Pyrimidine synthesis in Burkholderia cepacia ATCC 25416

K. LI AND T.P. WEST. 1995. Pyrimidine synthesis in the food spoilage agent Burkholderia cepacia ATCC 25416 was investigated. The five de novo pathway enzymes of pyrimidine biosynthesis were found to be active in B. cepacia ATCC 25416 and growth of this strain on uracil had an effect on the de novo enzyme activities. The in vitro regulation of aspartate transcarbamoylase activity in B. cepacia ATCC 25416 was studied and its activity was inhibited by PP_i, ATP, GTP, CTP and UTP. The enzymes cytidine deaminase, uridine phosphorylase and cytosine deaminase were found to be active in the salvage of pyrimidines in ATCC 25416. Overall, de novo pyrimidine synthesis in B. cepacia ATCC 25416 was regulated at the level of enzyme activity and its pyrimidine salvage enzymes differed from those found in B. cepacia ATCC 17759.     

Homologous expression,purification and characterization of a novel high-alkaline and thermal stable lipase from Burkholderia cepacia ATCC 25416

The lipase secreted by Burkholderia cepacia ATCC 25416 was particularly attractive in detergent and leather industry due to its specific characteristics of high alkaline and thermal stability. The lipase gene (lipA), lipase chaperone gene (lipB), and native promoter upstream of lipA were cloned. The lipA was composed of 1095 bp, corresponding to 364 amino acid residues. The lipB located immediately downstream of lipA was composed of 1035 bp, corresponding to 344 amino acid residues. The lipase operon was inserted into broad host vector pBBRMCS1 and electroporated into original strain. The homologous expression of recombinant strain showed a significant increase in the lipase activity. LipA was purified by three-step procedure of ammonium sulfate precipitation, phenyl-sepharose FF and DEAE-sepharose FF. SDS-PAGE showed the molecular mass of the lipase was 33 kDa. The enzyme optimal temperature and pH were 60 °C and 11.0, respectively. The enzyme was stable at 30–70 °C. After incubated in 70 °C for 1 h, enzyme remained 72% of its maximal activity. The enzyme exhibited a good stability at pH 9.0–11.5. The lipase preferentially hydrolyzed medium-chain fatty acid esters. The enzyme was strongly activated by Mg²⁺, Ca²⁺, Cu²⁺, Zn²⁺, Co²⁺, and apparently inhibited by PMSF, EDTA and also DTT with SDS. The enzyme was compatible with various ionic and non-ionic surfactants as well as oxidant H₂O₂. The enzyme had good stability in the low- and non-polar solvents.     

Construction of a deep-rough mutant of Burkholderia cepacia ATCC 25416 and characterization of its chemical and biological properties

Burkholderia cepacia is a bacterium with increasing importance as a pathogen in patients with cystic fibrosis. The deep-rough mutant Ko2b was generated from B. cepacia type strain ATCC 25416 by insertion of a kanamycin resistance cassette into the gene waaC encoding heptosyltransferase I. Mass spectrometric analysis of the de-O-acylated lipopolysaccharide (LPS) of the mutant showed that it consisted of a bisphosphorylated glucosamine backbone with two 3-hydroxyhexadecanoic acids in amide-linkage, 4-amino-4-deoxyarabinose (Ara4N) residues on both phosphates, and a core oligosaccharide of the sequence Ara4N-(1 --> 8) D-glycero-D-talo-oct-2-ulosonic acid (Ko)-(2 --> 4)3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The mutant allowed investigations on the biosynthesis of the LPS as well as on its role in human infection. Mutant Ko2b showed no difference in its ability to invade human macrophages as compared with the wild type. Furthermore, isolated LPS of both strains induced the production of tumor necrosis factor alpha from macrophages to the same extent. Thus, the truncation of the LPS did not decrease the biological activity of the mutant or its LPS in these aspects.     

