Purification and characterization of chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100

Burkholderia (formerly Pseudomonas) cepacia AC1100 mineralizes the herbicide 2,4,5-trichlorophenoxyacetate (2,4,5-T), and the first intermediate of 2,4,5-T degradation is 2,4,5-trichlorophenol. Chlorophenol 4-monooxygenase activity responsible for 2,4,5-trichlorophenol degradation was detected in the cell extract. The enzyme consisted of two components separated during purification, and both were purified to more than 95% homogeneity. The reconstituted enzyme catalyzed the hydroxylation of several tested chlorophenols with the coconsumption of NADH and oxygen. In addition to chlorophenols, the enzyme also hydroxylated some chloro-p-hydroquinones with the coconsumption of NADH and oxygen. Apparently, the single enzyme was responsible for converting 2,4,5-trichlorophenol to 2,5-dichloro-p-hydroquinone and then to 5-chlorohydroxyquinol (5-chloro-1,2,4-trihydroxybenzene). Component A had a molecular weight of 22,000 and contained flavin adenine dinucleotide. Component A alone catalyzed NADH-dependent cytochrome c reduction, indicating that it had reductase activity. Component B had a molecular weight of 58,000, and no catalytic activity has yet been shown by itself.     

Siderophore production by cystic fibrosis isolates of Burkholderia cepacia

Sixty-one Burkholderia cepacia isolates from patients with cystic fibrosis (CF) and four plant isolates were screened for production of the siderophores salicylic acid (SA), pyochelin, cepabactin, and ornibactins and fingerprinted by a PCR-based randomly amplified polymorphic DNA (RAPD) method. Of the 24 RAPD types determined, 22 (92%) were associated with isolates that produced SA, 21 (87%) were associated with isolates that produced ornibactins, 15 (60%) were associated with isolates that produced pyochelin, and 3 (12%) were associated with isolates that produced cepabactin. Of the 24 RAPD types plus 2 phenotypic variants of types 1 and 9, 3 were associated with isolates that produced all four siderophores, 8 were associated with isolates that produced three siderophores, 12 were associated with isolates that produced two siderophores, and 3 were associated with isolates that produced only one siderophore. These results suggest that the numbers and types of siderophores produced by CF isolates of B. cepacia correlate with RAPD type and that SA and ornibactins are the most prevalent siderophores produced.     

Changes in the regiospecificity of aromatic hydroxylation produced by active site engineering in the diiron enzyme toluene 4-monooxygenase

Pseudomonas mendocina KR1 toluene 4-monooxygenase is a multicomponent diiron enzyme. the diiron center is contained in the tmoA polypeptide of teh hydroxylase component [alphabetagamma)2,Mr approximately 212 kDa]. Product distribution studies reveal that the natural isoform is highly specific for para hydroxylation of toluene (kcat approximately 2 s-1 with respect to an alphabetagamma promoter), o-xylene (kcat approximately 0.8 s-1), m-xylene (kcat approximately 0.6 s-1), and other aromatic hydrocarbons. This degree of regioselectivity for methylbenzenes is unmatched by numerous other oxygenase enzymes. However, during the T4MO-catalyzed oxidation of p-xylene (kcat approximately 0.4 s-1), 4-methyl benzyl alcohol is the major product, showing that the enzyme could catalyze either aromatic or benzylic hydroxylation with the appropriate substrate. Site-directed mutagenesis has been used to study the contributions of tmoA active site residues Q141, I180, and F205 to the regiospecificity. Isoforms Q141C and F205I yielded shifts of regiospecificity away from p-cresol formation, with F205I giving an approximately 5-fold increase in the percentage of m-cresol formation relative to that of the natural isoform. The kcat of purified Q141C for toluene oxidation was approximately 0.2 s-1. Isoform Q141C also functioned predominantly as an aromatic ring hydroxylase during the oxidation of p-xylene, in direct contrast to the predominant benzylic hydroxylation observed for the natural isoform, while isoform F205I gave nearly equivalent amounts of benzylic and phenolic products from p-xylene oxidation. Isoform I180F gave no substantial shift in product distributions relativeto the natural isoform for all substrates tested. Upon the basis of a proposed active site model, both Q141 anf F205 are suggested to lie in a hydrophobic region closer to the FeA iron site, while I180 will be closer to FeB. These studies reveal that changes in the hydrophobic region predicted to be nearest to FeA can influence the regiospecificity observed for toluene 4-monooxygenase.     

