Analysis of transcription of the bph locus of Burkholderia sp. strain LB400 and evidence that the ORF0 gene product acts as a regulator of the bphA1 promoter

Fabrizio Beltramettia,1, Daniela Renierob,1, Silke Backhaus1 and Bernd Hofer1

German Research Centre for Biotechnology (GBF), Department of Environmental Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany1

Author for correspondence: Bernd Hofer. Tel: +49 531 6181467. Fax: +49 531 6181411. e-mail: bho{at}gbf.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although gene clusters for the degradation of biphenyls and polychlorobiphenyls have been extensively characterized, comparatively little is known about the regulation of their expression. In the present work, different aspects of transcription of the bph locus of the potent polychlorobiphenyl degrader Burkholderia sp. strain LB400 were investigated. An RNA blot analysis of the entire gene cluster revealed that the transcription of all genes encoding biphenyl catabolic enzymes responded similarly to the presence of biphenyl, succinate or a mixture of the two. One region of the locus, encompassing ORF0, was separately transcribed and differently regulated. A single start position was mapped for this monocistronic transcript. Synthesis of the adjacent RNA, encoding subunits of biphenyl dioxygenase, was strongly biphenyl-inducible. In this case, four major 5'-ends were mapped between 25 and 70 bp upstream of the start codon of gene bphA1. Sequence elements between approximately positions 710 and 1080 upstream were required in cis for full functioning of the respective promoter(s) (PbphA1). ORF0- mutants of strain LB400 retained the ability to grow on biphenyl, but showed decreased concentrations of bphA1A2 RNA and decreased lacZ expression in strains harbouring a reporter system with a bphA1–lacZ transcriptional fusion. This effect was compensated by the introduction of an intact ORF0 in trans, indicating that the ORF0 gene product mediates activation of PbphA1.

Keywords: aerobic bacteria, biphenyl catabolism, bph genes, transcriptional regulation

a Present address: Biosearch Italia SpA, Via R. Lepetit 34, 21040 Gerenzano (VA), Italy.

b Present address: Via Degli Aceri 8, 20030 Seveso (MI), Italy.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The bph locus of Burkholderia sp. strain LB400 encodes a pathway with the potential to break down a wide range of polychlorobiphenyls. The biphenyl dioxygenase of this catabolic route is able to attack a relatively broad spectrum of congeners including hexachlorobiphenyls (Bopp, 1986 ; Nadim et al., 1987 ; Haddock et al., 1995 ; Seeger et al., 1995 , 1999 ), and many of the dioxygenation products are further catabolized (Bedard & Haberl, 1990 ; Seeger et al., 1995 , 1999 ).

The bph gene cluster has been extensively characterized (Erickson & Mondello, 1992 ; Hofer et al., 1993 , 1994 ). The locus contains ten cistrons encoding enzymes for the degradation of biphenyls to benzoates, pyruvates and acetyl-CoA. It further harbours a glutathione transferase gene (bphK) and two ORFs, ORF0 and ORF1, of unknown function. This and similar gene clusters are frequently termed operons, but in most cases information on their transcription is scarce, and the formation of a single polycistronic mRNA is merely an assumption. One transcriptional study of the bph locus of strain LB400 identified three RNA 5'-ends in the 5'-terminal region of the gene cluster (Erickson & Mondello, 1992 ). On the basis of these data, the existence of three promoters (p1, p2 and p3) was postulated. p1 and p2, located directly upstream of the gene encoding the large subunit of biphenyl dioxygenase (bphA1), were reported to direct constitutive transcription, whereas p3, located upstream of ORF0, was described to be activated in the presence of biphenyl. In contrast to these findings, Furukawa and coworkers (Taira et al., 1992 ) reported the mapping of a single RNA start site within the 3'-terminal region of ORF0 of the highly similar bph locus of Pseudomonas pseudoalcaligenes KF707. However, no positive proof for functioning of any of the deduced promoters has been given. A two-component system regulating biphenyl catabolism in the Gram-positive strain Rhodococcus sp. M5 and a GntR-like transcriptional repressor of the bph gene cluster located on transposon Tn4371 have recently been identified (Labbé et al., 1997 ; Mouz et al., 1999 ), but data on the transcription of the bph gene cluster of strain LB400 remain scarce.

