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Journal of Bacteriology, March 2008, p. 1997-2003, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01781-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Phosphate Acquisition Components of the Myxococcus xanthus Pho Regulon Are Regulated by both Phosphate Availability and Development{triangledown}

David E. Whitworth,1* Antony B. Holmes,2 Alistair G. Irvine,2 David A. Hodgson,2 and David J. Scanlan2

Institute of Biological Sciences, Cledwyn Building, Aberystwyth University, Ceredigion SY23 3DD, United Kingdom,1 Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom2

Received 10 November 2007/ Accepted 26 December 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In many organisms, phosphatase expression and phosphate (P) uptake are coordinately regulated by the Pho regulon. In Myxococcus xanthus P limitation initiates multicellular development, a process associated with changes in phosphatase expression. We sought here to characterize the link between P acquisition and development in this bacterium, an organism capable of preying upon other microorganisms as a sole nutrient source. M. xanthus seems to possess no significant internal P stores, as reducing the P concentration to less than 10 µM retarded growth within one doubling time. Pyrophosphate, polyphosphate, and glyceraldehyde-3-phosphate could support growth as sole P sources, although many other P-containing biomolecules could not (including nucleic acids and phospholipids). Several Pho regulon promoters were found to be highly active during vegetative growth, and P limitation specifically induced pstSCAB, AcPA1, and pho3 promoter activity and repressed pit expression. Enhanced pstSCAB and pho3 promoter activities in a phoP4 mutant (in the presence of high and low concentrations of P) suggested that PhoP4 acts as a repressor of these genes. However, in a phoP4 background, the activities of pstSCAB remained P regulated, suggesting that there is additional regulation by a P-sensitive factor. Initiation of multicellular development caused immediate down-regulation of Pho regulon genes and caused pstSCAB and pho3 promoter activities to become P insensitive. Hence, P acquisition components of the M. xanthus Pho regulon are regulated by both P availability and development, with developmental down-regulation overriding up-regulation by P limitation. These observations suggest that when development is initiated, subsequent changes in P availability become irrelevant to the population, which presumably has sufficient intrinsic P to ensure completion of the developmental program.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The deltaproteobacterium Myxococcus xanthus exhibits a population-wide response to starvation, forming multicellular fruiting bodies within which sporulation occurs (4). This developmental program can be initiated by omitting phosphate (P) from the growth medium (13), and late events during development include expression of specific phosphatases. Phosphatase expression and P acquisition are coordinately regulated by the Pho regulon in many bacteria, including Escherichia coli, Bacillus subtilis, and Streptomyces lividans (8, 20, 22). The core of each Pho regulon comprises a two-component system (TCS) that responds to P limitation by induction of the pstSCAB operon (encoding a high-affinity P uptake system) and secreted phosphatases (which liberate P from extracytoplasmic P-containing compounds). TCSs typically comprise a sensor protein kinase (PhoR) and a partner response regulator protein (PhoB/P). Pho regulon response regulators serve as transcriptional activators when they are phosphorylated (8, 20, 22).

In M. xanthus seven TCS proteins (PhoRP1, PhoPR2, PhoPR3, and PhoP4) have been implicated in the regulation of phosphatase expression and late developmental events (3, 15, 17). Deleterious mutations in operons encoding the Pho1, Pho2, and Pho3 TCSs each cause a partial reduction in the activity of developmentally expressed phosphatases. The phenotypes of the pho2 and pho3 mutants are particularly similar and are also similar to the phenotype of the pho2 pho3 double mutant, suggesting that the Pho2 and Pho3 systems act in the same pathway (15). A phoP4 mutant exhibited almost complete abolition of phosphatase expression (17), implying that PhoP4 acts upstream of Pho1 and Pho2/Pho3 during late development. The phoP4 gene is immediately upstream of the M. xanthus pstSCAB operon, and disruption of phoP4 results in altered expression of pstSCAB (17). This suggests that PhoP4 has dual functions—regulating both P uptake and multicellular development. Several key questions thus arise. Is the Pho regulon sensitive to changes in P availability? What environmentally available P sources can be utilized for growth (M. xanthus can prey on other microorganisms as sole nutrient sources)? Is the Pho regulon regulated by developmental progression?


