Functional analysis of the Burkholderia cenocepacia J2315 BceAJ protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities
ABSTRACT The bceA(J) gene from the cystic fibrosis isolate Burkholderia cenocepacia J2315 encodes a 56-kDa bifunctional protein, with phosphomannose isomerase (PMI) and guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP) activities, a new member of the poorly characterised type II PMI class of proteins. Due to the lack of homology between the type II PMIs and the human PMI, this class of proteins are being regarded as interesting potential targets to develop new antimicrobials. The BceA(J) protein conserves the four typical motifs of type II PMIs: the pyrophosphorylase signature, the GMP active site, the PMI active site and the zinc-binding motif. After overproduction of BceA(J) by Escherichia coli as a histidine tag derivative, the protein was purified to homogeneity by affinity chromatography. The GMP activity is dependent on the presence of Mg(2+) or Ca(2+) as cofactors, while the PMI activity uses a broader range of divalent ions, in the order of activation Mg(2+) > Ca(2+) > Mn(2+) > Co(2+) > Ni(2+). The kinetic parameters K(m), V(max) and K(cat)/K(m) for the PMI and GMP activities were determined. Results suggest that the enzyme favours the formation of GDP-mannose instead of mannose catabolism, thus channelling precursors to the formation of glycoconjugates.
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- "Several of the enzymes involved in the process are encoded within the bce-I and bce-II gene clusters. Gene bceA encodes a bifunctional protein providing the first and third steps (phosphomannose isomerase and GDP-d-mannose pyrophosphorylase activities) for converting fructose-6-phosphate from central metabolism into GDP-d-mannose (Figure 2A; Sousa et al., 2007a, 2008). A bceA gene insertion mutant was still capable of producing half the amount of EPS of the parental strain B. cepacia IST408, showing that other enzymes producing GDP-dmannose are present in this microorganism (Sousa et al., 2007a). "
ABSTRACT: 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.Frontiers in Cellular and Infection Microbiology 12/2011; 1:16. DOI:10.3389/fcimb.2011.00016 · 2.62 Impact Factor
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- "These authors have found that, while the GMP domain of BceA J was putatively composed of α-helices interspaced by β-strands, the PMI domain of the protein was almost composed of β-strands. This predicted secondary structure is in good agreement with the occurrence of two distinct domains in type II PMIs (Sousa et al., 2008). Both the PMI and GMP enzyme activities were detected in all the purified type II PMIs mentioned before. "
ABSTRACT: This work was partially funded by FEDER and Fundação para a Ciência e Tecnologia (FCT), Portugal, through contract PTDC/EBB-BIO/098352/2008, and a post-doctoral grant to Sílvia A. Sousa. Christian G. Ramos acknowledges a doctoral grant from Fundação Calouste Gulbenkian (FCG).Biopolymers, 09/2010; , ISBN: 978-953-307-109-1
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- "This is a promising methodology that is worth to explore in the silencing of essential genes or genes involved in virulence of Bcc bacteria. Targeting of genes encoding components of the quorumsensing systems regulating the expression of virulence factors (Sokol et al. 2007), or the development of inhibitors of proteins involved in the synthesis of key virulence factors such as the EPS cepacian, as is the case of type II phosphomannose isomerases (Sousa et al. 2008a), also represent attractive targets for the development of novel therapeutics. Major regulatory proteins, as is the case of the Hfq small noncoding RNA chaperone (Sousa et al. 2010), also represent attractive targets. "
ABSTRACT: 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.Applied Microbiology and Biotechnology 04/2010; 87(1):31-40. DOI:10.1007/s00253-010-2528-0 · 3.81 Impact Factor