 mM D-[U-14C]glu- 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-

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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 mM 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 mM 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 4 mL.

Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry (MS)

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.

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 (cid:2)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 (cid:2)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 and then diluted with an appropriate volume of the matrix 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]. 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 the consequence of a reduction in glucose was not backbone synthesis.

R E S U L T S

OPGs isolated from the mutant are neutral 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 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 mM, 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

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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

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.

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.

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, by analogy with the mdoC gene that governs OPG succinylation in E. coli [14].

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 TOPPRED2 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.

opgC lies downstream from a locus similar to mdoGH of E.coli

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, The opgC1::Tn5TpMCS mutation was cloned into the pUC19 vector from genomic DNA using the trimethoprim resistance conferred by the transposon as a selection.

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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.

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 TOPPRED2 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.

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.

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, or opgGIH, 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.

[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, or opgH mutants were analyzed by thin layer chromatography, no spots corresponding to OPGs could be detected. These results 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

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Phylogenetic analysis of MdoH homologues

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. The W 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 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). 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

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

Table 2. OPG production in various opg mutants of R. sphaeroides 2.4.1.

OPG synthesis

OPG character

Strain

Chromosomal mutation

Plasmid

AP5 AP5 EG13 EG13 EG13 EG13 EG131 EG131 EG131 EG131 EG7 EG7 EG7 EG7 EG18 EG18 EG18 EG18 NFB4000 NFB4000 NFB4000

opgG5::W opgG5::W opgI13::kan opgI13::kan opgI13::kan opgI13::kan opgI131::kan opgI131::kan opgI131::kan opgI131::kan opgH7::kan opgH7::kan opgH7::kan opgH7::kan opgH18::kan opgH18::kan opgH18::kan opgH18::kan opgC1::Tn5 opgC1::Tn5 opgC1::Tn5

– pNFR20 – pGAK135 PGAK245 PGAK246 – pGAK135 pGAK245 pGAK246 – pGAK136 pNFR21 pUI8166 – pGAK136 pNFR21 pUI8166 – pUI8166 pNFR13

– + – – + + – – – – – + + + – + + + + + +

Absent Anionic Absent Absent Anionic Anionic Absent Absent Absent Absent Absent Neutral Neutral Anionic Absent Anionic Anionic Anionic Neutral Anionic Anionic

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. 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,

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5

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)

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.

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 failed to restore any OPG synthesis (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 synthesis is increased by a factor of 1.5 when mdoH+ 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

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[U-14C] glucose labeled OPGs

Fig. 7. DEAE-Sephacel anion exchange column chromatography pro- files of from strain NFB4245 (popgI+H+C+/mdoC mdoB). 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.

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. membrane proteins, with no sequence similarities, are probably involved in the transfer of succinyl residues in the through the cell membrane to OPGs present periplasmic space [14]. The open question was whether the R. sphaeroides genes can be expressed in E. coli, and what 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).

D I S C U S S I O N

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 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

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4

13. Talaga, P., Cogez, V., Wieruszeski, J.-M., Stahl, B., Lemoine, J., Osmoregulated periplasmic Lippens, G. & Bohin, J.-P. (2002) glucans of the free-living photosynthetic bacterium Rhodobacter sphaeroides. Eur. J. Biochem. 269, 2464–2472.

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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 where OPGs are the targets of 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.

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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]. As OPGs produced in R. 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.

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