Isolation of Burkholderia cepacia complex genomovars from waters

The aim of this study was to develop a selective enrichment broth as an aid for the isolation of Burkholderia cepacia complex (Bcc) bacteria from water. To allow growth of all nine genomovars, mixtures of two carbon sources had to be used, i.e. L-arabinose/D-cellobiose or L-arabinose/L-threonine. Selectivity was provided by polymyxin B and 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390). Following enrichment, Bcc bacteria were isolated on a diagnostic O/F agar supplemented with gentamicin. A preliminary bio-diversity study on 28 surface waters yielded five different genomovars, i.e. B. cepacia (genomovar I), B. multivorans, B. cenocepacia, B. vietnamiensis and B. anthina. Drinking waters did not contain Bcc bacteria. However, the genomovar pattern from a given sample varied with the enrichment broth used.     

Characterization of the Phthalate Permease OphD from Burkholderia cepacia ATCC 17616

The ophD gene, encoding a permease for phthalate transport, was cloned from Burkholderia cepacia ATCC 17616. Expression of the gene in Escherichia coli results in the ability to transport phthalate rapidly into the cell. Uptake inhibition experiments show that 4-hydroxyphthalate, 4-chlorophthalate, 4-methylphthalate, and cinchomeronate compete for the phthalate permease. An ophD knockout mutant of 17616 grows slightly more slowly on phthalate but is still able to take up phthalate at rates equivalent to that of the wild-type strain. This means that 17616 must have a second phthalate-inducible phthalate uptake system.     

Reductive catabolism of uracil and thymine by Burkholderia cepacia

Catabolism of uracil and thymine in Burkholderia cepacia ATCC 25416 was shown to occur using a reductive pathway. The first pathway enzyme, dihydropyrimidine dehydrogenase, was shown to utilize NADPH as its nicotinamide cofactor. Growth of B. cepacia on pyrimidine bases as the nitrogen source instead of on ammonium sulfate increased dehydrogenase activity at least 32-fold. The second and third reductive pathway enzymes, dihydropyrimidinase and N-carbamoyl-β-alanine amidohydrolase, respectively, exhibited activities elevated more than 21-fold when pyrimidine or dihydropyrimidine bases served as the nitrogen source rather than ammonium sulfate. The pathway enzyme activities were induced after growth on 5-methylcytosine. Received: 17 January 1997 / Accepted: 5 May 1997     

Small-scale production of Burkholderia cepacia ATCC21808 lipase adapted to high-throughput screening

The screening of variant libraries of recombinant Burkholderia cepacia ATCC21808 lipase generated in Escherichia coli is limited by expression difficulties that are mainly due to the formation of inclusion bodies. To circumvent these difficulties and provide an efficient small-scale screening protocol, the gene encoding the lipase from B. cepacia was expressed in various expression vectors. With the pFLAG-ATS-Lip-Hp construct, expression of up to 6807 U/L of culture was possible in Erlenmeyer flasks. The production protocol was miniaturized in 96 deep-well plates, yielding 1300 U/L of lipase in fusion with the FLAG tag. With this protocol, the activity was determined in less than 10 min for a full plate, with a coefficient of variance of about 25%. For validation, 18 mutants constructed by site-directed mutagenesis on position Valine 266 were screened. Nice variations of activity were detected and found to be in agreement with those obtained in Erlenmeyer flask cultures. The protocol enabled the identification of 5 mutants showing enhanced activity toward para-nitrophenyl butyrate.     

Isolation and transformation of uracil auxotrophs of the edible basidiomycete Pleurotus ostreatus

Uracil auxotrophs of Pleurotus ostreatus were isolated using the selectable marker, resistance to 5'-fluoro-orotic acid (5'-FOA). Two of the nine uracil auxotrophs obtained were transformed to prototrophy using plasmid pTRura 3-2 that contains the orotidine monophosphate decarboxylase (ura3) gene from Trichoderma reesei. Southern blot analyses of the transformants showed that the transforming DNA had integrated into the genome of the protoplasts. Using 2 x 10(7) protoplasts, this system gave a transformation efficiency of about 30 transformants per microg of DNA. Normal fruiting bodies were induced in the transformants by crossing them with wild-type monokaryons, and the basidiospores collected from these fruiting bodies showed a biased segregation rate to prototrophy. These results indicate the integrated DNA was stably inherited.     

