- Research article
- Open Access
The PhoBR two-component system regulates antibiotic biosynthesis in Serratia in response to phosphate
© Gristwood et al; licensee BioMed Central Ltd. 2009
- Received: 11 December 2008
- Accepted: 28 May 2009
- Published: 28 May 2009
Secondary metabolism in Serratia sp. ATCC 39006 (Serratia 39006) is controlled via a complex network of regulators, including a LuxIR-type (SmaIR) quorum sensing (QS) system. Here we investigate the molecular mechanism by which phosphate limitation controls biosynthesis of two antibiotic secondary metabolites, prodigiosin and carbapenem, in Serratia 39006.
We demonstrate that a mutation in the high affinity phosphate transporter pstSCAB-phoU, believed to mimic low phosphate conditions, causes upregulation of secondary metabolism and QS in Serratia 39006, via the PhoBR two-component system. Phosphate limitation also activated secondary metabolism and QS in Serratia 39006. In addition, a pstS mutation resulted in upregulation of rap. Rap, a putative SlyA/MarR-family transcriptional regulator, shares similarity with the global regulator RovA (regulator of virulence) from Yersina spp. and is an activator of secondary metabolism in Serratia 39006. We demonstrate that expression of rap, pigA-O (encoding the prodigiosin biosynthetic operon) and smaI are controlled via PhoBR in Serratia 39006.
Phosphate limitation regulates secondary metabolism in Serratia 39006 via multiple inter-linked pathways, incorporating transcriptional control mediated by three important global regulators, PhoB, SmaR and Rap.
- Secondary Metabolism
- Quorum Sense
- Histidine Kinase
Phosphate is an essential component of numerous biomolecules. Therefore, the control of intracellular phosphate concentrations is vital for bacterial survival. At least two major systems are involved in managing intracellular concentrations of inorganic orthophosphate (Pi), the preferred primary source of phosphate . When Pi is abundant, the low affinity Pit transporter appears to be primarily responsible for Pi uptake [2–4]. When Pi becomes limited, the high affinity Pst transport system (PstSCAB-PhoU) is activated, and takes over as the predominant Pi uptake system [5–8].
In Escherichia coli and other Enterobacteriaceae, the cellular response to Pi availability is mediated via the PhoBR two-component system. Under conditions of Pi limitation, the sensor histidine kinase PhoR is autophosphorylated . PhoR then activates its cognate response regulator, PhoB , which in turn activates expression of multiple genes, termed the Pho regulon, via direct binding to a conserved Pho box sequence found overlapping -35 regions in target gene promoters [10–12]. In E. coli, the Pho regulon is believed to consist of approximately 30 genes involved in the adaptation to survival under low Pi conditions, including pstSCAB-phoU and phoBR . Phosphate regulation is controlled via similar mechanisms in Bacillus subtilis and Streptomyces species, although the consensus Pho boxes are different in each system [13, 14]. Mutations in the pstSCAB-phoU operon result in constitutive activation of PhoR and hence, constitutive phosphorylation of PhoB [15, 16]. Therefore, pst mutants are proposed to mimic low Pi conditions.
Pi has been found to negatively regulate the biosynthesis of antibiotics and other secondary metabolites in multiple bacterial species (reviewed in ). However, the complex molecular mechanisms underlying the Pi mediated regulation of secondary metabolism are not well characterised. In this study we investigate the role of the PhoBR two-component system, and Pi availability, on the regulation of antibiotic production in the Gram-negative Enterobacteriaceae, Serratia sp. ATCC 39006 (Serratia 39006). Serratia 39006 synthesises the red, tripyrrole antibiotic, prodigiosin (Pig; 2-methyl-3-pentyl-6-methoxyprodigiosin) . The natural physiological role of Pig in the producing organism may be as an antimicrobial agent . In addition, Pig is of clinical interest due to the observed anticancer and immunosuppressive properties of this compound [20–22]. Serratia 39006 also produces the β-lactam antibiotic, carbapenem (Car; 1-carbapen-2-em-3-carboxylic acid) [23, 24]. Both the Pig and Car biosynthetic gene clusters have been characterised (pigA-O and carA-H, respectively) [25, 26].
