Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK
Correspondence
George P. C. Salmond
gpcs{at}mole.bio.cam.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the pigT ORF sequence reported in this paper is AJ973142.
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INTRODUCTION |
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Previous research in this laboratory has demonstrated numerous regulatory mechanisms, both genetic and environmental, involved in the control of secondary metabolite and (in some cases) exoenzyme production. Briefly, an N-acyl homoserine lactone (N-AHL) quorum-sensing (QS) system (SmaIR) functions, via a de-repression mechanism, to control Pig, Car and exoenzymes in response to increasing concentrations of N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL) synthesized by SmaI (Fineran et al., 2005; Slater et al., 2003
; Thomson et al., 2000
). QS controls the transcription of the Pig and Car biosynthetic genes by affecting expression of at least four other regulatory genes (carR, pigR, pigQ and rap) (Fineran et al., 2005
; Slater et al., 2003
).
Another key regulator is PigP, a novel putative DNA-binding protein, which controls expression of Pig and Car via modulation of at least seven other regulatory genes (carR, pigQ, pigR, pigS, pigV, pigX and rap) (Fineran et al., 2005). In addition, a GacAS family two-component system (PigQW) regulates Pig production via activation of the biosynthetic operon pigAO (Fineran et al., 2005
). We have also shown that Pig and Car levels are altered in response to phosphate concentration, sensed by the pstSCAB/phoBR system (Slater et al., 2003
). Our current model of secondary metabolite production involves an intricate hierarchical network of transcriptional regulation that integrates numerous cues, including cell density, phosphate and unknown signal(s), presumably detected by the sensor kinase PigW (Fineran et al., 2005
).
In the current study, we characterize a new physiological cue (gluconate) and a predicted sensor/effector (PigT) that regulate Pig production, in 39006, independently of the known regulators mentioned above. PigT is homologous to the Escherichia coli GntR protein, which is a repressor of the GntI group of genes required for gluconate utilization (Izu et al., 1997; Tong et al., 1996
). We demonstrate that PigT activates transcription of the Pig biosynthetic operon (pigAO), whereas addition of gluconate causes a reduction in transcription of pigAO. Furthermore, a putative PigT binding site was identified in the promoter of pigA, based on sequence similarity to the gnt operator site (Porco et al., 1997
). Therefore, PigT is predicted to activate pigAO transcription directly. We demonstrate that PigT increases expression of a pigA promoter cloned in E. coli and that this activation is inhibited by gluconate. The transcription profile of a chromosomal pigT : : lacZ fusion and primer extension of pigT demonstrated that pigT expression was maximal in exponential phase and decreased in stationary phase. The gluconate/PigT system represents a new, independent pathway involved in regulation of Pig production.
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METHODS |
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Construction of a plasmid (pTA38) that expresses native PigT.
A construct that enabled expression of native, untagged PigT was created as outlined below. The pigT gene was amplified by PCR, using primers PF103 and PF69, which contain EcoRI and PstI restriction sites, respectively. Additionally, primer PF103 contains a consensus ribosome-binding site (RBS, AGGAGGA). The PCR fragment of pigT was cloned into pQE-80L, previously digested with the enzymes EcoRI and PstI. The resulting plasmid, pTA38, was confirmed by DNA sequencing. Expression of plasmid pTA38 in both E. coli and 39006 hosts was induced with 1 mM IPTG.
Generation of a pigT : : mini-Tn5Sm/Sp mutant by transposon exchange mutagenesis.
Previously, we reported an efficient in vivo method based on phage transduction that allows exchange of a chromosomal transposon insertion for an alternative transposon (Fineran et al., 2005). Using this exchange method, strain HSPIG36S (pigT : : mini-Tn5Sm/Sp) was constructed from the parental strain HSPIG36 (pigT : : mini-Tn5lacZ1). The location of the new transposon was determined by PCR amplification across the insertion site using primers facing out of the transposon (LER1 and SP2 for mini-Tn5Sm/Sp) and the pigT-specific primers HS3 and HS4. PCR products in the expected size range were analysed further by sequencing and the point of insertion was determined. The pigT : : mini-Tn5Sm/Sp mutation was transduced into a clean LacA background using
OT8, and the nature of the transductants was confirmed by phenotype and PCR analysis.
Marker exchange mutagenesis of pigW.
