The genus Burkholderia includes strains pathogenic to animals and plants, bioremediators, or plant growth promoters. Genome sequence analyses of representative Burkholderia cepacia complex (Bcc) and non-Bcc strains for the presence of the bce-I gene cluster, directing the biosynthesis of the exopolysaccharide (EPS) cepacian, further extended this previously described cluster by another 9 genes. The genes in the bce-II cluster were named bceM to bceU and encode products putatively involved in nucleotide sugar precursor biosynthesis and repeat unit assembly, modification, and translocation across the cytoplasmic membrane. Disruption of the B. cepacia IST408 bceQ and bceR genes, encoding a putative repeat unit flippase and a glycosyltransferase, respectively, resulted in the abolishment of cepacian biosynthesis. A mutation in the bceS gene, encoding a putative acyltransferase, did not affect EPS production yield significantly but decreased its acetylation content by approximately 20%. Quantitative real-time reverse transcription-PCR experiments confirmed the induction of genes in the bce-I and bce-II clusters in a Burkholderia multivorans EPS producer clinical isolate in comparison to the level for its isogenic EPS-defective strain. Fourier Transform infrared spectroscopy analysis confirmed that the exopolysaccharide produced by 10 Burkholderia isolates tested was cepacian. The ability of Burkholderia strains to withstand desiccation and metal ion stress was higher when bacteria were incubated in the presence of 2.5 g/liter of cepacian, suggesting that this EPS plays a role in the survival of these bacteria by contributing to their ability to thrive in different environments.

A deficiency of plasmalogens, caused by impaired peroxisomal metabolism affects normal development and multiple organs in adulthood. Treatment options aimed at restoring plasmalogen levels may be relevant for the therapy of peroxisomal and non-peroxisomal disorders. In this study we determined the in vivo efficacy of an alkyl glycerol (AG), namely, 1-O-octadecyl-rac-glycerol, as a therapeutic agent for defects in plasmalogen synthesis. To achieve this, Pex7 knockout mice, a mouse model for Rhizomelic Chondrodysplasia Punctata type 1 characterized by the absence of plasmalogens, and WT mice were fed a control diet or a diet containing 2% alkyl-glycerol. Plasmalogen levels were measured in target organs and the biochemical data were correlated with the histological analysis of affected organs. Plasmalogen levels in all peripheral tissues of Pex7 KO mice fed the AG diet for 2 months normalized to the levels of AG fed WT mice. In nervous tissues of Pex7 KO mice fed the AG-diet, plasmalogen levels were significantly increased compared to control fed KO mice. Histological analysis of target organs revealed that the AG-diet was able to stop the progression of the pathology in testis, adipose tissue and the Harderian gland. Interestingly, the latter tissues are characterized by the presence of lipid droplets which were absent or reduced in size and number when ether-phospholipids are lacking, but which can be restored with the AAG treatment. Furthermore, nerve conduction in peripheral nerves was improved. When given prior to the occurrence of major pathological changes, the AG-diet prevented or ameliorated the pathology observed in Pex7 KO mice depending on the degree of plasmalogen restoration. This study provides evidence of the beneficial effects of treating a plasmalogen deficiency with alkyl-glycerol.

The Burkholderia cepacia complex (Bcc) is a group of 17 closely related species of the beta-proteobacteria subdivision that emerged in the 1980s as important human pathogens, especially to patients suffering from cystic fibrosis. Since then, a remarkable progress has been achieved on the taxonomy and molecular identification of these bacteria. Although some progress have been achieved on the knowledge of the pathogenesis traits and virulence factors used by these bacteria, further work envisaging the identification of potential targets for the scientifically based design of new therapeutic strategies is urgently needed, due to the very difficult eradication of these bacteria with available therapies. An overview of these aspects of Bcc pathogenesis and opportunities for the design of future therapies is presented and discussed in this work.

