INTRODUCTION

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Osmoregulated periplasmic glucans (OPGs) are a family of oligosaccharides found in the periplasmic space of gram-negative bacteria. Their common features are the presence of glucose as the sole constituent sugar and their increased levels in low-osmolarity media.

Nevertheless, they are very different in structure. Rhizobiaceae and related bacteria synthesize cyclic or branched cyclic glucans, whereas Enterobacteriaceae and related bacteria synthesize branched linear glucans. These structural differences are correlated with genes implicated in the synthesis of the glucose backbone. These genes can be divided into two classes which have no homology. The first class is represented by the very similar genes of Agrobacterium tumefaciens (chvA and chvB) and Sinorhizobium meliloti (ndvA and ndvB [7]), while the second class contains the very similar genes of Escherichia coli (mdoG and mdoH) and Pseudomonas syringae (orfI and hrpM [20]).

These glucans may be substituted with various residues, depending on the species considered. Surprisingly, there is no correlation between the glucosidic structure of OPGs and the nature of substituents. Cyclic OPGs of Brucella abortus (7), Ralstonia solanacearum, Xanthomonas campestris (28), and linear glucans of P. syringae (27) are devoid of substituents. OPGs of Azospirillum brasilense (cyclic glucans [1]) and Erwinia chrysanthemi (linear glucans [30]) are substituted only with succinyl residues, and cyclic OPGs of Bradyrhizobium japonicum are substituted only by phosphocholine (24). Cyclic glucans originating from S. meliloti and A. tumefaciens are substituted with both succinyl and phosphatidylglycerol residues (7), while cyclic OPGs of Rhodobacter sphaeroides are substituted with succinyl and acetate residues (29). OPGs originating from E. coli (linear glucans) are the only ones known to be substituted with three different substituents. These OPGs are substituted with phosphatidylglycerol and phosphatidylethanolamine derived from membrane phospholipids and by succinyl residues of unknown origin (17).

In E. coli, the glucosidic backbones are synthesized by the products of the mdoGH operon. They appear to contain 6 to 12 glucose residues per mole, with the principal species containing 8 to 9 glucose units (17). MdoG is a periplasmic protein of unknown function (which is nevertheless required for synthesis of glucans [18]), while MdoH is a transmembrane -1,2 glucosyl transferase acting on the cytoplasmic side of the inner membrane and is probably involved in the transfer of these glucans to the periplasmic space (10). The first characterized gene implicated in substitution was mdoB encoding the phosphoglycerol transferase I of E. coli (12, 16). This enzyme can transfer phosphoglycerol residues to an artificial acceptor, the -glucoside arbutin. Since arbutin cannot enter the cytoplasm, the transfer should occur on the periplasmic face of the inner membrane. Thus, one can conclude that glucan backbones are substituted after their translocation to the periplasmic space (6). Similar conclusions were proposed for the substitution of cyclic glucans in S. meliloti (7). This transfer leads to the formation of diacylglycerol, which is toxic for cells and immediately phosphorylated by the diacylglycerol-kinase (encoded by the dgk gene) to yield phosphatidic acid.

When a dgk strain was grown in presence of arbutin, diacylglycerol formation was stimulated and its subsequent accumulation stopped the growth of the dgk strain. This was the basis of the selection of mdoB mutants in which phosphatidylglycerol turnover was slowed sufficiently to allow growth (16).

More recently, Breedveld et al. (8) isolated a mutant unable to transfer phosphoglycerol to OPGs in S. meliloti. They used a thin-layer chromatographic screening protocol because the genetic selection described above was inefficient in this bacterium (8a).

In this paper, we describe the isolation of a new gene required for the substitution of OPGs by succinate in an E. coli mdoB strain by using a similar thin-layer chromatographic screening protocol.

	MATERIALS AND METHODS

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Bacterial strains and media. The E. coli strains and phage used in this study are described in Table 1. Bacteria were grown at 37°C with vigorous shaking in Luria broth (LB) or in minimal medium 63 supplemented with the required metabolites and glucose as the carbon source (21). Solid media were obtained by adding agar (15 g · l¹). When low-osmolarity medium was required, LB without NaCl or low-osmolarity medium was used (19).

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Transduction and transformation. Transduction with phage P1vir was carried out according to the method of Miller (21). E. coli cells were made competent and transformed by the calcium chloride technique (26).

Transposon mutagenesis. Random integration of Tn5 into the chromosome was obtained by using NK467 (Table 1) according to standard procedures (26).

Restriction enzymes, modification enzyme, and ligase. Restriction endonucleases (Eurogentec), the large (Klenow) fragment of DNA polymerase I, and ligase of T4 phage (Gibco BRL) were used according to manufacturer's recommendations.

