Distribution of Cepacian Biosynthesis Genes among Environmental and Clinical Burkholderia Strains and Role of Cepacian Exopolysaccharide in Resistance to Stress Conditions

Abstract

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.

Growth on mannitol-rich media elicits a genome-wide transcriptional response in Burkholderia multivorans that impacts on multiple virulence traits in an exopolysaccharide-independent manner

“…This differing bce induction between strains was confirmed independently by quantitative reverse transcriptase PCR (RT-PCR) analysis of bceB, confirming it to be significantly upregulated in C1576 (193-fold) but not elevated in ATCC 17616. This observation was striking, given that both C1576 and ATCC 17616 are known to produce cepacian upon growth on MYEM (P. Cescutti, Personal Communication; Ferreira et al, 2010), and disruption of the bceB gene in both strains abolishes detectable EPS production, highlighting the pivotal role of the bce gene cluster in the EPS biosynthesis of both strains. However, the role of the bce gene clusters in ATCC 17616 EPS production did not appear to arise from significant changes in their transcriptional activation.…”

“…Mannitol is one of several sugars and sugar alcohols that can induce the overproduction of exopolysaccharide (EPS) by members of the BCC, resulting in a profoundly mucoid phenotype (Bartholdson et al, 2008), and MYEM has been widely used in studies defining the biological and structural properties of this EPS. The most common EPS produced by members of the BCC is cepacian (Herasimenka et al, 2007), the biosynthetic enzymes for which are encoded by the bce-I and bce-II gene clusters (Moreira et al, 2003;Ferreira et al, 2010). Whilst EPS has been identified as a putative virulence factor within the BCC (Conway et al, 2004;Cunha et al, 2004;Bylund et al, 2006), it has been reported that an inverse correlation exists between the amount of EPS produced and the rate of lung function decline in CF patients, with the presence of nonmucoid isolates linked to the greatest decline in the patients studied (Zlosnik et al, 2011).…”

Mechanisms Controlling the Expression of the Exopolysaccharide of Burkholderia and Role in Niche Adaptation

“…One common feature across the genus is the ability to produce an exopolysaccharide (EPS) termed cepacian. This ability is confirmed by the presence of the bce biosynthetic genes in all Burkholderia sequenced genomes with the exception for Burkholderia mallei (Ferreira et al, 2010). Cepacian is composed of a branched acetylated heptasaccharide repeat-unit with D-glucose, D-rhamnose, D-mannose, D-galactose and D-glucuronic acid in the ratio of 1:1:1:3:1 (Cescutti et al, 2000).…”

“…The last step of cepacian biosynthesis comprises polymerization and export of the polysaccharide to the extracellular environment. A multienzyme complex including a repeat-unit translocase, a polysaccharide polymerase, an outer membrane protein, among others are involved in this process (Ferreira et al, 2010;Moreira et al, 2003). Insights into the biosynthetic steps will be covered in this chapter.…”

“…Cepacian is composed of a branched acetylated heptasaccharide repeat-unit with D-glucose, D-rhamnose, D-mannose, D-galactose and D-glucuronic acid in the ratio of 1:1:1:3:1 (Cescutti et al, 2000). Cepacian biosynthesis starts with the synthesis of the nucleotide sugar precursors, catalyzed by isomerases, mutases, epimerases, among other enzymes (Ferreira et al, 2010). This step is followed by the assembly of the repeat-units by the sequential addition of sugars to an isoprenoid lipid by dedicated glycosyltransferases.…”