Search

The Burkholderia cepacia complex (Bcc) emerged as problematic opportunistic pathogens to cystic fibrosis (CF) patients. Although several virulence factors have been identified in Bcc, the knowledge of their relative contribution to Bcc pathogenicity remains scarce. In this work, we describe the identification and characterization of a B. cepacia IST408 mutant containing a disruption in the hfq gene. In other bacteria, Hfq is a global regulator of metabolism, acting as an RNA chaperone involved in the riboregulation of target mRNAs by small regulatory non-coding RNAs (sRNAs). The B. cepacia Hfq protein was overproduced as a histidine-tagged derivative, and we show evidence that the protein forms hexamers and binds sRNAs. When provided in trans, the B. cepacia IST408 hfq gene complemented the Escherichia coli hfq mutant strain GS081. Our results also show that the B. cepacia hfq mutant is more susceptible to stress conditions mimicking those faced by Bcc bacteria when infecting the CF host. In addition, the B. cepacia hfq mutant and two hfq mutants derived from B. dolosa and B. ambifaria clinical isolates also exhibited a reduced ability to colonize and kill the nematode Caenorhabditis elegans, used as an infection model. These data, together with the conservation of Hfq orthologues among Bcc, strongly suggest that Hfq plays a major role in the survival of Bcc under stress conditions, contributing to the success of Bcc as CF pathogens. DOI 10.1099/mic.0.035139-0

Bacterial type IV secretion systems (T4SS) perform two fundamental functions related to pathogenesis: the delivery of effector molecules to eukaryotic target cells, and genetic exchange. Two T4SSs have been identified in Burkholderia cenocepacia K56-2, a representative of the ET12 lineage of the B. cepacia complex (Bcc). The plant tissue watersoaking (Ptw) T4SS encoded on a resident 92 kb plasmid is a chimera composed of VirB/D4 and F-specific subunits, and is responsible for the translocation of effector(s) that have been linked to the Ptw phenotype. The bc-VirB/D4 system located on chromosome II displays homology to the VirB/D4 T4SS of Agrobacterium tumefaciens. In contrast to the Ptw T4SS, the bc-VirB/D4 T4SS was found to be dispensable for Ptw effector(s) secretion, but was found to be involved in plasmid mobilization. The fertility inhibitor Osa did not affect the secretion of Ptw effector(s) via the Ptw system, but did disrupt the mobilization of a RSF1010 derivative plasmid. DOI 10.1099/mic.0.033043-0

Burkholderia cenocepacia is highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalysed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome of B. cenocepacia contains three predicted ugd genes: [ugd.sub.BCAL2946], [ugd.sub.BCAM0855] and [ugd.sub.BCAM2034], all of which were individually inactivated. Only inactivation of [ugd.sub.BCAL2946] resulted in increased sensitivity to polymyxin B and this sensitivity could be overcome when either [ugd.sub.BCAL2946] or [ugd.sub.BCAM0855] but not [ugd.sub.BCAM2034] was expressed from plasmids. The growth of a conditional [ugd.sub.BCAL2946] mutant, created in the [DELTA][ugd.sub.BCAM0855] background, was significantly impaired under non-permissive conditions. Growth could be rescued by either [ugd.sub.BCAL2946] or [ugd.sub.BCAM0855] expressed in trans, but not by [ugd.sub.BCAM2034]. Biochemical analysis of the purified, recombinant forms of [ugd.sub.BCAL2946] and [ugd.sub.BCAM0855] revealed that they are soluble homodimers with similar in vitro Ugd activity and comparable kinetic constants for their substrates UDP-glucose and [NAD.sup.+]. Purified [Ugd.sub.BCAM2034] showed no in vitro Ugd activity. Real-time PCR analysis showed that the expression of [ugd.sub.BCAL2946] was 5.4- and 135-fold greater than that of [ugd.sub.BCAM0855] and [ugd.sub.BCAM2034], respectively. Together, these data indicate that the combined activity of [Ugd.sub.BCAL2946] and [Ugd.sub.BCAM0855] is essential for the survival of B. cenocepacia but only the most highly expressed ugd gene, [ugd.sub.BCAL2946], is required for polymyxin B resistance.

