- Research article
- Open Access
Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2
© Hamlin et al; licensee BioMed Central Ltd. 2009
- Received: 8 May 2009
- Accepted: 18 October 2009
- Published: 18 October 2009
Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2.
Translational fusions of the PA degradation gene promoters with eGFP were constructed and introduced in B. cenocepacia K56-2. eGFP expression was observed when the reporter strains were grown in minimal media containing glycerol and PA or other compounds expected to proceed through the PA pathway, and in synthetic CF medium (SCFM). Addition of succinate or glucose to the PA containing medium repressed eGFP expression. To show that BCAL0210, a putative TetR-type regulator gene encodes a regulator for the PA genes in B. cenocepacia, we developed a BCAL0210 insertional mutant reporter strain. Results show that these strains exhibit fluorescence regardless of the presence of PA in the culture.
The PA catabolic genes of B. cenocepacia K56-2 are induced by PA and other related compounds, are negatively regulated by PaaR (named herein), a TetR-type regulator, and are subjected to catabolic repression by glucose and succinate. As the PA catabolic pathway of B. cenocepacia appears to be induced during growth in synthetic cystic fibrosis medium (SCFM), further research is necessary to determine the relevance of this pathway in CF-like conditions and in other host-pathogen interactions.
- Cystic Fibrosis
- Luria Bertani
- Inverted Repeat
- Chronic Granulomatous Disease
- Phenylacetic Acid
The Burkholderia cepacia complex (Bcc) is a group of Gram negative bacteria that comprises at least fifteen taxonomically related species [1, 2]. Bcc strains occupy multiple niches from soil to humans as they have emerged as opportunistic pathogens in patients with cystic fibrosis (CF), chronic granulomatous disease, and other medical conditions associated with a compromised immune system [1, 3]. Burkholderia species have evolved large genomes that allow them to deal with a variety of nutrient sources, predation and competition. The three chromosomes of B. cenocepacia, one of the most common species found in CF patients , encode a broad array of catabolic functions. Yet, the contribution of these metabolic capacities to colonization and survival in the host has not been established.
The phenylacetic acid (PA) catabolic pathway is the central route where catabolism of many aromatic compounds converge and are directed to the Krebs cycle . It comprises of four steps, namely the PA-CoA ligation-activation performed by PaaK , the hydroxylation step for which the PaaABCDE enzymatic complex is responsible , the enoyl-CoA isomerization/hydration, ring opening performed by PaaG and PaaZ, , and the β-oxidation step carried out by PaaF and PaaH, .
Previously, we initiated the functional characterization of the PA catabolic pathway of B. cenocepacia K56-2  and demonstrated that interruption of putative PA-CoA ring hydroxylation activity, but not the lower steps of PA degradation, resulted in an attenuated pathogenic phenotype in the Caenorhabditis elegans model of infection. Here, we report that the PA catabolic genes of B. cenocepacia K56-2 are induced by PA, are negatively regulated by PaaR, a TetR-type regulator and are subjected to catabolic repression by glucose and succinate.
Translational reporter plasmids containing PA catabolic gene promoters are responsive to PA and related compounds
In addition, we sought to determine whether the PA genes were activated in response to Synthetic Cystic Fibrosis Medium (SCFM), a chemically defined medium formulated according to the contents of CF sputum . Our results show that P paaA reporter activity increases approximately 5-fold when cells are grown in SCFM (Figure 2).
The PA catabolic genes are subject to catabolic repression by TCA intermediates and sugars
Insertional mutagenesis of BCAL0210 results in increased expression of PA-inducible genes
In order to determine if paaABCDE and BCAL0211-BCAL0210 were part of the same transcriptional unit, a transcriptional analysis was performed. Total RNA was isolated from B. cenocepacia cells grown with LB containing 1 mM PA and subjected to RT-PCR using specific primers. Results show that the paaA, paaB, paaC, paaD and paaE genes are contained on a single transcript and are thus co-regulated at the transcriptional level (Figure 4B). Primers were unable to generate an amplicon between paaE and BCAL0211 although an amplicon was generated between BCAL0211 and BCAL0210, indicating that they are located on the same transcript. Taken together these results demonstrate that paaABCDE and BCAL0211-BCAL0210 are two separate transcriptional units.