In this report we describe a Northern blot analysis of transcription of the entire bph locus, an exact mapping of RNA 5'-ends in the 5'-terminal region by primer extension analysis, and the demonstration of promoter function using a mono-copy promoter-probe system in strain LB400. Furthermore, we investigated the expression of ORF0 and its involvement in the regulation of transcription from the promoter upstream of bphA1, the first gene of the locus encoding a polypeptide of a biphenyl catabolic enzyme.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids, media and growth conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. Burkholderia strains were grown at a temperature of 30 °C in L medium (Bopp et al., 1983 ) or M9 minimal medium (Sambrook et al., 1989 ) supplemented with trace elements (Bauchop & Elsden, 1969 ) and either succinate (20 mM) or biphenyl (5 mM nominal concentration), or both, as carbon sources. Escherichia coli strains were grown at 37 °C on LB (Sambrook et al., 1989 ). Media were supplemented with ampicillin (100 µg ml-1), kanamycin (50 µg ml-1) or chloramphenicol (10 µg ml-1) when required.


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Table 1. Strains and plasmids used in this study

 
Constructions of plasmids and strains.
Recombinant DNA techniques were performed according to standard methods (Sambrook et al., 1989 ). Plasmid DNA was isolated with the QIAprep Spin Miniprep Kit (Qiagen) and sequenced using a Taq Dyedeoxy terminator cycle sequencing kit and an automatic DNA sequencer model 373A (Applied Biosystems), according to the manufacturer’s instructions. Oligonucleotides (Table 2) were synthesized by GIBCO. For cloning, E. coli strain INV{alpha}F' was used, unless otherwise indicated. For conjugal transfers E. coli CC118[{lambda}pir] was used as donor and E. coli HB101(pRK600) as helper strain (Young & Poulis, 1978 ). In the newly constructed plasmids, the orientation of the cloned genes was the same as the orientation of the lacZ gene (for pCR2.1, pUC19, pK18mobsacB and pUJ8 derivatives) or of the Kmr gene (for pUTminiTn5Km derivatives). In detail, plasmids and strains were constructed as follows.


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Table 2. Oligonucleotides used in this study

 
LB400ORF0FS.
The 1·2 kb EcoRI fragment of pCR120 (construction described below) containing ORF0 was cloned in EcoRI-digested pUC19, yielding pUC120. Linearization of pUC120 with BglII, filling-in of the ends, and religation generated pUC120FS. The EcoRI fragment of pUC120FS was then cloned into the EcoRI site of pK18mobSacB (Schäfer et al., 1994 ), yielding plasmid pK18120FS. The wild-type copy of ORF0 in strain LB400 was replaced by homologous recombination after transfer of this suicide plasmid by a triparental mating (Young & Poulis, 1978 ). Kmr colonies able to utilize glutamate were selected. Kms double-crossover recombinants were selected after addition of 10% sucrose to agar plates as previously described (Schäfer et al., 1994 ). Successful exchange of ORF0 was verified by PCR amplification with primers CIOP1R and CIOP4RBIS (which anneal to regions external to the cloned fragment) followed by incubation with BglII. For further analysis, the PCR fragment of a BglII- clone was sequenced with primers CIOP1R and CIOP3. Additionally, a 2·35 kb PCR fragment encoding ORF0 and part of bphA1 was amplified from LB400ORF0FS genomic DNA by use of primers CIOP0 and CIOP5 and cloned in pCR2.1, generating pCRFS24. The integrity of the insert was verified by sequencing. The lack of a full-length ORF0 protein was verified by SDS-PAGE analysis of 35S-labelled proteins of E. coli BL21[DE3](pLysS, pCRFS24) (data not shown).

LB400[TnEE28] and LB400ORF0FS[TnEE28].
(See also Fig. 1 and Table 4.) The 2·8 kb EcoRI fragment containing the ORF0 and bphA1 genes of strain LB400 was excised from pAIA1 (B. Hofer & S. Backhaus, unpublished) and inserted into the EcoRI site upstream of the promoterless trp–lacZ fusion in plasmid pUJ8, yielding pUJEE28. This transcriptional fusion was excised as a 6·9 kb NotI fragment and cloned downstream of a transcriptional terminator into the respective site of pUTminiTn5Km, generating pUTTnEE28. This suicide plasmid was used for monocopy insertion of the reporter gene fusion into the genomes of strains LB400 and LB400ORF0FS as described by de Lorenzo et al. (1990) . Kmr colonies able to utilize glutamate were selected.



