©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Divergent Transcription and a Remote Operator Play a Role in Control of Expression of a Nopaline Catabolism Promoter in Agrobacterium tumefaciens(*)

Ferenc Marincs (§) , Derek W. R. White

From the (1) Plant Molecular Genetics Laboratory, AgResearch, Grasslands Research Centre, Tennent Drive, Private Bag 11008, Palmerston North 5320, New Zealand

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The nocP-nocR divergent gene arrangement of the nopaline catabolism (noc) operon of the Agrobacterium tumefaciens Ti plasmid pTiT37 was examined with respect to the expression of the nocP promoter. Under repressive conditions, i.e. in the absence of nopaline, four distinct levels of P expression were observed. The lowest level of expression, i.e. full repression, was detected in the presence of the NocR repressor, together with the remote noc operator and productive transcription from the divergent nocR promoter. The next level was observed in the absence of either the NocR protein or of the operator or of both. The third level was detected when abortive transcription from the nocR promoter occurred, irrespective of the presence or absence of the NocR protein. The highest level of P expression was observed in the absence of both productive transcription from P and the operator sequence, whether or not the NocR protein was present. Under inductive conditions, i.e. in the presence of nopaline, expression of P was activated if both the NocR protein and the operator were present. Absence of either NocR or the operator resulted in lack of inducibility of the nocP promoter. Transcription from the divergent nocR promoter had no influence on the activation of P. It was also found that the absence of the operator affected plasmid supercoiling in vivo. The results suggest that DNA topology has a role in the regulation of the nocP promoter.


INTRODUCTION

Remote operators, which are situated outside of the -65 to +20 region of the regulated promoter, are found in many prokaryotic operons, for example in ara, deo, gal, and lac(1) . These operators, however, have one or more remote or proximal (between the -65 and +20 positions) counterparts, and interaction of a regulatory protein with these multiple sites modulates expression of the regulated promoter (2, 3, 4, 5) . However, in those operons where only a single operator is involved, the position of the operator is proximal to or slightly remote from the promoter, as has been shown for promoters regulated by the Fur, LexA, MetJ, and PurR proteins (1) . In these cases, gene expression is repressed or activated by direct contact between the regulatory protein and the RNA polymerase (6). However, regulation of gene expression by a single remote operator, which is more than 70 bp() upstream of the -35 hexamer of a 70 promoter, has not yet been reported to date.

In this respect, the position of a putative operator (O ), which we have localized in the noc operon of the Agrobacterium tumefaciens Ti plasmid pTiT37, is unusual. In this operon, two genes, nocP (formerly called nocB) and nocR, are divergently transcribed by nonoverlapping promoters. The NocR protein alternatively represses or activates expression of P, depending on the absence or presence of nopaline, respectively (7, 8) . The putative operator is located 3 bp downstream of the -10 hexamer of the nocR promoter and centered 137 bp upstream of the -35 hexamer of the regulated nocP promoter. Its deletion resulted in failure of NocR binding in vitro(8) . There are no other binding sites for the NocR protein in the noc promoter region (7) , and no regulatory sites exist in the coding regions of either the nocP or the nocR genes (8) .

This report addresses the role of the putative operator in the regulation of the nocP promoter. The results demonstrate that in the absence of the inducer, expression of P is much higher in the absence than in the presence of the operator, when the NocR repressor is present. Furthermore, different levels of P expression were observed depending upon productive or abortive transcription from the divergent nocR promoter and presence or absence of the operator sequence, even in the absence of the NocR regulatory protein. In the presence of the inducer, P was activated only in the presence of both NocR and the operator. Transcription from the adjacent nocR promoter did not perturb the activation. The noc operator was also found to affect plasmid supercoiling in vivo. The results suggest that expression of P can be controlled through a single remote operator and that at least repression of the nocP promoter may be interpreted in terms of alteration of local DNA topology.