A conserved Inverted Repeat is necessary for negative control of P paaA
Bacterial Strains and Plasmids
Strain or plasmid
Reference or source
B. cenocepacia strains
ET12 clone related to J2315, CF clinical isolate
E. coli strains
F-, ϕ 80 lac ZΔM15 endA1 recA1 hsdR 17(rK-mK+)supE 44 thi- 1 ΔgyrA 96 (ΔlacZYA-arg F)U169 relA 1
araD Δ(lac pro) argE (Am) recA 56 Rifr nalA λ pir
ori r6K, ΩTpr mob+
oricolE1, RK2 derivative, Kmr mob+ tra+
pGPΩTp, internal fragment from BCAL0210
pJH1, eGFP replaced promoter with multiple cloning site
pJH2, BCAL0211 promoter region (P BCAL0211 )
pJH2, paaZ promoter region (P paaZ )
pJH2, paaA promoter region (P paaA )
pJH2 paaH promoter region (P paaH )
pJH7, ACCGACCGGTCGGT → TAGATGTATCTCAG
pJH7, ACCGACCGGTCGGT → TAGATGTGGTCGGT
pJH7, ACCGACCGGTCGGT → ACCGACCATCTCAG
Because PaaX is involved in the regulation of upstream pathways of PA catabolism in other microorganisms through binding a conserved PaaX box [23, 24] we searched for the consensus IR sequence in the genome of B. cenocepacia. A position weight matrix (PWM)  of the conserved IR present in the promoter regions of the paaA, paaH and paaZ plus the divergent promoter of paaF and BCAL0211 was constructed (Additional file 2) and used to search the entire genome sequence of B. cenocepacia J2315. The coordinate positions of sequences detected up to a cut off score of 17.0 are listed (Additional file 3). The top scores for the search were the ones for the paaZ, paaF, paaA and paaH inverted repeats while BCAL0211 IR scored lower at 12.0. Other sequences with scores that ranked from 18.41 to 17.37 did not locate in putative promoters or between -10 and -35 regions, likely representing false positives. We concluded that the 15 bp IR sequences are specific to the PA catabolic gene clusters.
In contrast to what has been observed in E. coli and Pseudomonas putida , the PA genes of B. cenocepacia K56-2 are organized into three gene clusters. We hypothesize that this arrangement may allow regulation of gene expression at different levels. The observation that eGFP expression driven by P paaA is roughly 3-fold stronger than either the P paaH or P paaZ promoters (Figure 1) is suggestive of a higher requirement for the product of the PaaABCDE enzymatic complex than the other intermediates. This could be simply due to the optimal kinetic coupling between the different steps or that the product of the ring hydroxylation complex is used in a second pathway with a yet unknown biological function. The presence of a poly(A) tract upstream of the paaA -35 element (Figure 5A) that resembles an UP element  may likely account for the increased activity.
Activity of PpaaA and IR mutated derivatives as a result of growth in M9 minimal media containing glycerol or PA.
Mean fluorescence/O.D.600 ± SD with indicated carbon sources
187 ± 33
1096 ± 107
1579 ± 10
1062 ± 15
1345 ± 111
1026 ± 52
2159 ± 111
1503 ± 60
Catabolic repression of aromatic compound degradation by TCA intermediates and glucose has been described in the β-proteobacterium Acidovorax sp. , and P. putida  respectively. In accordance with these data we found that the PA catabolic pathway of B. cenocepacia K56-2 is subject to catabolic repression by glucose and succinate (Figure 3). Interestingly, P paaA is induced after 18 h of growth in SCFM probably as a result of the presence of phenylalanine (Figure 2). This observation is consistent with the recently reported B. cenocepacia global gene expression response to SCFM, which shows the induction of the PA catabolic pathway . Whether this finding is relevant for pathogenesis of Bcc in the CF lung environment remains an unexplored point of interest.
We show that the PA gene promoters are responsive to PA, SCFM, and other compounds expected to proceed via the PA pathway. We also show the PA gene promoters are negatively regulated by PaaR, a TetR-type regulator, and are subjected to catabolic repression by succinate and glucose.