Production of secondary metabolites in Serratia 39006 is controlled by a hierarchial network of regulators . This includes a LuxIR-type quorum sensing (QS) system (SmaIR) [25, 28, 29], which allows gene expression to be regulated in response to cell density via the production and detection of low molecular weight signal molecules . In Serratia 39006, the N-acyl homoserine lactone (AHL) synthase SmaI produces two signalling molecules, N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL), with BHL being the major product . At low cell density, SmaR acts as a transcriptional repressor of target genes [28, 29]. At high cell density, and hence high BHL/HHL levels, SmaR binds BHL/HHL, resulting in decreased DNA-binding affinity with a consequent alleviation of repression. QS controls secondary metabolism in Serratia 39006 via at least four other regulatory genes (carR, pigQ, pigR and rap) [28, 29]. The putative SlyA/MarR-family transcriptional regulator, Rap (regulator of antibiotic and pigment), is an activator of Pig and Car production in Serratia 39006 . Rap shares similarity with the global transcriptional regulator RovA (r egulator o f v irulence) from Yersina spp. [32–34]. More than 20 additional genes have been shown to regulate secondary metabolism in Serratia 39006, and these are predicted to be responding to additional environmental stimuli [19, 27, 35, 36].
Previously, we demonstrated that, in Serratia 39006, mutations within genes predicted to encode homologues of the E. coli PstSCAB-PhoU phosphate transport system resulted in over-production of both Pig (10-fold) and Car (2-fold), at the level of transcription of the biosynthetic genes . In this study we investigate further the molecular mechanism by which these effects are occurring. We demonstrate that secondary metabolism in Serratia 39006 is upregulated in response to mutations in PstSCAB-PhoU or Pi limitation, via the PhoBR two-component system. In addition, we provide evidence that expression of the smaI, pigA and rap genes are activated via PhoBR in Serratia 39006. Hence, we propose a model in which Pi limitation increases secondary metabolism in Serratia 39006 via multiple, inter-linked pathways, incorporating the global transcriptional regulators PhoB, SmaR and Rap.
Sequence analysis of the pstSCAB-phoU operon in Serratia 39006
The Serratia 39006 pstS gene was predicted to encode a protein most similar to PstS from the enteric bacteria Erwinia carotovora ssp. atroseptica SCRI1043 (Eca 1043) (82% identity/90% similarity). The putative protein product encoded by pstC shared 90% identity and 95% similarity with PstC of Eca 1043. The pstA gene is predicted to encode a protein most similar to PstA of Eca 1043 (87% identity/92% similarity). The predicted protein encoded by pstB was most similar to PstB of Eca 1043 (88% identity/91% similarity). Finally, phoU was predicted to encode a protein most similar to PhoU of Eca 1043 (94% identity/98% similarity).
Isolation and sequence analysis of phoBR mutants of Serratia 39006
Mutations in the pstSCAB-phoU operon are thought to mimic growth in limiting phosphate, and hence result in constitutive activation of the Pho regulon . We previously showed that Pig, Car and AHL production were increased in the pstS mutant . A possible explanation for this effect is that pigA, carA and smaI are regulated via the Serratia 39006 Pho regulon.
Random transposon insertions in the phoBR operon were isolated based on their lack of hyperpigmentation when grown on Pi-limiting media. Growth on Pi-limiting media results in increased Pig production in the wild-type (WT; throughout this manuscript WT refers to the LacA parental strain) . Potential phoBR mutants were then checked for their loss of alkaline phosphatase activity (phoA, encoding alkaline phosphatase, is a conserved Pho regulon gene [1, 37]) and the sequence of the operon was determined, as described in Methods. The phoB gene was predicted to encode a 229 amino acid (aa) protein with highest similarity to PhoB from Eca 1043 (96% identity/98% similarity). The phoR gene was located 28 bp downstream of phoB, and was predicted to encode a 440 aa protein sharing the highest degree of similarity to Eca 1043 PhoR (87% identity/90% similarity).
