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Osmoregulated periplasmic glucans in Proteobacteria

Jean-Pierre Bohin
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb09075.x 11-19 First published online: 1 May 2000

Abstract

Large amounts of osmoregulated periplasmic glucans (OPGs) are found in the periplasmic space of Proteobacteria. Four families of OPGs are described on the basis of structural features of the polyglucose backbone. Depending on the species considered, OPGs can be modified to various extent by a variety of substituents. Genes governing the backbone synthesis are identified in a limited number of species. They belong to three unrelated families. OPG synthesis is subject to osmoregulation and feedback control. Osmoregulation can occur at the level of gene expression and/or at the level of enzyme activity. Mutants defective in OPG synthesis have a highly pleiotropic phenotype, indicative of an overall alteration of their envelope properties. Mutants of this kind were obtained as attenuated or avirulent derivatives of plant or animals pathogen. Thus, OPGs appear to be important intrinsic components of the Gram-negative bacterial envelope, which can be essential in extreme conditions found in nature, and especially when bacteria must interact with an eukaryotic host.

Keywords
  • Bacterial envelope
  • Periplasmic space
  • Osmoregulation
  • Pathogenicity
  • Glycosyltransferase

1 Introduction

The envelope of Gram-negative bacteria is characterized by a specialized cell compartment, the periplasmic space, located between the internal compartment, the cytoplasm, from which it is separated by the cell membrane, and the external environment from which it is separated by the outer membrane (reviewed in [1]). The periplasm forms a concentrated gel-like matrix containing the murein sacculus, a variety of specialized proteins found exclusively in this compartment, and the osmoregulated periplasmic glucans (OPGs) found at least in all the Proteobacteria tested. OPGs exhibit quite different structures among various species but they share several common characteristics: (i) they are oligosaccharides made of a limited number of units (5–24); (ii) D-glucose is the only constituent sugar; (iii) glucose units are linked, at least partially, by β-glycosidic bonds; (iv) glucan concentration in the periplasm increases in response to a decrease of environmental osmolarity.

The first report of OPG structures is probably the description by McIntire et al. [2], in 1942, of cyclic β,1-2 glucans found in culture filtrates of a crown-gall organism (Agrobacterium tumefaciens). In this work, the glucans were essentially thought as a particular sub-class of exopolysaccharides. The second discovery of OPGs came thirty years later during the study of phospholipid turn-over in Escherichia coli by E.P. Kennedy's group [3]. In this bacterium, the rapid phosphatidylglycerol turn-over is associated with the transfer of sn-1-phosphoglycerol to a new class of oligosaccharides, named as a consequence ‘membrane-derived oligosaccharides’ (MDOs, reviewed in [4]). Finally, the relation between the linear β-glucans found in the periplasm of E. coli and the cyclic β-glucans found in the periplasm of A. tumefaciens was established in 1986 with the demonstration that the syntheses of both kind of molecules are osmoregulated [5].

2 Glucan backbone structures

OPGs were described in various species of the alpha, beta and gamma subdivisions of the Proteobacteria. No information is available for other Gram-negative bacteria. Beyond their common features OPGs show an unexpected structural diversity. Four families can be distinguished on the basis of backbone organization (Fig. 1 and Table 1).

1

Four families of structures found in OPGs of Proteobacteria.