Bacterial strains, nematode strains and growth conditions
Bacterial strains and plasmids are listed in Table 1. B. cenocepacia K56-2 was grown at 37°C in Luria Bertani (LB) or M9 minimal medium with 5 mM PA or 25 mM of the indicated carbon sources, supplemented as required, with 100 μg/ml trimethoprim (Tp), 50 μg/ml gentamicin (Gm) and 200 μg/ml chloramphenicol (Cm). E. coli was grown at 37°C in LB medium supplemented with 50 μg/ml Tp, 40 μg/ml kanamycin (Km) or 20 μg/ml Cm.
Reporter activity assays
96-well microplates containing 150 μl of M9 minimal media supplemented with indicated carbon source(s) were inoculated with 2 μl from an overnight culture grown in LB, washed with PBS and adjusted to an O.D. 600 of 2.0 with M9 minimal salts. 96-well microplates were incubated at 37°C with shaking at 200 rpm. eGFP protein has excitation/emission wavelengths of 488/509 . Relative fluorescence, defined as the ratio between arbitrary fluorescence and optical density at 600 nm (O.D.600) was measured with a Biotek Synergy 2 plate reader, using excitation 485/20 and emission 528/20 filter sets. O.D. 600 values were converted to 1 cm path length O.D. 600 using a standard curve.
BLAST searches of the genome sequence of B. cenocepacia strain J2315 were performed with the B. cenocepacia Blast Server at Sanger Institute http://www.sanger.ac.uk/cgi-bin/blast/submitblast/b_cenocepacia. J2315 belongs to the same clonal lineage as strain K56-2 . Gene clusters were visualized with Artemis software  and VectorNTI software (Invitrogen). PWM scores were calculated manually  (Additional file 2) as described by Hertz and Stormo  and Schnieder and Stephens . Identification of binding sites using this PWM was achieved using the Target Explorer . For TCOFFEE analysis  the default substitution matrix was used, with a gap opening penalty of -10 and a gap extension penalty of -1.
Molecular Biology techniques
Recombinant DNA techniques were carried out as previously described . DNA ligase (New England Biolabs) was used as recommended by the manufacturers. E. coli DH5α cells were transformed using the calcium chloride protocol  and electroporation was used for transformation of E. coli SY327 cells . Reporter plasmids were constructed in E. coli and conjugation into B. cenocepacia K56-2 was accomplished by triparental mating  with E. coli DH5α carrying the helper plasmid pRK2013 . DNA was amplified using a PTC-221 DNA engine (MJ Research) or an Eppendorf Mastercycler ep gradient S thermal cycler with Taq DNA polymerase, Phusion High-Fidelity PCR Kit or Proofstart DNA polymerase (Qiagen) (New England Biolabs). Amplification conditions were optimized for each primer pair and are available upon request. PCR products and plasmids were purified with QIAquick purification kit (Qiagen) and QIAprep Miniprep kit (Qiagen), respectively.
RNA isolation methods and RT-PCR analysis
For RNA isolation, bacteria were grown in LB supplemented with 1 mM PA. Cells were harvested during early log phase (O.D. 600 = 0.3) and lysed in TE buffer pH 8.0 containing 400 μl/ml lysozyme for 5 minutes at room temperature. RNA was recovered with the RNeasy Mini kit (Qiagen), and samples eluted into (Diethyl Pyrocarbonate) DEPC treated water. Total RNA was visualized in a 1% agarose gel in TAE buffer. Residual DNA was removed by on column treatment with DNase I (15 min, room temperature), in DNase buffer (Qiagen). The RNA was then used as a template in reverse transcription (RT) or stored at -20°C until use. Reverse transcription was performed by SuperScript RT First-Strand synthesis using relevant gene specific primers (Additional file 1). The resultant cDNA was PCR amplified using gene specific primers (Additional file 1), and the conditions optimized for each reaction. For every PCR, the appropriate controls with water and RNA in the absence of RT were included to ensure that the amplicons obtained were a result of cDNA and not of contaminating genomic DNA.