EXPERIMENTAL PROCEDURES

Construction of plasmids pNOC100, pNOC32, pNOC12, and pNOC12o has been described previously (7, 8) . To construct pNOC860, the operator-deleted nocR promoter fragment (P) was excised from pNOC12o by digestion with HindIII+BamHI and was inserted in front of the promoterless -glucuronidase (gusA) gene of pBI101.3 (9) . The P-gusA fusion was then cloned upstream of the P -luc fusion of pNOC100. The wild type and the operator-deleted nocR promoters from pNOC12 and pNOC12o were cloned into pNOC100, yielding pNOC202 and pNOC301, respectively. For these procedures standard DNA manipulation techniques were used (10) . Plasmids were routinely maintained in Escherichia coli DH5 and were conjugated into the pTiT37-carrying A208 (11) and the Ti plasmid-free C58C1 (12) A. tumefaciens strains by triparental mating (13) .

Induction and measurement of the reporter enzymes were performed as described previously (8) , except that p-nitrophenyl--D-glucuronide was used as the substrate for -glucuronidase.

To examine their topoisomers, plasmids pNOC12 and pNOC12o were introduced into the E. coli strain HB101. Bacteria were grown in Luria-Bertani media at 37 °C to the mid-log phase and treated with oxolinic acid at 50 µg/ml final concentration for 20 min. Plasmid DNAs were isolated by the modified alkaline lysis method of Saunders and Burke (14) . To separate plasmid topoisomers, electrophoresis of the samples were done on a 1% agarose gel in TBE buffer (10) , in the presence of 24 µg/ml chloroquine at 40 V for 20 h at room temperature followed by staining with ethidium bromide.


RESULTS

A CATGNCATG direct repeat (Fig. 1) has been identified as the only binding site for the NocR protein in the nocP-nocR promoter region and has been proposed to act as an operator in expression of the nocP promoter regulated by the NocR protein (8) . To investigate the role of this sequence, two plasmids, pNOC32 and pNOC860 (Fig. 2), were used. Since both plasmids contain the luc and gusA reporter genes fused to the nocP and nocR promoters, respectively, transcriptional activity of the promoters can be extrapolated by measuring activities of the Luc and GusA enzymes. Plasmid pNOC32 has the wild type promoter region, whereas in pNOC860 the putative operator is deleted.


Figure 1: Sequence of the nocP-nocR promoter region. The start codons, the -35 and -10 hexamers are underlined. The direct repeats of the operator are double underlined. An alternating purine-pyrimidine sequence overlapping with the operator is in bold and lowercase labels the base pair which is out of alternation. The sequence presented here is available from GeneBank under the accession number L04475.




Figure 2: Structure of the pNOC plasmids used in this study (B). The top part (A) shows the arrangement of the nocP-nocR region in the Ti plasmid pTiT37. Genes are labeled by differently shaded boxes. In pNOC860, pNOC301, and pNOC12o the position of the deleted sequence is labeled by a black box. The flanking regions of the plasmids are not shown, and genes are not drawn to scale.



In the A. tumefaciens strain A208, supplementing the NocR protein from trans, a 15-fold higher expression of P was detected in the mutant pNOC860 plasmid than in the wild type plasmid pNOC32, in the absence of nopaline. Since the presence of the direct repeat is needed for binding of NocR in vitro(8) , the higher expression of P of pNOC860 in a NocR background could be due to the failure of NocR binding and indicates the operator role of the CATGNCATG sequence.

As the NocR protein is not needed for expression of the isolated nocP promoter, we concluded previously that NocR is a repressor in the absence of nopaline (8) . Expression of P in the absence of the operator (strain A208/pNOC860), however, was only 16-17% of the constitutive expression of the isolated nocP promoter (data not shown). This observation is contradictory to the rule that deleting the binding site of a repressor results in constitutive expression of the regulated gene (15) . Therefore, we performed the reverse experiment, i.e. the wild type pNOC32 plasmid was introduced into a NocR strain, C58C1. Since the NocR protein autoregulates its own synthesis (8) , the 9-fold higher expression of P in strain C58C1/pNOC32 () confirmed the absence of the NocR protein. The lack of the nocR gene in strain C58C1 was also confirmed by Southern hybridization (data not shown). In this proven NocR strain, expression of the nocP promoter was similar to that of strain A208/pNOC860. Furthermore, in the nocRO double mutant C58C1/pNOC860 strain, similar expression of P was observed to that which was detected in the A208/pNOC860 and the C58C1/pNOC32 strains (). These results revealed that the nocP promoter is expressed equally in either the absence of the NocR protein or in the absence of the operator or in the absence of both. Therefore, there is another factor which caused the relatively low expression of P under these circumstances.