Strain PIG62L (pigW : : lacZKm) was generated by a marker exchange strategy. Firstly, primers PF95 and PF96, containing XbaI and SalI restriction sites, respectively, were used to PCR-amplify a fragment of the pigW gene. This fragment was ligated into pBluescript II KS+digested with XbaI and SalI, and the resulting construct (pTA35) was confirmed by DNA sequencing. Secondly, a promoterless lacZ gene and a Km resistance cassette were amplified from plasmid pUTmini-Tn5lacZ1 using primers PF97 and PF98, which contain NcoI and NheI restriction sites, respectively. The lacZKm fragment was ligated to specific NcoI and NheI sites internal to the pigW gene in pTA35, generating plasmid pTA36. Next, the entire pigW : : lacZKm fragment was excised from plasmid pTA36 on the unique XbaI and SalI restriction sites and ligated into the marker exchange vector pKNG101 digested with XbaI and SalI, generating plasmid pTA37. Marker exchange with plasmid pTA37 was performed using a sucrose selection protocol similar to that described elsewhere (Kaniga et al., 1991). The pigW mutant was confirmed by PCR, sequencing and phenotypic analysis.
Construction of pigA promoter : : lacZ fusions and assay conditions.
The pigA promoter was delineated into four different size fragments and cloned into the promoterless lacZ plasmid pRW50 (Lodge et al., 1992). The plasmids, named pTA15, pTA30, pTA31 and pTA32 in order of decreasing size, were created by cloning BamHI/HindIII-digested PCR products [generated with forward primers HS78, PF70, PF71 and PF72, respectively, and the reverse primer HS79 (Table 2
, Fig. 3
)] into BamHI/HindIII-digested pRW50. The nature of all plasmids was confirmed by DNA sequencing.
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-Galactosidase assays.
-Galactosidase activity in bacterial cultures grown in liquid media was determined using ONPG as substrate, as described elsewhere (Miller, 1972
). Enzyme activity was expressed as the initial reaction rate per ml of sample per OD600 of the bacterial culture tested (
A420 min1 ml1 OD6001). Results presented are the mean±standard deviation (SD) of three independent experiments, unless stated otherwise.
Bioassays of prodigiosin, carbapenem, N-AHLs, exoenzymes and motility.
The assays for Pig and Car were performed as described previously (Slater et al., 2003). Pig production was plotted as (A534 ml1 OD6001)x50. Detection of N-AHLs was performed using the Serratia LIS bioassay described in Thomson et al. (2000)
. Activity of pectate lyase and cellulase was analysed on agar plates containing the corresponding substrates, as described elsewhere (Andro et al., 1984
). Motility was assessed on tryptone swarm agar (TSA) plates (10 g Bacto tryptone l1, 5 g NaCl l1 and 3 g agar l1). Overnight bacterial cultures were adjusted to an OD600 of 0·2, 3 µl was spotted onto the plates, and halo size was examined after growth for 16 h at 30 °C.
Primer extension and RNA studies.
A hot-acidic phenol method (Aiba et al., 1981) was used to extract total RNA from 39006. Primer extension analysis for the pigA transcript was performed as described previously (Slater et al., 2003
) using primer HS34. All primer extension reactions were performed with 25 µg total RNA and 0·2 pmol of the appropriate 32P-labelled primer. Primer extension analysis of pigT was performed using primer gntR SS(2), which anneals 122 bp downstream of the predicted pigT translational start site. mRNA transcripts were quantified using ImageJ, a densitometry program available at http://rsb.info.nih.gov/nih-image/Default.html.
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RESULTS |
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To demonstrate that the transposon disruption in pigT was responsible for the Pig phenotype, complementation was performed. Plasmid pTA38, which expresses native PigT, was able to complement the Pig phenotype in HSPIG36, whereas a pQE-80L control could not (compare Fig. 1e with Fig. 1f
). Therefore, PigT affects both swimming motility and Pig production in 39006 and shares sequence similarity with GntR from E. coli.
PigT is a transcriptional regulator of Pig
To examine the impact of PigT on Pig production in more detail, Pig was assessed throughout growth in LB in the pigT mutant and compared to that of the WT (Fig. 2a). Pig production was approximately 10 % of the levels observed in the WT. To enable construction of double chromosomal mutants, strain HSPIG36S (pigT : : mini-Tn5Sm/Sp) was constructed (see Methods). HSPIG36S was shown to have the same swimming motility and Pig production defects as HSPIG36. Expression of a chromosomal pigA : : lacZ fusion was studied throughout growth in the WT and pigT mutant backgrounds (Fig. 2b
), and was found to be reduced to approximately 30 % of WT levels. In addition, primer extension studies demonstrated that the pigAO transcript in the pigT mutant was similarly reduced compared to that of the WT (Fig. 2c
). The pigT mutant was unaffected for Car and BHL/HHL production when examined throughout growth in LB (data not shown). These results were consistent with a model in which PigT regulates Pig production by controlling the transcription of the biosynthetic genes (pigAO).