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture conditions, media, and bacterial strains. Cultures of M. xanthus were incubated at 30°C. Double Casitone yeast (DCY) medium was used as a rich medium for maintenance and manipulation of strains (2% Casitone, 0.2% yeast extract, 10 mM Tris [pH 8.0], 8 mM MgSO4). A1W defined medium is the same as A1 (2) except that it is supplemented with 1 mM potassium chloride. A1W lacking 1 mM potassium phosphate was designated A1W-P. The starvation media used were TM (10 mM Tris [pH 8.0], 8 mM MgSO4) and TPM (TM with 1 mM potassium phosphate). For developmental assays, TM and TPM were solidified with 1.5% agarose. Phosphate levels in media were determined using the method of Itaya and Ui (9). The M. xanthus strains used in this study were wild-type strain DK1622 (10) and phoP4 mutant VP963 (16). VP963 carries an unmarked in-frame deletion of phoP4 engineered in a DK1622 background. The Escherichia coli strain used for plasmid construction and P1-mediated transduction was MC1061 [hsdR mcrB araD139 {Delta}(araABC-leu)7679 galU galK rpsL thi {Delta}lacX74 (lacIPOZY)].

Manipulation and physiological assays of M. xanthus. P1-mediated transduction of plasmids from E. coli into M. xanthus was performed by using the method described by Hodgson (6). Transductants were verified by their production of LacZ (as demonstrated by production of indigo upon incubation on medium containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside [X-Gal]) and by successful PCR amplification of the promoter-lacZ unit. LacZ expression assays were performed as described by Hodgson (6) in triplicate using triplicate biological samples. During analysis, only promoter activity changes that were twofold or greater were considered significant. Multicellular development assays were performed as described by Higgs and Merlie (5) in triplicate. Assay reagents were obtained from Sigma.

DNA manipulation and plasmid construction. Table 1 shows the plasmids utilized in this study. Promoter regions were amplified by PCR using the oligonucleotide primers shown in Table 2. PCR products were introduced into the pCR2.1-TOPO vector (Invitrogen) by using the manufacturer's instructions. Promoter regions were then subcloned into pDAH274 (Table 1) using unique restriction sites (BamHI and EcoRI) engineered into the amplification oligonucleotide primers (Table 2). Screening for plasmids containing inserts was performed using the screening primers shown in Table 2 and a PCR protocol with annealing at 50°C and 30 cycles. All plasmids were verified by DNA nucleotide sequencing. Fine chemicals and enzymes were obtained from Sigma and Fermentas, respectively, and were used according to the suppliers' protocols.


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  		TABLE 1. Plasmids used

 

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  		TABLE 2. Oligonucleotide primers

 

   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
M. xanthus can acquire P from a variety of P-containing compounds. Initially, we determined if M. xanthus required inorganic P for growth or whether it could obtain P from more complex molecules. A modified form of the defined minimal medium A1 (2) was obtained by replacing potassium phosphate with potassium chloride (A1W-P), thus eliminating P from the medium (see Materials and Methods). By monitoring the growth rate by determining changes in the optical density at 600 nm, it was observed that M. xanthus wild-type strain DK1622 exhibited no significant growth in A1W-P (Fig. 1) compared to growth in A1W (A1W-P which was supplemented with 1 mM potassium phosphate; doubling time, ~24 h). The kinetics of growth in A1W were virtually identical to those in A1 (2). Supplementing A1W-P with different concentrations of potassium phosphate showed that growth could be supported at a rate similar to that in A1W by phosphate concentrations as low as 10 µM. However, at a P concentration of 1 µM or lower, the growth rate was reduced to the growth rate in A1W-P.


Figure 1
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  		FIG. 1. Growth kinetics of M. xanthus on different P sources. Changes in cell number were monitored by recording the optical density at 600 nm at different times after inoculation (at zero time) into minimal medium (A1W-P) with different P sources. Optical density values were normalized to 0.1 at day 1, and the values are the averages of three independent experiments (the standard deviation was within 15% of the sample mean in all cases, and error bars are not shown). Pyrophosphate (PPi), polyphosphate (PolyP), and glyceraldehyde-3-phosphate (Gly-3-P) could support growth, while glucose-6-phosphate (Glu-6-P) could not.