Plasmid construction. For the sequencing of a fragment of the mdoC gene, a genomic DNA fragment of the mdoC1::Tn5 strain NFB1897 was cloned in the BamHI site of plasmid pUC8. This restriction enzyme cut once in the Tn5 DNA outside the gene, conferring kanamycin resistance to the transposon. Kanamycin-resistant clones harboring a plasmid containing a 16-kb DNA insert were isolated on plates containing kanamycin and ampicillin.

DNA purification. Standard procedures (26) were used for genomic DNA extraction, large scale plasmid isolation, and rapid analysis of recombinant plasmids.

DNA sequencing. DNA sequences were determined with the Sequenase version 2.0 kit (US Biochemical Corporation) except for the Tn5 insertion point, where the oligonucleotide 5'-CATGGAAGTCAGATCCTGG-3' (Eurogentec), corresponding to the end of the two IS50 of Tn5, was used as a primer.

Thin-layer chromatographic screening method. Each Tn5 mutant generated from NFB732 was screened as described by Breedveld et al. (8) and modified as follows: 4 ml of an overnight culture of E. coli grown in LB without NaCl was centrifuged and washed with 1 ml of water, and the pellet was resuspended in 250 µl of 80% ethanol (vol/vol). The cell suspension was then heated for 30 min at 70°C and centrifuged for 10 min at 15,000 × g at room temperature, and the supernatant (containing OPGs) was dried under a vacuum.

Extraction of OPGs. Cultures (200 ml) of E. coli were grown overnight in LB without NaCl. OPGs were extracted by the charcoal adsorption procedure (19). Pyridine extract obtained by this procedure was chromatographed on a Biogel P4 column (Bio-Rad). The column (1.5 by 68 cm) was equilibrated with 0.5% acetic acid and eluted at a rate of 15 ml · h¹ in the same buffer. Fractions (2.5 ml) containing oligosaccharides were pooled, concentrated by rotary evaporation, and desalted on a Biogel P2 column (Bio-Rad), and fractions containing OPGs were pooled and lyophilized. Sugar content was determined colorimetrically by using the phenol-sulfuric acid reagent procedure (11).

Determination of succinate content from OPGs. One milligram of OPG was dissolved in 200 µl of 0.5 M NaOH and incubated at 100°C for 30 min to liberate the succinyl residues from OPG. Glucosidic backbones were removed by absorption on 50 mg of charcoal, and the charcoal was then washed three times with 0.5 ml of water. The four supernatants (2.5 ml) were pooled, concentrated by rotary evaporation, and desalted on a minicolumn of Dowex AG 50 × 8, 20- to 50-mesh (Bio-Rad) on H⁺ form.

Determination of neutral and anionic characteristics of OPGs. Strains were grown in low-osmolarity medium (5 ml) supplemented with 0.24 mM D-[U-¹⁴C]glucose (125 MBq mmol¹). Glucans extracted as described above were desalted on a PD10 column (Pharmacia) equilibrated with a Tris-HCl 10 mM [pH 7.4] buffer. OPG-containing fractions were pooled and chromatographed on a DEAE-Sephacel column (1.5 by 38 cm; Pharmacia) equilibrated with 10 mM Tris-HCl [pH 7.4] and eluted with the same buffer containing 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.

	RESULTS AND DISCUSSION

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Isolation of a mutant with altered phenotype of OPGs. A random Tn5 mutagenesis was performed in the mdoB strain NFB732 (deficient for the phosphoglycerol transferase I). In mdoB strains, OPGs lack phosphoglycerol substituents and succinyl residues are the only anionic substituents. Mild alkali treatment of OPGs removes selectively these succinyl residues. Glucans of NFB732 devoid of anionic substituents were substituted only with phosphoethanolamine residues and can be easily distinguished by a thin-layer chromatographic method (described in Materials and Methods). As expected, migration of glucans is greatly reduced after mild alkali treatment (Fig. 1, lane 3) compared to that of untreated glucans (Fig. 1, lane 1). Glucans extracted from 996 random Tn5 insertion mutants of NFB732 were analyzed by this chromatographic screening.

View larger version (48K): [in this window] [in a new window]   FIG. 1.   Thin-layer chromatographic analysis of OPGs extracted from NFB732 (mdoB, lanes 1 and 2) or NFB1864 (mdoB mdoC, lanes 3 and 4). Extracts were applied directly to thin-layer chromatography plates (lanes 1 and 3) or first subjected to mild alkali treatment to remove succinyl substituents (lanes 2 and 4).

