1 Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1
2 Department of Medicine, University of Western Ontario, London, Ontario, Canada N6A 5C1
Correspondence
Miguel A. Valvano
mvalvano{at}uwo.ca
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF317697.
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INTRODUCTION |
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Burkholderia cepacia is an aerobic, Gram-negative, catalase-positive bacterium found in soil and water environments (Coenye & Vandamme, 2003). Strains identified as B. cepacia belong to a group of at least nine closely related species or genomovars, collectively referred to as the B. cepacia complex (Coenye & Vandamme, 2003
). B. cepacia complex strains have become medically important multidrug-resistant opportunistic pathogens, particularly in patients with cystic fibrosis and chronic granulomatous disease (Govan et al., 1996
; Govan & Deretic, 1996
; Speert et al., 1994
). Infections in cystic fibrosis patients by B. cepacia complex bacteria are often associated with increased morbidity and mortality. Some infected patients also succumb to a rapidly progressive necrotizing pneumonia, termed the cepacia syndrome (Bals et al., 1999
; Govan & Vandamme, 1998
; Govan & Deretic, 1996
; Tablan et al., 1985
; Tummler & Kiewitz, 1999
). Chronic inflammation of the lungs and airways in cystic fibrosis presumably contributes to the release of ROS, resulting in damage to lung tissue (Bals et al., 1999
). The survival and persistence of B. cepacia in this highly oxidative environment sharply contrasts with the observation that B. cepacia complex isolates are killed by oxidative mechanisms of neutrophils (Speert et al., 1994
).
Previously, we have shown that B. cepacia complex strains can survive intracellularly in both macrophages and amoebae (Lamothe et al., 2004; Marolda et al., 1999
; Saini et al., 1999
). In addition, B. cepacia complex strains can survive within a murine macrophage cell line in the presence of an oxidative burst, and reduced nitric oxide production (Saini et al., 1999
). These observations suggest that bacterial resistance or adaptation to oxidative damage may play a role in the infectivity and persistence of B. cepacia complex strains within the airways of patients with cystic fibrosis (Valvano et al., 2005
). In fact, the degree of survival of several B. cepacia complex isolates in the presence of exogenous H2O2 can be directly correlated to the level of catalase activity (Lefebre & Valvano, 2001
). On average, Burkholderia cenocepacia strains produced the highest levels of catalase activity, and displayed an increased survival upon challenge with H2O2 (Lefebre & Valvano, 2001
).
More than 80 % of the B. cepacia complex strains that are isolated in Canada from patients with cystic fibrosis are identified as B. cenocepacia, and they all belong to a single lineage, ET12, that has been shown to be transmissible among patients (Speert et al., 2002). All B. cenocepacia isolates examined in our study produce a bifunctional catalase/peroxidase (Lefebre & Valvano, 2001
). In this work, we identified two genes from one of these isolates (strain C5424) that encode the functional catalase/peroxidase enzymes KatA and KatB. We demonstrate that a katA-deficient mutant exhibits a carbon-source-dependent growth defect, and increased sensitivity to H2O2 under iron limitation. A katB-deficient mutant, in contrast, showed reduced growth and hypersensitivity to H2O2 under all conditions tested. We provide evidence demonstrating that KatB is the major catalase/peroxidase enzyme in C5424, while KatA is a specialized catalase/peroxidase that plays a novel functional role to protect critical tricarboxylic acid (TCA) cycle enzymes.
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METHODS |
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PCR amplifications.
PCR amplifications were performed in a PTC-0200 DNA engine (MJ Research) with either Pwo polymerase (Roche) or Taq polymerase (Qiagen), using the supplied Q solution for G+C-rich templates, and B. cenocepacia chromosome as a template. The DNA sequences of the oligonucleotide primers are indicated in Table 2. The specific conditions for PCR amplification were optimized for each primer pair, and they are available from the authors upon request. PCR amplification products were separated in 0·7 or 1·2 % agarose gels, and purified using the QiaQuick gel extraction system (Qiagen).
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Southern blot analysis.
The 473 bp amplicon (katA probe) was labelled directly with DIG-11-UTP using the kat-NT and kat-CT primers (Table 2) and a PCR labelling kit (Roche), as recommended by the manufacturer. B. cenocepacia genomic DNA was isolated, and individually digested with SalI, NsiI and BamHI. Southern blot analysis of genomic DNA was conducted as described elsewhere. Briefly, DNA was separated on a 0·5 % agarose gel, and transferred to a nitrocellulose membrane by capillary action. The membrane was incubated with the katA probe under high-stringency conditions (50 %, v/v, formamide at 42 °C). Hybridization signals were detected by chemiluminescence with disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD) as recommended by the manufacturer (Roche).