 
We then sought to identify substances that could replace P in A1W and still support growth by adding them individually to A1W-P. We chose substances that might be available to M. xanthus either because of lysis of prey organisms or because of their presence in the soil environment. The substances tested included phosphatidylethanolamine, phosphatidylcholine, phytic acid, phosphonates (methylphosphoric acid, phosphonomethyl glycine, 3- N-phosphomethyl glycine, phosphonoacetic acid, 2-amino-3-phosphonopropionic acid, and 2-aminoethylphosphonic acid), RNA, DNA, deoxynucleoside triphosphate, nucleoside triphosphate, ATP, glucose-6-phosphate, glyceraldehyde-3-phosphate, glycerol-3-phosphate, sodium pyrophosphate, and sodium polyphosphate. Enough of each compound was added to ensure that phosphoryl groups were present at a molar equivalent of ≥1 mM. A substance was classified as a potential phosphate source for M. xanthus if its inclusion in A1W-P allowed the optical density at 600 nm (or number of CFU) of a culture to at least double after 7 days of incubation. When this criterion was used, only pyrophosphate, polyphosphate, glycerol-3-phosphate, and glyceraldehyde-3-phosphate were classified as potential P sources for M. xanthus (Fig. 1).

Identification of candidate Pho regulon phosphatases. Five phosphatase activities have been identified previously in M. xanthus (23). Two of them were vegetatively expressed magnesium-dependent activities (one with an acidic pH optimum and one with an alkaline pH optimum), and three were magnesium-independent activities that were induced during development (one acidic, one alkaline, and one neutral). In order to identify candidate genes that might encode previously observed phosphatase activities, we searched the predicted protein sequences derived from the M. xanthus genome using BLASTP (1) with the sequences of experimentally characterized phosphatases described previously as query sequences. The five most promising candidate genes were predicted to encode phosphatases with the correct pH optima, and these phosphatases were designated AcPA1, AcPA2, AlPA1, AlPA2, and NPA (acidic, acidic, alkaline, alkaline, and neutral, respectively). AlPA1 is a homologue of the PhoD alkaline phosphatase (COG3540), AlPA2 is predicted to be a member of the alkaline phosphate superfamily (COG1524), AcPA1 is homologous to the SurE acid phosphatase (COG0946), and AcPA2 is homologous to the NapD acid phosphatase, while NPA is homologous to the neutral phosphatase of Staphylococcus aureus (GenBank accession number P21222) and is predicted to be a member of the COG0637 group (phosphatases and phosphohexomutases).

Construction of Pho gene reporter strains. Based on previous experimental analysis and the bioinformatic analysis described above, 13 candidate Pho regulon promoter regions/operons were identified for further analysis; these regions/operons included the five phosphatase genes, four TCS operons, the pstSCAB operon, a pit operon, and the genes encoding PAPK and polyphosphate kinase (PPK) (Table 3). To characterize expression of promoters under different conditions, each promoter region was cloned upstream of a promoterless lacZ gene. This was achieved by PCR amplification of a region of M. xanthus genomic DNA originating immediately upstream of the start codon of the gene/operon of interest and extending upstream for ~1 kb. The exception was the pho3 operon, where an open reading frame was found 9 bp directly upstream of phoR3 (MXAN_6415, an ompA-related protein precursor gene), which was in turn preceded by an 86-bp intergenic region. The pho3 operon was therefore assumed to include MXAN_6415, phoR3, and phoP3, and the promoter probe was designed accordingly. PCR products were introduced into pCR2.1-TOPO (Invitrogen) and then directionally subcloned into pDAH274 using unique restriction sites (BamHI and EcoRI) engineered into the oligonucleotide primers used for amplification. Tables 1 and 2 show the plasmids and primers utilized. The resulting plasmids (promoter probes) were introduced into M. xanthus wild-type strain DK1622 by P1-mediated transduction (see Materials and Methods) with selection for kanamycin resistance. Kanamycin-resistant transductants contained promoter probe plasmids integrated into the chromosome by homologous recombination (i.e., merodiploids with tandem duplication of the cloned promoter regions). LacZ production by the resulting transductants was assumed to mirror the activity of the relevant promoter in its natural context. Unfortunately, efforts to produce promoter probes for the phoP4 and ppk genes were unsuccessful. To ensure robust analysis, only changes in LacZ expression greater than twofold were considered physiologically relevant.


