Laboratoire de Microbiologie, Université Joseph Fourier, BP 53, 38041 Grenoble Cedex-9, France1
Laboratoire de Génétique des Prokaryotes, Université Libre de Bruxelles, IBBM 12, rue de Pr R. Jeneer et J. Brachet, 6041 Gosselies, Belgium2
Department of Genetics, The John Innes Centre, Norwich NR4 7UH, UK3
Author for correspondence: Sébastien Mouz. Tel: +33 4 76 88 92 10. e-mail: sebastien.mouz{at}ibs.fr
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ABSTRACT |
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Keywords: sigma-54, rpoN mutant, bph genes, Tn4371
Abbreviations: BP, biphenyl
The GenBank accession number for the sequence determined in this work is AJ131690.
a Present address: Institut de Biologie Structurale J. P. Ebel, Laboratoire dIngéniérie des Macromolécules, 41, Rue Jules Horowitz, 38027 Grenoble Cedex 1, France.
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INTRODUCTION |
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The Tn4371 transposon carries biphenyl genes (bph) which allow Ralstonia metallidurans (formerly Alcaligenes eutrophus; see Goris et al., 2001 ) strains such as CH34, and other Ralstonia species, to use biphenyl (BP) as sole carbon and energy source (Springael et al., 1993
; and unpublished results). The BP catabolic pathway consists of an upper and a lower pathway. They respectively convert BP into 2,4-dihydroxypentadienoate and benzoate and further degrade 2,4-dihydroxypentadienoate into acetyl-CoA and pyruvate, which enter the tricarboxylic cycle. In R. metallidurans CH34, the genes involved in benzoate degradation are chromosomal.
54 consensus binding sites were found upstream of the Tn4371 bph genes and the closely related genes in KKS102 (Merlin et al., 1997
; Kikuchi et al., 1994
). Consistent with these observations, in contrast to its wild-type parent H16/RP4::Tn4371, a
54-deficient R. eutropha strain, HF09/RP4::Tn4371, cannot grow with BP as the sole carbon and energy source (Merlin et al., 1997
). In R. metallidurans CH34, most Tn4371 bph genes are however transcribed from a single
70 promoter (Mouz et al., 1999
).
We have now cloned and sequenced the CH34 rpoN and surrounding genes, inactivated the rpoN gene by disruption, and used transcriptional fusions to look for promoters in the sequenced region. We isolated a CH34 rpoN-deficient mutant, which was tested for autotrophic growth, urease and proline oxidase expression. These were all 54 dependent as reported earlier for R. eutropha strains or/and other bacterial species (Römermann et al., 1988
; Totten et al., 1990
; Kohler et al., 1994
). Tn4371 was introduced into the CH34 rpoN strain to test for
54 dependence of the transposon bphC gene expression and for growth on BP as the sole carbon and energy source.
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METHODS |
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DNA manipulations.
Plasmid DNA was extracted from E. coli as described by Birnboim & Doly (1979) and from R. metallidurans as described by Kado & Liu (1981)
. The genomic library of R. metallidurans CH34 was constructed in the broad-host-range vector pLAFR3 (Staskawicz et al., 1987
) and subcloning of fragments from cosmids or other plasmids was performed in pBluescript SKII+, pIJ2925 or pBBRIMCS vector. Plasmid constructs were transformed into E. coli XL1Blue (Inoue et al., 1990
) and recovered on LB plates supplemented with the appropriate antibiotic and with 55 mg X-Gal l-1 and 65 mg IPTG l-1. Nucleotide sequencing was performed on a Pharmacia ALF sequencer or by Genome express (Grenoble, France). The nucleotide sequences were analysed with the GCG software (Genetics Computer Group Package, version 7, April 1991, Madison, WI, USA) using FASTA and BLAST and the non-redundant GenBank-EMBL database (no. 109).
PCR amplification.