View this table:
1

OPG structures and biosynthetic genes distributions among Proteobacteria

Bacterial speciesOPGa FamilyGlucose residues per molBondsSubstituentsbGenomec sequencingMdoGd similarity (%)MdoHd similarity (%)NdvBd similarity (%)
Gamma subdivision
E. coliI5–12β-1,2P-GroC100100<5
β-1,6P-Etn30
Suc
S. typhiP9194
30
E. chrysanthemiI5–12β-1,2SucN8271
β-1,6Ace
P. aeruginosaP7365
P. syringaeI6–13β-1,2NoneN6862
β–1,6
Shewanella putrefaciensP3823
Vibrio choleraeP3522
H. influenzaeC<5<5<5
Y. pestisP
X. campestrisIV16β-1,2NoneN
α-1,6
Beta subdivision
R. solanacearumIV13β-1,2NoneN
α-1,6
Bordetella pertussisP
Neisseria gonorrhoeaeP
N. europeaeP8890
Alpha subdivision
R. sphaeroidesIV18β-1,2SucN
α-1,6Ace
Rhodobacter capsulatusP30
B. abortusII17–25β-1,2NoneN51
S. melilotiII17–25β-1,2P-GroN100
Suc
MeMal
B. japonicumIII10–13β-1,3P-ChoN
β-1,6
A. brasilenseIII12–13β-1,3SucN11
β-1,6
β-1,4
α-1,3
Caulobacter crescentusP19
Rickettsia prowazekiiC<5<5<5
  • aSee text for further detail.

  • bAbbreviations: P-Gro, phosphoglycerol; P-Etn, phosphoethanolamine; P-Cho, phosphocholine; Ace, acetyl; Suc, succinyl; MeMal, methylmalonyl.

  • cGenome sequencing project: C, complete; P, in progress, N, not available.

  • dProtein similarities were determined by comparing Blast2 scores (tblastn, protein sequence against genomic DNA translated in all six reading frames). Scores for MdoG, MdoH, and NdvB, were 1017, 1650 and 5644, respectively.

2.1 Family I

OPGs of E. coli are heterogeneous in size. They appear to range from 5 to 12 glucose residues, with the principal species containing 8 or 9 glucose residues [4]. The structure is highly branched, the backbone consisting of β-1,2-linked glucose units to which the branches are attached by β-1,6 linkages. Very similar structures (with only slight differences in the degree of polymerization) were found for the OPGs produced by other members of the gamma sub-division of the Proteobacteria: the closely related Enterobacteriaceae Erwinia chrysanthemi (V. Cogez, P. Talaga and J.-P. Bohin, unpublished results) and the more distantly related species Pseudomonas syringae[6].

2.2 Family II

Among members of the family Rhizobiaceae, periplasmic glucans are cyclic and heterogeneous in size (reviewed in [7]). Agrobacterium, Rhizobium, Sinorhizobium and Brucella species synthesize periplasmic glucans with similar structures. In these genera, periplasmic glucans are composed of a cyclic β-1,2-glucan backbone containing 17–25 glucose residues. Much larger molecules (up to 40 glucose units) were detected within cultures of a strain of Sinorhizobium meliloti[7].

2.3 Family III

Extracts of Bradyrhizobium spp. revealed the presence of β-1,6- and β-1,3-cyclic glucans containing 10–13 glucose units per ring [7,8]. NMR spectra very similar to those observed for Bradyrhizobium were obtained with OPGs extracted from Azospirillum brasilense; however three distinct glucans (I, II, and III) were separated by high-performance anion-exchange chromatography [9](and unpublished observation). The three glucans consist of a cyclic structure. Glucan I is made of 12 glucose units linked by 3 β-1,3, 8 β-1,6 and one β-1,4 linkages. Glucan II is derived from the glucan I by the addition of a glucose linked by an α-1,3 linkage, and glucan III is derived from glucan II by the addition of a 2-O-methyl group onto the α-linked glucose unit. Thus, the OPGs of the family III differ from those of the family II not only by the nature of the glycosidic linkage but also by a strict control of the ring size.

2.4 Family IV

Ralstonia solanacearum[10], Xanthomonas campestris[10,11] and Rhodobacter sphaeroides (Talaga, P., Cogez, Wieruszeski, J.-M., Stahl, B., Lemoine, J., Lippens, G., and Bohin, J.-P., unpublished) synthesize OPGs of very similar structural features. These OPGs are cyclic and they have a unique degree of polymerization (DP=13, 16, and 18, respectively). One linkage is α-1,6 whereas all the other glucose residues are linked by β-1,2 linkages. The presence of this α-1,6 linkage induces structural constraints in this kind of molecules, which contrast with the very flexible structures of the cyclic all β-1,2 OPGs of the family II [12].

