Copper biosorption by Pseudomonas cepacia and other strains

Biosorption of copper by Pseudomonas cepacia was found to be dependent on added copper concentration. Copper uptake by the cells was rapid over the range of copper concentrations tested and complete within the first 10 min of incubation time. The effect of pH on copper uptake by P. cepacia was determined using overlapping buffers over the pH range 3–8, and copper biosorption from a 10 mM copper solution was greatest at pH 7. Copper uptake (measured by analysis of cell digests) was unaffected by cyanide and azide (up to 30 mM) and by incubation of cells with a 10 mM copper solution at 4 °C. Evidence from these results suggested that copper uptake by P. cepacia cells involves surface binding and not intracellular accumulation by active transport. Biosorption of copper by various Pseudomonas isolates from metal-contaminated environments agreed well with copper biosorption by Pseudomonas strains from the National Collection of Type Cultures (NCTC).     

Iron acquisition mechanisms of the Burkholderia cepacia complex

The Burkholderia cepacia complex (Bcc) is comprised of at least 10 closely related species of Gram-negative proteobacteria that are associated with infections in certain groups of immunocompromised individuals, particularly those with cystic fibrosis. Infections in humans tend to occur in the lungs, which present an iron-restricted environment to a prospective pathogen, and accordingly members of the Bcc appear to possess efficient mechanisms for iron capture. These bacteria specify up to four different types of siderophore (ornibactin, pyochelin, cepabactin and cepaciachelin) that employ the full repertoire of iron-binding groups present in most naturally occurring siderophores. Members of the Bcc are also capable of utilising some exogenous siderophores that they are not able to synthesise. In addition to siderophore-mediated mechanisms of iron uptake, the Bcc possess mechanisms for acquiring iron from haem and from ferritin. The Bcc therefore appear to be well-equipped for life in an iron-poor environment. An erratum to this article can be found at     

Ornibactins—a new family of siderophores from Pseudomonas

Novel linear hydroxamate/hydroxycarboxylate siderophores from strains of Pseudomonas cepacia were isolated and named ornibactins. The ornibactins represent modified tetrapeptide siderophores, possessing the sequence l-Orn¹(N -OH, N -acyl)-d-threo-Asp(-OH)-l-Ser-l-Orn⁴(N -OH, N -formyl)-1,4-diaminobutane. The N -acyl groups of Orn¹(N -OH, N -acyl) may vary and represent the three acids 3-hydroxybutanoic acid, 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid, leading to a mixture of three different ornibactins, designated according to their acyl chain length as ornibactin-C4, ornibactin-C6 and ornibactin-C8. Each of the siderophores is accompanied by a small amount of a more hydrophilic component with a 16 a.m.u. higher mass. The structure elucidation was based on results from gas chromatography amino acid analysis, electrospray mass spectrometry, and one- and two-dimensional nuclear magnetic resonance techniques.     

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     

Construction of a recA mutant of Burkholderia (formerly Pseudomonas), cepacia

A recA mutant was constructed of a soil isolate of Burkholderia cepacia, strain ATCC 17616. Prior to mutagenesis, the recA gene was cloned from this strain by its ability to complement the methyl methanesulfonate sensitivity of an Escherichia coli recA mutant. Sequence analysis of the strain showed high sequence similarity (94% nucleic acid and 99% amino acid identity) with the recA gene previously cloned from a clinical isolate of B. cepacia, strain JN25. The subcloned recA gene from B. cepacia ATCC 17616 restored UV resistance and recombination proficiency to recA mutants of E. coli and Pseudomonas aeruginosa, as well as restoring the ability of D3 prophages to be induced to lytic growth from a RecA⁻ strain of P. aeruginosa. The recA mutant of B. cepacia ATCC 17616 was constructed by λ-mediated Tn5 mutagenesis of the cloned recA gene in E. coli, followed by replacement of the Tn5-interrupted gene for the wild-type allele in the chromosome of B. cepacia by marker exchange. The RecA⁻ phenotype of the mutant was demonstrated by the loss of UV resistance as compared to the parental strain. Southern hybridization analysis of chromosomal DNA from the mutant indicated the presence of Tn5 in the recA gene, and the location of the Tn5 insertion in the recA allele was identified by nucleotide sequence analysis. A test using the recA mutant to see if acquired resistance to d-serine toxicity in B. cepacia might be a result of RecA-mediated activities proved negative; nevertheless, RecA activity potentially contributes to the overall genomic plasticity of B. cepacia and a recA mutant will be useful in bioengineering of this species. Received: 24 January / Received revision: 11 July 1997 / Accepted: 25 August 1997     