Cloning of katA and katB genes.
SalI- and NsiI-digested DNA fragments from B. cenocepacia C5424 were purified from an agarose gel, and used to construct a genomic library by ligation to SalI- and NsiI-cleaved pKS-Bluescript, respectively. Ligation mixes were transformed into E. coli DH5, and transformants were plated on LB agar plates with ampicillin and 0·2 % (w/v) X-Gal and 2 mM IPTG. Colonies with a white colour phenotype were pooled into groups of 50. Plasmid DNA was extracted from each pool, and screened by Southern blot hybridization using the katA probe, as described above. Pools yielding positive signals were further split into smaller groups, and rescreened until plasmids from individual colonies hybridizing to the probe were isolated. These experiments resulted in the isolation of plasmids pML25, pML26 and pML30, which span the katA gene and its flanking sequences (Fig. 1
, Table 1
). The DNA inserts in these plasmids were fully sequenced, and the sequence has been deposited in GenBank under accession no. AF317697. The coding region of the katA gene was amplified by PCR using the katA-NT and katA-CT primers (Table 2
). The 2·2 kb product was treated with mung bean nuclease, digested with EcoRI, and ligated into pMLBAD digested with EcoRI and SmaI. The resulting plasmid, pMLBAD-katA, carries the katA under control of the ParaBAD promoter (Fig. 1
, Table 1
). Plasmid pKMBAD-katA, also carrying the katA gene under the control of the ParaBAD promoter, and conferring chloramphenicol resistance, was constructed for the complementation of the C5424 katA-defective mutant (see below) by digesting pMLBAD-katA with EcoRI and PstI, and ligating the resulting 2·3 kb fragment carrying the complete katA gene into pKMBAD.
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Functional complementation of the E. coli catalase-deficient strain UM2.
Catalase activity in strain UM2 alone, or harbouring pMLBAD-katA, pKMBAD-katA or pKMBAD-katB, was assessed on agar plates by visualizing bubbles upon the addition of 3 % H2O2 solution to the edge of colonies.
Construction of katA and katB mutants.
pSUP202-Tp, a modified version of the suicide plasmid pSUP202 (Simon et al., 1983) that carries the dhfr gene encoding trimethoprim resistance, was used to disrupt katA in C5424 by a single cross-over event. An internal fragment of katA was amplified by PCR using primers Amut-NT and Amut-CT (Table 2
). The product was ligated into the EcoRV site of pSUP202-Tp, which disrupts the tetracycline-resistance gene in the plasmid. Transformants carrying plasmids with the internal katA fragment were selected for resistance to trimethoprim and sensitivity to tetracycline, and screened by restriction digest to identify pML31. pGP
Tp, a derivative of pGP704 that carries the Pir-dependent R6K origin of replication and the dhfr gene flanked by terminator sequences, was used to disrupt katB. An internal fragment of katB was amplified with Bmut-NT and Bmut-CT primers. The product was ligated into the SmaI site of pGP
Tp, and transformed into E. coli SY327. Trimethoprim-resistant colonies were screened by restriction digestion and PCR to confirm the presence and orientation of the PCR product to identify pML102. Both pML31 and pML102 (Fig. 1
, Table 1
) were transferred into B. cenocepacia strain C5424 by tri-parental mating, as described above. Exconjugants containing either pML31 or pML102 that had integrated into the C5424 genome were selected on LB agar supplemented with trimethoprim and gentamicin (to remove E. coli donor and helper strains). The integration of both suicide plasmids was confirmed by Southern blot hybridization using either katA- or katB-specific probes, allowing identification of the katA- and katB-deficient mutant strains MDL1 and MDL2, respectively.
Resistance to H2O2.
Bacterial resistance to H2O2 was assessed as described previously (Lefebre & Valvano, 2001). Briefly, cultures were grown to stationary phase in LB broth with and without
,
'-dipyridyl, and approximately 107 cells were incubated with 10 mM H2O2 for 30 min at room temperature. Aliquots were removed, plated on LB agar, and the survival rate was calculated using the following equation: percentage survival = (no. colonies in treated sample/no. colonies at time 0)x100. Also, H2O2 resistance was assessed by a disk diffusion inhibition assay. Briefly, overnight cultures were diluted to an OD600 of 0·4, and inoculated into molten top agar. Sterile disks (8 mm) were treated with 20 µl H2O2 dilutions (ranging from 0 to 100 mM), and placed on the agar. Zones of inhibition were measured following 2436 h incubation at 37 °C.