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Fig. 1. Overview of the transcriptional analysis of the 5'-terminal region of the bph locus. A, genes and ORFs. B, location of probes used. C, location of elongation primers used. D, location of mapped transcripts: a, ORF0 RNA; b, bphA1A2 RNA. E, transposon constructs employed to identify bph segments important for promoter function. GATC indicates a 4 bp insertion in ORF0. The numbers refer to positions in the bph locus (Erickson & Mondello, 1992 ). Numbering of the gene cluster starts at the first base-pair of the 5'-terminal EcoRI site. Abbreviations: E, EcoRI; Bg, BglII; lacZ, ß-galactosidase-encoding gene.

 

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Table 4. Promoter activity of transcriptional bph/reporter gene fusions in strain LB400 background

 
LB400[TnBg2000].
(See also Fig. 1 and Table 4.) A 2·05 kb bph fragment was excised from pUJEE28 with BglII, filled in and inserted into the SmaI site of pUJ8, generating pUJBg2000. This transcriptional fusion was then subcloned, yielding pUTTnBg2000, and finally integrated into strain LB400, as described above.

LB400[Tn120], -[TnPC55], -[TnPI87], -[TnPI45], -[TnPI75] and -[TnPI29].
(See also Fig. 1 and Table 4.) The promoter fragments 120, PC55, PI87, PI45, PI75 and PI29 were generated by PCR using as template pDD5301 and as primer pairs CIOP1R and CIOP4R, CIOP0 and CIOPPC, CIOP2 and CIOP4R, CIOP3R and CIOP4R, CIOP2 and CIOP10, or CIOP3R and CIOP10, respectively. The amplification products were cloned in pCR2.1 using the TA cloning Kit (Invitrogen) according to the manufacturer’s instructions. The resulting plasmids were pCR120, pCRPC55, pCRPI87, pCRPI45, pCRPI75 and pCRPI29. The integrity of the inserts was verified by sequencing. The amplification products were excised from the respective pCR plasmids as EcoRI fragments, subcloned and integrated into strain LB400 as described above. The resulting subclones were pUJ120, pUJPC55, pUJPI87, pUJPI45, pUJPI75 and pUJPI29 in the pUJ series and pUTTn120, pUTTnPC55, pUTTnPI87, pUTTnPI45, pUTTnPI75 and pUTTnPI29 in the pUTTn series.

LB400[TnFS24] and LB400ORF0FS[TnFS24].
(See also Fig. 1 and Table 5.) The 2·4 kb bph segment of pCRFS24 (described above) was excised with EcoRI, subcloned as above, yielding pUJFS24 and pUTTnFS24, and finally integrated into the genomes of the two recipients.


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Table 5. Promoter activity of transcriptional bph/reporter gene fusions in ORF0 diploid LB400 mutants with different ORF0 genotypes

 
LB400[TnlacZ].
The 4·3 kb NotI fragment of pUJ8 was cloned into NotI-digested pUTminiTn5Km, yielding pUTTnlacZ. The mini-transposon was then integrated into the genome of LB400 as described above.

Northern blot analysis.
Strain LB400 and its derivatives were grown on biphenyl or succinate as described above. At OD600 0·5, total RNA was extracted with the RNeasy Midi Kit (Qiagen) according to the supplier’s instructions. Aliquots of 30 µg of RNA (determined photometrically) were denatured at 100 °C in the presence of formaldehyde (2 M) and 50% formamide, separated on 1% agarose/10% formaldehyde gels (Sambrook et al., 1989 ), and blotted onto a Byodine B Transfer Membrane 0·45 µm (PALL) (Sambrook et al., 1989 ). Probes were generated by PCR using pDD5301 as template and the primer pairs given in Table 3. Digoxigenin-labelled nucleotides were incorporated into gel-purified probes using the Random Primed DNA Labelling Kit Dig (Boehringer Mannheim) according to the manufacturer’s instructions. Membranes were hybridized at 50 °C and signals were detected with anti-digoxigenin antibodies conjugated with alkaline phosphatase (Boehringer Mannheim) according to the supplier’s instructions. RNA lengths were estimated using as reference the Dig-labelled RNA molecular weight marker I (Boehringer Mannheim).


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Table 3. Probes used for detection of potential bph transcripts

 
Primer extension analysis.
Strain LB400 was grown and total RNA was extracted as described under Northern blot analysis. Primers CIOPPC and CIOP4R (Table 2) (each 20 pg) were end-labelled by incubation with [{gamma}-32P]rATP and 1 U T4 polynucleotide kinase (Promega) at 37 °C for 40 min (Sambrook et al., 1989 ). Then 10–20 pg of the labelled primers were hybridized with 25 µg RNA at 50 °C for 20 min and extended with 1 U AMV reverse transcriptase (Promega) at 42 °C for 40 min (Sambrook et al., 1989 ). Reference sequence ladders were generated using the same primers with the Deaza G/A T7Sequencing Mixes Kit (Pharmacia Biotech) in accordance with the supplier’s instructions. Primer extension products and sequence ladders were run on sequencing gels (Sambrook et al., 1989 ).