Genomic complexity and plasticity of Burkholderia cepacia

Burkholderia cepacia has attracted attention because of its extraordinary degradative abilities and its potential as a pathogen for plants and for humans. This bacterium was formerly considered to belong to the genus Pseudomonas in the gamma-subclass of the Proteobacteria, but recently has been assigned to the beta-subclass is based on rrn gene sequence analyses and other key phenotypic characteristics. The B. cepacia genome is comprised of multiple chromosomes and is rich in insertion sequences. These two features may have played a key role in the evolution of novel degradative functions and the unusual adaptability of this bacterium.     

Subspecific differentiation of Burkholderia cepacia isolates in cystic fibrosis

While ceramide has emerged as a potent signal transducer, inconsistencies in the kinetics of ceramide generation, or its absence, in response to stimuli have led to confusion and skepticism as to its potential role in apoptosis or proliferation. Here we show that in U937 and HL60 myeloid leukemia cells and in normal skin fibroblasts, cell-permeant ceramides can trigger neutral sphingomyelinase activation, sphingomyelin hydrolysis, and endogenous ceramide generation regardless of Bcl2 overexpression. These observations identify neutral sphingomyelinase as a novel target for ceramide and show that this positive feedback mechanism is responsible for signal propagation, as exemplified by mitogen-activated protein kinase activation in daunorubicin-treated cells. This study provides insight into a fundamental process of cell biology. Indeed, such a sustained ceramide-mediated signal throughout the apoptotic process would ensure self-destruction, perhaps by overriding evolutionary conserved primal cell survival mechanisms.     

Transmission and prevalence of Burkholderia cepacia in Welsh cystic fibrosis patients

EDTA-dependent pseudothrombocytopenia (PTCP) consists of an inappropriate low platelet count caused by autoantibodies present in the serum samples reacting with platelets only in EDTA-anticoagulated blood. By using immunoprecipitation and Western blot techniques, we studied the immunochemical specificity of platelet agglutinating autoantibodies in the serum samples of 10 patients with PTCP. Furthermore, to evaluate a possible role of PTCP-associated IgG autoantibodies in increased platelet turnover, we assayed the plasma glycocalicin (GC) level and calculated the GC index for every patient. Our results provide direct evidence that an epitope located on platelet membrane glycoprotein IIb is recognized by PTCP-associated IgG antibodies; moreover GC levels in patients with EDTA-dependent PTCP were similar to control levels, thus excluding an increased platelet turnover. We conclude that antiplatelet antibodies directed against platelet cryptantigens are unlikely to have a major role in the increased removal of cells from circulation.     

Burkholderia cepacia bacteremia: a retrospective analysis of 70 episodes

Burkholderia cepacia, a widespread gram-negative environmental bacillus associated with nosocomial infections, is considered to be of relatively low virulence and rarely to cause invasive disease. We retrospectively analyzed the risk factors, clinical manifestations, antimicrobial susceptibilities, and prognostic factors of B. cepacia bacteremia cases. From 1982 through 1995, 70 episodes of bacteremia due to B. cepacia occurred in 52 patients at the National Taiwan University Hospital. The overall case fatality rate was 11%. Sixty-four episodes were nosocomial infections. The common predisposing conditions were stay in an intensive care unit (61%) and invasive procedures, including urinary catheter (54%), intravenous catheter (70%), and intubation (57%). Three episodes involved polymicrobial bacteremia. In 41 episodes in which the infectious focus was identified, the respiratory tract was the most common portal of entry (17/41) followed by intravascular catheters (11/41). Most strains tested were susceptible to ceftazidime (95%), piperacillin (93%), minocycline (85%), and cefotaxime (82%); but most were resistant to aminoglycosides, tetracycline, carbenicillin, and ticarcillin. For empirical therapy of B. cepacia bacteremia, ceftazidime or piperacillin should be the drug of choice.     