Isolation and characterization of a cyanide-utilizing Burkholderia cepacia strain

A bacterium that utilizes cyanide as a nitrogen source was isolated from soil after enrichment in a liquid medium containing potassium cyanide (10mM) and glucose (1.0%, w/v). The strain could tolerate and grow in potassium cyanide at concentrations of up to 25mM. It could also utilize potassium cyanate, potassium thiocyanate, linamarin and a range of aliphatic and aromatic nitriles. The isolate was tentatively identified as Burkholderia cepacia strain C-3. Ammonia and formic acid were found in the culture supernatant of the strain grown on fructose and potassium cyanide, no formamide was detected, suggesting a hydrolytic pathway for the degradation of cyanide. The cyanide-degrading activity was higher in early and the stationary phase cells. Crude cell extracts of strain C-3 grown on nutrient broth exhibited cyanide-degrading activity. The characteristics of strain C-3 suggest that it would be useful in the bioremediation of cyanide-containing waste.     

Isolation and characterization of a novel haloacid permease from Burkholderia cepacia MBA4

Burkholderia cepacia MBA4 is a bacterium that can utilize 2-haloacids as carbon and energy sources for growth. It has been proposed that dehalogenase-associated permease mediates the uptake of haloacid. In this paper, we report the first cloning and characterization of such a haloacid permease. The structural gene, designated deh4p, was found 353 bases downstream of the dehalogenase gene deh4a. Quantitative analysis of the expression of deh4p showed that it was induced by monochloroacetate (MCA), to a level similar to the MCA-induced level of deh4a. The nucleotide sequence of deh4p was determined, and an open reading frame of 1,656 bp encoding a putative peptide of 552 amino acids was identified. Deh4p has a putative molecular weight of 59,414 and an isoelectric point of 9.88. Deh4p has the signatures of sugar transport proteins and integral membrane proteins of the major facilitator superfamily. Uptake of [(14)C]MCA into the cell was Deh4p dependent. Deh4p has apparent K(m)s of 5.5 and 8.9 muM and V(max)s of 9.1 and 23.1 nmol mg(-1) min(-1) for acetate and MCA, respectively. A mutant with a transposon-inactivated haloacid operon failed to grow on MCA even when deh4a was provided in trans.     

Isolation and Characterization of a Novel Haloacid Permease from Burkholderia cepacia MBA4

Burkholderia cepacia MBA4 is a bacterium that can utilize 2-haloacids as carbon and energy sources for growth. It has been proposed that dehalogenase-associated permease mediates the uptake of haloacid. In this paper, we report the first cloning and characterization of such a haloacid permease. The structural gene, designated deh4p, was found 353 bases downstream of the dehalogenase gene deh4a. Quantitative analysis of the expression of deh4p showed that it was induced by monochloroacetate (MCA), to a level similar to the MCA-induced level of deh4a. The nucleotide sequence of deh4p was determined, and an open reading frame of 1,656 bp encoding a putative peptide of 552 amino acids was identified. Deh4p has a putative molecular weight of 59,414 and an isoelectric point of 9.88. Deh4p has the signatures of sugar transport proteins and integral membrane proteins of the major facilitator superfamily. Uptake of [¹⁴C]MCA into the cell was Deh4p dependent. Deh4p has apparent K_ms of 5.5 and 8.9 μM and V_maxs of 9.1 and 23.1 nmol mg⁻¹ min⁻¹ for acetate and MCA, respectively. A mutant with a transposon-inactivated haloacid operon failed to grow on MCA even when deh4a was provided in trans.     