Construction of insertional mutant BCAL0210 of B. cenocepacia K56-2
BCAL0210 was disrupted using single crossover mutagenesis with plasmid pGPÙTp, a derivative of pGP704 that carries the dhfr gene flanked by terminator sequences . Briefly, an internal 300-bp fragment of BCAL0210 was PCR amplified using appropriate primers (Additional file 1). The PCR-amplified was digested with Xba I and EcoR I respectively, cloned into the Xba I and EcoR I digested vector and maintained in E. coli SY327. The resulting plasmids (Table 1) were conjugated into B. cenocepacia strain K56-2 by triparental mating. Conjugants that had the plasmid integrated into the K56-2 genome were selected on LB agar plates supplemented with Tp 100 μg/ml and Gm 50 μg/ml. Integration of the suicide plasmids was confirmed by colony PCR, using primer SC025 that anneals to the R6K origin of replication of pGPÙTp, and primers upstream of the expected site of insertion (Additional file 1). All mutant strains were confirmed by sequencing PCR-amplified DNA fragments containing the insertion site.
Construction of eGFP translational fusion plasmids
To create pJH1, digestion with Xba I/Nde I of pSCrhaB4 resulted in a 784 bp fragment containing eGFP, which was cloned into the same sites in pAP20  such that eGFP is under control of the constitutive dhfr promoter. E. coli transformants were selected with 20 μg/ml chloramphenicol. The plasmid was conjugated into B. cenocepacia K56-2 by tri-parental mating with E. coli helper strain containing plasmid pRK2013. As B. cenocepacia is intrinsically resistant to Gm, in all conjugations Gm was added to the final transfer to eliminate donor E. coli. To create pJH2, pJH1 was then PCR amplified using divergently oriented primers (Additional file 1) containing multiple restriction sites on the 5' ends such that the self-ligated product of the reaction has a multiple cloning site in place of the original promoter. Growth rates for B. cenocepacia K56-2 with or without pJH2 were similar (data not shown). DNA fragments corresponding to paaZ from -420 to +90 (510 bp), paaA from -396 to +84 (480 bp), and paaH from -327 to +72 (399 bp) of B. cenocepacia K56-2 chromosomal DNA were amplified and cloned into pJH2 to create pJH6, pJH7, and pJH8 respectively.
Construction of site directed plasmid mutants
The plasmids pJH10, pJH11 and pJH12 were constructed by plasmid PCR mutagenesis to contain mutations in the entire, left or right region of the conserved IR in the paaA core promoter. Appropriate phosphorylated primers (Additional file 1) were used to divergently amplify template pJH7 (containing the paaA promoter), and each contained mismatch mutations on their 5' ends. Plasmids were self-ligated, transformed into E. coli DH5α and then conjugated into B. cenocepacia wild type. Mutations were verified by sequence analysis (The Centre for Applied Genomics, Toronto).
Nucleotide accession number
The nucleotide sequence of translational fusion vector pJH2 is deposited in GenBank under accession no. FJ607244.
We thank Julian Parkhill and Mathew Holden for allowing us access to the draft annotation of B. cenocepacia J2315, and Ann Karen Brassinga for critically reading the manuscript. JNRH was supported by a graduate scholarship from the Manitoba Health Research Council (MHRC). RAMB is supported by a Manitoba Graduate Scholarship. This study was supported by the NSERC grant N° 327954.
