Article

Functional analysis of the Burkholderia cenocepacia J2315 BceAJ protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities.

IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisbon, Portugal.
Applied Microbiology and Biotechnology (Impact Factor: 3.81). 07/2008; 80(6):1015-22. DOI: 10.1007/s00253-008-1612-1
Source: PubMed

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.

0 Followers
 · 
204 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The bacterial plant pathogen Xylella fastidiosa produces biofilm that accumulates in the host xylem vessels, affecting disease development in various crops and bacterial acquisition by insect vectors. Biofilms are sensitive to the chemical composition of the environment, and mineral elements being transported in the xylem are of special interest for this pathosystem. Here, X. fastidiosa liquid cultures were supplemented with zinc and compared with non-amended cultures for determining the effect of Zn on growth, biofilm, and exopolysaccharide (EPS) production in batch and flow culture conditions. Results show that Zn reduces growth and biofilm production in both conditions. However, in microfluidic chambers under liquid flow and with constant bacterial supplementation (closer to conditions inside the host), a dramatic increase in biofilm aggregates was seen in the Zn-amended medium. Biofilms formed under these conditions were strongly attached to surfaces and were not removed by media flow. This phenomenon was correlated with increased EPS production in stationary phase cells grown under high Zn concentrations. Zn did not cause greater adhesion to surfaces by individual cells. Additionally viability analyses suggest that X. fastidiosa may be able to enter the viable but non-culturable state in vitro, and Zn can hasten the onset of this state. Together, these findings suggest that Zn can act as a stress factor with pleiotropic effects on X. fastidiosa and indicate that, although Zn could be used as a bactericide treatment, it could trigger the undesired effect of stronger biofilm formation upon re-inoculation events.
    Applied and Environmental Microbiology 11/2013; DOI:10.1128/AEM.02998-13 · 3.95 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 01/2011; 1:16. DOI:10.3389/fcimb.2011.00016 · 2.62 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A multidisciplinary approach is being used to identify and validate virulence factors and determinants of bacteria of the Burkholderia cepacia complex (Bcc). Bcc is a group of problematic opportunistic pathogenic bacteria, particularly among cystic fibrosis patients, due to their easy transmissibility, resistance to multiple antibiotics and potential to cause a fatal necrotizing pneumonia. Genes encoding a Type II phosphomannose isomerase (BceA J ), an outer membrane protein A (Bcn-OmpA), and an acyl carrier protein (ACP) were already cloned and functionally characterized, envisaging their exploitation as new targets for the rational design of novel therapeutic strategies to fight infections caused by these emergent multi-drug resistant human pathogens.
    Bioengineering (ENBENG), 2011. ENBENG 2011. 1st Portuguese Meeting inBioengineering (ENBENG), 2011. ENBENG 2011. 1st Portuguese Meeting in; 01/2011