3 Glucan backbone synthesis

Up to the present, relatively limited information is available about the genes necessary for OPG backbone biosynthesis. This is due in part to the difficulty to observe a phenotype correlating with lack of OPG biosynthesis (see below). Three distinct sets of genes were characterized: (i) in bacteria producing OPGs of the family I (E. coli, P. syringae and E. chrysanthemi); (ii) in bacteria producing OPGs of the family II (A. tumefaciens, S. meliloti, Sinorhizobium fredii and Brucella abortus); (iii) in bacteria producing OPGs of the family III (Bradyrhizobium japonicum and A. brasilense). Mutants were first characterized in several pioneer organisms (E. coli, P. syringae, A. tumefaciens, S. meliloti) and complementation experiments were used to obtain the homologous genes in closely related organisms. Today, since many bacterial genome sequencing projects are completed (18) or on the way to be completed soon (85; see Genomes On Line Database, !!http://geta.life.uiuc.edu/~nikos/genomes.html) one can address the question of OPG gene conservation (Table 1).

3.1 The mdo gene family

In E. coli, the two genes forming the mdoGH operon are necessary for OPG biosynthesis ([4], and references therein). MdoH, the mdoH gene product, is a 97-kDa protein spanning the inner membrane. MdoH consists of three large cytoplasmic regions linked by eight transmembrane segments [13]. This protein is necessary to a glucosyltransferase activity, which allows the in vitro production of linear β-1,2 polyglucose chains from the precursor UDP-glucose, if the acyl carrier protein is present [4]. Actually, the MdoH central domain shows structural features of a family 2-glycosyltransferase (B. Henrissat, personal communication; see Carbohydrate-Active enZYmes, http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html). To explain the presence of eight transmembrane segments in MdoH, we have postulated that they could form a channel for OPG translocation to the periplasm during the synthesis [13]. MdoG is a 56-kDa periplasmic protein whose function has not been established but is necessary to the polymerization. A tempting hypothesis would be that MdoG cooperates with MdoH, probably by catalyzing the addition of branches to the linear backbone emerging on the periplasmic side of the inner membrane. Genes closely related to mdoGH have been isolated in P. syringae (see references in [4]) and E. chrysanthemi (F. Page, S. Altabe, N. Hugouvieux-Cotte-Pattat, J.-M. Lacroix, J. Robert-Baudouy, and J.-P. Bohin, unpublished data). Systematic search for sequence similarity with MdoG or MdoH, including sequencing projects in progress (Infobiogen Website, http://www.infobiogen.fr, 29 December 1999, last date accessed) revealed the presence of conserved genes in various bacteria of the gamma subdivision of Proteobacteria (Table 1) and the levels of similarity found corroborate the generally accepted phylogenetic tree (data not shown). However, several species of the gamma subdivision, whose genomes are completely (or virtually) sequenced, do not show any similarity (for example, Haemophilus influenzae or Yersinia pestis; Table 1). In contrast, highly conserved genes are found in Nitrosomonas europeae (a member of the beta subdivision) and some similar sequences are found in members of the alpha subdivision whose sequencing is not complete. Despite the fact that data concerning the presence and, if so, the structures of OPGs in these species are not available, we can make some speculations: (i) different genes could have evolved to allow synthesis of similar structures (among the gamma subdivision, for example); (ii) alternatively, closely related genes could have evolved to allow the synthesis of different structures (OPGs of the families I and IV, for example).

During the search for genes encoding proteins homologous to MdoG, two paralogous genes were found in E. coli and in its close relative Salmonella typhi. The E. coli gene was named mdoD because it influences the sizes of OPG molecules. When it was inactivated on the chromosome, OPGs with a higher degree of polymerization were observed (Y. Lequette and J.-P. Bohin, unpublished data).