ß-Galactosidase assays.
Bacterial cultures were grown on succinate or biphenyl to an OD600 of 0·5. Aliquots (500 µl) were permeabilized with chloroform and SDS and processed as described by Miller (1972) .

Detection of proteins.
35S-labelling of plasmid-encoded gene products was carried out in E. coli BL21[DE3](pLysS) as previously described (Hofer et al., 1993 ). Plasmids used were pCR120, pCRPI29 and pCRFS24. SDS-PAGE analysis has also been described (Hofer et al., 1993 ).

Computational methods.
Database searches and sequence alignments were performed with the BLASTN, BLASTP (Altschul et al., 1990 ) and BESTFIT (Devereux et al., 1984 ) programs.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Analysis of transcription of the bph locus by probe hybridization
Transcription of the entire bph locus was investigated by Northern analysis using RNA extracted from strain LB400 after growth in minimal medium containing succinate, biphenyl or a mixture of the two as sole carbon source(s). Thirteen double-stranded probes complementary to all genes or ORFs of the bph locus (Table 3) were generated by PCR. Only probe S1, which is complementary to the 5'-end of the locus upstream of ORF0 (Fig. 1), gave no signal, indicating that this DNA region is not expressed. The ORF0 probe S2 (Fig. 1) detected a transcript of about 1·35 kb (Fig. 2a) which was designated ORF0 RNA. Probes S3, SA2 and S4, containing parts of bphA1, bphA2 and ORF1, respectively (Fig. 1), all detected the same RNA with a length of about 2·4 kb (Fig. 2b). This transcript is subsequently designated bphA1A2 RNA. The other probes visualized a variety of relatively short RNAs. The majority of these transcripts were even shorter than the corresponding genes. This finding strongly suggests a degradation of larger transcripts. The concentrations of all bph transcripts, with the single exception of ORF0 RNA, responded similarly to growth of strain LB400 on the different carbon sources. Large differences in amounts were observed in the order biphenyl-grown>biphenyl/succinate-grown>succinate-grown (Fig. 2b). These results suggest that transcription of the entire bph locus downstream of ORF0 is similarly regulated and that at least two mechanisms operate, up-regulation by biphenyl (and possibly other carbon sources) and down-regulation by succinate (and probably other carbon sources). ORF0 RNA was found in approximately equal concentrations in succinate- and biphenyl/succinate-grown cells and in moderately enhanced concentration in biphenyl-grown cells. The different biphenyl responses of the ORF0 and bphA1A2 RNAs indicate that ORF0 is transcribed independently from the other bph genes and that the two RNAs do not represent processing products of the same precursor.



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Fig. 2. Northern blot analysis of Burkholderia sp. strain LB400 transcripts. RNA samples were extracted from cells grown on succinate (S), biphenyl (B) or a mixture of the two (S+B). Aliquots of 30 µg were separated on 10% formaldehyde/1% agarose gels and visualized by hybridization with different probes. The sizes of the main transcripts are indicated. RLM: RNA length marker. (a) Probe S2 (ORF0); (b) probe S3 (bphA1).

 
Mapping of RNA 5'-ends
RNA 5'-ends were mapped in the 5'-terminal region of the bph locus by primer extension on RNA extracted from cells grown in minimal medium with the addition of either biphenyl or succinate. Since ORF0 RNA was not detected with probe S1, its 5'-end should be located within a few hundred base-pairs upstream of ORF0. As shown in Fig. 3(a), primer CIOPPC detected a single 5'-terminus located 89 bp upstream of the start codon of ORF0, which corresponds to C522 of the published sequence (Erickson & Mondello, 1992 ). The signal was moderately enhanced by growth on biphenyl. Promoter activity of a fragment encompassing this region (see Table 4), below) and the absence of transcripts hybridizing with the probe S1 (Table 3) are consistent with the interpretation that the mapped 5'-end represents a genuine transcriptional start site rather than an RNA terminus formed by processing.