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PigT is predicted to act directly at the pigA promoter
The transcriptional hierarchy data described above indicate that pigT does not control the transcription of the other regulators examined, consistent with the notion that PigT directly activates pigAO transcription. Examination of the sequence 5' of pigAO revealed a potential PigT binding site (ATGTTACTGGTAACTG) centred at 78·5 relative to the pigA transcriptional start site (Fig. 3a) (Slater et al., 2003
). The bold type represents the two conserved half sites of the predicted PigT/GntR binding sites. This predicted binding site is based on the E. coli GntR palindromic consensus binding sequence (ATGTTA-(N4, GC-rich)-TAACAT) (Porco et al., 1997
) present in the promoter regions of GntR-regulated genes. The position of this binding site suggests that PigT may be binding directly to the pigAO promoter region and functioning as a Class I activator to regulate transcription (Busby & Ebright, 1999
).
To test if PigT directly activates transcription of the Pig biosynthetic operon, the pigA promoter was cloned into the low-copy, promoterless lacZ expression vector pRW50 (Lodge et al., 1992). Promoter delineation studies were performed on the pigA promoter to determine regions required for PigT activation and promoter activity (see Methods). The results showed that, in an E. coli gntR mutant (YU563), the presence of PigT caused an increase in transcription from the pigA promoter (Fig. 3b
). Furthermore, the shortest construct (pTA32), which still possessed the 35 and 10 elements, but not the predicted PigT binding site, was not activated by PigT. These results suggest that PigT directly activates transcription of pigAO and this activation requires a 150 bp region 5' of the pigA promoter. Within this region there is an inverted repeat sequence similar to the E. coli GntR box, which we predict is a PigT box.
We had reported previously that transposon insertions within the ROP1 (repressor of pigment) region of the pigA promoter in 39006 resulted in a hyper-pigmented phenotype and an increase in pigA : : lacZ expression (Slater et al., 2003). It was suggested that the ROP1 region might (1) be a repressor binding site, (2) be important for DNA conformation or (3) encode a small RNA that acts negatively on Pig production. Deletion of the ROP1 region had little effect on promoter activity in E. coli (Fig. 3b
). Furthermore, PigT was still able to activate pigA promoter constructs that lacked the ROP1 region, indicating that ROP1 is not required for the PigT-mediated activation of pigA. Thus, the DNA region between 193 and 44 inclusive is required for PigT-mediated activation of pigA transcription.
Expression of pigT is maximal in exponential phase
To assess the expression of pigT, -galactosidase activity was measured from a chromosomal pigT : : lacZ fusion throughout growth (Fig. 4a
). In addition, primer extension analysis of the pigT transcript was performed (Fig. 4b
). These experiments showed that levels of the pigT transcript peak in exponential phase (6 h) and then decline as the cultures reach stationary phase (12 h).
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DISCUSSION |
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In E. coli, GntR negatively regulates the GntI group of genes involved in gluconate utilization. However, when gluconate is present in the medium, GntR is thought to bind gluconate (or one of its metabolic intermediates), and repression of the GntI genes is alleviated (Izu et al., 1997; Tong et al., 1996
). The gluconate-sensing role of GntR, and the sequence similarity to PigT, prompted us to examine the effects of gluconate on secondary-metabolite biosynthesis in 39006.
Gluconate, even in the presence of glucose or in complex media (LB), could decrease Pig production in 39006 (Figs 1 and 5a). The gluconate-mediated decrease in Pig production was accompanied by a reduction in pigA transcription, even if gluconate was added after the onset of pigmentation (Fig. 5b
).
These data suggested a model whereby PigT mediates gluconate repression of Pig production. Based on genetic and biochemical data from E. coli GntR, it is clear that GntR represses gene expression by binding to DNA in the promoter regions of GntI genes in the absence of gluconate (Peekhaus & Conway, 1998). However, in 39006, PigT-activated Pig production is hypothesized to be deactivated by the addition of gluconate. It was possible that PigT was controlling transcription of pigAO indirectly by repressing a repressor of Pig. Therefore, to try and identify a potential regulatory intermediate, the effects of a pigT mutation on the transcription of multiple known secondary metabolite regulators were examined. However, the effect of PigT on Pig production was independent of all the regulators investigated. PigT did not control transcription of any of those tested and, likewise, no regulator examined controlled expression of pigT. This result suggested that PigT might directly activate transcription of the pigAO operon.