3.2 The ndv gene family

In S. meliloti, two linked genes are present: ndvB and ndvA (Table 1) ([7], and references therein). NdvB is a very large cytoplasmic membrane protein with a molecular mass of 319 kDa, of which only the 60% N-terminal end is actually required for glucan synthesis. The NdvB enzymatic activity can be assayed efficiently in vitro. Unpurified membrane preparations from ndvB+ strain are able to catalyze the formation of cyclic β-1,2 glucans from UDP-glucose and a high molecular-mass membrane protein is labeled when radioactive UDP-glucose is present. NdvA is a 67-kDa cytoplasmic protein which shares amino acid sequence similarity with several ATP-binding cassette transporters. The greatest similarity is found with the type I secretion protein HlyB. For this reason it was proposed that NdvA function is the translocation of the cyclic molecules toward the periplasm, and eventually to the external milieu, since several strains secrete OPGs in their environment. Consistent with this hypothesis, it has been shown that ndvA mutants are impaired in OPG secretion and in some OPG modifications which would occur in the periplasmic space (see below) [14]. Very similar genes were found in A. tumefaciens (chvB and chvA) and in S. fredii (ndvB), and B. abortus (cgs) [15]. These genes are strictly homologous and can complement OPG biosynthetic deficiency of ndvB mutants of S. meliloti. All these genes are involved in the synthesis of OPGs of the family II and systematic search for similarity with NdvB in available sequence data did not reveal sequences with a significant similarity. There is one exception: the cviB gene necessary for OPG synthesis in A. brasilense[16], which synthesizes OPGs of the family III (Table 1). One should notice also that low-resolution sequencing of R. sphaeroides chromosome II revealed the presence of a locus similar to ndvB [17], but the published data are still very incomplete.

3.3 A third gene family

Using the hypothesis that the different species of OPGs were functional homologues in different bacteria, Bhagwat et al. identified a cosmid clone from B. japonicum which complemented several defective characters (see below) of a S. meliloti ndvB mutant (see references in [18]). This clone carried the genetic information for synthesis of OPGs of the family III and two linked genes (ndvB and ndvC) were characterized. NdvB (102 kDa) and NdvC (62 kDa) are probably two membrane-bound proteins. When ndvB was inactivated, no OPGs were synthesized. In contrast, when ndvC was inactivated normal amounts of OPGs were produced, but their structures contained only β,1–3 linkages. NdvB, which does not share significant similarities with the namesake protein of S. meliloti, belongs to the same glycosyltransferase family as MdoH. NdvC shows significant similarities with several yeast genes involved in glucan metabolism. Thus, NdvB could be responsible for the primary polymerization of β,1-3-glucan molecules and NdvC could catalyze the formation of β,1-6 linkages by addition of new glucose units or by a rearrangement of the previous molecules.

4 Glucan substitution

In many, but not all, species the OPG backbones can be modified to various extents with a variety of substituents (Table 1). These substituents appear to belong to two classes: (i) residues originating from the membrane phospholipids like phosphoglycerol, phosphoethanolamine, or phosphocholine; (ii) residues originating from the intermediary metabolism like acetyl, succinyl and methylmalonyl. For the latter, acyl-coenzymes A are the likely donor molecules but this has not been demonstrated.

Phosphoglycerol transfer was shown to occur in the periplasmic compartment both in E. coli[4] and in S. meliloti[14]. In E. coli, mdoB encodes a membrane bound protein which transfers phosphoglycerol residues at the outer face of the cellular membrane to the artificial acceptor arbutin but not to soluble molecules of OPGs [4]. Thus, it was proposed that phosphoglycerol residues are transferred to nascent OPG molecules, still linked to the membrane. In S. meliloti, the cgmB gene encodes a soluble protein which shows only very limited similarity with MdoB [14]. Moreover, this enzyme can transfer phosphoglycerol residues to purified molecules of OPGs. Thus, the two proteins appear very different in the way they modify OPGs.