PhoB regulates expression of pstC in Serratia 39006
In E. coli, the pst operon is activated via direct binding of PhoB to a conserved Pho box upstream of pstS [10–12]. As Serratia 39006 is a member of the Enterobacteriaceae, we identified potential Pho boxes based on the E. coli consensus sequence. A potential Pho box was identified within the pstS promoter region of Serratia 39006, centred 122 bp upstream of the pstS start codon (Fig. 1B). This suggested that, as could be expected based on regulation of the pstSCAB-phoU genes in other bacteria, the pstSCAB-phoU genes in Serratia 39006 may be regulated by PhoB. A putative Pho box was also identified upstream of phoB (Fig 1B), centred 68 bp upstream of the phoB start codon, suggesting that phoBR may be auto-regulated via the putative Pho box.
β-Glucuronidase activity produced from a chromosomal pstC::uidA transcriptional fusion was measured in the presence or absence of a secondary mutation in phoB. The pstC::uidA fusion strain does not contain a functional Pst transporter and is therefore believed to mimic low phosphate conditions. These data showed that, in the presence of functional PhoB, pstC was expressed constitutively throughout growth (Fig. 1C). Expression was dramatically reduced following inactivation of phoB, indicating that PhoB activates expression of the pst operon in Serratia 39006 (Fig. 1C).
Insertions within phoBR abolish upregulation of secondary metabolism and QS in the pstS mutant
Insertions within phoBR abolish transcriptional upregulation of pigA and smaI in the pstS mutant
Insertions within pstSCAB-phoU result in increased transcription of rap
PhoB activates expression from the pigA and rap promoters in an E. coli system
Pi regulates secondary metabolism and QS in Serratia 39006
There are multiple studies identifying environmental factors that effect Pig production in Serratia spp., including the effects of salt concentration, temperature, oxygen availability and multiple metal ion concentrations . However, the molecular mechanism underlying most of these responses has not been elucidated. Here, we investigate the molecular mechanism by which Pi limitation affects secondary metabolism in the enteric bacteria Serratia 39006.
It was previously shown that a pstS mutation in Serratia 39006 resulted in the upregulation of QS and secondary metabolism . Here, we demonstrate that these effects are occurring via the PhoBR two-component system, since a secondary mutation in phoBR abolished the effects of a pstS mutation. In addition, we confirm that QS and secondary metabolism are upregulated in response to Pi limitation, and that this is occurring primarily via the PstSCAB-PhoU transport system. We also demonstrate that expression of rap is upregulated in response to a pstS mutation. Rap is an activator of Pig and Car, and a repressor of surfactant production and swarming motility, in Serratia 39006 [19, 29]. Rap shares similarity with the SlyA/MarR-family global transcription factor, RovA, which regulates genes required for host colonization in Yersinia spp. [32–34]. Therefore, our results indicate that three global transcriptional regulators, Rap, SmaR and PhoB, are involved in mediating the effects of Pi limitation on secondary metabolism in Serratia 39006.
A mutation of the pstSCAB-phoU genes resulted in a clear increase in Pig and AHL production, and a clear increase in pigA, smaI and rap transcription. However, following Pi limitation, the effects on secondary metabolism and gene expression were less dramatic. The degree of activation of Pig and AHL production, and pigA transcription, was approximately 35% lower following Pi limitation than the levels of activation observed in a pstS mutant. In addition, a clear increase in rap transcription was not observed following Pi limitation. It is possible that this reduced effect is due to the fact that a pstS mutant is constitutively mimicking extreme Pi limitation. However, when WT cells are transferred to phosphate limiting media, there may be phosphate carry over from the initial inoculum, and the cells may utilise existing intracellular phosphate stores, for example inorganic polyphosphate, before phosphate starvation occurs. As the increase in rap transcription in a pstS mutant is below 2-fold, we believe that a 35% reduction in activation, in response to Pi limitation, may be undetectable. An alternative explanation could be that rap is induced via PhoBR, but not in response to Pi limitation. Previously, PhoBR has been shown to activate expression of the asr (acid shock RNA) gene, but Pi limitation did not activate asr expression . In addition, there is also evidence that PhoB can be activated by non-partner histidine kinases, in the absence of PhoR . This has lead to the hypothesis that PhoBR may activate genes in response to a variety of environmental cues, in addition to Pi limitation .