The Tn5 insertion is responsible for the altered phenotype of OPGs. The Tn5 mutation from NFB1864 was transferred by P1 phage generalized transduction into strains DF214 and JM83 and their mdoB derivatives. After selection of transductants for kanamycin resistance, OPGs were extracted and subjected to the thin-layer chromatographic analysis. Strains containing both mdoB and mdoC mutations produced glucans with the same migration pattern as those of NFB1864, indicating that the Tn5 insertion was responsible for the altered phenotype of OPGs (data not shown).

OPGs isolated from the mdoC mutant are neutral. Specific labeling of OPGs was obtained by growing cells of strain DF214 (pgi, zwf) and its derivatives, NFB734 (mdoB), NFB1919 (mdoC), and NFB1933 (mdoB, mdoC), in the presence of radioactive glucose. They produced the same amount of OPGs when grown in a low-osmolarity medium and a similar reduction (six to seven times) was observed when the osmolarity was increased by the addition of 0.3 M NaCl. The anionic or neutral character of OPGs was confirmed by a DEAE-Sephacel chromatographic analysis of radiolabeled glucans. OPGs extracted from DF214 (mdo⁺, Fig. 2) were separated into five subfractions, named I to V (III, IV, and V correspond to A, B, and C, respectively, in Van Golde et al. [31]). Subfractions II to V were eluted at increasing concentrations of NaCl and correspond to anionic replaced glucans. Phosphoglycerol and phosphoethanolamine substitutions of each subfraction were previously analyzed by using radiolabeled precursors (22, 31). Phosphoglycerol and phosphoethanolamine were present in subfractions III to V, and low levels of phosphoethanolamine were detected in subfractions I and II, while phosphoglycerol was hardly detected in subfraction II. Subfractions III and IV were further characterized (31). Both were heterogeneous and contained succinyl ester substituents. At pH 7.4, the anionic character is conferred only by phosphoglycerol and succinyl substituents. If OPGs were homogeneously sized, one could consider that subfraction II to V correspond to increasing negative charges (1, 2, 3, and 4) attached to the glucosidic backbone. However, the OPG backbones are heterogeneous in size, with a degree of polymerization ranging from 6 to 12. Thus, subfraction II to V should correspond to various charge-to-mass ratios due to various levels of substitution of heterogeneously sized backbones.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   DEAE-Sephacel anion exchange column chromatography profiles of [U-14C] glucose labeled OPGs from strains DF214 (mdo+), NFB734 (mdoB), NFB1933 (mdoB mdoC), and NFB1919 (mdoC). Ionic strength was increased by steps of 0.05 M NaCl at fractions indicated by the arrows. Fractions (4 ml) were collected and aliquots were counted. Peak I, neutral glucans; peaks II to V, anionic glucans. The number on the right of each peak refers to the percentage of the total for the corresponding subfraction.

Tn5 insertion lies within a gene adjacent to the mdoGH operon. The mdoC1::Tn5 mutation was cloned into the pUC8 plasmid vector from the genomic DNA by selecting for the kanamycin resistance conferred by the transposon. The DNA sequence read from the resulting plasmid started within the IS50 DNA and continued in the mdoC DNA. Comparison of this sequence and the E. coli genomic DNA data (5, 23) allowed us to localize mdoC as the previously unassigned gene, named b1047, just upstream from the mdoGH operon (19).

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Genetic organization of the mdoC region of the E. coli chromosome (top) and restriction map of the 8-kb insert of pNF239 and its derivatives (bottom). Only relevant restriction sites are shown; arrows and T indicate transcriptional units and transcriptional terminators, respectively. PstI* and colE1 are part of the insert originating from the colE1 vector (9). Horizontal empty bars indicate the structure of the various inserts of the plasmids.

Nucleotide sequence analysis of the mdoC gene. The Tn5 was found inserted at nucleotide 1107787 (5) (GenBank accession no. U00096). It is 377 nucleotides downstream from a putative start codon ATG (nucleotide 1108164). The corresponding open reading frame, encoding a 385-amino-acid polypeptide, ends at a TAA codon (nucleotide 1107007). Fifty-five nucleotides downstream from the TAA, two inverted repeats followed by a stretch of six Ts characteristic of an intrinsic transcription terminator are found.

Amino acid sequence analysis of MdoC. The MdoC protein (385 amino acids) exhibits stretches of hydrophobic amino acids over its entire length: 10 transmembrane segments are predicted by the TopPred2 program (26a, 33). The exoH gene, encoding a succinyl-transferase of 370 amino acids, is implicated in the synthesis of exopolysaccharides and is essential for the nodulation process of Rhizobium meliloti. ExoH, like MdoC, is essentially hydrophobic, and 7 or 11 transmembrane segments were proposed (3, 13). Nevertheless, no amino acid similarities between these two proteins were found.