A simple assay for fluorescent siderophores produced by Pseudomonas species and an efficient isolation of pseudobactin

Several iron binding metabolites (siderophores) of Pseudomonas fluorescens B10 (JL-3133) have been detected using C₁₈ reverse phase HPLC coupled with photodiode array detection methods. This analysis utilized a volatile mobile phase of 90% 20 mm NH₄HCO₃/10% MeOH, pH 6.5. It has been shown to be applicable to other P. fluorescens strains for the detection of related metabolites. Direct scale-up of the analytical HPLC conditions allowed for the efficient preparative isolation of pseudobactin, the principle siderophore produced by P. fluorescens B10 (JL-3133).     

Quorum Sensing in Burkholderia cepacia: Identification of the LuxRI Homologs CepRI

Burkholderia cepacia has emerged as an important pathogen in patients with cystic fibrosis. Many gram-negative pathogens regulate the production of extracellular virulence factors by a cell density-dependent mechanism termed quorum sensing, which involves production of diffusible N-acylated homoserine lactone signal molecules, called autoinducers. Transposon insertion mutants of B. cepacia K56-2 which hyperproduced siderophores on chrome azurol S agar were identified. One mutant, K56-R2, contained an insertion in a luxR homolog that was designated cepR. The flanking DNA region was used to clone the wild-type copy of cepR. Sequence analysis revealed the presence of cepI, a luxI homolog, located 727 bp upstream and divergently transcribed from cepR. A lux box-like sequence was identified upstream of cepI. CepR was 36% identical to Pseudomonas aeruginosa RhlR and 67% identical to SolR of Ralstonia solanacearum. CepI was 38% identical to RhlI and 64% identical to SolI. K56-R2 demonstrated a 67% increase in the production of the siderophore ornibactin, was protease negative on dialyzed brain heart infusion milk agar, and produced 45% less lipase activity in comparison to the parental strain. Complementation of a cepR mutation restored parental levels of ornibactin and protease but not lipase. An N-acylhomoserine lactone was purified from culture fluids and identified as N-octanoylhomoserine lactone. K56-I2, a cepI mutant, was created and shown not to produce N-octanoylhomoserine lactone. K56-I2 hyperproduced ornibactin and did not produce protease. These data suggest both a positive and negative role for cepIR in the regulation of extracellular virulence factor production by B. cepacia.     

Molecular cloning and expression of perhydrolase genes from Pseudomonas aeruginosa and Burkholderia cepacia in Escherichia coli

Two bacterial perhydrolase genes, perPA and perBC, were cloned from Pseudomonas aeruginosa and Burkholderia cepacia, respectively, using PCR amplification with primers designed to be specific for conserved amino acid sequences of the already-known perhydrolases. The amino acid sequence of PerPA was identical to a putative perhydrolase of P. aeruginosa PAO1 genome sequences, whereas PerBC of B. cepacia was a novel bacterial perhydrolase showing similarity of less than 80% with all other existing perhydrolases. Most importantly, the perPA gene was expressed as a soluble intracellular form to an extent of more than 50% of the total protein content in Escherichia coli. Two perhydrolase enzymes were confirmed to exhibit the halogenation activity towards Phenol Red and monochlorodimedone. These results suggested that we successfully obtained the newly identified members of the bacterial perhydrolase family, expanding the pool of available perhydrolases.     