It may not be entirely accurate to describe the effect of a pstS mutation, or Pi limitation, on QS as 'upregulation'. For QS to function correctly, it is the absolute concentrations of the AHL signal molecule that is critical, not the amount per cell . Due to the growth defect observed following a pstS mutation or Pi limitation, the amount of AHL per cell is increased, but the absolute value remains comparable to WT/Pi excess conditions. Therefore, it may be more accurate to state that the upregulation of smaI transcription, following pstS mutation or Pi limitation, allows maintenance of QS regulon control despite the reduced growth rate. This idea is supported by the fact that although carR, pigQ, pigR and rap are all regulated by QS in Serratia 39006 [28, 29], only rap transcription is upregulated in response to a pstS mutation. Our experiments indicate that, in response to a pst mutation, rap is activated independently of QS, and that activation may be mediated via PhoB.
Activation of carA expression, following pstS mutation, was previously reported to be dependent on the upregulation of QS . However, as Rap is also an activator of carA transcription , it is possible that Rap, rather than QS, is responsible for the activation of carA following a pstS mutation. We propose that a dual mechanism, involving (1) the alleviation of SmaR repression at lower cell density, via upregulation of smaI, and (2) increased levels of Rap via PhoB mediated transcriptional activation, is responsible for the increase in carA expression following pstS mutation. In the absence of AHL (and hence constitutive SmaR repression), carA transcription is essentially abolished  and hence, further activation by Rap, in response to a pstS mutation, might not be possible.
Multiple studies have linked Pi limitation to enhanced secondary metabolite production . However, the complex molecular mechanisms underlying phosphate-mediated regulation have proven difficult to elucidate. Extensive studies in Streptomyces species have shown that PhoPR (PhoBR) activates secondary metabolism in response to Pi limitation, including biosynthesis of undecylprodigiosin, a tripyrrole closely related to Pig [40, 41]. However, in Streptomyces, inactivation of PhoP or deletion of phoPR also activates secondary metabolism . In contrast, deletion of phoB and/or phoR in Serratia 39006 had no impact on secondary metabolism, demonstrating clear differences between the regulatory mechanisms employed by these distantly related bacteria. Although the requirement for increased secondary metabolism under conditions of phosphate limitation is unclear, it has been proposed that enhanced secondary metabolism allows the production of compounds which may, for example, directly antagonise other microorganisms or act as signalling molecules, thereby providing producing organisms with a competitive advantage under nutrient deprived conditions [40, 42, 43].
In conclusion, we have established that via the global transcriptional regulators PhoB, SmaR and Rap, multiple inter-linked pathways are acting to upregulate secondary metabolism in Serratia 39006 under conditions of Pi limitation, highlighting the importance of Pig and Car production under these conditions.
Bacterial strains, plasmids, phage and culture conditions
Bacterial strains and plasmids are listed in Additional File 1[44–49]. Serratia sp. ATCC 39006 derivative strains were grown at 30°C and E. coli strains were grown at 37°C in Luria broth (LB; 5 g l-1 yeast extract, 10 g l-1 bacto tryptone and 5 g l-1 NaCl), minimal media (0.1% w/v (NH4)2SO4, 0.41 mM MgSO4, 0.2% w/v glucose, 40 mM K2HPO4, 14.7 mM KH2PO4, pH 6.9–7.1) or in phosphate limiting (PL) media (0.1% w/v (NH4)2SO4, 0.41 mM MgSO4, 0.2% w/v glucose, 0.1 M HEPES, pH 6.9–7.1 ± 5 mM KH2PO4) in shake flasks at 300 rpm, or on plates supplemented with 1.5% (w/v) agar (LBA). For the phoBR mutagenesis screen, Serratia 39006 was grown on PGM agar plates (5 g l-1 bacto peptone, 1% v/v glycerol and 1.5% w/v agar). Bacterial growth (OD600) was measured in a Unicam Heλios spectrophotometer at 600 nm. When required, media were supplemented with antibiotics at the following final concentrations; kanamycin 50 μg ml-1 (Km), spectinomycin 50 μg ml-1 (Sp), ampicillin 100 μg ml-1 (Ap), and tetracycline 35 μg ml-1 (Tc). The generalised transducing phage ϕOT8 was used for transduction of chromosomal mutations as described previously .