土壤中产脂肪酶洋葱伯克霍尔德菌的分离与鉴定

洋葱伯克霍尔德菌(Burkholderia cepacia)在生物防治、生物降解等农业领域有着广泛的应用,它产生的脂肪酶则在有机合成、精细化工等领域潜力巨大。采用改良的TB-T平板筛选法从土壤中初步筛选出300株洋葱伯克霍尔德菌,然后用脂肪酶活性检测平板对300株菌进行筛选,最终获得6株脂肪酶产量高的菌,通过发酵发现6株菌均有较好的产脂肪酶能力。随后通过16S rDNA比对的方法将6株全部鉴定为B.cepacia。在此基础上,采用HaeⅢ-recA RFLP和基因种特异性PCR对6株菌进行了基因种鉴定,结果表明JWT16、G63YL、WJ158和JWT137属于Burkholderia cenocepacia菌,JWP9属于Burkhold-eria vietnamiensis,JWT267则属于Burkholderia multivorans。     

A Nonhemolytic Phospholipase C from Burkholderia cepacia

Burkholderia cepacia is an opportunistic pathogen that causes serious pulmonary infections in cystic fibrosis patients. Although several potential virulence factors—a protease, lipase, and two phospholipases C (one hemolytic and one nonhemolytic)—have been identified, only two, the protease and the lipase, have been described in detail. The goal of this study was to purify and characterize a nonhemolytic phospholipase C secreted by B. cepacia strain Pc224c. The enzyme was concentrated from culture supernatants and purified by polyacrylamide gel electrophoresis. The 54-kDa protein was stable in the presence of sodium dodecyl sulfate (up to 10%) and at 4°, 22°, and 37°C; it was, however, inactivated at 100°C. The enzyme bound to glass, chromatography matrices, and polyvinylidene difluoride and cellulose membranes, suggesting that it is hydrophobic.  In a genetic approach, primers based on conserved sequences of a B. cepacia Pc69 hemolytic phospholipase C and both the Pseudomonas aeruginosa hemolytic and nonhemolytic proteins were designed to identify the Pc224c nonhemolytic phospholipase C gene. One polymerase chain reaction product was identified; it was sequenced and the sequence compared with sequences in the BLAST database. The best match was the Pseudomonas aeruginosa hemolytic phospholipase C. Ten additional B. cepacia strains were screened for the gene by Southern hybridization; five had the 4-kb band, suggesting that these strains have a similar form of the PLC gene. Nine of the ten strains reacted with the probe, suggesting that similar sequences were present, but in another form. Received: 13 October 1998 / Accepted: 6 November 1998     

Flagellin administration protects respiratory tract from Burkholderia cepacia infection

Burkholderia cepacia is an important pathogen that often causes pneumonia in immunocompromised individuals. Here, it was demonstrated that the TLR5 agonist flagellin could locally activate innate immunity. This was characterized by rapid expressions of IL-1beta, TNF-alpha, and iNOS mRNA and a delay in the expression of IL-10 mRNA. A significant elevation in the IL-1beta, TNF-alpha, and nitric oxide levels was also noted. In the respiratory tract, flagellin induced neutrophil infiltration into the airways, which was observed by histopathological examination and confirmed by the neutrophil count and level of myeloperoxidase activity. This was concomitant with a high activity of alveolar macrophages that engulfed and killed B. cepacia in vitro. The flagellin mucosal treatment improved the B. cepacia clearance in the mouse lung. Thus, the present findings illustrate the profound stimulatory effect of flagellin on the lung mucosal innate immunity, a response that needs to be exploited therapeutically to prevent the development of respiratory tract infection by B. cepacia.     

Cellular aspects of Burkholderia cepacia infection

The Gram-negative bacterium Burkholderia cepacia has recently emerged as an important opportunistic pathogen in humans. This review focuses on the cellular aspects of B. cepacia infection and the dynamics of the B. cepacia-host cell interaction, including recent advances in our understanding of the ability of B. cepacia to adhere to, enter, and survive intracellularly within human cells.