The biosynthesis of the exopolysaccharide (EPS) cepacian by Burkholderia cepacia complex strains requires the 16.2-kb bce cluster of genes. Two of the clustered genes, bceD and bceF, code for two proteins homologous to phosphotyrosine phosphatases and tyrosine kinases, respectively. We show experimental evidence indicating that BceF is phosphorylated on tyrosine and that the conserved lysine residue present at position 563 in the Walker A ATP-binding motif is required for this autophosphorylation. It was also proved that BceD is capable of dephosphorylating the phosphorylated BceF. Using the artificial substrate p-nitrophenyl phosphate (PNPP), BceD exhibited a V max of 8.8 mol of PNPP min ؊1 mg ؊1 and a K m of 3.7 mM PNPP at 30°C. The disruption of bceF resulted in the abolishment of cepacian accumulation in the culture medium, but 75% of the parental strain's EPS production yield was still registered for the bceD mutant. The exopolysaccharide produced by the bceD mutant led to less viscous solutions and exhibited the same degree of acetylation as the wild-type cepacian, suggesting a lower molecular mass for this mutant biopolymer. The size of the biofilm produced in vitro by bceD and bceF mutant strains is smaller than the size of the biofilm formed by the parental strain, and this phenotype was confirmed by complementation assays, indicating that BceD and BceF play a role in the establishment of biofilms of maximal size.

Biofilms are communities of microbial cells that are encapsulated within a self-produced polymeric matrix. The matrix is critical to the success of biofilms in diverse habitats; however, many details of the composition, structure, and function remain enigmatic. Biofilms formed by the Gram-positive bacteriumBacillus subtilisdepend on the production of the secreted film-forming protein BslA. Here, we show that a gradient of electron acceptor availability through the depth of the biofilm gives rise to two distinct functional roles for BslA and that these roles can be genetically separated through targeted amino acid substitutions. We establish that monomeric BslA is necessary and sufficient to give rise to complex biofilm architecture, whereas dimerization of BslA is required to render the community hydrophobic. Dimerization of BslA, mediated by disulfide bond formation, depends on two conserved cysteine residues located in the C-terminal region. Our findings demonstrate that bacteria have evolved multiple uses for limited elements in the matrix, allowing for alternative responses in a complex, changing environment.

Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens infecting hosts such as cystic fibrosis (CF) patients. Long-term Bcc infection of CF patients' airways has been associated with emergence of phenotypic variation. Here we studied two Burkholderia multivorans clonal isolates displaying different morphotypes from a chronically infected CF patient to evaluate trait development during lung infection. Expression profiling of mucoid D2095 and non-mucoid D2214 isolates revealed decreased expression of genes encoding products related to virulence-associated traits and metabolism in D2214. Furthermore, D2214 showed no exopolysaccharide production, lower motility and chemotaxis, and more biofilm formation, particularly under microaerophilic conditions, than the clonal mucoid isolate D2095. When Galleria mellonella was used as acute infection model, D2214 at a cell number of approximately 7¾10 6 c.f.u. caused a higher survival rate than D2095, although 6 days post-infection most of the larvae were dead. Infection with the same number of cells by mucoid D2095 caused larval death by day 4. The decreased expression of genes involved in carbon and nitrogen metabolism may reflect lower metabolic needs of D2214 caused by lack of exopolysaccharide, but also by the attenuation of pathways not required for survival. As a result, D2214 showed higher survival than D2095 in minimal medium for 28 days under aerobic conditions. Overall, adaptation during Bcc chronic lung infections gave rise to genotypic and phenotypic variation among isolates, contributing to their fitness while maintaining their capacity for survival in this opportunistic human niche.