Succinyl transfer was shown to occur also in the periplasmic compartment of S. meliloti (see ref. in [14]). In E. coli, localization of the succinylation was not determined but the mdoC gene, which is necessary for succinylation of OPGs, encodes a protein with 10 predicted transmembrane segments. Consequently, it was proposed that this protein could catalyze the transfer of succinyl residues from the cytoplasmic side of the membrane to the nascent glucan backbones on the periplasmic side of the membrane [19]. If this is true, one can postulate the existence of a protein complex where the enzymes necessary for backbone synthesis and the enzymes necessary for backbone modification could work coordinately (Fig. 2).

2

OPG biosynthetic complex of E. coli: a working model.

In E. coli, the OPGs produced by the biosynthetic apparatus are very heterogeneous, both at the level of the degree of polymerization and of branching, and this variety of backbones can further be modified by one, two, or three residues of phosphoglycerol, and/or succinyl, and/or phosphoethanolamine. Thus, a majority of the OPG molecules have a high anionic character (up to five negative charges), while a minority of them is neutral.

5 Regulation of OPG biosynthesis

OPG biosynthesis appears to be subjected to regulation at two distinct levels. First, OPGs are synthesized in abundance when the medium osmolarity of the medium is very low (below or around 100 mOsm, depending on the lowest osmolarity tolerated by the bacterial species considered). For E. coli grown in low osmolarity medium, OPGs represent about 5% of the dry weight of E. coli cells, but OPG content drops dramatically when cells are grown in media with higher osmolarities (Fig. 3), even if an osmoprotectant like betaine is present [20]. When the medium osmolarity is high (over 600 mOsm) OPG represent only about 0.5% of the cellular dry weight. This osmoregulation was observed, more or less, in every bacterial species tested, including R. sphaeroides, R. solanacearum, and E. chrysanthemi (unpublished observations), with the exception of Brucella strains (see references in [21]) and S. meliloti strain GR4.

3

Osmotic regulation of OPG synthesis in E. coli[20].

When the osmolarity of the E. coli growth medium is decreased suddenly, OPG synthesis increases, but almost one generation time is necessary before the OPG content of the cells reaches its characteristic level [20]. Then, if the osmolarity of the growth medium is increased suddenly, OPG synthesis stops quickly but, since no OPG degradation is observed, several generations are necessary to adjust by dilution the OPG content of the cells [20]. This stability of OPGs was also reported in the case of S. meliloti[7]. Thus, a delay between stress and response is characteristic of the OPG content modulations.

Osmoregulation of the polyglucose biosynthetic activities appears to occur at a transcriptional and/or at a post-translational level depending on the bacterial species. The membrane-bound activities found in extracts of S. meliloti and E. coli do not vary with osmolarity of the growth medium, but these activities are very sensitive to ionic strength [21]. Consequently, it was postulated that OPG synthesis decreases in cells growing at high osmolarity as a consequence of ion accumulation into the cytoplasm. However, in E. coli, transcription of the mdoGH operon was observed to follows the same osmotically controlled response as the OPG synthesis, and, in A. brasilense, the level of glucosyltransferase activity present in membrane preparations of A. brasilense is strongly reduced when the bacteria were grown in medium of high osmolarity [21]. Thus, in different bacteria, regulation could occur either or both at the level of gene expression and protein activity.

A recent study of the cgmB transcripts in S. meliloti revealed that transcription of this gene (which encodes a phosphoglycerol transferase) is strongly enhanced in medium of low osmolarity, whereas an earlier study has indicated that phosphoglycerol transfer is inhibited at the level of enzyme activity when cells are grown at high osmolarity [14].

A second level of regulation is a feedback control of OPG synthesis. This was demonstrated with the study of an E. coli strain (pgi, zwf) unable to synthesize OPGs if glucose is not added to the medium. After addition of glucose, OPGs accumulate rapidly until their amount reaches a plateau corresponding to that observed in a wild-type strain and this same feedback control is observed regardless of medium osmolarity [4,20]. A similar conclusion was reached with the study of factors influencing OPG excretion by Rhizobium leguminosarum. Some strains, in particular environmental conditions (the presence of high NaCl concentrations, for example), continuously secrete OPGs which cannot any longer participate to the feedback control, and thus accumulate up to a g/l concentration in the medium (see reference in [7])

A simple hypothesis is that regulation of OPG synthesis maintains a constant concentration of these compounds in the periplasmic space, the volume of which increases as the medium osmolarity decreases [1].