Diversity analysis of Burkholderia cepacia complex in the water bodies of West Lake, Hangzhou, China

A survey of Burkholderia cepacia complex (Bcc) species was conducted in water bodies of West Lake in China. A total of 670 bacterial isolates were recovered on selective media. Out of them, 39.6% (265 isolates) were assigned to the following species: Burkholderia multivorans, Burkholderia cenocepacia recA lineage IIIA, IIIB, Burkholderia stabilis, Burkholderia vietnamiensis, and Burkholderia seminalis while B. cenocepacia is documented as a dominant Bcc species in water of West Lake. In addition, all Bcc isolates tested were PCR negative for the cblA and esmR transmissibility marker genes except B. cenocepacia IIIB A8 which was positive for esmR genelater. The present study raises great concerns on the role of West Lake as a “reservoir” for potential Bcc pathogenic strains.     

Response of the cyanobacterium,Oscillatoria tenuis,to low iron environments: the effect on growth rate and evidence for siderophore production

Cyanobacteria vary in their ability to grow in media contaning low amounts of biologically available iron. Some strains, such as Oscillatoria tenuis, are well adapted to thrive in low-iron environments. We investigated the mechanism of iron scavenging in O. tenuis and found that this cyanobacterium has a siderophore-mediated iron transport system that differs significantly from the traditional hydroxamate-siderophore transport system reported from other cyanobacteria. Unlike other cyanobacteria, this strain produces two types of siderophores, a hydroxamate-type siderophore and a catechol-type siderophore. Production of these two siderophores is expressed at two different iron levels in the medium, suggesting two different iron regulated uptake systems. We compared the production of each siderophore with the growth rate of the culture and found that the production of the catechol siderophore enhances the growth rate of the cyanobacterium, whereas the cells maintain lower than maximal growth rates when only the hydroxamate-type siderophore is being produced.Abbreviation EDDA ethylene diamine di-(o-hydroxyphenylacetic acid)     

Regulation of Expression of the Nonhemolytic Phospholipase C of Burkholderia cepacia

Burkholderia cepacia is an opportunistic pathogen that causes serious pulmonary infections in cystic fibrosis patients. We have purified and partially characterized one potential virulence factor for the organism—a nonhemolytic phospholipase C—and we studied the effect of iron restriction and choline and phosphate concentrations on the expression of phospholipase C. Iron limitation did not affect expression, the effect of choline was variable, and high phosphate concentrations repressed expression. Experiments with heat-treated spent culture supernatants suggested that autoinducers affected the expression of the phospholipase and two other potential virulence factors, a protease and a lipase. We screened 26 B. cepacia isolates for autoinducer activity: 11 induced violacein production in the autoinducer-deficient mutant Chromobacterium violaceum CV026. Spent supernatants from two strains, one that was positive in the C. violaceum assay and one that was negative, were tested for inducing early expression of phospholipase C, protease, and lipase in homologous and heterologous cultures. Expression of all three enzymes was increased or induced at an earlier stage in the growth curve in every case, suggesting not only that autoinducers were involved in the regulation of the expression of these enzymes, but also that the autoinducers were of two different classes. Received: 13 May 1999 / Accepted: 14 June 1999     

Growth kinetics of EDTA biodegradation by Burkholderia cepacia

A pure culture of an EDTA-degrading strain was isolated from the Taiwan environment. It was identified as Burkholderia cepacia, an aerobic bacterium, elliptically shaped with a length of 5–15 m. The degradation assay showed that the degradation efficiency of Fe-EDTA by B. cepacia was approximately 91%. Evaluation of kinetic parameters showed that Fe-EDTA degradation followed substrate inhibition kinetics. This is evident from the decrease in specific growth rate with an increase in the initial substrate concentration greater than 500 mg/l. To estimate the kinetic parameters – _max, K_S and K_I, five substrate–inhibition models were used. From the results of non-linear regression, the value of _max ranged from 0.150 to 0.206 d^–1, K_S from 74 to 87 mg/l, and K_I from 890 to 2289 mg/l. The five models were found to underestimate the maximum specific growth rate by 1.5–3.7. Therefore, predictions based on these models would result in lower predicted value than those from the experimental kinetic data.     