One possible explanation for this phenomenon is that competition of closely spaced promoters, like P and P, for the RNA polymerase influences their expression (16) . However, despite the 9-fold variation observed between expression of the nocR promoter in strains A208/pNOC860, C58C1/pNOC32, and C58C1/pNOC860, only a 1.3-1.5-fold difference was observed between expression of P in the same strains. This result indicates that there is no competition between the nocP and nocR promoters and that expression of P is independent of the strength of P.

An alternate explanation for the low level expression of P observed in the absence of the NocR protein is that the transcription process from the nocR promoter influences expression of the divergent nocP promoter. Transcription generates negative supercoils behind the transcribing RNA polymerase in a topologically closed domain (17, 18, 19) , and the generated torsional stress may affect functions of the nearby sequences (19, 20, 21, 22) . The supercoil-generating effect of the transcription, however, depends on the transcript length (23) . To study this aspect in the noc operon, we truncated the gusA gene from the nocR promoter in plasmid pNOC32 to produce plasmid pNOC202 (Fig. 2). As a consequence of the lack of the gusA gene, transcription from the nocR promoter is abortive in plasmid pNOC202, resulting in a much shorter transcript compared with plasmid pNOC32 in which productive transcription occurs. Plasmid pNOC202 was then introduced into both the NocR C58C1 and NocR A208 strains. A 4.7-fold higher expression of P was observed in the absence (pNOC202) than in the presence (pNOC32) of the gusA gene (). In the NocR A208 strain, however, deletion of the gusA gene resulted in a 47-fold increase of expression of the nocP promoter (: A208/pNOC202 versus A208/pNOC32). The absolute levels of P expression, however, were the same in either the A208/pNOC202 or the C58C1/pNOC202 strains (). These results demonstrate that in the absence of the gusA gene, expression of the nocP promoter is independent of the NocR protein, even in the presence of the operator, and that the presence of the gusA gene is needed for full repression of P by the NocR protein. Furthermore, the presence of the gusA gene fused to the nocR promoter has reduced expression of the nocP promoter, even in the absence of the NocR protein.

To determine how the abortive transcription from P affects expression of P in the absence of the operator, a plasmid, pNOC301 (Fig. 2), which lacks both the operator and the gusA gene, was constructed and introduced into the NocR A208 and NocR C58C1 strains. Similar levels of P expression were detected in either the presence or the absence of the NocR protein (). The expression of P in pNOC301, however, was 3.5-fold higher than in pNOC202 and about 10-fold higher than in pNOC860, in either the A208 or the C58C1 strains (). These results indicated that the enhanced expression of P in the operator-deleted plasmid pNOC301 is independent of the NocR protein and simply due to the absence of the operator sequence. Comparison of the data also revealed that absence of the operator sequence did not influence expression of P when the gusA gene is fused to the nocR promoter (pNOC860 versus pNOC32, in strain C58C1). In contrast, deletion of the operator resulted in a 3.5-fold increase of P expression in the absence of the gusA gene (pNOC31 versus pNOC202, in strain C58C1).

The results described earlier in this section suggested that supercoils generated by the transcription of an adjacent gene may repress expression of P. Furthermore, the presence of the operator did or did not affect expression of P, depending on abortive or productive transcription of P, respectively. This suggested that the operator influences DNA topology, depending on the supercoil level of the neighboring sequences. To study this, two plasmids, pNOC12 and pNOC12o (8) , containing and lacking the operator sequence, respectively, were introduced into the E. coli strain HB101 which is wild type for both gyrase and topoisomerase I. Plasmid DNAs exist in a negatively supercoiled form inside the bacterial cells (24) and can be isolated in this form. When plasmids pNOC12 and pNOC12o were isolated from untreated bacteria, no difference was observed in their topoisomer distribution (Fig. 3, lanes 1 and 2). In contrast, treating the strains with a gyrase inhibitor, oxolinic acid (25) , resulted in a shift of the topoisomers of both plasmids (Fig. 3, lanes 3 and 4), indicating relaxation of the DNA as the consequence of inhibition of gyrase by oxolinic acid. Despite the relaxation of both plasmids, the operator-deleted mutant plasmid pNOC12o became less relaxed compared with the operator-containing plasmid, pNOC12 (Fig. 3, lane 3 versus lane 4).