6 Phenotypes of OPG defective mutants (Opg)

Mutants defective in OPG synthesis were obtained during the screening or the selection of attenuated or avirulent mutants of plant or animal pathogens. This was the case for chv mutants of A. tumefaciens and for hrpM mutants of P. syringae which, in addition, are unable to elicit the hypersensitive reaction (necrosis of plant cells resulting in the containment and death of invading bacteria) in nonhost plants. Similarly, ndv mutants of S. meliloti form defective nodules on alfalfa. One of three highly attenuated mutants of Salmonella typhimurium possesses a MudJ insertion in a gene homologous to mdoB[22]. One of several Pseudomonas aeruginosa PA14 mutants severely impaired in their virulence toward Caenorhabditis elegans possesses a TnphoA insertion in a gene homologous to mdoH[23]. This mutant has also a dramatic effect in the mouse model, causing no mortality, and is severely affected in its ability to grow in Arabidopsis leaves.

In other bacterial species, site-directed mutagenesis was used to inactivate OPG biosynthetic genes previously identified. In B. japonicum, mutations of the ndvB gene which result in total absence of OPGs led to the formation of ineffective nodules on soybean plants whereas mutations in the ndvC gene which result in formation of structurally distinct OPGs (predominantly β-1,3) do not affect nodulation. In E. chrysanthemi, mutations in both the mdoG and the mdoH homologues result in a complete loss of virulence (F. Page, S. Altabe, N. Hugouvieux-Cotte-Pattat, J.-M. Lacroix, J. Robert-Baudouy, and J.-P. Bohin, unpublished data).

Despite some differences in detail when comparing various bacterial species, mutants unable to synthesize OPGs have a highly pleiotropic phenotype, which indicates a probable overall alteration of their envelope properties. Opg mutants of A. tumefaciens and S. meliloti are severely impaired for growth in hypoosmotic media [7], whereas growth of similar mutants in E. coli[4] or E. chrysanthemi (unpublished data) is only slightly affected. Whatever the species considered, chemotaxis and motility are altered and this phenotype was reported to be the consequence of a reduced synthesis of flagellin. In E. coli, Opg mutants have a different porin composition with increased amount of OmpC, they are resistant to endogenously produced lysis protein of phage MS2, and they produce increased amount of colanic acid capsular polysaccharide [4,24]. According to the proposed model, activation of the capsular polysaccharide biosynthetic (cps) gene expression is accomplished through interactions, either directly or indirectly, between OPGs, RcsC (a sensor), and RcsB (a positive regulator) [24]. Mucoid phenotypes of Opg mutants were also observed in members of the family Rhizobiaceae [7] and in E. chrysanthemi (unpublished observation). Finally, in this latter organism, absence of OPGs results in hypersensitivity to biliary salts and reduced production of secreted enzymes (unpublished data).

Several groups isolated suppressor mutations able to restore a wild-type phenotype for Opg mutants. The data reported support the idea that OPG deficiency may affect structural organization of the cell envelope. E. coli chemotactic pseudorevertants were mutated in the ompB locus, mainly involved in regulation of the outer membrane porins OmpC and OmpF [25]. Nevertheless, this locus is not involved in regulation of OPG synthesis. Similarly, pseudorevertants of S. meliloti ndv mutants were selected for restoration of osmotolerance, motility, or symbiosis. Pseudorevertants for vegetative properties regained only slight symbiotic ability while symbiotic pseudorevertants were unrestored for vegetative properties and were still highly impaired in the first steps of the symbiotic interaction [7]. Partial suppression of the symbiotic defect was also observed after introduction in ndv mutants of cosmids which contained exo genes that are necessary for exopolysaccharide production. However, the basis of this partial suppression remains obscure [7]. In some instances, treatments applied to Opg mutants can partially restore wild-type functions. In A. tumefaciens, stability of the VirB10 protein, a component of the T-DNA transfer machinery, is affected by temperature and osmolarity. VirB10 instability is exacerbated in chvB mutant and virulence of chvB mutants was partially restored by incubation at low temperature [26].