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Fig. 3. Mapping of RNA 5'-ends upstream of ORF0 and bphA1: autoradiograms of extensions of primers CIOPPC (a) and CIOP4R (b). RNA was extracted from strain LB400 growing in minimal medium supplemented with either succinate (lane S) or biphenyl (lane B). Lanes T through A contain the respective sequencing reactions. The position of the first base of each major mRNA species relative to the first base-pair of the respective cistron is indicated at the left margin.

 
The different biphenyl responses of the bphA1A2 and ORF0 RNAs and the annealing of bphA1A2 RNA with probes S3, S2A and S4, but not with probe S2 (Fig. 1), indicate that the promoter for this transcript is located within a region of 200–300 bp upstream of the bphA1 gene. As shown in Fig. 3(b), primer extension experiments performed with oligonucleotide CIOP4R detected four major 5'-ends located 25, 41, 47 and 70 bp upstream of the start codon of bphA1 [corresponding to G1414, T1398, G1392 and T1368 in the published sequence (Erickson & Mondello, 1992 )]. All four signals were strongly enhanced by growth on biphenyl. An overview of the location of the ORF0 and bphA1A2 RNAs is given in Fig. 1(D).

Demonstration of promoter function and characterization of required DNA lengths
In order to verify and quantitate the activity of the promoters suggested by primer elongation and to identify the lengths of the DNA regions required for full promoter function, we fused different fragments (shown in Fig. 1) containing the probable start sites of ORF0 and bphA1A2 RNA to a lacZ reporter gene located on an artificial transposon. The resulting transposons were then inserted into the LB400 genome. The use of transposons ensures that the reporter system closely mimics the natural situation by increasing the promoter copy number only from one to two or leaving it unchanged, if mutants are used in which the wild-type promoter is inactivated. At least three individual clones resulting from the insertion of each reporter transposon were used for the measurement of LacZ activity, which was determined after growth of the strains on either biphenyl or succinate (Table 4. Only gross values are listed because a true background subtraction is not possible. The fusion of bph bp 90–664, harbouring the mapped PORF0, (LB400[TnPC55]) showed a low level of increase in promoter activity after growth on biphenyl. This result is in accordance with the data obtained in the Northern blot and primer extension analyses. In contrast, LB400[TnEE28], a fusion with bph bp 1–2855, harbouring the mapped PORF0 and PbphA1 (which may consist of several overlapping promoters), showed a significantly higher biphenyl-responsiveness of transcription. Transcription from PORF0 is probably not measured with this construct, as our results suggest its termination within the cloned fragment (cf. above). Somewhat higher LacZ activities were measured for biphenyl and succinate when the 5'- and 3'-ends of the cloned fragment were shifted to positions 364 and 1605, respectively (LB400[Tn120]). This may be due to deletion of the terminator for ORF0 RNA or a consequence of the different fusions between bph DNA and reporter gene (see following section). In any case, all of the other subfragments fused to the reporter system showed no significant promoter activity. Surprisingly, this includes the large segments encompassing bp 724–1605 and 785–2839. The similar behaviour of shorter segments renders it improbable that silencer-like sequences are responsible for the inactivity of long segments. This suggests that the presence in cis of sequences between positions 364 and 724 is important for proper functioning of PbphA1.

Analysis of ORF0 translation and mutational analysis of the role of the ORF0 gene product for transcription from the bphA1 promoter
ORF0 encodes a hypothetical protein of 245 amino acids and 27·8 kDa. Having demonstrated the synthesis of a specific ORF0 RNA in strain LB400, we investigated the translation of ORF0. A PCR-generated fragment containing ORF0 was cloned in E. coli in a phage T7 RNA polymerase-dependent expression system (plasmid pCR120). This allows the specific visualization of insert-encoded proteins, as their synthesis proceeds under conditions that suppress host transcription. Using 35S-labelling, we were able to visualize after SDS-PAGE a protein with a mobility corresponding to 27·5 kDa whose synthesis was dependent on the cloned fragment (Fig. 4).



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Fig. 4. SDS-PAGE analysis of expression of ORF0. The products of genes downstream of a phage T7 late promoter of the vector were visualized by labelling with 35S-labelled amino acids and autoradiography of the gel. Lanes 1 and 4, size markers (molecular masses indicated); lane 2, plasmid pCRPI29; lane 3, plasmid pCR120 harbouring ORF0.