- Mahenthiralingam E, Urban TA, Goldberg JB: The multifarious, multireplicon Burkholderia cepacia complex. Nat Rev Microbiol. 2005, 3 (2): 144-156. 10.1038/nrmicro1085.PubMedView ArticleGoogle Scholar
- Vanlaere E, Lipuma JJ, Baldwin A, Henry D, De Brandt E, Mahenthiralingam E, Speert D, Dowson C, Vandamme P: Burkholderia latens sp. nov., Burkholderia diffusa sp. nov., Burkholderia arboris sp. nov., Burkholderia seminalis sp. nov. and Burkholderia metallica sp. nov., novel species within the Burkholderia cepacia complex. Int J Syst Evol Microbiol. 2008, 58 (Pt 7): 1580-1590. 10.1099/ijs.0.65634-0.PubMedView ArticleGoogle Scholar
- Valvano MA, Keith KE, Cardona ST: Survival and persistence of opportunistic Burkholderia species in host cells. Curr Opin Microbiol. 2005, 8 (1): 99-105. 10.1016/j.mib.2004.12.002.PubMedView ArticleGoogle Scholar
- Holden MT, Seth-Smith HM, Crossman LC, Sebaihia M, Bentley SD, Cerdeno-Tarraga AM, Thomson NR, Bason N, Quail MA, Sharp S, Cherevach I, Churcher C, Goodhead I, Hauser H, Holroyd N, Mungall K, Scott P, Walker D, White B, Rose H, Iversen P, Mil-Homens D, Rocha EP, Fialho AM, Baldwin A, Dowson C, Barrell BG, Govan JR, Vandamme P, Hart CA, Mahenthiralingam E, Parkhill J: The genome of Burkholderia cenocepacia J2315, an epidemic pathogen of cystic fibrosis patients. J Bacteriol. 2009, 191 (1): 261-277. 10.1128/JB.01230-08.PubMed CentralPubMedView ArticleGoogle Scholar
- Luengo JM, Garcia JL, Olivera ER: The phenylacetyl-CoA catabolon: a complex catabolic unit with broad biotechnological applications. Mol Microbiol. 2001, 39 (6): 1434-1442. 10.1046/j.1365-2958.2001.02344.x.PubMedView ArticleGoogle Scholar
- Ferrandez A, Minambres B, Garcia B, Olivera ER, Luengo JM, Garcia JL, Diaz E: Catabolism of phenylacetic acid in Escherichia coli. Characterization of a new aerobic hybrid pathway. J Biol Chem. 1998, 273 (40): 25974-25986. 10.1074/jbc.273.40.25974.PubMedView ArticleGoogle Scholar
- Fernandez C, Ferrandez A, Minambres B, Diaz E, Garcia JL: Genetic characterization of the phenylacetyl-Coenzyme A oxygenase from the aerobic phenylacetic acid degradation pathway of Escherichia coli. Appl Environ Microbiol. 2006, 72 (11): 7422-7426. 10.1128/AEM.01550-06.PubMed CentralPubMedView ArticleGoogle Scholar
- Ismail W, El-Said Mohamed M, Wanner BL, Datsenko KA, Eisenreich W, Rohdich F, Bacher A, Fuchs G: Functional genomics by NMR spectroscopy. Phenylacetate catabolism in Escherichia coli. Eur J Biochem. 2003, 270 (14): 3047-3054. 10.1046/j.1432-1033.2003.03683.x.PubMedView ArticleGoogle Scholar
- Law RJ, Hamlin JN, Sivro A, McCorrister SJ, Cardama GA, Cardona ST: A functional phenylacetic acid catabolic pathway is required for full pathogenicity of Burkholderia cenocepacia in the Caenorhabditis elegans host model. J Bacteriol. 2008, 190 (21): 7209-7218. 10.1128/JB.00481-08.PubMed CentralPubMedView ArticleGoogle Scholar
- Lefebre MD, Valvano MA: Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates. Appl Environ Microbiol. 2002, 68 (12): 5956-5964. 10.1128/AEM.68.12.5956-5964.2002.PubMed CentralPubMedView ArticleGoogle Scholar
- Kanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000, 28 (1): 27-30. 10.1093/nar/28.1.27.PubMed CentralPubMedView ArticleGoogle Scholar
- Kanehisa M, Goto S, Hattori M, Aoki-Kinoshita KF, Itoh M, Kawashima S, Katayama T, Araki M, Hirakawa M: From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res. 2006, D354-7. 10.1093/nar/gkj102. 34 DatabaseGoogle Scholar
- Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y: KEGG for linking genomes to life and the environment. Nucleic Acids Res. 2008, D480-4. 36 DatabaseGoogle Scholar
- Palmer KL, Aye LM, Whiteley M: Nutritional cues control Pseudomonas aeruginosa multi-cellular behavior in cystic fibrosis sputum. J Bacteriol. 2007, 189 (22): 8079-8087. 10.1128/JB.01138-07.PubMed CentralPubMedView ArticleGoogle Scholar
- Martinez-Blanco H, Reglero A, Luengo JM: Carbon catabolite regulation of phenylacetyl-CoA ligase from Pseudomonas putida. Biochem Biophys Res Commun. 1990, 167 (3): 891-897. 10.1016/0006-291X(90)90607-O.PubMedView ArticleGoogle Scholar
- Bruckner R, Titgemeyer F: Carbon catabolite repression in bacteria: choice of the carbon source and autoregulatory limitation of sugar utilization. FEMS Microbiol Lett. 2002, 209 (2): 141-148.PubMedView ArticleGoogle Scholar
- Aranda-Olmedo I, Ramos JL, Marques S: Integration of signals through Crc and PtsN in catabolite repression of Pseudomonas putida TOL plasmid pWW0. Appl Environ Microbiol. 2005, 71 (8): 4191-4198. 10.1128/AEM.71.8.4191-4198.2005.PubMed CentralPubMedView ArticleGoogle Scholar
- Cardona ST, Mueller C, Valvano MA: Identification of essential operons in Burkholderia cenocepacia with a rhamnose inducible promoter. Applied and Environmental Microbiology. 2006, 72 (4): 2547-2555. 10.1128/AEM.72.4.2547-2555.2006.PubMed CentralPubMedView ArticleGoogle Scholar
- Ma D, Alberti M, Lynch C, Nikaido H, Hearst JE: The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals. Mol Microbiol. 1996, 19 (1): 101-112. 10.1046/j.1365-2958.1996.357881.x.PubMedView ArticleGoogle Scholar
- Su CC, Rutherford DJ, Yu EW: Characterization of the multidrug efflux regulator AcrR from Escherichia coli. Biochem Biophys Res Commun. 2007, 361 (1): 85-90. 10.1016/j.bbrc.2007.06.175.PubMed CentralPubMedView ArticleGoogle Scholar
- Ramos JL, Martinez-Bueno M, Molina-Henares AJ, Teran W, Watanabe K, Zhang X, Gallegos MT, Brennan R, Tobes R: The TetR family of transcriptional repressors. Microbiol Mol Biol Rev. 2005, 69 (2): 326-356. 10.1128/MMBR.69.2.326-356.2005.PubMed CentralPubMedView ArticleGoogle Scholar
- Ferrandez A, Garcia JL, Diaz E: Transcriptional regulation of the divergent paa catabolic operons for phenylacetic acid degradation in Escherichia coli. J Biol Chem. 2000, 275 (16): 12214-12222. 10.1074/jbc.275.16.12214.PubMedView ArticleGoogle Scholar
- del Peso-Santos T, Bartolome-Martin D, Fernandez C, Alonso S, Garcia JL, Diaz E, Shingler V, Perera J: Coregulation by phenylacetyl-coenzyme A-responsive PaaX integrates control of the upper and lower pathways for catabolism of styrene by Pseudomonas sp. strain Y2. J Bacteriol. 2006, 188 (13): 4812-4821. 10.1128/JB.00176-06.PubMed CentralPubMedView ArticleGoogle Scholar
- Kim HS, Kang TS, Hyun JS, Kang HS: Regulation of penicillin G acylase gene expression in Escherichia coli by repressor PaaX and the cAMP-cAMP receptor protein complex. J Biol Chem. 2004, 279 (32): 33253-33262. 10.1074/jbc.M404348200.PubMedView ArticleGoogle Scholar
- Wasserman WW, Sandelin A: Applied bioinformatics for the identification of regulatory elements. Nat Rev Genet. 2004, 5 (4): 276-287. 10.1038/nrg1315.PubMedView ArticleGoogle Scholar
- Ross W, Gosink KK, Salomon J, Igarashi K, Zou C, Ishihama A, Severinov K, Gourse RL: A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science. 1993, 262 (5138): 1407-1413. 10.1126/science.8248780.PubMedView ArticleGoogle Scholar