The fragment containing a part of the rpoN gene from R. metallidurans CH34 was amplified (95 °C for 1 min, 55 °C for 1 min and 72 °C for 1·5 min; 30 cycles) using primers rpoN3 (5'-GAGTGCCTGGCCCTGCAGTTG-3') and rpoN4 (5'-GGAGATGGTTGACTCGTGTAA-3'), providing a 550 bp amplified product. The PCR mixture (50 µl total volume) contained 2 ng template DNA, 50 pmol primers, 0·2 mmol dNTPs and 1 unit Vent DNA polymerase (New England Biolabs). The resulting DNA fragment was purified by gel electrophoresis on a 1·2% agarose gel, using Gene Clean kits (Bio 101). The recovered fragment was cloned in the pCR-Script SK+ plasmid (Stratagene) to be sequenced. Plasmid pECG549, which was used to complement the CH34 rpoN mutant, carries a wild-type CH34 rpoN gene, obtained by PCR amplification of CH34 genomic DNA (95 °C for 1 min, 59 °C for 1 min and 68 °C for 2 min; 30 cycles) using primers rpoN7 (5'-GCTCGAGCATCAGCGAAGGCACCGTGCTGGC-3') and rpoN10 (5'-GGAAGCTTCGCAGCGGCGGCGTGATGTCGAGG-3'). rpoN7 annealed in the coding sequence of orf263 so as to most likely cover the entire putative promoter region of the rpoN gene. The PCR mixture (50 µl total volume) contained 2 ng template DNA, 50 pmol primers, 0·2 mmol dNTPs, 5 µl of the enzyme buffer, 5 µl of the Enhancer buffer and 1 unit Platinum Taq DNA polymerase (Gibco BRL). The 1787 bp amplified fragment was cloned in the plasmid pCR-Script SK+ (Stratagene). The resulting plasmid was digested with restriction enzymes XhoI and HindIII, which cleaved the rpoN7 and rpoN10 primers at their 5' extremities. The XhoIHindIII fragment was purified and ligated into plasmid pBBRIMCS digested with the same restriction enzymes to generate pECG549. The 3190 bp fragment used to construct the plasmid pECG550 (see Fig. 1) was amplified (95 °C for 1 min, 59 °C for 1 min and 68 °C for 4 min; 30 cycles) using primers rpoN13 (5'-GCTCTAGACGTCGTTCTACATGATCGTCG-3') and rpoN14 (5'-GGATCCGCTACGCCATGGTTGTTCTCG-3'). These respectively flank the EcoRV and the second SmaI restriction sites illustrated in Fig. 1(a)
.
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Construction of an rpoN mutant.
To construct a CH34 rpoN mutant, the tetracycline-resistance cassette (tet) originating from the plasmid pBBRIMCSTc (Kovach et al., 1995 ) was isolated as an EcoRI fragment and inserted into the plasmid pIJ2925 (Janssen & Bibb, 1993
) to be flanked by two BglII sites. The BglII fragment was recovered after digestion and inserted between the two BglII sites present in the rpoN coding region carried by plasmid pECG534 (Fig. 1a
), a pBluescript SKII+ derivative which carries a 1·8 kb EcoRVSalI fragment recovered from the pECG500 cosmid. In the resulting plasmid, pECG548 (Fig. 1a
), the rpoN gene was disrupted and deleted of 28 codons (Ser151 to Ile178, thus including the first Leu residue of the Leu-zipper in motif II).
To recombine the rpoN::Tc mutation in the CH34 rpoN chromosomal gene, pECG548 was linearized by digestion with BstXI and introduced into CH34 by electroporation using a Bio-Rad Gene Pulser. Preparation of electrocompetent cells and electroporation were done as described by Taghavi et al. (1994) . The presence of the rpoN::Tc mutation in the chromosome of the transformants obtained was checked by Southern blot hybridization using the 1·1 kb EcoRVBglII fragment from pECG545 (Table 1
) and the EcoRI fragment containing the tet gene from the pBBRIMCSTc plasmid as probes.
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RESULTS |
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Sequencing of the rpoN gene and surrounding region of R. metallidurans CH34
Subcloning and sequencing of the appropriate region of the pECG500 30 kb insert provided a 4643 bp sequence including the 1500 bp rpoN gene and five surrounding ORFs (Fig. 1a; GenBank accession number AJ131690). Conceptual translation of CH34 rpoN provided a product with all expected features of
54, a Gln-rich N-terminal region I with a Leu-zipper, an acidic region II, a region III covering two-thirds of the protein with a second Leu-zipper proximal at the N-terminal end and the DNA binding helixturnhelix motif near the C-terminus. The rpoN box, ARRTVAKYR, most characteristic of
54 proteins, was present between residues Val469 and Gln479.
As in other bacteria where it has been analysed, the CH34 rpoN gene was flanked by a series of conserved ORFs, two (orf155 and orf263) upstream and three (orf130, orf95 and orf151) downstream of rpoN. Comparison with the corresponding region in other bacteria revealed high similarity with ORFs present in known rpoN clusters and the function of which remains elusive. A potential purine-rich ribosome-binding site, but no typical promoter sequence, could be detected upstream of each of the CH34 ORFs analysed. Except for orf95, these displayed the mol% G+C and codon usage typical of Ralstonia protein-coding regions (Liesegang et al., 1993 ; Table 2
).
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CH34 orf130 was similar to genes located at the same position downstream of rpoN in other bacteria. Their predicted products are usually considered as rpoN expression modulators, although this property has been demonstrated only for Klebsiella pneumoniae orf95 (Merrick & Coppard, 1989 ).