 
The results presented above suggest that parts of the ORF0 region may fulfil an important function for PbphA1 in cis. Therefore, a potential role of the ORF0 protein for this promoter was not investigated by deletion analysis, but by introduction of a frameshift mutation (insertion of 4 bp) at codon 59. This should lead to premature termination of the mutant ORF0 after 63 codons, i.e. within the segment encoding its potential helix–turn–helix motif. The wild-type ORF0 was exchanged in strain LB400 for the mutant analogue, yielding strain LB400ORF0FS. The replacement was verified by PCR amplification followed by restriction and DNA sequencing (for details see Methods). This mutant retained the ability to grow on biphenyl. For a quantitative analysis of bph expression, the mutant ORF0/PbphA1 region was fused to the lacZ reporter gene (yielding TnFS24, Fig. 1) and introduced into the genomes of strains LB400 and LB400ORF0FS. For comparison, lacZ expression from the reporter transposon carrying a wild-type ORF0/PbphA1 region (TnEE28) was measured in the same two backgrounds. Three individual transposon mutants of each strain were examined. The mean values of LacZ activity are given in Table 5. The levels of lacZ expression from TnEE28 were not significantly different in the two recipients. In both types of mutants, growth on biphenyl relative to succinate led to a significant increase in LacZ activity. This was also observed for mutants LB400[TnFS24] which possess an intact ORF0 allele in the chromosome. This demonstrates that the frameshift does not significantly interfere with the functioning of PbphA1. Similar to LB400[Tn120] (Table 4), LB400[TnFS24] showed higher LacZ activity than LB400[TnEE28] after growth on both carbon sources, indicating that the additional bph DNA in TnEE28 influences the level of lacZ expression. TnEE28 contains a complete bphA1 gene in front of lacZ while TnFS24 contains a truncated bphA1 gene. This or other features of the additional DNA segment, e.g. leading to formation of different RNA secondary and tertiary structures, may very well influence such phenomena as mRNA processing or ribosome binding which ultimately would affect the efficiency of translation. Relative to the LB400 background, the LB400ORF0FS background reduced lacZ expression from TnFS24 approximately twofold after growth on succinate, and five- to sixfold after growth on biphenyl. This indicates a positive influence of the ORF0 protein on PbphA1, particularly during biphenyl catabolism. These results were confirmed by analysis of the concentration of bphA1A2 RNA in strain LB400 and in mutants LB400ORF0FS and LB400ORF0FS[TnEE28] after growth on biphenyl (data not shown). The concentration was strongly reduced in strain LB400ORF0FS, but was similar to wild-type levels in strain LB400ORF0FS[TnEE28]. The results of both experimental approaches show that the ORF0 gene product mediates, either directly or indirectly, up-regulation of transcription from PbphA1.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Northern analysis of transcription of the bph locus of Burkholderia sp. strain LB400 revealed that the transcription of all genes encoding biphenyl catabolic enzymes responded similarly to the presence of biphenyl, succinate or a mixture of the two. It also showed that transcription of these genes was subject to at least two types of regulatory mechanisms, up-regulation by growth on biphenyl and down-regulation by growth on succinate (and probably other carbon sources). This was indicated by the intermediate concentrations of bph RNAs in the presence of both carbon sources. The analysis furthermore demonstrated that one region of the locus, encompassing ORF0, was separately transcribed and differently regulated. The RNA blotting analysis detected a variety of relatively small transcripts. With the exception of ORF0, bphA1, bphA2, ORF1, bphK and bphD the major transcripts appeared to have sizes even smaller than the genes from which the respective probes were derived. Moreover, sometimes a given probe detected relatively high amounts of additional shorter RNAs. These observations suggest a processing of longer transcripts and incomplete inhibition of RNase activity during work-up. The formation of numerous bph transcripts may in part also be due to the existence of several promoters and terminators. We note that evidence has recently been presented for the existence of at least five promoters in the closely related bph locus of P. pseudoalcaligenes KF707 (Watanabe et al., 2000 ).