Pro-inflammatory effects of Burkholderia cepacia on cystic fibrosis respiratory epithelium

Burkholderia cepacia causes pulmonary infection with high mortality in cystic fibrosis (CF) patients which is likely to involve interaction with respiratory epithelium. In this study the pro-inflammatory properties of B. cepacia were examined using a range of respiratory epithelial cell lines. B. cepacia and cell-free culture supernatants were used to stimulate cell lines with (SigmaCFTE29o- and IB3) and without (A549) the CF transmembrane conductance regulator mutation (CFTR), together with corrected cell lines (C38 and S9). Interleukin (IL)-6 and IL-8, but not GM-CSF or IL-1beta, were released from all the cell lines whereas PGE(2) (prostaglandin E(2)) was released from the A549, IB3 and S9 cell lines only. Nuclear factor (NF)-kappaB activation preceded cytokine release and suppression of NF-kappaB activity diminished cytokine release. These studies indicated that B. cepacia secretory products are potent pro-inflammatory agents for respiratory epithelium and suggest functional CFTR is not required for cytokine or prostanoid responses.     

Detection and classification of hyperfine-shifted 1H, 2H, and 15N resonances of the Rieske ferredoxin component of toluene 4-monooxygenase

T4MOC is a 12.3 kDa soluble Rieske ferredoxin that is obligately required for electron transfer between the oxidoreductase and diiron hydroxylase components of toluene 4-monooxygenase from Pseudomonas mendocina KR1. Our preliminary 1H NMR studies of oxidized and reduced T4MOC [Markley, J. L., Xia, B., Chae, Y. K., Cheng, H., Westler, W. M., Pikus, J. D., and Fox, B. G. (1996) in Protein Structure Function Relationships (Zaidi, Z., and Smith, D., Eds.) pp 135-146, Plenum Press, London] revealed the presence of hyperfine-shifted 1H resonances whose short relaxation times made it impractical to use nuclear Overhauser effect (NOE) measurements for assignment purposes. We report here the use of selective isotopic labeling to analyze the hyperfine-shifted 1H, 2H, and 15N signals from T4MOC. Selective deuteration led to identification of signals from the four Hbeta atoms of cluster ligands C45 and C64 in the oxidized and reduced forms of T4MOC. In the reduced state, the Curie temperature dependence of the Hbeta protons corresponded to that predicted from the simple vector spin-coupling model for nuclei associated with the localized ferric site. The signal at 25.5 ppm in the 1H spectrum of reduced T4MOC was assigned on the basis of selective 2H labeling to the His Hepsilon1 atom of one of the cluster ligands (H47 or H67). This assignment was corroborated by a one bond 1H-13C correlation (at 25.39 ppm 1H and 136.11 ppm 13C) observed in spectra of [U-13C]T4MOC with a 1H-13C coupling constant of approximately 192 Hz. The carbon chemical shift and one bond coupling constant are those expected for 1Hepsilon1-13Cepsilon1 in the imidazolium ring of histidine and are inconsistent with values expected for cysteine 1Halpha-13Calpha. The His Hepsilon1 proton exhibited weak Curie temperature dependence from 283 to 303 K, contrary to the anti-Curie temperature dependence predicted from the spin coupling model for nuclei associated with the localized ferrous site. A 1H peak at -12.3 ppm was observed in spectra of reduced T4MOC; this signal was found to correspond to a hydrogen (probably in an H-bond to the cluster) that exchanged with solvent with a half-time of about 2 days in the oxidized state but with a much longer (undetectable) half-time in the reduced state. These results with T4MOC call into question certain 1H assignments recently reported on the basis of NOE measurements for the comparable Rieske ferredoxin component of an evolutionarily related alkene monooxygenase from Xanthobacter sp. Py2 [Holz, R. C., Small, F. J., and Ensign, S. A, (1997) Biochemistry 36, 14690-14696]. Selective 15N labeling was used to identify hyperfine-shifted 15N NMR signals from the backbone nitrogens of all four cluster ligands (C45, H47, C64, and H67), from the Nepsilon2 atoms of the two histidine ligands (H47 and H67), and from nonligand Gln and Ala residues (Q48 and A66) present in the cluster-binding motif of T4MOC in the oxidized and reduced states. The results indicate that the Ndelta1 of each of the two ligand histidines of T4MOC are ligated to an iron atom and reveal a pattern of H-bonding to the Rieske [2Fe-2S] center involving four (H47, Q48, A66, and H67 of T4MOC) of the five backbone amide H-bonds expected on the basis of comparison with the crystal structures of other related Rieske proteins; the fifth backbone amide (I50 of T4MOC) failed to exhibit a hyperfine shift. This anomaly may arise from the lack of an associated disulfide in T4MOC, a fundamental structural difference between the three types of Rieske proteins that may be related to functional diversity in this protein family.     