The role of the orf151 orthologues is slightly better understood. They would belong to a small family of PTS IIA enzymes, which transport fructose and mannitol. However, different functions have been assigned to these gene products in different bacteria, such as a requirement for their presence for normal growth in minimal medium supplemented with the amino acids proline, arginine or histidine in P. aeruginosa (Jin et al., 1994 ), the inhibition of nif gene expression in K. pneumoniae (Merrick & Coppard, 1989
) and the catabolic repression by glucose of the
54-dependent promoter Pu which directs transcription of the toluene-degradation tol operon in P. putida (Cases et al., 1999
).
CH34 orf155, located upstream of orf263, shared location, orientation and some similarity (63% over 50 amino acids) with the putative E. coli orf185, the function of which remains unknown (Powell et al., 1995 ).
Transcriptional organization of the CH34 rpoN region
To detect promoters located upstream and downstream of the rpoN gene, we constructed a series of transcriptional fusions with the bphC gene from transposon Tn4371. The BphC protein, a 2,3-dihydroxybiphenyl dioxygenase, converts dihydroxybiphenyl into 2-hydroxy-6-oxo-6-phenylhexa-2,4-pentadienoate, a yellow compound, easily quantified as A434. The bphC gene was cloned in the broad-host-range vector pBBRIMCS (Kovach et al., 1994 ) providing the control plasmid (pECG610; Mouz et al., 1999
). Four different restriction fragments straddling various portions of the rpoN region were cloned upstream of the pECG610 bphC gene (see Fig. 1b
). All the resulting plasmids (pECG344347) and pECG610 were transferred into R. metallidurans CH34 by triparental mating. Expression of BphC was measured in the transconjugants, in minimal medium as described previously (Mouz et al., 1999
).
PECG544 contained a 730 bp fragment, which covered orf155 and orf263. CH34/pECG544 produced background levels of BphC, indicating that orf263 did not have its own promoter and was probably cotranscribed with at least the upstream orf155. BphC activity in CH34/pECG545, whose plasmid carried an insert covering the distal half of orf263 and the 5' end of rpoN, was high, suggesting that the rpoN gene was transcribed from its own promoter. pECG546 carried the 594 bp SalI fragment that covers the end of the rpoN gene and extends into downstream orf130. In CH34/pECG546, BphC specific activity was again significantly higher than the background, suggesting the presence of another promoter between rpoN and orf130. The insert in pECG547 covered the end of rpoN, orf130, orf95 and the beginning of orf151. CH34/pECG547 produced twice as much BphC as CH34/pECG546. Another promoter could thus be located upstream of either orf95 or orf151. The last construct, pECG550, with an insert covering half of orf263, rpoN, orf130, orf95 and the beginning of orf151, produced BphC levels similar to those found with pECG547. Hence, the rpoN transcript did not appear to extend into downstream ORFs, making a possible polarity resulting from disruption of the rpoN gene unlikely.
The same plasmids were introduced and tested in the mutant CH34rpoN (see below for the construction of the mutant). As shown in Fig. 1(b), BphC activities were not significantly different from those seen in the wild-type with the respective plasmid. Nor were they affected by the nature of the nitrogen source (data not shown). Disruption of the rpoN gene and depletion of
54 would thus appear unlikely to have secondary consequences resulting from an effect of the protein on other genes in the rpoN region.
Characterization of the CH34 rpoN mutant
The CH34 rpoN gene was disrupted by insertion of a tet gene cassette (see Methods). CH34rpoN::Tc was tested for autotrophic growth as previously described for an rpoN mutant of R. eutropha H16 (Römermann et al., 1988 ). Autotrophic growth was abolished as expected (data not shown). Expression of urease and proline oxidase, two enzymes responsible for the assimilation of the poor nitrogen sources urea and proline, and under the control of
54 in other bacteria, including R. eutropha, were measured in CH34rpoN::Tc and wild-type CH34 grown with different nitrogen sources. Table 3
shows that urease activity increased when CH34 grew on the poor nitrogen sources, proline or nitrate, rather than on ammonium or glutamine. Proline oxidase was induced when proline was present in the growth medium, but also decreased twofold in the presence of 0·4% ammonium (Table 3
). Thus, both urease and proline oxidase activities responded to the nature of the available nitrogen source in R. metallidurans CH34. In CH34rpoN::Tc, only background levels of urease and proline oxidase were detected whatever the nitrogen source (Table 4
). Thus, in the absence of ammonium,
54 activated expression of enzymes involved in the assimilation of poor nitrogen sources. Consistent with these results, when cells were grown on proline, the generation time of the rpoN mutant was more than twice as long as that of the wild-type CH34 (475 vs 188 min). This differed from results reported for R. eutropha H16 rpoN mutants (Römermann et al., 1988
), which grew faster than wild-type H16 in minimal medium with proline, suggesting that in R. eutropha H16,
54 represses rather than stimulates proline utilization. When introduced in CH34rpoN::Tc, the plasmid pECG549, a pBBRIMCS derivative carrying the sole rpoN gene expressed from its own promoter (see Methods for more details) restored autotrophic growth (data not shown) and most of the urease and proline oxidase activity (see Table 4
). The slightly lower activities observed in the complemented mutant could result from some instability of the complementing plasmid. Alternatively, overproduction of
54 could sequester the core RNA polymerase and hence reduce the availability of RNA polymerase
70 to transcribe the put (proline degradation) and ure (urease degradation) operon, which most likely require
70.