Interestingly, a putative PigT binding site, based on that demonstrated for GntR (Porco et al., 1997), was identified upstream of the 35 element in the pigA promoter (Fig. 3a
). Furthermore, promoter delineation experiments in E. coli implied that PigT only activated pigA promoter constructs that contained a 150 bp region (from 193 to 44) which includes this putative PigT binding site (Fig. 3b
). Based on this genetic evidence, our model, that PigT directly activates expression of pigAO in the absence of gluconate, contradicts the conventional role of GntR, which functions as a repressor of gluconate metabolism genes in E. coli. However, in our model, the binding of PigT (like GntR) to DNA is dependent on the absence of gluconate. We suggest that the binding of gluconate to PigT results in PigT being unable to bind DNA, and therefore activation of transcription is prevented. Interestingly, we have evidence that, in E. coli, GntR can activate the cloned pigA promoter, and this activation can be inhibited by the presence of gluconate (P. Fineran, unpublished results). This suggests that, E. coli GntR can also be an activator of gene expression if the placement of the GntR binding site is permitting. It remains to be determined whether PigT functions analogously to GntR by regulating genes involved in uptake and utilization of gluconate. However, both the sequence context of pigT, with respect to genes potentially involved in gluconate utilization, and the presence of a putative PigT binding site in the promoter of the putative gluconokinase (data not shown), indicate that PigT may have a role in regulating gluconate metabolism.
The pigA promoter studies raised a few interesting points about the regulation of the pigAO operon. Firstly, the transcriptional start site of pigA has been mapped by primer extension, and putative 10 and 35 elements identified (Slater et al., 2003). A TAAAGA
TAAAGG substitution in the predicted 10 element resulted in a greater than 50 % reduction in pigA promoter activity (data not shown), supporting this assignment. Secondly, interrogation of the sequence 5' of pigA has revealed a predicted CRP binding site centred at 64·5, which overlaps one half of the predicted PigT binding site (Fig. 3a
). Preliminary data demonstrated an increase in pigA promoter expression in an E. coli crp mutant (P. Fineran, unpublished results). However, the role of CRP requires further investigation. Interestingly, the gntT promoter in E. coli is also regulated by both GntR and CRP (Peekhaus & Conway, 1998
). Furthermore, it is interesting that, in a previous mutagenesis screen, we isolated a predicted novel adenylate cyclase mutant (pigR) with decreased Pig (Fineran et al., 2005
).
Primer extension and expression of a chromosomal pigT : : lacZ fusion was used to assess the transcription profile of pigT throughout growth. pigT was maximally expressed during midlate exponential phase, but was attenuated during the transition into stationary phase (Fig. 4a, b). This transcript profile is consistent with a role for PigT as a major activator of pigA, and hence Pig, levels of which increase dramatically in midlate exponential phase (Fig. 2a, b
).
Our model of PigT directly activating the pigAO operon (and hence Pig production) and inhibition of this interaction in the presence of gluconate is based on the following lines of evidence: (1) PigT shares significant sequence similarity with E. coli GntR (77 % identity/81 % similarity), which has been shown biochemically to respond to gluconate and alter its DNA binding affinity accordingly (Peekhaus & Conway, 1998); (2) addition of gluconate or mutation of pigT had similar effects on Pig production and transcription of pigAO (Figs 2 and 5
), but no effect on Car and BHL/HHL synthesis; (3) epistasis experiments demonstrated that gluconate could not further repress pigmentation in a pigT mutant (Fig. 6
); (4) finally, in an E. coli gntR mutant, PigT-mediated activation of the pigA promoter was inhibited by gluconate (Fig. 6
).
The physiological benefit of 39006 repressing Pig in the presence of gluconate is unknown. Little is known of the true habitat(s) of 39006, but it is possible that gluconate is present in a niche where Pig is not required. GntR homologues have been associated with the regulation of various phenotypes in bacteria. For example, a GntR homologue regulates production of antibiotics and the onset of aerial mycelia and spore formation in Streptomyces coelicolor (Sprusansky et al., 2003).
The current investigation has increased our understanding of the physiological and genetic regulation of the biosynthesis of a tripyrrole antibiotic pigment, prodigiosin (Pig). We have identified a new physiological cue (gluconate) that represses Pig production and characterized the potential sensor of this signal (PigT). The gluconate/PigT pathway represents a system for control of pigmentation that is apparently completely independent of the regulatory network of secondary metabolite production that we have recently characterized in some detail (Fineran et al., 2005; Slater et al., 2003
; Thomson et al., 1997
, 2000
). Fig. 7
highlights some of the important physiological and genetic factors that modulate Pig biosynthesis in 39006, including the PigT/gluconate system. Both the physiological rationale and molecular mechanism driving this gluconate-mediated regulation of the red, tripyrrole antibiotic warrant further investigation.
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ACKNOWLEDGEMENTS |
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Received 3 June 2005;
revised 21 September 2005;
accepted 27 September 2005.
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