Oligonucleotide primers used in this study
pigA, primer extension oligo
smaI, primer extension oligo
carA, primer extension oligo
F primer for KmR gene of miniTn5 Km1
R primer for KmR gene of miniTn5 Km1
Random primed PCR primer 1
Random primed PCR primer 2
Random primed PCR primer 3
pstSCAB region sequencing primer
pstSCAB region sequencing primer
pstSCAB region sequencing primer
pstSCAB region sequencing primer
phoU sequencing primer
phoU sequencing primer
F primer for pTG27, smaI promoter construct
R primer for pTG27, smaI promoter construct
R primer for pTA14, rap promoter construct
F primer for pTA14, rap promoter construct
F primer for pTA74, PhoB expression construction
R primer for pTA74, PhoB expression construction
F primer for sequencing phoR
R primer for sequencing phoR
F phoR primer
R phoR primer
pstS sequencing primer
pstA sequencing primer
pstB sequencing primer
pstS sequencing primer
pstA sequencing primer
pstS sequencing primer
pstS sequencing primer
pBluescript II KS+ sequencing primer
pBluescript II KS+ sequencing primer
Sequencing of the pstSCAB-phoU operon
Preliminary sequence analysis indicated the mini-Tn5 Sm/Sp insertions in strains ROP2 and KHC5 were in pstS and pstA respectively . To determine the full sequence of pstS and its surrounding genes, a Serratia 39006 Pst I sub-genomic library was created in pBluescript II KS+. One clone containing pstS was analysed further and was named pPST1. The pst region was sequenced via a 'primer walking' technique using primers PST1, PST2, PST3, PST4, PST5, PSTSLN, PSTSRN. To complete the pstSCAB-phoU operon, a 2.1 kbp region of pstSCA was PCR amplified with the primers NW244 and NW245, and then sequenced using primers NW244, NW245, NW246 and NW247. Random primed PCR was used to extend the phoU sequence obtained from primer walking of pPST1, as described previously . Gene specific primer NW250 was used in two separate random primed PCR reactions, one with PF106, PF107, PF108 , and a second with NW225, NW226, NW227. The products generated were then amplified with the nested primer PF109 or NW251, respectively and the resulting PCR products sequenced with primer NW251.
Transposon mutagenesis screen for phoBR mutants
To isolate phoBR mutants, Serratia 39006 strain LacA was subjected to a random transposon mutagenesis by conjugation with E. coli S17–1 λpir harbouring plasmid pUTmini-Tn5 Km1 as described previously . Ten thousand mutants were picked onto glucose minimal medium plates and replica-plated onto PGM agar Colonies that did not exhibit a hyper-pigmented phenotype were selected, based on the rationale that if hyper-pigmentation was not induced in response to Pi limitation, it might be due to an insertion in phoBR (strains BR1 and BR9 were isolated using this screen). The pstS::miniTn5Sm/Sp was transduced into non-Pi responsive mutants, and non-hyperpigmented mutants were then selected (strains RBR1 and RBR9 were selected following this screen). This suggested that these uncharacterised insertions had disrupted a regulatory element(s) common to pstS mutants and Pi limitation effects. The possibility that phoBR had been disrupted was investigated further by measuring alkaline phosphatase activity, encoded by phoA, which is a well conserved member of enteric Pho regulons . Mutants RBR1 and RBR9 did not produce elevated levels of alkaline phosphatase as observed in the pstS mutant (data not shown). Sequence analysis, described below, confirmed that the insertions in BR1 and BR9 were within phoR and phoB respectively.