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Biphenyl utilization by R. metallidurans CH34rpoN::Tc
Transposon Tn4371 carries bph genes responsible for the mineralization of BP (Springael et al., 1993 ; Merlin et al., 1997
). Merlin et al. (1997)
observed that in contrast to R. eutropha H16/RP4::Tn4371, which grows on minimal medium supplemented with BP as a sole carbon source, HF09, an rpoN mutant of H16 (Friedrich et al., 1981
), provided with the same plasmid, does not. This suggested that expression of the Tn4371 bph genes could be under the control of
54. CH34/RP4::Tn4371 and the corresponding rpoN::Tc strain were grown in parallel in TRIS-minimal medium supplemented with either gluconate or gluconate and BP. BphC activity (the product of the Tn4371 bphC gene) was measured in all cultures. Both strains expressed the same BphC specific activity (data not shown), ruling out a direct or indirect effect of
54 in bphC transcription. Nevertheless, when provided with BP as the sole carbon and energy source in liquid cultures where the BP concentration is low, CH34rpoN/RP4::Tn4371 grew very poorly compared to CH34/RP4::Tn4371 (generation time 24·1 h vs 9·3 h). The two strains also displayed different growth patterns when streaked (or spread; not shown) on TRIS-agar plates, where BP was provided as the sole carbon source in the form of crystals stuck to the plate lid. While the parent strain formed colonies through the whole streak (or plate), the rpoN derivative only grew under the BP crystals (Fig. 2
). The discrepancy between CH34rpoN/RP4::Tn4371, which displayed reduced growth on BP, and HF09/RP4::Tn4371, which displayed no growth on that same carbon source, could be explained by our observation that HF09, in contrast to CH34rpoN, failed to grow on benzoate, a BP degradation product.
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DISCUSSION |
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This sort of breach in the conservation of the rpoN region is not restricted to Ralstonia. An additional orf203 was found just downstream of the rpoN gene in Caulobacter crescentus (Janakiraman & Brun, 1997 ). A 1·6 kb non-coding region is present between rpoN and orf130 homologues in Rhizobium etli (Michiels et al., 1998
). In the Gram-positive bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides, the rpoN gene is located in a cluster of nif genes and hence is not flanked by similar ORFs (Masepohl et al., 1988
; Meijer & Tabita, 1992
).
Transcriptional organization in the rpoN regions is less conserved than the order of the genes. In E. coli and P. aeruginosa, rpoN seems to be cotranscribed with downstream genes. In Acinetobacter calcoaceticus (Ehrt et al., 1994 ) and in P. putida (Kohler et al., 1994
), rpoN is transcribed independently of the downstream ORFs, two of which are presumably cotranscribed. Our results indicated that in R. metallidurans CH34 the ORFs downstream of rpoN were transcribed independently of rpoN, a situation resembling that in A. calcoaceticus and P. putida. However, in CH34, both downstream ORFs could be transcribed independently, which could be related to the presence of the supplementary orf95 between orf130 and orf151.
In R. metallidurans CH34, the Tn4371 bph genes are transcribed from a 70-like promoter (Mouz et al., 1999
), ruling out a direct role for
54 in that process. Here, we found the same BphC activity in the rpoN mutant and the wild-type strain, ruling out an indirect role of
54 in the regulatory pathway of the expression of the bph operon. Nevertheless, CH34rpoN::Tc carrying the plasmid RP4::Tn4371 only grew in the close vicinity of BP crystals (Fig. 2
), where the BP concentration is likely to be the highest. In addition, that strain grew slowly in liquid cultures where BP concentration remains low due to poor solubility. BP, a hydrophobic molecule, is usually considered as diffusing into cells without the need for a transporter. The existence of a high-affinity transporter necessary for uptake of BP at low concentration and which could be directly or indirectly controlled by
54 could provide an explanation for our observations. Experiments are in progress to test that hypothesis.
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ACKNOWLEDGEMENTS |
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Received 20 March 2001;
accepted 27 March 2001.
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