Inactivation of 4-oxalocrotonate tautomerase by 2-oxo-3-pentynoate

The compound, 2-oxo-3-pentynoate, has been synthesized and tested as an inhibitor of the enzyme 4-oxalocrotonate tautomerase. The enzyme is rapidly and irreversibly inactivated by the acetylenic product analogue in a time-dependent fashion. The enzyme displays saturation kinetics and is protected from inactivation by the presence of substrate. These observations are consistent with inactivation taking place at the active site. Partial reactivation ( approximately 18%) occurs by incubating the inactivated enzyme with 10 mM hydroxylamine (pH 7.3). The partition ratio, determined to be approximately 0.4, suggests that the inactivation of 4-OT by 2-oxo-3-pentynoate shows half-of-the-sites stoichiometry. The same phenomenon is observed in the inactivation of 4-OT by 3-bromopyruvate and can be explained by examination of the crystal structure. Mass spectral analysis shows that a single residue is modified on the enzyme which has been localized to the nine residue amino-terminal fragment Pro-1 to Glu-9. It can be reasonably concluded that Pro-1 is the site of covalent attachment. Inactivation of 4-OT can occur by either a Michael addition of 4-OT to C-4 of 2-oxo-3-pentynoate or by the enzyme-catalyzed rearrangement of 2-oxo-3-pentynoate to an allene derivative which alkylates Pro-1. These results provide the foundation for the use of 2-oxo-3-pentynoate in future mechanistic studies and as a ligand in an inactivated 4-OT complex that can be studied by X-ray crystallography. Finally, 2-oxo-3-pentynoate is an acetylene analogue of a variety of 2-oxo acids and as such may have general utility as an inhibitor of reactions that bind and process these compounds.     

Analysis of transmission of Burkholderia cepacia isolates in an intrahospital by randomly amplified polymorphic DNA-PCR method

Strains of Burkholderia cepacia isolated in our hospital from November 1995 to September 1996 were classified with randomly amplified polymorphic DNA-PCR (RAPD-PCR) and conventional biochemical tests (ID test.NF-18, API20NE, and Neg Combo 4J kit), and intrahospital isolates of B. cepacia were analysed. During the period 28 strains from inpatients and 2 from medical apparatus were isolated. Twenty four of 28 (85.7%) were from sputum. In 1996 from January to February, 20 strains were detected from 8 inpatients, and two strains were from the nebulizers at the Trauma and Critical Care Center (TCC). With typing of B. cepacia by conventional methods no epidemiological relations among isolates were found. However, DNA patterns of original isolates from the nebulizers at TCC by RAPD-PCR were identical with those of isolates in sputa from patients in other wards who had stayed at TCC, indicating that TCC was an initial source of transmission and the strain was transmitted with the patients to the wards. These results suggest that RAPD-PCR method might be a useful tool to analyse an epidemiological survey for intrahospital transmission of isolate.     

Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov

We performed an integrated genotypic and phenotypic analysis of 128 strains of the genera Burkholderia, Ralstonia, and Pseudomonas in order to study the taxonomic structure of Burkholderia cepacia and its relationships with other Burkholderia species. Our data show that presumed B. cepacia strains isolated from cystic fibrosis patients belong to at least five distinct genomic species, one of which was identified as Burkholderia vietnamiensis. This group of five phenotypically similar species is referred to as the B. cepacia complex. The name Burkholderia multivorans is proposed for one of these genomic species, which was formerly referred to as B. cepacia genomovar II; the remaining B. cepacia groups are referred to as genomovars I, III, and IV, pending additional differential phenotypic tests. The role and pathogenic potential of each of these taxa, particularly in view of the potentially fatal infections in cystic fibrosis patients, need further evaluation. The data presented also demonstrate that Pseudomonas glathei and Pseudomonas pyrrocinia should be reclassified as Burkholderia species.     

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.     

Novel pathway for conversion of chlorohydroxyquinol to maleylacetate in Burkholderia cepacia AC1100

Burkholderia cepacia AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and beta-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.     

Inactivation of bovine liver 2-keto-4-hydroxyglutarate aldolase by cyanide in the presence of aldehydes

Kinetic data show that the irreversible inactivation of liver 2-keto-4-hydroxyglutarate aldolase observed when the enzyme is incubated with an aldehydic substrate (or substrate analogue) in the presence of cyanide is a biphasic process and can, under certain conditions, involve a direct interaction between the enzyme and cyanide. The kinetic data are consistent with a scheme consisting of three competing reactions: (1) irreversible addition of cyanide to the enzyme-substrate Schiff base intermediate, (2) reversible cyanohydrin formation between cyanide and the aldehydic substrate (or substrate analogue), and (3) an interaction of cyanide with the enzyme which is not substrate dependent. Approximately 0.4 mol of cyanide is associated with 1 mol (120 000 g) of enzyme when 2-keto-4-hydroxyglutarate aldolase is incubated with [14-C]-cyanide followed by exhaustive dialysis; an ionic attachment possibly at a carboxylate binding site, is suggested. Whereas native enzyme, not treated with cyanide, has ten Nbs2-titratable sulfhydryl groups, approximately one less such group reacts with Nbs2 when the aldolase is incubated with cyanide (in the absence of aldehydic substrate). It is suggested that the binding of cyanide results in a conformational change of the enzyme; conformational changes in the presence of cyanide are confirmed by circular dichroism spectra.     

Outcome of Burkholderia (Pseudomonas) cepacia colonisation in children with cystic fibrosis following a hospital outbreak

BACKGROUND: While there are reports on the outcome in adults and teenagers with cystic fibrosis of colonisation with Burkholderia (Pseudomonas) cepacia, there is little information in children. METHODS: In December 1991 only one of 115 children with cystic fibrosis attending a paediatric centre was colonised with B cepacia. Over the next 12 months there was a rapid increase with 23 (20%) becoming colonised; eighteen (79%) of these became colonised in hospital at a time that overlapped with the admission of a B cepacia positive child. Three different bacteriocin types were isolated, with one type (S22/PO) being present in 17 (74%) patients. The outcome for children who became colonised with B cepacia was compared with that in 33 children who continued to be colonised with Pseudomonas aeruginosa alone. RESULTS: Children colonised with B cepacia were older and more poorly nourished than those colonised with P aeruginosa, but did not have poorer pulmonary function. After colonisation, the forced expiratory volume in one second (FEV1) deteriorated between consecutive annual tests, with the average deterioration being greater in those with higher initial levels. Five children with B cepacia died from respiratory failure although none showed a fulminant deterioration. Introduction of segregation measures within hospital led to a dramatic decrease in the number of newly colonised patients. CONCLUSIONS: This study provides further evidence for person-to-person spread of B cepacia and confirms the effectiveness of simple isolation measures in interrupting spread. Colonisation with B cepacia and P aeruginosa in children is associated with a more rapid decline in lung function and a significantly increased mortality compared with cases colonised with P aeruginosa alone.     

