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
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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 trplacZ 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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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 manufacturers 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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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 suppliers 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 manufacturers instructions. Membranes were hybridized at 50 °C and signals were detected with anti-digoxigenin antibodies conjugated with alkaline phosphatase (Boehringer Mannheim) according to the suppliers instructions. RNA lengths were estimated using as reference the Dig-labelled RNA molecular weight marker I (Boehringer Mannheim).
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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)
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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.
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RESULTS |
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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 90664, 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 12855, 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 7241605 and 7852839. 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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DISCUSSION |
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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
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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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 ShineDalgarno 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 & OGara, 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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ACKNOWLEDGEMENTS |
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Received 12 January 2001;
revised 17 April 2001;
accepted 24 April 2001.