As mentioned earlier, OPGs can be recovered in the external medium under certain conditions. However, addition of purified OPGs to the inoculum, cannot restore attachment and virulence or symbiotic ability of Rhizobiaceae Opg mutants. In contrast, these properties can be restored by pretreatment of the plants with purified rhicadhesin, a calcium-binding protein located on the bacterial cell surface [27].

7 Potential applications

OPGs of the family II possess the interesting capacity to form inclusion complexes with hydrophobic guest molecules (reviewed in [7]). Actually, these large and flexible cyclic molecules can adopt numbers of different structural conformations among which some exhibit a cavity compatible with inclusion. This capacity suggests that these molecules may find applications in food or pharmaceutical industries. Obviously, this capacity is not expected from OPGs of the family I. Moreover, recent structural studies on the glucan produced by R. solanacearum reveals that this cyclic constrained molecule presents a limited number of conformations and would have a very small cavity compatible with the presence of no more than one molecule of water (D. Horvath, G. Lippens, J.-M. Wieruszeski, and J.-P. Bohin, unpublished data). Finally, as far as no unique technical applications can be attributed to OPGs, the cost of industrial production remains certainly a limitation to their potential applications.

8 Speculation

OPGs appear to be important intrinsic components of the Gram-negative bacterial envelope, which can be essential in extreme conditions found in nature, especially when bacteria must interact with an eukaryotic host. But, what is the fundamental function of these compounds? E.P. Kennedy has proposed that OPG function as periplasmic osmoprotectants on the basis of their anionic character, where OPGs would participate in a Donnan equilibrium through the outer membrane [4]. However, in several cases OPGs are not substituted or are substituted by neutral residues. Consequently, osmoprotection is not likely to be the sole, or even primary function. OPGs may have a structural role in the envelope organization as carbohydrate molecules interacting with other structural components like phospholipids and/or peptidoglycan (see discussion of this point in [26]).

OPGs may also have a function as informational molecules sensed by specific proteins present in the periplasmic compartment or bound to one or the other membrane. OPGs were thus proposed as a possible signal for two E. coli inner membrane sensor proteins: EnvZ [25] and RcsC [24]. As discussed previously, when bacteria are shifted from a diluted medium to a concentrated environment, OPGs are not degraded but are instead diluted by the successive cell divisions. During the first steps of infection, bacteria which would have been exposed previously to media of low osmolarities, would possess, in their periplasmic space, OPG concentrations higher than characteristic for their new environment. If the OPG concentration could be monitored by one or several sensor proteins, it could be used as a kind of internal counter of the number of cell division, and, indirectly, of bacteria at very low density population. This could control the expression of a set of genes according to the increase of cell population and it would be, in some way, the counterpart of the quorum sensing systems involved in gene expression control at high cell density.

Finally, since OPGs can be released from the bacteria to their environment, sensing mechanisms could have evolved in potential plant hosts. This would be, perhaps, analogous to the signaling role played by lipid A with animal cells. For example, OPGs produced by B. japonicum may function as suppressors of a host defense response [18].

Acknowledgements

I thank all the present and former members of the Groupe de Génétique des Enveloppes Bactériennes for their work on this topic. I am also very grateful to our colleagues in various laboratories all around the world who have accepted a friendly collaboration. Special thanks are due to G. Lippens and J.-M. Wieruszeski. Work in my laboratory was supported by grants from the Centre National de la Recherche Scientifique, the Université des Sciences et Technologies de Lille, the Région Nord-Pas de Calais and the Ministère de l'Enseignement Supérieur et de la Recherche.

Footnotes

  • 1 This article is dedicated to the memory of Barbara J. Bachmann for her invaluable contribution to bacterial genetics as curator of the Escherichia coli Genetic Stock Center.

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View Abstract