Variation in Flagellin Genes and Proteins of Burkholderia cepacia

The majority of isolates of Burkholderia cepacia, an important opportunistic pathogen associated with cystic fibrosis, can be classified into two types on the basis of flagellin protein size. Electron microscopic analysis indicates that the flagella of strains with the larger flagellin type (type I) are wider in diameter. Flagellin genes representative of both types were cloned and sequenced to design oligonucleotide primers for PCR amplification of the central variable domain of B. cepacia flagellin genes. PCR-restriction fragment length polymorphism analysis of amplified B. cepacia flagellin gene products from 16 strains enabled flagellin type classification on the basis of product size and revealed considerable differences in sequence, indicating that the flagellin gene is a useful biomarker for epidemiological and phylogenetic studies of this organism.Burkholderia cepacia (formerly Pseudomonas cepacia; a member of the rRNA group II pseudomonads) has emerged as an increasingly important opportunistic pathogen, particularly in relation to patients suffering from cystic fibrosis (CF) (15). Acquisition of B. cepacia, often occurring after lengthy colonization with Pseudomonas aeruginosa, can lead to the rapid deterioration or death of CF patients, and this organism appears to be transmissible between patients (14). There is considerable evidence that some strains of B. cepacia are more virulent than others and that the outcome of colonization by a particular strain can vary from rapidly fatal septicemia to maintenance of stable respiratory function (16). A number of factors have been implicated in the greater virulence of some strains. These include adhesion to respiratory mucin (31, 32) and the presence of cable pili (33).Motility in B. cepacia is by means of polar flagella. Flagella, each consisting of a flagellin filament, hook, and basal body, have been implicated as invasive virulence factors for a number of bacteria (28), including P. aeruginosa (11). Unlike P. aeruginosa, which appears to sit in microcolonies in the viscid mucus, leading to progressive lung damage with episodes of acute debilitating exacerbation, some strains of B. cepacia cause rapidly fatal pneumonia in CF patients (15), suggesting that they may have the ability to move through the mucus. Because of their location on the outside of bacterial cells, flagellins have been targeted in vaccine design. Brett et al. (4) demonstrated that flagellin-specific antisera were capable of protecting diabetic rats from challenge with strains of Burkholderia pseudomallei (another member of rRNA group II). In a recent study, an O-polysaccharide moiety of B. pseudomallei was covalently linked to the flagellin protein from the same strain. O-polysaccharide–flagellin conjugates elicited a high-level immunoglobulin G response capable of protecting diabetic rats from challenge with a heterologous strain of B. pseudomallei (5).Two distinct flagellin protein molecular mass groups in B. cepacia have been reported by Montie and Stover (23). In this previous study, type I flagellins were reported as having a molecular mass of 31 kDa while the molecular mass of type II flagellins was reported as 44 to 46 kDa. This early study, using a limited number of isolates, suggested that with regard to flagellin, B. cepacia is analogous to another CF pathogen, P. aeruginosa, in which two flagellin antigenic types distinguishable by protein or gene size are found (43). Several representatives of the heterologous a-type and homologous b-type fliC loci of P. aeruginosa (encoding flagellins) have been sequenced (37). In addition, PCR amplification of flagellin genes coupled with restriction fragment length polymorphism (RFLP) analysis can be used as a method for differentiating between clinical isolates of P. aeruginosa (7, 43). In this paper we report the development of a similar approach to the study of populations of B. cepacia and discuss the divergence of a highly variable gene, the flagellin gene (fliC), within populations of B. cepacia.     