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  		TABLE 3. Putative operons involved in the regulation of P uptake in M. xanthus

 
Vegetative expression of Pho regulon promoters. All 11 promoter probes were introduced into M. xanthus wild-type strain DK1622, and LacZ production during vegetative growth in shaken cultures (in DCY rich medium; P concentration, ~8 mM) was determined (Fig. 2). Several Pho regulon promoters were active during vegetative growth. The NPA, pit, pstSCAB, pho1, pho3, and PAPK gene promoters had high levels of activity (LacZ specific activity, >100 U), while the pho2 and AcPA1 gene promoters had lower levels of activity (average specific activity, ~35 U). The remaining promoters were either inactive or initiated transcription at very low levels (average specific activity, <20 U).


Figure 2
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  		FIG. 2. Promoter (LacZ) specific activities during vegetative growth (1 day after inoculation at an optical density at 600 nm of 0.1), during the stationary phase (5 days after inoculation), and after 2 days of incubation in starvation medium with phosphate (TPM) and starvation medium lacking phosphate (TM).

 
For all promoters except the NPA gene promoter, LacZ expression did not change significantly upon the transition to stationary phase or upon introduction into starvation medium, whether phosphate was present or not (TPM and TM, respectively [see Materials and Methods]). However, the NPA gene promoter was activated approximately twofold by starvation and upon the transition to stationary phase (Fig. 2).

P-dependent changes in Pho regulon gene expression. To test the effect of P on expression of Pho regulon genes, wild-type M. xanthus strains containing the 11 promoter probes were subcultured into A1W and A1W-P, and LacZ production was then assayed after a suitable period of adaptation (2 days). For most promoters, there was a slight reduction in LacZ expression in A1W compared to rich medium, probably as a consequence of the reduced growth rate in A1W. However, the NPA gene promoter exhibited greater activity in A1W than in DCY medium, presumably reflecting the reduced levels of nutrients in A1W compared to DCY medium. Most promoter activities were not significantly different in A1W and A1W-P (Fig. 3). However the AcPA1, pho3, and pstSCAB gene promoters were induced around fourfold, twofold, and fourfold, respectively, by incubation in A1W-P, while the pit promoter activity was reduced twofold in A1W compared to A1W-P (Fig. 3).


Figure 3
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  		FIG. 3. Promoter (LacZ) specific activities with P limitation. Strains containing different promoter probes were incubated in A1W and A1W-P. The specific activities of P-responsive promoters (AcPA1 gene, pho3, pstSCAB, and pit promoters) were also determined for a phoP4 mutant.

 
To determine whether P responsiveness was PhoP4 dependent, the pho3, pit, AcPA1, and pstSCAB gene promoter probes were introduced into phoP4 mutant VP963, and LacZ expression was assayed in A1W. Expression from the pstSCAB promoter was threefold higher in the phoP4 mutant than in the wild-type background (Fig. 3), suggesting that PhoP4 represses pstSCAB expression in the presence of high levels of P and that the induction of pstSCAB upon P depletion is a consequence of loss of PhoP4-mediated repression. However, expression from the pstSCAB promoter was still enhanced in A1W-P compared to A1W (Fig. 3), suggesting that another factor (factor X) activates pstSCAB expression under P-limiting conditions. The AcPA1 and pho3 gene promoters also remained P responsive in a phoP4 background, but the pit promoter was no longer down-regulated by P limitation (Fig. 3). This suggests that PhoP4 does not regulate the AcPA1 or pho3 gene but rather represses expression of the pit operon under P-limiting conditions.

Developmental expression of Pho regulon promoters. Phosphatase activities are developmentally regulated in M. xanthus, and we therefore tested whether Pho regulon genes were also developmentally regulated. The pstSCAB, pho3, pit, and PAPK gene promoters were switched off during early development in TM, as shown by an exponential decrease in the LacZ specific activity with time (Fig. 4). Other Pho regulon promoters were relatively unaffected by development. Changes in most promoter activities were not significantly affected by the presence of P (TM compared to TPM); the only exception was the NPA gene promoter, which was transiently up-regulated during early development, but only in the presence of P (Fig. 4). Thus, the P responsiveness of the Pho regulon pho3, AcPA1, pit, and pstSCAB components is lost during multicellular development.