Determination of catalase/peroxidase activity.
Crude cell-free lysates of wild-type, katA and katB mutant strains were generated from cultures grown to exponential and stationary phases. Catalase activities within the lysates were measured spectrophotometrically, and also assessed by native PAGE to identify bands with catalase and peroxidase activity, as described by Katsuwon & Anderson (1992) and Lefebre & Valvano (2001)
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Determination of aconitase activity.
Aconitase activity was measured from crude cell lysates under aerobic conditions, as described elsewhere (Gruer & Guest, 1994). Briefly, cultures were grown in LB broth, or LB broth supplemented with 100 µM
,
'-dipyridyl, 50 µM ferrous sulphate or 1 % (w/v) arabinose, as required, and harvested in late stationary phase. Cells were resuspended in Tris/citrate buffer (20 mM citrate, pH 8), and lysed by sonic disruption. Lysates were cleared by centrifugation, and total protein content was determined using the Bio-Rad system, with BSA as a standard. Aconitase activity was assayed spectrophotometrically at room temperature by monitoring the production of cis-aconitate as an increase of absorbance at 240 nm, following the combination of 0100 µg total protein with 20 µM isocitrate. Specific aconitase activity was calculated as the change in absorbance per min per mg protein through the linear portion of the curves. For inactivation of AcnB activity, crude cell lysates were incubated with either 1 mM EDTA or 100 µM
,
'-dipyridyl for 60 min prior to the assay. Reconstitution of AcnB activity was conducted by incubating cell lysates with 1 mM DTT and 1 mM Fe(NH4SO4)2 at 0 °C for 30 min prior to the assay.
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RESULTS |
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Another catalase/peroxidase gene, designated katB, was identified in the genome of the related strain J2315 (Fig. 1b). The predicted KatB protein is also homologous to typical catalase/peroxidase of the HPI class, and shows 82·9 % similarity and 76 % identity to KatA at the amino acid level. A putative oxyR gene is located 621 bp upstream of katB, and is transcribed in the same direction. The organization of these genes resembles that of the katG locus in the related species Burkholderia pseudomallei (Loprasert et al., 2002
). Using katBNT and katBCT primers, we confirmed by PCR that katB was also present in the strain C5424 (data not shown).
To determine whether katA and katB encode functional proteins, pML26 and pKMBADkatB (Fig. 1) were transformed into E. coli strain UM2, which carries mutations in both katE and katG genes (Table 1
). Restoration of catalase activity in the transformants was assessed by applying 3 % H2O2 to the border of colonies, and inspecting them for bubble formation due to oxygen release. The plasmid pKMBAD-katB complemented the catalase deficiency of the E. coli double-catalase mutant UM2, indicating that KatB is functional. In contrast, initial experiments did not show complementation of the catalase deficiency in UM2(pML26) cells. Lack of complementation could be due to either poor expression of katA from a B. cenocepacia promoter in E. coli, or the absence of a promoter region in the cloned fragment. Therefore, we cloned the coding region of katA under the control of the arabinose-inducible ParaBAD promoter of pMLBAD, generating the plasmid pMLBAD-katA. In the presence of arabinose, the catalase-deficient phenotype of E. coli UM2(pMLBAD-katA) was restored, indicating that the B. cenocepacia KatA protein is functional in E. coli when appropriately expressed.
A katA-deficient mutant of strain C5424 shows reduced resistance to exogenous H2O2 under conditions of iron limitation
We generated a katA mutant carrying a targeted integration of the suicide plasmid pML31 into the katA gene, which was designated MDL1 (Table 1). The site-specific integration of pML31 in B. cenocepacia MDL1 was independently confirmed by Southern blot hybridization and by PCR analysis of genomic DNA (data not shown). Mutant and parental strains were grown to late stationary phase in LB broth, and LB broth supplemented with either 100 µM FeSO4 (to promote oxidative stress conditions) or 100 µM of the iron chelator
,
'-dipyridyl, and then assessed for sensitivity to exogenous H2O2. Both strains showed no significant differences in the levels of H2O2 resistance when grown in LB broth alone or LB broth supplemented with FeSO4 (Fig. 2
, and data not shown). In contrast, MDL1 displayed decreased survival (a decrease of approx. 2 log units compared to the parent) after H2O2 challenge under iron limitation (Fig. 2
). The survival defect of MDL1 was corrected by pMLBAD-katA as long as the plasmid-encoded katA gene was induced by adding arabinose to the growth medium (Fig. 2
). These experiments demonstrate that the sensitivity of MDL1 to H2O2 challenge under iron limitation is associated with the lack of KatA function. Furthermore, this phenotype was complemented by pMLBAD-katA, indicating that it was due to the disruption of the katA gene, and not caused by a polar effect on downstream genes, or a secondary mutation elsewhere.
