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.

Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGH operon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgH gene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.Osmoregulated periplasmic glucans (OPGs) are a family of oligosaccharides found in the periplasmic space of gram-negative bacteria. Their two common features are the presence of glucose as the sole constituent sugar and their increased level in media of low osmolarity (5).Members of the family Enterobacteriaceae and related bacteria synthesize a family of linear and branched OPGs that are variously substituted. The linear backbone is constituted by glucose units joined by ␤,1-2 linkages, and the branches are made of one glucose unit linked to the main chain by a ␤,1-6 linkage. In Escherichia coli, the backbone, containing 7 to 13 glucose units, is substituted with phosphoglycerol, phosphoethanolamine, and succinyl residues (19). In Erwinia chrysanthemi, the backbone contains 5 to 12 glucose units substituted with succinyl and acetyl residues (9), and in Pseudomonas syringae, the backbone, consisting of 6 to 13 glucose units, is not substituted (38). In E. coli, the OPG backbone is synthesized by the products of the mdoGH operon located in the vicinity of pyrC, a gene involved in the biosynthesis of uracil (6). In this bacterium, the defect in OPG synthesis does not confer an easily selectable phenotype in laboratory conditions. Thus, the mdoGH locus was cloned using the linked selectable genetic marker pyrC (23).Many factors are involved in the virulence of pathogenic bacteria, and OPGs appear to be among them. In P. syringae pv. syringae, the causal agent of brown spot disease of the common bean (Phaseolus vulgaris), the hrpM mutant, obtained after transposon mutagenesis, was isolated because it failed to incite disease on the host plant and to cause the hypersensitive reaction on a non-host plant such as tobacco (29). The hrpM mutant does not synthesize OPGs, and the hrpM locus complements the OPG synthesis defect of the mdoH200::Tn10 mutant of E. coli. The amino acid sequences of HrpM and MdoH are 75.5% identical and 87.5% similar (25). More recently, a transposon insertion in a gene similar to hrpM/mdoH was isolated because it severely reduces the virulence in Pseudomonas aeruginosa PA14, an opp...

Dickeya dadantii is a plant-pathogenic enterobacterium responsible for the soft rot disease of many plants of economic importance. We present here the sequence of strain 3937, a strain widely used as a model system for research on the molecular biology and pathogenicity of this group of bacteria. Dickeya dadantii, formerly Erwinia chrysanthemi (11), is the causative agent of soft rot disease in a wide range of plant species, including many economically important crops (10). Soft rot results from the maceration of plant tissues following degradation of pectin, the major component of primary cell walls (7). D. dadantii is a devastating opportunistic pathogen in storage organs and fleshy tissues, particularly when compromised by bruising, excess water, low oxygen levels, or high temperatures. D. dadantii is also associated with systemic infections, vascular disorders, foliar necroses, and latent infections in growing plants. We sequenced and annotated the complete genome of Dickeya dadantii strain 3937, a strain widely used as a model system for research on the molecular biology and pathogenicity of this group of bacteria. Two whole-genome shotgun libraries were prepared with plasmid pHOS2 with target insert sizes of 2 to 3 kb and 10 to 12 kb. We collected approximately 67,000 dual-end sequences, 67% from small-insert clones and 33% from the larger insert library. Sequences were assembled into contigs using the Celera assembler (9), and this assembly was transferred to SeqMan II (Lasergene) for finishing. Primer walking was employed to close gaps covered by clones available from the shotgun libraries. The remaining gaps were closed by sequencing PCR products generated using primers designed from the ends of assembled and ordered contigs. PCR products spanning each rRNA operon were sequenced separately to resolve sequence differences between copies. We used Glimmer 2.0 (3) for initial prediction of protein coding regions. We added, deleted, and revised endpoints of genes based on comparisons to other genomes, genes, and proteins in the NCBI databases. tRNA sequences were identified using tRNAscan-SE (8) with additional examination to identify specific tRNAs not distinguishable by their anticodons alone. rRNA genes were identified by comparison to other enterobacterial sequences using

The murI gene of Escherichia coli was recently identified on the basis of its ability to complement the only mutant requiring D-glutamic acid for growth that had been described to date: strain WM335 of E. coli B/r (P. Doublet, J. van Heijenoort, and D. Mengin-Lecreulx, J. Bacteriol. 174:5772-5779, 1992). We report experiments of insertional mutagenesis of the murI gene which demonstrate that this gene is essential for the biosynthesis of D-glutamic acid, one of the specific components of cell wall peptidoglycan. A special strategy was used for the construction of strains with a disrupted copy of murI, because of a limited capability of E. coli strains grown in rich medium to internalize D-glutamic acid. The murI gene product was overproduced and identified as a glutamate racemase activity. UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala), which is the nucleotide substrate of the D-glutamic-acid-adding enzyme (the murD gene product) catalyzing the subsequent step in the pathway for peptidoglycan synthesis, appears to be an effector of the racemase activity.