Figure 4
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  		FIG. 4. Promoter (LacZ) specific activities at different times during development in the absence of phosphate (TM). The specific activities of the NPA gene, pho3, pstSCAB, and pit promoters during development in starvation medium with phosphate (TPM) were also determined. T, time (in days).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The Pho regulon of M. xanthus appears to be far more complex than other prokaryotic examples, with seven TCS regulatory proteins rather than the usual two. Additionally, the regulon regulates (and is regulated by) two discrete (although interdependent) physiological processes (P availability and uptake and multicellular development). While expression of P uptake genes is induced by P limitation, P starvation at high cell densities on a solid substrate can initiate progression through the developmental program, a process that shuts down expression of many P acquisition genes.

If a population of M. xanthus cells is transferred to medium lacking P, growth ceases within one doubling time (~24 h in A1W). This suggests that the amounts of phosphate stored in the cells (usually polyphosphate) are small. This agrees with the results of polyphosphate assays, which show that polyphosphate levels are low in exponentially growing cells, although polyphosphate transiently accumulates during early development (24). Cessation of growth required the concentration of P to be reduced below 10 µM and was unaffected by previous incubation in different media, whether the medium was rich or minimal (DCY medium or A1W) (unpublished data). M. xanthus is able to grow with other microorganisms as sole nutrient sources (16). We therefore tested a range of P-containing biomolecules that might be expected to be released by lysis of prey to determine their abilities to act as sole P sources. Surprisingly, nucleic acids, phospholipids, and nucleotides could not act as sole P sources in our assays, nor could glucose-6-phosphate. We also tested phytic acid and phosphonates, as phytase and phosphonate transporter genes have been annotated in the M. xanthus genome (MXAN_5326 and MXAN_5978, respectively), although no homologues of phosphonate metabolism genes could be identified.

Our assays did show that pyrophosphate, polyphosphate, glycerol-3-phosphate, and glyceraldehyde-3-phosphate could be utilized by M. xanthus as sole P sources. With polyphosphate, there was a lag phase prior to exponential growth longer than that observed with P itself (Fig. 1). This suggests that there is a delay in utilization, which may be a consequence of the requirement for accumulation of secreted polyphosphatases prior to uptake of liberated P or due to a requirement for de novo expression of polyphosphatase genes. Genes encoding four pyrophosphatases are predicted to be present in the M. xanthus genome (MXAN_0862, MXAN_1671, MXAN_2473, and MXAN_5577), and one of them (MXAN_1671) is predicted to encode a V-type H+-translocating pyrophosphatase. There is no homologue of the gene encoding the UhpT transporter of hexose phosphates in the M. xanthus genome sequence. The glpT gene of M. xanthus has been characterized (14) and shown to be developmentally regulated, and it is induced early in development (0 to 24 h). Glycerol-3-phosphate was found to inhibit swarming and aggregation, and disruption of glpT caused cells to aggregate even on rich medium (14). The ability to utilize glycerol-3-phosphate and glyceraldehyde-3-phosphate as sole P sources suggests that there is uptake of glycerol-3-phosphate by cells, which can be readily produced from glyceraldehyde-3-phosphate, presumably liberated by extracellular lipolysis of prey organisms. As the doubling time of our cultures in A1W was ~24 h and M. xanthus doubles every ~3 h in rich medium, care must be taken when our conclusions are extrapolated to rich medium. However, it is still not clear to what degree utilization of different P sources occurs during predation. It is likely that multiple P sources are utilized simultaneously, and it would be interesting to quantify the relative contributions of different prey biomolecules to M. xanthus P uptake.