Primer extension analysis mapped the 5'-end of the monocistronic ORF0 RNA to C522, 89 bp upstream of the start codon of ORF0. This deviates by only 6 bp from the approximate position determined by Erickson & Mondello (1992) using S1 nuclease mapping and makes it unlikely that the detected RNA terminus is an artefact of either mapping technique. Moreover, the lack of transcription further upstream of ORF0 is consistent with the location of a promoter in this region. Thus we conclude that ORF0 RNA is initiated at C522. Inspection of the sequence around this site shows that the region from position -13 to position +8 has a very high AT content of 86 mol%. Watanabe et al. (2000) found that also the ORF0 of the closely related bph locus of P. pseudoalcaligenes KF707 is transcribed into a monocistronic RNA and mapped its 5'-end to position 106 upstream of the ORF0 start codon. A comparison of the two promoter regions (Fig. 5a) shows an overall sequence identity of about 30%. In both cases, at appropriate spacings upstream of the mapped RNA start positions, limited similarities, typical for positively regulated promoters, are found to the -10 and -35 consensus motifs of {sigma}70 promoters. Whilst KF707 shows a perfect match only with the -35 consensus sequence, LB400 shows an almost perfect match only with the -10 consensus motif. An unusually high local sequence identity of 60% exists in the region between the mapped RNA start sites. Within this region, the binding motif for GntR-type transcriptional regulators as defined by Watanabe et al. (2000) is perfectly conserved. Indeed, these authors demonstrated in vitro binding of the ORF0 protein of strain KF707 to a 38 bp DNA fragment encompassing this region. This suggests that ORF0 of strain LB400 could be subject to the same type of (auto-)regulation. In agreement with this, a similar two- to threefold enhancement of ORF0 transcription by growth on biphenyl relative to succinate was observed with both organisms. We note that this contrasts with previous data for LB400, which indicated a much larger difference in favour of biphenyl-grown cells (Erickson & Mondello, 1992 ).



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Fig. 5. Sequence alignments of promoter regions of the bph loci of Burkholderia sp. strain LB400 and P. pseudoalcaligenes KF707. Asterisks between sequences indicate nucleotide identity. (a) Promoter regions upstream of ORF0. The ORF0 start codons are boxed. Mapped start nucleotides are marked by arrows. Potential -10 and -35 recognition motifs are over- and underlined, respectively; matches with their consensus sequences are highlighted by dots. Highly conserved nucleotides of GntR-type regulator recognition sites are marked by three connected boxes. (b) Promoter regions upstream of bphA1. The ORF0 stop codons are boxed. Mapped start nucleotides are marked by arrows; the numbers indicate their distances from the bphA1 start codons. The RpoN ({sigma}54) recognition motif is marked by two connected boxes; note that the correct spacing of 10 nucleotides is only found in the LB400 sequence.

 
Using a transcriptional fusion with lacZ, we demonstrated in strain LB400 promoter activity of the region upstream of bphA1. This is the first gene of the locus that encodes a catabolic enzyme. The respective primer extension experiment yielded four major signals corresponding to positions -25, -41, -47 and -70 upstream of the bphA1 start codon (Fig. 5b). Erickson & Mondello (1992) determined the approximate positions of RNA 5'-ends of this region by S1 nuclease mapping. They did not observe the terminus at position -25, but otherwise their results agree well with ours. Thus at least three of the termini are unlikely to be artefacts of the specific mapping technique used. Only a single RNA 5'-end was found in the corresponding bph region of strain KF707, which contains about 85% of identical residues. It was mapped at position -99 relative to the start of bphA1 (Fig. 5b). Unfortunately, we were unable to narrow down the promoter region much by deletion analysis. Thus, it cannot be said with certainty whether the mapped RNA 5'-ends originate from processing or transcription or both. We note, however, that the length and localization of bphA1A2 RNA and the absence of RNA of the same biphenyl responsiveness annealing to probe S2 are consistent with the view that the mapped ends represent transcriptional start sites. Overlapping promoters, depending on different regulatory factors, have previously been described and appear to be useful in maintaining appropriate levels of mRNA under various physiological conditions (Nørregaard-Madsen et al., 1994 ; Kallipolitis & Valentin-Hansen, 1998 ). Inspection of the regions upstream of the mapped start sites revealed no high degree of similarity with known prokaryotic promoter consensus sequences. The RpoN ({sigma}54) recognition motif (GG-N10-GC) (Ishimoto & Lory, 1989 ; Totten et al., 1990 ), positioned 1 bp closer to the putative start site than normally observed, is found only in strain LB400 (Fig. 5b). However, a detailed comparison with the well-characterized RpoN-dependent mop (Schirmer et al., 1997 ), xyl (Abril et al., 1991 ; Perez-Martin & de Lorenzo, 1996 ) and dmp (Shingler et al., 1993 ; Fernandez et al., 1994 ) promoters showed no further similarities. When strain LB400 was grown on succinate as sole carbon source, the same 5'-ends were detected for bphA1A2 RNA. This indicates that the change in carbon sources does not lead to a switch in promoter(s) used and is in agreement with the results of Erickson & Mondello (1992) . However, a major difference from this previous work is once more the biphenyl/succinate-responsiveness of transcription. We note that all of the different techniques employed by us, particularly the RNA blotting and primer extension analyses, indicated strongly enhanced transcription from this region in the presence of biphenyl.