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MDL2 did not form isolated colonies on agar plates, and the growth was mainly restricted to zones of high cell density, such as the initial points of streaking (data not shown). Since it was difficult to assess H2O2 sensitivity in MDL2 by enumerating c.f.u. as with MLD1, we used instead a disk diffusion inhibition assay. Zones of inhibition in top agar lawns containing C5424, MDL1 and MDL2 were measured after exposure to sterile filter disks impregnated with 20 µl H2O2 solution (at concentrations ranging from 0 to 100 mM). C5424 and MDL1 showed comparable levels of inhibition, with zones of 8 and 9 mm, respectively, after challenge with 100 mM H2O2, but no inhibition zones were detected at lower H2O2 concentrations. In contrast, MDL2 showed hypersensitivity to H2O2, with zones of growth inhibition of 10 mm diameter appearing at concentrations as low as 2·5 mM. MDL2 also showed reduced growth in LB broth, as the strain required roughly 60 h to reach stationary phase (OD600 2·0), in contrast to 2430 h for either C5424 (parental) or MDL1 (katA mutant). However, the growth defect in MDL2 was not related to iron availability, as it was not affected by the addition of exogenous iron or the presence of
,
'-dipyridyl (data not shown).
Complementation experiments to restore catalase/peroxidase function in MDL2 were conducted using pKMBAD-katA and pKMBAD-katB. In both cases, catalase/peroxidase activity, as determined by native PAGE, was restored in the presence of arabinose. MDL2(pKMBAD-katB) exhibited a single band of catalase activity that co-migrated with the activity seen in the parental C5424, indicating that KatB is the major catalase/peroxidase in the strain (Fig. 3, lane 6). In contrast, MDL2(pKMBAD-katA) showed three distinct bands of weak catalase activity, one of which co-migrated with the band of activity associated with KatB (Fig. 3
, lane 4). All bands were a result of KatA expression, as they were absent from MDL2, and from MDL2-pKMBAD-katA grown in the presence of glucose (Fig. 3
, lanes 3 and 5). Analysis of the peroxidase activity by native PAGE revealed identical banding profiles to those seen with the catalase-specific stain (data not shown). Based on the phenotypic characterization of the katB mutant MDL2, we conclude that KatB is the major catalase/peroxidase in C5424, which is required for full resistance to H2O2 exposure, while KatA is a minor catalase/peroxidase, which is only required under iron limitation.
MDL1 shows reduced growth in the presence of some substrates of the TCA cycle
Despite the reduced viability of the MDL1 katA mutant under iron limitation in the presence of H2O2, its growth rate in LB under iron limitation, but in the absence of exogenously added H2O2, was comparable with that of the parental C5424 (data not shown). Further experiments using a defined minimal medium supplemented with various carbon sources showed that the growth rate of MDL1 was reduced in the presence of succinate, but not affected in the presence of glucose or glycerol, irrespective of the ,
'-dipyridyl concentration (Fig. 4
, and data not shown). Since succinate is catabolized via the TCA cycle, we hypothesized that detoxification of the ROS generated by the biochemical reactions of this cycle (Messner & Imlay, 1999
) could require the function of KatA. In the presence of glucose or glycerol, enough ATP for bacterial growth would be generated through the terminal steps of glycolysis (Oexle et al., 1999
), effectively bypassing TCA cycle reactions and the requirement for KatA. Thus, we examined the growth rates of C5424 and MDL1 in TM medium supplemented with various carboxylic acids that are catabolized through the TCA cycle. The results show that the growth of the mutant was compromised in cultures supplemented with citrate, 2-oxoglutarate, succinate or pyruvate (Fig. 4
). The growth differences observed with MDL1 relative to the parental strain were less obvious for cultures in the presence of fumarate. Furthermore, addition of exogenous iron to the TM medium alleviated the growth deficiency, while the MDL1 grew very poorly in the presence of 25 µM
,
'-dipyridyl (Fig. 4
, and data not shown), confirming that the growth defect was linked to both the availability of cellular iron and the particular carbon source supplied.