- Flannagan RS, Aubert D, Kooi C, Sokol PA, Valvano MA: Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo. Infect Immun. 2007, 75 (4): 1679-1689. 10.1128/IAI.01581-06.PubMed CentralPubMedView ArticleGoogle Scholar
- Saenger W, Orth P, Kisker C, Hillen W, Hinrichs W: The tetracycline repressor-A paradigm for a biological switch. Angew Chem Int Ed Engl. 2000, 39 (12): 2042-2052. 10.1002/1521-3773(20000616)39:12<2042::AID-ANIE2042>3.0.CO;2-C.PubMedView ArticleGoogle Scholar
- Ohtsubo Y, Goto H, Nagata Y, Kudo T, Tsuda M: Identification of a response regulator gene for catabolite control from a PCB-degrading beta-proteobacteria, Acidovorax sp. KKS102. Mol Microbiol. 2006, 60 (6): 1563-1575. 10.1111/j.1365-2958.2006.05197.x.PubMedView ArticleGoogle Scholar
- Yoder-Himes DR, Chain PS, Zhu Y, Wurtzel O, Rubin EM, Tiedje JM, Sorek R: Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing. Proc Natl Acad Sci USA. 2009, 106 (10): 3976-3981. 10.1073/pnas.0813403106.PubMed CentralPubMedView ArticleGoogle Scholar
- Cormack BP, Valdivia RH, Falkow S: FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996, 173 (1 Spec No): 33-38. 10.1016/0378-1119(95)00685-0.PubMedView ArticleGoogle Scholar
- Darling P, Chan M, Cox AD, Sokol PA: Siderophore production by cystic fibrosis isolates of Burkholderia cepacia. Infect Immun. 1998, 66 (2): 874-877.PubMed CentralPubMedGoogle Scholar
- Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B: Artemis: sequence visualization and annotation. Bioinformatics. 2000, 16 (10): 944-5. 10.1093/bioinformatics/16.10.944.PubMedView ArticleGoogle Scholar
- Hertz GZ, Stormo GD: Identifying DNA and protein patterns with statistically significant alignments of multiple sequences. Bioinformatics. 1999, 15 (7-8): 563-577. 10.1093/bioinformatics/15.7.563.PubMedView ArticleGoogle Scholar
- Schneider TD, Stephens RM: Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 1990, 18 (20): 6097-6100. 10.1093/nar/18.20.6097.PubMed CentralPubMedView ArticleGoogle Scholar
- Sosinsky A, Bonin CP, Mann RS, Honig B: Target Explorer: An automated tool for the identification of new target genes for a specified set of transcription factors. Nucleic Acids Res. 2003, 31 (13): 3589-3592. 10.1093/nar/gkg544.PubMed CentralPubMedView ArticleGoogle Scholar
- Notredame C, Higgins DG, Heringa J: T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000, 302 (1): 205-217. 10.1006/jmbi.2000.4042.PubMedView ArticleGoogle Scholar
- Sambrook J, Russell DW: Molecular cloning: A laboratory manual. 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, 3Google Scholar
- Cohen SN, Chang AC, Hsu L: Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA. 1972, 69 (8): 2110-2114. 10.1073/pnas.69.8.2110.PubMed CentralPubMedView ArticleGoogle Scholar
- Miller VL, Mekalanos JJ: A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988, 170 (6): 2575-2583.PubMed CentralPubMedGoogle Scholar
- Craig FF, Coote JG, Parton R, Freer JH, Gilmour NJ: A plasmid which can be transferred between Escherichia coli and Pasteurella haemolytica by electroporation and conjugation. J Gen Microbiol. 1989, 135 (11): 2885-2890.PubMedGoogle Scholar
- Figurski DH, Helinski DR: Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA. 1979, 76 (4): 1648-1652. 10.1073/pnas.76.4.1648.PubMed CentralPubMedView ArticleGoogle Scholar
- Mahenthiralingam E, Coenye T, Chung JW, Speert DP, Govan JR, Taylor P, Vandamme P: Diagnostically and experimentally useful panel of strains from the Burkholderia cepacia complex. J Clin Microbiol. 2000, 38 (2): 910-913.PubMed CentralPubMedGoogle Scholar
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