During development three phosphatase activities are induced, while two further activities are expressed during vegetative growth (23). Developmental phosphatase expression is significantly reduced in pho2 and pho3 mutants and pho2 pho3 double mutants (15). However, the developmental defects due to pho2 and pho3 mutations and pho2 pho3 double mutations are not apparent when exogenous P is supplied (15). This suggests that developmental phosphatases are important in P scavenging late in development. We utilized bioinformatics to identify the five genes most likely to encode these activities, expecting to see changes in promoter activity during development. To our surprise, four of the promoters were almost completely inactive and not affected by development, while the fifth appeared to be induced by nutrient limitation and transiently activated early in development, but only in the presence of P. This suggests that either we were unsuccessful in identifying the genes encoding the developmental and vegetative phosphatase activities described here (this could have been the case if, for instance, the phosphatase activities belong to phosphoprotein phosphatases) or the phosphatase activities are regulated posttranslationally. One putative phosphatase promoter (the AcPA1 promoter) was activated by P limitation, so AcPA1 may be a Pho regulon phosphatase. However, AcPA1 is regulated only by P availability and not by development.

Several other promoters were found to be highly active during vegetative growth, including the PAPK gene, pho1, pho3, pit, and pstSCAB promoters. For the pho1 promoter, we observed a typical expression activity of ~180 U of LacZ, which agreed well with previous studies (3). The previously reported down-regulation of pho1 promoter activity during development (3) was also observed (Fig. 4), with lower activities in media containing P (data not shown). We expected to see significant promoter activity for the pit promoter during growth with high P levels; however, the levels of activity were rather low. In contrast, the activities of the pstSCAB and PAPK gene promoters were remarkably high. It is particularly intriguing that the PAPK gene promoter activity was so high, given that the substrate for the gene product (polyphosphate) is essentially absent from cells during vegetative growth. Perhaps the PAPK gene is expressed at high levels to keep polyphosphate levels low during vegetative growth. This might be the case if polyphosphate acts as an alarmone signal for activation of the Lon protease rather than as a phosphate storage molecule in M. xanthus, as suggested by Zhang et al. (24). In B. subtilis pstSCAB promoter activity is induced ~5,000-fold upon P limitation, from ~0 to ~4,500 U of LacZ (18). In contrast, P limitation in M. xanthus increases the pstSCAB promoter activity from ~150 to ~650 U of LacZ (Fig. 3). Thus, in M. xanthus pstSCAB expression is unusually high in the presence of P. This may suggest that M. xanthus has adapted to a lifestyle or environment where free P is typically rare and where high constitutive expression of the pstSCAB operon would confer a selective advantage. It would be interesting to know what concentrations of P are typically found surrounding cells engaged in predation in the natural environment.

It was reported previously that a phoP4 mutant exhibited levels of pstSCAB transcripts different than those in the wild-type strain (17). The vegetative expression of the pstSCAB promoter probe in a phoP4 mutant was approximately threefold greater than that in the wild type, which suggested that PhoP4 represses expression of pstSCAB in the presence of high P levels and that P limitation relieves repression by PhoP4. However, pstSCAB promoter activity remained P responsive in the phoP4 mutant (Fig. 3), and there was a further fivefold induction of promoter activity upon P limitation. Thus, the pstSCAB promoter is regulated by PhoP4 (negatively) and also by a separate P-responsive "factor X." The only regulatory operon of the Pho regulon to be transcriptionally regulated by P availability is pho3 (Fig. 3), and the Pho3 system might therefore be factor X. Alternatively, factor X could be PhoU, which is encoded at the end of the pstSCAB operon.

The pho3 and AcPA1 promoters were also induced by P limitation (twofold and fourfold, respectively), while the pit promoter was twofold less active under P-limiting conditions (Fig. 3). Induction of the AcPA1 gene and pho3 was not phoP4 dependent. However, the down-regulation of the pit genes in the absence of P was phoP4 dependent (Fig. 3). Thus, it seems that PhoP4 acts as a repressor of pstSCAB expression independent of P, but under P-limiting conditions PhoP4 also represses expression of the pit operon. The AcPA1 gene and pho3 promoters are P responsive but are not affected by a phoP4 deletion, suggesting that another factor (factor X?) regulates induction of these promoter activities upon P limitation.