Figure 3: Effect of the noc operator on plasmid supercoiling. Lanes 1 and 2, plasmids pNOC12o and pNOC12, respectively, isolated from untreated E. coli strain HB101. Lanes 3 and 4, plasmids pNOC12o and pNOC12, respectively, isolated after oxolinic acid treatment. Direction of the migration is from top to bottom. At the applied chloroquine concentration, the topoisomers which were less relaxed (i.e. more supercoiled) before the electrophoresis migrated more slowly.



Activities of the nocP and nocR promoters were also determined in all of the constructed strains under inductive conditions, i.e. in the presence of nopaline (). The results revealed that the presence of both the NocR protein and the operator is needed for activation of the nocP promoter. Absence of either resulted in failure to induce of P. In contrast to the repression, activation of P, however, was nearly the same in either the presence or the absence of a productive transcription from the nocR promoter (only a 1.09-fold difference was observed). Nopaline did not influence expression of the nocR promoter (), in agreement with our previous report (8) .


DISCUSSION

We analyzed the expression of the nocP promoter of the A. tumafaciens Ti plasmid pTiT37, and two main points should be highlighted from the results described above.

First, we have found that productive transcription from the adjacent nocR promoter represses expression of the divergent nocP promoter, even in the absence of the NocR regulatory protein. In previous studies, only activation of a mutant promoter in a topoisomerase I mutant strain has been reported when a divergent transcription unit was placed upstream of the promoter (26, 27, 28) .

Second, results from different experiments, in good correlation with each other, revealed that an upstream sequence, which is the operator for the nocP promoter in the presence of the NocR regulatory protein, modulates both expression of P and DNA supercoiling, even in the absence of NocR. To our knowledge no similar effects have been reported for a bacterial operator to date. The mechanism by which the noc operator affects DNA topology is not clear yet. However, since an 18-bp alternating purine-pyrimidine putative Z-DNA forming sequence overlaps the noc operator (Fig. 1), a B-Z or Z-B transition is possible, as it has been demonstrated previously that B to Z transition of synthetic purine-pyrimidine (alternating dG-dC) sequences influences the supercoil level of DNA (29) .

Full repression of the nocP promoter was observed only in the presence of the NocR protein, its binding site, and productive transcription from the divergent nocR promoter. Thus it is possible that a mutual interaction between binding of the NocR protein to the remote operator and DNA supercoiling has a role in repression of the nocP gene. Our results suggest that repression of the nocP promoter is influenced by changes in the local supercoiling level. The sensitivity of P to supercoiling is not unexpected, because it has the features, namely a 19-bp intervening region between the -35 and -10 hexamers and G-C pairs in the -10 hexamer (Fig. 1), which have been found in supercoil-sensitive promoters (30, 31) . However, in contrast to the repression, activation of the nocP promoter was not sensitive to the transcription from the adjacent nocR promoter. This observation suggests that repression and activation of the nocP promoter may occur by different molecular mechanisms. This proposal is supported by the observation that insertion of an extra 47 bp into plasmid pNOC32 between the regulated nocP promoter and the operator did not influence repression, but dramatically decreased activation of P (data not shown).

In conclusion, our results suggest that gene expression can be regulated through a single operator at a distance and that DNA supercoiling is one part of the cellular repertoire by which gene expression is regulated in naturally found divergent promoter pairs.

  
Table: Expression of the nocP and nocR promoters in Agrobacterium



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 64-6-356-8019; Fax.: 64-6-351-8032; E-mail: agpfxm@pnv.palm.cri.nz.

The abbreviation used is: bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Richard Biggs, Brigitta Dudás, and Michael McManus for their critical reading of the manuscript and va Vincze for the plasmid pBI101.3.


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