Colonization behavior of bacterium Burkholderia cepacia inside the Oryza sativa roots visualized using green fluorescent protein reporter

Colonization behavior of endophytic bacteria Burkholderia cepacia strains RRE-3 and RRE-5 was studied in the seedlings of rice variety NDR97 using confocal laser scanning microscopy under controlled laboratory and greenhouse conditions. For studying colonization pattern, bacterial strains were tagged with pHRGFPGUS plasmid. The role of bacterial strains (both gfp/gus-tagged and untagged) in growth promotion was also studied. After coming into contact with the host root system the bacteria showed an irregular spreading. Dense colonization was observed on the primary and secondary roots and also on the junction of emergence of the lateral roots. Results showed that the colonization pattern of Burkholderia cepacia strains was similar to that of other endophytic bacteria isolated from non-legumes. Burkholderia cepacia got entry inside the root at the sites of emergence of lateral roots, without formation of infection threads as in the case of symbiotic rhizobacteria. Observations suggested that the endophytic bacterial strains RRE-3 and RRE-5 entered inside the rice roots in a progressive manner. Bacteria were found to line up along the intercellular spaces of adjoining epidermal cells adjacent to the lateral root junction, indicating endophytic colonization pattern of Burkholderia cepacia strains. Experiments with the rice seedlings inoculated with RRE-3 and RRE-5 strains revealed that both strains enhanced plant growth considerably when observed under laboratory and greenhouse conditions and produced significantly higher plant biomass. No considerable difference was observed between the gfp/gus-tagged and non-gfp/gus-tagged strains in the plant growth experiments both in the laboratory and greenhouse conditions.     

日化产品中洋葱伯克霍尔德氏菌复合群(Bcc)的分类和神秘伯克霍尔德氏菌的耐药性研究

【目的】对从2020–2022年不同日化产品中分离的29株洋葱伯克霍尔德氏菌复合群(Burkholderia cepacia complex,Bcc)进行分类和分型,另将2020年前来源于日化产品中6株被鉴定为Burkholderia lata的菌株进行分类更正。探究神秘伯克霍尔德氏菌(Burkholderia aenigmatica)的耐药性。【方法】本文主要应用多位点分型研究方法(multilocus sequence typing,MLST),PCR扩增atpD、gltB、gyrB、recA、lepA、phaC和trp B 7个管家基因片段,将测序结果与MLST数据库中的数据比对分析,获得菌株各管家基因的编号和ST型(sequence type),对本检测中心分离自日化产品的Bcc进行分型;利用多位点序列分析(multilocus sequence analysis,MLSA),结合MLST中等位基因的核苷酸序列构建进化树,从而对Bcc进行系统发育分析和鉴定。利用最小抑菌浓度法(minimum inhibitory concentration,MIC)测定Bcc对常见防腐剂(1,...     

Time‐kill studies with a ceftazidime‐treated mixed culture consisting of Pseudomonas aeruginosa,Burkholderia cepacia and Staphylococcus aureus

So far, time‐kill analyses were mostly done with isolates of bacteria. Here, we used a mixed culture consisting of Pseudomonas aeruginosa, Burkholderia cepacia, and Staphylococcus aureus to investigate the impact of ceftazidime treatment. Following an earlier study with the same strains, the influence of different ceftazidime concentrations as well as repeated ceftazidime treatment was tested. In order to assess the influence of substrate competition, which might be relevant to interpret mixed‐culture time‐kill studies, the major metabolites of the chemically defined cultivation medium were measured additionally. Time‐kill experiments were conducted in shake flasks with the chemically defined and modified medium M199. The cell concentration in the mixed culture was analyzed on the single‐species level using a quantitative terminal restriction fragment (qT‐RFLP) method. The amount of gluconate produced in mixed culture positively correlated with increased ceftazidime concentrations (5, 15, 30, 60 mg/L). Burkholderia cepacia developed resistance after repeated ceftazidime addition and reached the highest cell concentration of the three cultivated strains. Pseudomonas aeruginosa showed a pronounced regrowth phase after removal of ceftazidime, while growth of S. aureus was not influenced by medium exchange. In conclusion, growth of B. cepacia was dominant in the ceftazidime‐treated mixed culture over the observed time range, due to low susceptibility against ceftazidime as well as advantages in substrate usage.     