It appears that the promoters of P acquisition genes are turned off early during development, independent of P availability. In addition, P-responsive promoters become P unresponsive during starvation and indeed during development. This suggests that starvation induces a response that makes subsequent availability of P irrelevant. Presumably at such a point sufficient P is available to the population to allow completion of the developmental program, and the population may commit to completing this program at an early stage. This has parallels with the process of sporulation in B. subtilis, during which accumulation of phosphorylated Spo0A leads to down-regulation of the Pho regulon ~2 h after starvation (7). We also observed that the profile of promoter activities during development was not affected by mutation of phoP4, suggesting that the primary role of PhoP4 is in the regulation of P acquisition rather than in developmental events. However, this is difficult to reconcile with the dominance of the phoP4 developmental phenotype over the phenotypes of pho1, pho2, or pho3 mutants, unless PhoP4 modifies the activity of Pho1, Pho2, and Pho3 posttranscriptionally.

Finally, since four PhoP homologues are involved in the Pho regulon of M. xanthus, we attempted to find Pho boxes (PhoP binding sites), as defined by similarity to the consensus Pho box sequences of E. coli, B. subtilis, and cyanobacteria (11, 12, 21), in Pho gene promoter regions. No sequences that significantly resembled known Pho boxes could be identified in these regions. This perhaps should not have been surprising, as there would presumably be four sets of Pho box sequences (one per PhoP homologue) and an uncertain degree of sequence similarity.

In summary, it seems that the Pho regulon of M. xanthus has several novel features: the regulon is bifunctional, regulating both P acquisition and multicellular development (responding to P availability and developmental progression); it involves four transcriptional regulators, one of which acts as a repressor; and during starvation-induced fruiting body formation the regulon becomes insensitive to P levels.


   ACKNOWLEDGMENTS
 
D.E.W. was funded by Biotechnology and Biological Sciences Research Council grant BBD0039891. A.G.I. and A.B.H. were funded by the Engineering and Physical Sciences Research Council through the MOAC Doctoral Training Centre at the University of Warwick.

We thank Mitchell Singer (University of California, Davis) for providing the phoP4 mutant VP963.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Biological Sciences, Cledwyn Building, Aberystwyth University, Ceredigion SY23 3DD, United Kingdom. Phone: (44) 1970 621828. Fax: (44) 1970 622350. E-mail: dew{at}aber.ac.uk Back