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DISCUSSION |
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We also demonstrate that although the cloned katA encodes a functional catalase/peroxidase, the katA insertional mutant did not show any detectable reduction in the enzyme activity under the conditions tested, and displayed a growth defect only in the presence of carbon sources that are metabolized through the TCA cycle. The katA mutant was also sensitive to H2O2, but only when bacterial cells were grown under iron limitation. The phenotypes of the katA mutant were corrected by complementation with the intact katA gene, confirming that they were due to the absence of the KatA protein. The katA gene is located on the second-largest chromosome of the clonally related strain J2315, and is part of a putative three-gene operon that also includes a bromoperoxidase and quinone oxidoreductase genes. The location of the katA gene, and its association with genes encoding enzymes involved in redox reactions, suggest a specialized role for KatA.
The iron chelator -
'-dipyridyl can enter the bacterial cytoplasm, and, under these conditions, some enzymes of the TCA cycle, like AcnB, can be rapidly demetallated (Varghese et al., 2003
). We reasoned that a similar situation could occur in B. cenocepacia C5424, such that KatA could be required to counteract the effects of iron depletion on one or more TCA cycle enzymes. Consistent with this notion, we observed reduced growth of the katA mutant in low-iron-containing minimal medium supplemented with various carbon sources that are metabolized through the TCA cycle, especially with citrate and succinate, while no growth defect was found with glucose or glycerol. Our results demonstrating that the aconitase activity is significantly reduced in the katA mutant, but not affected in the parental strains or in the katB mutant, suggest that KatA could be involved, directly or indirectly, in stabilizing this enzyme under conditions of iron limitation and TCA substrates in the growth medium.
The reduction in aconitase activity associated with MDL1 was clearly linked with the loss of KatA expression, since complementation of the KatA defect could restore aconitase activity to wild-type levels. Therefore, we propose that in B. cenocepacia C5424, and possibly other B. cepacia complex strains, KatA may contribute to maintain the function or stability of aconitase under iron limitation. An inspection of the genome sequence of the related B. cenocepacia strain J2315 revealed the presence of genes encoding very good homologues of the AcnA and AcnB proteins from E. coli, suggesting they may have similar properties. Determination of the aconitase activity in B. cenocepacia C5424 and the katA mutant in the presence of excess iron and reducing conditions (Table 3) revealed an aconitase form that has similar biochemical properties to AcnB, and its activity does not appear to be affected in the katA mutant. Thus, it is possible that KatA acts on another form of aconitase, which may resemble the E. coli AcnA. In E. coli, AcnB is rapidly inactivated when cellular iron pools are low or under oxidative stress (Varghese et al., 2003
). In contrast, AcnA synthesis is induced by oxidative stress and iron limitation, but it appears to require an unidentified cellular component for stability (Varghese et al., 2003
). Insertional inactivation of the B. cenocepacia acnA and acnB candidate genes will permit us to directly address this possibility.
The catalase activity of the KatA protein is negligible, since it could only be detected in the katB mutant when the katA gene was overexpressed under a regulatable strong promoter, and also by complementation of the E. coli double-catalase mutant UM2. Our working model predicts that the KatA protein contributes to the stability of an AcnA-like protein itself, or that of another factor involved in maintaining the normal function of the TCA cycle enzymes in the presence of iron limitation. However, it is puzzling why KatB, which is presumably present in large quantities, cannot compensate for the KatA defect. One possible explanation is that KatA carries out its protective role in a fashion that is independent of enzymic function. If this is the case, KatA must presumably possess specific structural motifs, absent or modified in KatB, that are required for this role. Experiments to directly test this possibility, and to elucidate the differential regulation of katA and katB expression, are under way in our laboratory.
In summary, we have determined that B. cenocepacia C5424 carries two catalase/peroxidases that share substantial similarity in their amino acid sequences, but play different functional roles. Future studies are required to further characterize the relationship between KatA and the function of aconitase and possibly other enzymes associated with cellular metabolism, as well as to determine whether both KatA and KatB activities are required for bacterial survival in vivo.
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ACKNOWLEDGEMENTS |
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Received 14 October 2004;
revised 19 January 2005;
accepted 22 February 2005.
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