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.

FIG. 1.

Genetic organization of the bce gene cluster directing the biosynthesis of cepacian by Burkholderia bacteria. In representative strains of the species B. xenovorans, B. phymatum, B. phytofirmans, and B. graminis, the bce genes are clustered together in the same genomic region (a), while, in representative strains of the Burkholderia cepacia complex, comprising B. pseudomallei, B. oklahomensis, and B. thailandensis, the bce genes are split into two regions 155 to 314 kb apart (b). Strains from B. mallei have the bce-II cluster only. The locus tags for each gene in the B. vietnamiensis G4 genome are as follows: for bceA, Bcep1808_4200; for bceB, Bcep1808_4201; for bceC, Bcep1808_4202; for bceD, Bcep1808_4203; for bceE, Bcep1808_4204; for bceF, Bcep1808_4205; for bceG, Bcep1808_4206; for bceH, Bcep1808_4207; for bceI, Bcep1808_4208; for bceJ, Bcep1808_4209; for bceK, Bcep1808_4210; for bceM, Bcep1808_4471; for bceN, Bcep1808_4472; for bceO, Bcep1808_4473; for bceP, Bcep1808_4474; for bceQ, Bcep1808_4475; for bceR, Bcep1808_4476; for bceS, Bcep1808_4477; for bceT, Bcep1808_4479; and for bceU, Bcep1808_4480.

FIG. 2.

EPS production by Burkholderia strains. Cells from different Burkholderia species (a) and from B. cepacia IST408 (•), B. cepacia IST408 bceR::pIS58-2 (▪), B. multivorans ATCC 17616 (▴), and B. multivorans ATCC 17616 bceS::pSF71-8 (▵) (b) were grown in MM for 3 days at 30°C and EPSs quantified by dry weight after ethanol precipitation. Data represent the means of results from at least three independent experiments. Error bars show standard deviations. Phenotype of B. cepacia IST408 bceR::pIS58-2 harboring pMLBAD (c) or pIS94-1 containing the bceR gene (d) grown in solid MM supplemented with 1% arabinose.

FIG. 3.

FTIR analysis of the purified EPS produced by Burkholderia cepacia IST408 (a) and B. phytofirmans PsJN (b), showing similar spectra. Peaks: 1 and 2, carboxylic and/or hydroxyl C-O; 3 and 4, C-H; 5, water; 6, carbonyl; 7, C-C bonds; 8, water and hydroxyl groups of the EPS.

FIG. 4.

Quantitative real-time RT-PCR analysis of the relative transcript abundances in B. multivorans D2095 with respect to B. multivorans D2214 after 17 h of growth (a) and B. cepacia IST408 with respect to bceQ::pIS58-1 after 24 h of growth (b). For each gene, the data were standardized to values obtained for the internal control gene gyrB. The results were obtained from three independent experiments. Error bars represent standard deviations.

FIG. 5.

Protective role of EPS against desiccation and iron ion stress. Cells from overnight grown cultures of B. xenovorans LB400 (▪), B. multivorans ATCC 17616 (▴), and B. cepacia IST408 (•) were harvested by centrifugation and exposed to desiccation (a) or 50 mM ferrous sulfate (b) at 30°C in the presence (closed symbols) or absence (open symbols) of 2.5 g/liter of cepacian. The remaining viable bacterial counts were determined at different time points by determining the numbers of CFU. The data represents the means of results from three independent experiments. Error bars show standard deviations.

FIG. 6.

Pathway leading to the nucleotide-sugar precursors for cepacian biosynthesis by Burkholderia and model for the assembly and export of the EPS. With the exception of BceP, all the Bce proteins have confirmed or putative roles in EPS biosynthesis, as described in the text. Abbreviations: Glc, glucose; GlcA, glucuronic acid; Gal, galactose; Rha, rhamnose; Man, mannose; GDP, guanosine-5′-diphosphate; UDP, uridine-5′-diphosphate; PGM, phosphoglucomutase; UGE, UDP-glucose epimerase; PMM, phosphomannomutase; UGP, UDP-glucose pyrophosphorylase; PGI, phosphoglucose isomerase; GMP, GDP-d-mannose pyrophosphorylase; UGD, UDP-glucose dehydrogenase; PMI, phosphomannose isomerase; GRS, GDP-rhamnose synthetase.