Purification and properties of a cyanide-degrading enzyme from Burkholderia cepacia strain C-3

A cyanide-hydrolysing enzyme from Burkholderia cepacia strain C-3 isolated from soil was purified to electrophoretic homogeneity by ammonium sulphate precipitation and column chromatography on HiTrap Q (DEAE-agarose) and phenyl-Sepharose HP. The enzyme was purified 48-fold with a 0.8% yield and a final specific activity of 26.8 u/mg protein. The purified enzyme was observed as a single polypeptide band of molecular mass 38 kDa during both denaturing and non-denaturing gel electrophoresis. Enzymatic activity was optimal at pH 8.0–8.5 and at 30–35 °C. Activity was stimulated by Mo²⁺, Sn²⁺, and Zn²⁺, and inhibited by Al³⁺, Co²⁺, Cu²⁺ and Hg²⁺. The enzyme was specific for cyanide and thiocyanate with formate and ammonia as the main products from KCN degradation. Its K _m and V _max values were 1.4 mM and 15.2 u/mg protein, respectively. Apparent substrate inhibition occurred at cyanide concentrations greater than 2 mM.     

Intraspecific metabolic diversity among strains ofBurkholderia cepacia isolated from decayed onions,soils, and the clinical environment

A collection of 218 strains ofBurkholderia cepacia (including 18% strain replicates) was assembled from organic soils, decayed onions, and clinical sources. Each strain was characterized for virulence to onion, catabolic ability using the Biolog GN microtiter plate, and several other behaviors. Overall test reproducibility was estimated at 98%. The results obtained using the Biolog GN system corresponded well to those obtained using standard methods. Three coefficients of resemblance (Gower similarity, pattern difference, and Jaccard similarity) were calculated and clustered by the group-average method. The sorted matrices and phenograms, while giving evidence of an underlying phenetic structure to theB. cepacia nomenspecies, gave little evidence of sorting by broad source of isolation. Strains isolated from within fields or samples were frequently found to be similar, however, strains isolated from fields with similar cropping histories were not. The Gower-transformed centroids of ordained clusters were projected in a principal coordinate system and estimates of disjunction were calculated. Strains ofB. cepacia were shown to be non-uniformly distributed in taxonomic space. Strains isolated by serial dilution on onion slices formed a tight phenetic cluster which includes the type strain of the nomenspecies and that of a synonymous group (Pseudomonas multivorans); the strains in this phenon were generally virulent to onion and were partially differentiated from others by pectolytic behavior and by the production of diffusible pigment on King's medium A. Further characterization should better resolve the taxonomy of the nomenspecies.     

Specificity of siderophore-mediated transport of iron in rhizobia

The trihydroxamate siderophore, hydroxamate K, has been purified from culture filtrates of iron-deficient Rhizobium leguminosarum biovar viciae MNF710. The iron complex has a molecular weight of 828 and an absorption maximum at 443 nm (_M=1510). ⁵⁵Fe complexed to purified hydroxamate K was taken up by MNF710, its hydroxamate-negative mutant MNF7102 and Rhizobium leguminosarum biovar trifolii WU95 via an iron-regulated transport system, but Rhizobium meliloti U45 failed to take up the iron-siderophore complex under any conditions. A similar pattern of iron uptake was observed with ferrioxamine B. MNF710, MNF7102, U45 and WU95 all transported ⁵⁵Fe-ferrichrome but only the first three strains took up ⁵⁵Fe-ferrichrome A. All these ⁵⁵Fe-trihydroxamate uptake systems were ironregulated in MNF710, MNF7102 and WU95. In contrast, uptake of ⁵⁵Fe-rhodotorulate, a dihydroxamate, was essentially constitutive in all four organisms. Similarly, uptake of ⁵⁵Fe-citrate and ⁵⁵Fe-nitrilotriacetic acid was constitutive. None of the strains took up ⁵⁵Fe complexed with enterobactin or with pyoverdins from Pseudomonas aeruginosa ATCC15692 (PAO1) and Pseudomonas fluorescens ATCC17400.