Sequencing of the phoBR operon
To determine the site of the transposon insertion in strain BR1, chromosomal DNA was digested with Eco RV and ligated into pBluescript II KS+. The ligation was used as template in a single-primer-site PCR using primers KML and KMR that anneal to the 5' and 3' ends of mini-Tn5 Km1 respectively in combination with primers T3 and T7. Sequencing of the resultant PCR products revealed that BR1 contained an insertion within a gene similar to phoR from E. coli. A further PCR using chromosomal DNA from the BR9 mutant with primers PHORL and PHORR (homologous to phoR 5' and 3' ends) and primers KML and KMR demonstrated that BR9 contained an insertion within a gene with similarity to phoB from E. coli. To further confirm the phoBR sequence, PCR products of phoB and phoR were generated with primer pairs PF154/PF155 and PF180/PF182 respectively and sequenced on both strands from independent products.
Construction of a plasmid (pTA74) that expresses native PhoB
A construct that enabled expression of native, untagged PhoB was created as outlined below. The phoB gene was amplified by PCR, using primers PF154 and PF155, which contain Eco RI and Hin dIII restriction sites, respectively. Additionally, primer PF154 contains a consensus ribosome-binding site (RBS, AGGAGGA). The PCR fragment of phoB was cloned into pQE-80L, previously digested with the enzymes Eco RI and Hin dIII. The resulting plasmid, pTA74, was confirmed by DNA sequencing. Expression of plasmid pTA74 in E. coli was induced with 1 mM IPTG.
Construction of promoter::lacZ fusions and assay conditions
Plasmid pTA15 was constructed as described previously . The rap and smaI promoter regions were cloned into the promoterless lacZ plasmid pRW50  to give the plasmid constructs pTA14 and pTG27, respectively. Plasmid pTG27 was constructed by cloning an Eco RI/Hin dIII digested PCR product (generated using forward primer OTG124 and reverse primers OTG125) into Eco RI/Hin dIII digested pRW50. Plasmid pTA14 was constructed by cloning an Eco RI/Hin dIII digested PCR product (generated using forward primer PF43 and reverse primer PF42) into Eco RI/Hin dIII digested pRW50. All constructs were confirmed by DNA sequencing.
Promoter activity assays were performed in E. coli DH5α cells as described in . Briefly, DH5α cells were transformed with the promoter::lacZ construct (pTA14, pTA15 or pTG27) and either pTA74 (encoding native PhoB) or the empty vector control, pQE-80L. The resulting strains were grown in LB containing Ap, Tc and 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). At late exponential phase, 1 ml samples were assayed for β-galactosidase activity.
Prodigiosin, carbapenem, AHL, β-galactosidase, β-glucuronidase and alkaline phosphatase assays
The assays for Pig and Car were performed as described previously . Pig production was plotted as (A534 ml-1 OD600-1). Detection of AHLs was performed using the Serratia LIS bioassay described in . β-Galactosidase activity was determined as described previously  and was represented as (ΔA420 min-1 ml-1 OD600-1). β-Glucuronidase activity was determined as for β-galactosidase activity except that reactions were performed in GUS buffer (50 mM NaPO4, 1 mM EDTA, 5 mM DTT, pH 7.0), using the substrate p-nitrophenyl β-glucuronide (PNPG; 10 mM), and measured at A405. β-Glucuronidase activity was represented as (ΔA405 min-1 ml-1 OD600-1). Alkaline phosphatase activity was assayed as described previously . Results presented are the mean ± the standard deviation of three independent experiments, unless stated otherwise.
Primer Extension and RNA studies
RNA was extracted from Serratia 39006 and primer extension analysis for the pigA and smaI transcripts was performed as described previously [28, 29]. All primer extension reactions were performed with 25 μg of total RNA and 0.2 pmol of the appropriate 32P-labelled primer. Oligonucleotide primers HS34 and HS36 were used in primer extension reactions for pigA and smaI respectively.
We thank all members of the Salmond group for helpful discussions, I. Foulds for technical assistance and Corinna Richter for the identification of strain PCF58A9. This work was supported by the BBSRC, UK. TG and LE were supported by BBSRC studentships.
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