{triangledown} Published ahead of print on 4 January 2008. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
  2. Bretscher, A. P., and D. Kaiser. 1978. Nutrition of Myxococcus xanthus, a fruiting myxobacterium. J. Bacteriol. 133:763-768.[Abstract/Free Full Text]
  3. Carrero-Lérida, J., A. Moraleda-Muñoz, R. García-Hernández, J. Pérez, and J. Muñoz-Dorado. 2005. PhoR1-PhoP1, a third two-component system of the family PhoRP from Myxococcus xanthus: role in development. J. Bacteriol. 187:4976-4983.[Abstract/Free Full Text]
  4. Diodati, M. E., R. E. Gill, L. Plamann, and M. Singer. 2007. Initiation and early developmental events, p. 43-76. In D. E. Whitworth (ed.), Myxobacteria: multicellularity and differentiation. ASM Press, Washington, DC.
  5. Higgs, P. I., and J. P. Merlie, Jr. 2007. Myxococcus xanthus: cultivation, motility and development, p. 465-478. In D. E. Whitworth (ed.), Myxobacteria: multicellularity and differentiation. ASM Press, Washington, DC.
  6. Hodgson, D. A. 1993. Light-induced carotenogenesis in Myxococcus xanthus: genetic analysis of the carR region. Mol. Microbiol. 7:471-488.[CrossRef][Medline]
  7. Hulett, F. M., J. Lee, L. Shi, G. Sun, R. Chesnut, E. Sharkova, M. F. Duggan, and N. Kapp. 1994. Sequential action of two-component genetic switches regulates the PHO regulon in Bacillus subtilis. J. Bacteriol. 176:1348-1358.[Abstract/Free Full Text]
  8. Hulett, F. M. 1996. The signal-transduction network for Pho regulation in Bacillus subtilis. Mol. Microbiol. 19:933-939.[CrossRef][Medline]
  9. Itaya, K., and M. Ui. 1966. A new micromethod for the colorimetric determination of inorganic phosphate. Clin. Chim. Acta 14:361-366.[CrossRef][Medline]
  10. Kaiser, D. 1979. Social gliding is correlated with the presence of pili in Myxococcus xanthus. Proc. Natl. Acad. Sci. USA 76:5952-5956.[Abstract/Free Full Text]
  11. Liu, W., and F. M. Hulett. 1998. Comparison of PhoP binding to the tuaA promoter with PhoP binding to other Pho-regulon promoters establishes a Bacillus subtilis Pho core binding site. Microbiology 144:1443-1450.[Abstract]
  12. Makino, K., M. Amemura, S.-K. Kim, A. Nakata, and H. Shinagawa. 1994. Mechanism of transcriptional activation of the phosphate regulon in Escherichia coli, p. 5-12. In A. Torriani-Gorini, E. Yagil, and S. Silver (ed.), Phosphate in microorganisms: cellular and molecular biology. ASM Press, Washington, DC.
  13. Manoil, C., and D. Kaiser. 1980. Guanosine pentaphosphate and guanosine tetraphosphate accumulation and induction of Myxococcus xanthus fruiting body development. J. Bacteriol. 141:305-315.[Abstract/Free Full Text]
  14. Moraleda-Muñoz, A., J. Carrero-Lérida, A. L. Extremera, J. M. Arias, and J. Muñoz-Dorado. 2001. Glycerol-3-phosphate inhibits swarming and aggregation of Myxococcus xanthus. J. Bacteriol. 183:6135-6139.[Abstract/Free Full Text]
  15. Moraleda-Muñoz, A., J. Carrero-Lérida, J. Pérez, and J. Muñoz-Dorado. 2003. Role of two novel two-component regulatory systems in development and phosphatase expression in Myxococcus xanthus. J. Bacteriol. 185:1376-1383.[Abstract/Free Full Text]
  16. Pham, V. D., C. W. Shebelut, M. E. Diodati, C. T. Bull, and M. Singer. 2005. Mutations affecting predation ability of the soil bacterium Myxococcus xanthus. Microbiology 151:1865-1874.[Abstract/Free Full Text]
  17. Pham, V. D., C. W. Shebelut, I. R. Jose, D. A. Hodgson, D. E. Whitworth, and M. Singer. 2006. The response regulator PhoP4 is required for late developmental events in Myxococcus xanthus. Microbiology 152:1609-1620.[Abstract/Free Full Text]
  18. Qi, Y., Y. Kobayashi, and F. M. Hulett. 1997. The pst operon of Bacillus subtilis has a phosphate-regulated promoter and is involved in phosphate transport but not in regulation of the Pho regulon. J. Bacteriol. 179:2534-2539.[Abstract/Free Full Text]
  19. Scanlan, D. J., S. A. Bloye, N. H. Mann, D. A. Hodgson, and N. G. Carr. 1990. Construction of lacZ promoter probe vectors for use in the cyanobacterium Synechococcus R2 PCC7942: application to the identification of CO2 regulated promoters. Gene 90:43-49.[CrossRef][Medline]
  20. Sola-Landa, A., R. S. Moura, and J. F. Martin. 2003. The two-component PhoR-PhoP system controls both primary metabolism and secondary metabolite biosynthesis in Streptomyces lividans. Proc. Natl. Acad. Sci. USA 100:6133-6138.[Abstract/Free Full Text]
  21. Su, Z., V. Olman, and Y. Xu. 2007. Computational prediction of Pho regulons in cyanobacteria. BMC Genomics 8:156-167.[CrossRef][Medline]
  22. Wanner, B. L. 1993. Gene regulation by phosphate in enteric bacteria. J. Cell. Biochem. 51:47-54.[CrossRef][Medline]
  23. Weinberg, R. A., and D. R. Zusman. 1990. Alkaline, acid, and neutral phosphatase activities are induced during development in Myxococcus xanthus. J. Bacteriol. 172:2294-2302.[Abstract/Free Full Text]
  24. Zhang, H., N. N. Rao, T. Shiba, and A. Kornberg. 2005. Inorganic polyphosphate in the social life of Myxococcus xanthus: motility, development, and predation. Proc. Natl. Acad. Sci. USA 102:13416-13420.[Abstract/Free Full Text]

Journal of Bacteriology, March 2008, p. 1997-2003, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01781-07
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