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex, a group of metabolically versatile bacteria that have emerged as opportunistic pathogens in cystic fibrosis and immunocompromised patients. Previously a screen of transposon mutants in a rat pulmonary infection model identified an attenuated mutant with an insertion in paaE, a gene related to the phenylacetic acid (PA) catabolic pathway. In this study, we characterized gene clusters involved in the PA degradation pathway of B. cenocepacia K56-2 in relation to its pathogenicity in the Caenorhabditis elegans model of infection. We demonstrated that targeted-insertion mutagenesis of paaA and paaE, which encode part of the putative PA-coenzyme A (CoA) ring hydroxylation system, paaZ, coding for a putative ring opening enzyme, and paaF, encoding part of the putative beta-oxidation system, severely reduces growth on PA as a sole carbon souree. paaA and paaE insertional mutants were attenuated for virulence, and expression of paaE in trans restored pathogenicity of the paaE mutant to wild-type levels. Interruption of paaZ and paaF slightly increased virulence. Using gene interference by ingested double-stranded RNA, we showed that the attenuated phenotype of the paaA and paaE mutants is dependent on a functional p38 mitogen-activated protein kinase pathway in C. elegans. Taken together, our results demonstrate that B. cenocepacia possesses a functional PA degradation pathway and that the putative PA-CoA ring hydroxylation system is required for full pathogenicity in C. elegans.

The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysaccharide (EPS) when onion tissue was provided as the sole nutrient. EPS production was not species-specific, was observed in isolates from both clinical and environmental sources, and did not correlate with the ability to cause maceration of onion tissue. Chemical analysis suggested that the onion components responsible for EPS induction were primarily the carbohydrates sucrose, fructose and fructans. Additional sugars were investigated, and all alcohol sugars tested were able to induce EPS production, in particular mannitol and glucitol. To investigate the molecular basis for EPS biosynthesis, we focused on the highly conserved bce gene cluster thought to be involved in cepacian biosynthesis. We demonstrated induction of the bce gene cluster by mannitol, and found a clear correlation between the inability of representatives of the Burkholderia cenocepacia ET1 2 lineage to produce EPS and the presence of an 11 bp deletion within the bceB gene, which encodes a glycosyltransferase. Insertional inactivation of bceB in Burkholderia ambifaria AMMD results in loss of EPS production on sugar alcohol media. These novel and surprising insights into EPS biosynthesis highlight the metabolic potential of the Bcc and show that a potential virulence factor may not be detected by routine laboratory culture. Our results also highlight a potential hazard in the use of inhaled mannitol as an osmolyte to improve mucociliary clearance in individuals with cystic fibrosis.

Burkholderia cenocepacia is an opportunistic pathogen causing serious infections in patients with cystic fibrosis. The widespread distribution of this bacterium in the environment suggests that it must adapt to stress to be able to survive. We identified in B. cenocepacia K56-2 a gene predicted to encode RpoE, the extra-cytoplasmic stress response regulator. The rpoE gene is the first gene of a predicted operon encoding proteins homologous to RseA, RseB, MucD and a protein of unknown function. The genomic organization and the co-transcription of these genes were confirmed by PCR and RT-PCR. The mucD and rpoE genes were mutated, giving rise to B. cenocepacia RSF24 and RSF25, respectively. While mutant RSF24 did not demonstrate any growth defects under the conditions tested, RSF25 was compromised for growth under temperature (44 [degrees]C) and osmotic stress (426 mM NaCI). Expression of RpoE in trans could complement the osmotic growth defect but exacerbated temperature sensitivity in both RSF25 and wild-type K56-2. Inactivation of rpoE altered the bacterial cell surface, as indicated by increased binding of the fluorescent dye calcofluor white and by an altered outer-membrane protein profile. These cell surface changes were restored by complementation with a plasmid encoding rpoE. Macrophage infections in which bacterial colocalization with fluorescent dextran was examined demonstrated that the rpoE mutant could not delay the fusion of B. cenocepacia-containing vacuoles with lysosomes, in contrast to the parental strain K56-2. These data show that B. cenocepacia rpoE is required for bacterial growth under certain stress conditions and for the ability of intracellular bacteria to delay phagolysosomal fusion in macrophages.

A lipase-catalyzed parallel kinetic resolution of mixtures of [beta]-D/L-nucleosides is developed. Computer modeling of the transition-state analogues during acylation of [beta]-D- and [beta]-L-2' -deoxycytidine with Pseudomonas cepacia lipase (PSL-C) is used for describing the high selectivity, where PSL-C has favored the 3E-O-levulination of the [beta]-D enantiomer and the 5' -OH group is acylated in 2E-deoxy-[beta]-L-cytidine.

A combination of high-performance liquid chromatography, mass spectrometry, and bioassays were used to determine if the diffusible signal factor (DSF) produced by Burkholderia cenocepacia (BDSF) is conserved in other members of the Burkholderia cepacia complex (Bcc). The data provide evidence for the important roles of DSF family molecules which are highly conserved bacterial cell-cell communication signals towards the ecology of the organisms that produce them.

A study was conducted to analyze the salt anions that influence the activity of Pseudomonas cepacia lipase both in aqueous and in nonaqueous media (NAM). The results showed that the decrease of the activation energy caused by the stabilization of the transition state due to kosmotropic anions might be responsible for specific anion enzyme activation/deactivation in NAM.