The genus Burkholderia comprises more than 60 species able to adapt to a wide range of environments such as soil and water, and also colonize and infect plants and animals. They have large genomes with multiple replicons and high gene number, allowing these bacteria to thrive in very different niches. Among the properties of bacteria from the genus Burkholderia is the ability to produce several types of exopolysaccharides (EPSs). The most common one, cepacian, is produced by the majority of the strains examined irrespective of whether or not they belong to the Burkholderia cepacia complex (Bcc). Cepacian biosynthesis proceeds by a Wzy-dependent mechanism, and some of the B. cepacia exopolysaccharide (Bce) proteins have been functionally characterized. In vitro studies showed that cepacian protects bacterial cells challenged with external stresses. Regarding virulence, bacterial cells with the ability to produce EPS are more virulent in several animal models of infection than their isogenic non-producing mutants. Although the production of EPS within the lungs of cystic fibrosis (CF) patients has not been demonstrated, the in vitro assessment of the mucoid phenotype in serial Bcc isolates from CF patients colonized for several years showed that mucoid to non-mucoid transitions are relatively frequent. This morphotype variation can be induced under laboratory conditions by exposing cells to stress such as high antibiotic concentration. Clonal isolates where mucoid to non-mucoid transition had occurred showed that during lung infection, genomic rearrangements, and mutations had taken place. Other phenotypic changes include variations in motility, chemotaxis, biofilm formation, bacterial survival rate under nutrient starvation and virulence. In this review, we summarize major findings related to EPS biosynthesis by Burkholderia and the implications in broader regulatory mechanisms important for cell adaptation to the different niches colonized by these bacteria.

The bacterial tyrosine-kinase (BY-kinase) family comprises the major group of bacterial enzymes endowed with tyrosine kinase activity. We previously showed that the BceF protein from Burkholderia cepacia IST408 belongs to this BY-kinase family and is involved in the biosynthesis of the exopolysaccharide cepacian. However, little is known about the extent of regulation of this protein kinase activity. In order to examine this regulation, we performed a comparative transcriptome profile between the bceF mutant and wild-type B. cepacia IST408. The analyses led to identification of 630 genes whose expression was significantly changed. Genes with decreased expression in the bceF mutant were related to stress response, motility, cell adhesion, and carbon and energy metabolism. Genes with increased expression were related to intracellular signaling and lipid metabolism. Mutation of bceF led to reduced survival under heat shock and UV light exposure, reduced swimming motility, and alteration in biofilm architecture when grown in vitro. Consistent with some of these phenotypes, the bceF mutant demonstrated elevated levels of cyclic-di-GMP. Furthermore, BceF contributed to the virulence of B. cepacia for larvae of the Greater wax moth, Galleria mellonella. Taken together, BceF appears to play a considerable role in many cellular processes, including biofilm formation and virulence. As homologues of BceF occur in a number of pathogenic and plant-associated Burkholderia strains, the modulation of bacterial behavior through tyrosine kinase activity is most likely a widely occurring phenomenon. Bacterial tyrosine kinases (BY-kinases) are best characterized for regulating the biosynthesis and the export of bacterial extracellular carbohydrate polymers in both Gram-positive and Gram-negative bacteria. These proteins comprise two domains: a transmembrane activator domain with a large extracellular loop and an intracellular catalytic domain encompassing the ATPbinding site and tyrosine-rich region (1). These two domains can either be linked in a single polypeptide, such as in Proteobacteria and Actinobacteria, or split into two separate proteins encoded by adjacent genes, as in the Firmicutes (1). Resolution of the BYkinase tridimensional structures showed no resemblance to their mammalian counterparts, and therefore these proteins are seen as good targets for antibacterial drug design (2).A number of studies have shown the importance of BY-kinases in polysaccharide biosynthesis, mainly as components of a multienzymatic complex responsible for polysaccharide polymerization and export (3). Moreover, several enzymes involved in sugarnucleotide biosynthesis and repeat unit formation have also been identified as phosphorylation targets of BY-kinases. For example, Escherichia coli Wzc and Bacillus subtilis PtkA BY-kinases were found to phosphorylate UDP-glucose dehydrogenase enzymes (4, 5), EpsD from Streptococcus thermophilus (6) and Wzc of Klebsiella pneumoniae (7) were found to phosphorylate the undecaprenylphosphate glycosyltransf...