Full activity of the bphA1 promoter in biphenyl-grown cells required more than 700 bp upstream of the start of transcription. Regulatory regions that map hundreds of base-pairs distant from their cognate promoter have been described in other systems such as the deo promoters of E. coli (Valentin-Hansen et al., 1986 ) or the algD promoter of Pseudomonas aeruginosa (Kato & Chakrabarty, 1991 ). With respect to the dependence of PbphA1 on the ORF0 protein (see below), we note that regions with 5/6 or 6/6 matches, respectively, with the binding motif for GntR-type transcriptional regulators are found approximately 180 bp upstream of the ORF0 stop codons in the bph loci of strains LB400 and KF707. If this sequence acts in concert with the motif upstream of ORF0 or if the latter acts alone, e.g. via a DNA looping mechanism, this gives a rationale for the observed dependence of PbphA1 on sequences between positions 364 and 724.

Five base-pairs upstream of the start codon of ORF0 a potential Shine–Dalgarno sequence is found. We demonstrated translation of ORF0 in the heterologous E. coli system. As mentioned previously (Hofer et al., 1996 ; SWISS-PROT database entry P37335), the sequence of the gene product resembles those of transcriptional regulators, specifically of the GntR family (Haydon & Guest, 1991 ); an alignment is shown in Fig. 6. A strain LB400 mutant in which ORF0 was inactivated by a frameshift mutation showed a significant decrease in biphenyl-induced transcription of bphA1A2 RNA which was compensated by the presence of a second, intact ORF0. This demonstrates that the ORF0 gene product exerts a positive effect on PbphA1 and provides a rationale for the biphenyl-dependent induction of BphC activity observed in biphenyl-utilization-deficient recipients after introduction of the bph locus of strain LB400 (Dowling & O’Gara, 1994 ; Hofer et al., 1996 ). Negative regulation by GntR-like proteins has recently been reported for gene clusters encoding phenol (Arai et al., 1999 ) and biphenyl (Mouz et al., 1999 ) catabolism. However, also positive regulation has been described for members of the GntR family, such as GlcC (Pellicer et al., 1996 , 1999 ), LuxZ (GenPept database accession no. AAD00703) and MatR (GenPept database accession no. AAF28803). Recently, evidence has been obtained that the closely related protein encoded by the ORF0 of P. pseudoalcaligenes KF707 (82% sequence identity with the ORF0 protein of strain LB400) acts as a positive regulator for the transcription of its own gene and of all or most of the genes of the bphX0X1X2X3D region (Watanabe et al., 2000 ). Our results show that although the ORF0 protein of strain LB400 is not absolutely required for the transcription from PbphA1, it significantly enhances the activity of this promoter.



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Fig. 6. Sequence alignment of the two domains of the ORF0 protein from Burkholderia sp. strain LB400 with related transcriptional regulatory proteins from biphenyl-degrading bacteria and with the prototype GntR protein from Bacillus subtilis. Orf0-KF707, ORF0 protein from P. pseudoalcaligenes KF707 (Watanabe et al., 2000 ); Orf0-B4, ORF0 protein from Pseudomonas sp. strain B4 (NCBI protein database accession no. CAB93964); BphS-Tn4371, transposon-encoded biphenyl regulatory protein (Mouz et al., 1999 ). Domains according to the ProDom database (http://protein.toulouse.inra.fr/prodom.html) are indicated by square brackets. Residues identical in all sequences are indicated by §, residues identical in all sequences from biphenyl-degrading strains are indicated by *. A consensus sequence for the helix–turn–helix (HTH) DNA-binding region, derived from 40 sequences, is shown in boldface; residues identical with the consensus sequence are highlighted in the individual sequences.

 
Finally, we note that transcription of the bph gene clusters in strains LB400 and KF707, though similar, appears not to be identical. Thus, after inactivation of ORF0, only strain LB400 retained its ability to use biphenyl as sole carbon source. Furthermore, while the induction of ORF0 expression by growth on biphenyl relative to growth on succinate was two- to threefold in both bacteria, the induction of bphD in strain KF707 (no data are presently available for the other bph genes of KF707) was in the same range, but was significantly higher in strain LB400.


   ACKNOWLEDGEMENTS
 
F.B. was supported by a grant from FEMS and from Consorzio Interuniversitario Biotecnologie (CIB) and is grateful to Jean Armengaud for helpful discussions.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 12 January 2001; revised 17 April 2001; accepted 24 April 2001.