A melanin pigment purified from an epidemic strain of Burkholderia cepacia attenuates monocyte respiratory burst activity by scavenging superoxide anion

The acquisition of Burkholderia cepacia in some cystic fibrosis patients is associated with symptoms of acute pulmonary inflammation that may be life threatening. The ability of lipopolysaccharide (LPS) from B. cepacia to prime a monocyte cell line for enhanced superoxide anion generation was investigated and compared with the priming activities of LPSs from Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Escherichia coli. The human monocyte cell line MonoMac-6 (MM6) was primed overnight with different LPSs (100 ng/ml), and the respiratory burst was triggered by exposure to opsonized zymosan (125 micrograms/ml). Superoxide generation was detected by enhanced chemiluminescence with Lucigenin. B. cepacia LPS was found to prime MM6 cells to produce more superoxide anion than P. aeruginosa or S. maltophilia LPS, and this priming response was CD14 dependent. In addition, the inhibition of respiratory burst responses in monocytes by a bacterial melanin-like pigment purified from an epidemic B. cepacia strain was investigated. The melanin-like pigment was isolated from tyrosine-enriched media on which B. cepacia had been grown and was purified by gel filtration, anion ion-exchange chromatography, and ethanol precipitation. The scavenging potential of the melanin-like pigment for superoxide anion radical (*O2-) generated during the respiratory burst was confirmed with superoxide produced from a cell-free system with xanthine-xanthine oxidase and detected by electron paramagnetic resonance spectroscopy with the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide. The addition of melanin during the LPS priming stage had no effect on the subsequent triggering of the respiratory burst, but melanin inhibited *O2- detection when added at the triggering stage of the respiratory burst. We conclude that melanin-producing B. cepacia may derive protection from the free-radical-scavenging properties of this pigment.     

MS2 Inactivation by Chloride-Assisted Electrochemical Disinfection

An electrochemical (EC) disinfection system employing an iridium–antimony–tin-coated titanium anode and direct current was used to inactivate bacteriophage MS2 in synthetic solutions with sodium chloride addition. The inactivation data fit the modified Chick–Watson (n ≠ 1) model well. The model indicates that, although better disinfection could be achieved with increases in salt content, contact time, and applied current, these three parameters influence the EC disinfection of MS2 in distinct manners and to different degrees. Compared with chlorination, our EC disinfection system exhibited superior inactivation capability especially with a longer contact time or in the presence of ammonium. The formation of trihalomethanes and haloacetic acids in the EC system was smaller than that from chlorination but a large formation of chlorate ions was observed. These differences indicate that the EC system is likely to produce other potent oxidants that enhance inactivation and alter disinfection by-product formation.     

Biodiversity of a Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages

A Burkholderia cepacia population naturally occurring in the rhizosphere of Zea mays was investigated in order to assess the degree of root association and microbial biodiversity at five stages of plant growth. The bacterial strains isolated on semiselective PCAT medium were mostly assigned to the species B. cepacia by an analysis of the restriction patterns produced by amplified DNA coding for 16S rRNA (16S rDNA) (ARDRA) with the enzyme AluI. Partial 16S rDNA nucleotide sequences of some randomly chosen isolates confirmed the ARDRA results. Throughout the study, B. cepacia was strictly associated with maize roots, ranging from 0.6 to 3.6% of the total cultivable microflora. Biodiversity among 83 B. cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique with two 10-mer primers. An analysis of RAPD patterns by the analysis of molecular variance method revealed a high level of intraspecific genetic diversity in this B. cepacia population. Moreover, the genetic diversity was related to divergences among maize root samplings, with microbial genetic variability markedly higher in the first stages of plant growth; in other words, the biodiversity of this rhizosphere bacterial population decreased over time.