Regulation of stalk elongation by phosphate in Caulobacter crescentus

Abstract

In Caulobacter crescentus, stalk biosynthesis is regulated by cell cycle cues and by extracellular phosphate concentration. Phosphate-starved cells undergo dramatic stalk elongation to produce stalks as much as 30 times as long as those of cells growing in phosphate-rich medium. To identify genes involved in the control of stalk elongation, transposon mutants were isolated that exhibited a long-stalk phenotype irrespective of extracellular phosphate concentration. The disrupted genes were identified as homologues of the high-affinity phosphate transport genes pstSCAB of Escherichia coli. In E. coli, pst mutants have a constitutively expressed phosphate (Pho) regulon. To determine if stalk elongation is regulated by the Pho regulon, the Caulobacter phoB gene that encodes the transcriptional activator of the Pho regulon was cloned and mutated. While phoB was not required for stalk synthesis or for the cell cycle timing of stalk synthesis initiation, it was required for stalk elongation in response to phosphate starvation. Both pstS and phoB mutants were deficient in phosphate transport. When a phoB mutant was grown with limiting phosphate concentrations, stalks only increased in length by an average of 1.4-fold compared to the average 9-fold increase in stalk length of wild-type cells grown in the same medium. Thus, the phenotypes of phoB and pst mutants were opposite. phoB mutants were unable to elongate stalks during phosphate starvation, whereas pst mutants made long stalks in both high- and low-phosphate media. Analysis of double pst phoB mutants indicated that the long-stalk phenotype of pst mutants was dependent on phoB. In addition, analysis of a pstS-lacZ transcriptional fusion showed that pstS transcription is dependent on phoB. These results suggest that the signal transduction pathway that stimulates stalk elongation in response to phosphate starvation is mediated by the Pst proteins and the response regulator PhoB.

FIG. 1

Model of the Pho regulon and organization of pst and pho genes of Caulobacter. (A) The Caulobacter life cycle and effect of phosphate starvation. The life cycle of swarmer cells is depicted. The newborn swarmer cell spends an obligatory period of its life cycle as a chemotactically competent polarly flagellated cell unable to initiate DNA replication. Stalk synthesis is initiated at the pole that previously contained the flagellum coincidently with the initiation of DNA replication during the swarmer-to-stalked cell differentiation. The new stalked cell elongates, initiates cell division, and synthesizes a flagellum at the pole opposite the stalk, giving rise to an asymmetric predivisional cell. Cell division yields a stalked cell that can immediately initiate a new cell cycle and a swarmer cell. Phosphate starvation yields elongated cells with long stalks. (B) Model of the Pho regulon. This model is adapted from work with E. coli (52). The PstSCAB proteins form the high-affinity phosphate transport system. When phosphate is in excess, the Pst complex represses the autophosphorylation of the histidine kinase PhoR. PhoU is required to inhibit the expression of the Pho regulon, but is not required for phosphate transport by the Pst system. Deletion of phoU has deleterious effects on growth, and these effects are dependent on phoB (15). When cells are starved for phosphate, the Pst complex releases PhoR, which autophosphorylates and transfers the phosphate residue to PhoB. PhoB∼P binds to the Pho box sequences of promoters (−10 and bent arrow) and activates the transcription of most genes of the Pho regulon. In a few cases, binding of PhoB-∼P represses transcription. We hypothesize that PhoB∼P activates the transcription of a gene or genes whose expression results in an increase in stalk synthesis. (C) Organization of the pst-pho gene cluster. Genes are represented by arrows, and the sites of transposon insertion in the different mutants are represented by “lollipop” structures. The thick line labeled PCR under the region between pstC and pstA indicates the PCR product that was obtained with oligonucleotides from the end of the phoR-pstC sequence contig and the beginning of the pstA-pstB-phoU-phoB sequence contig. The pstS gene is shown below the pst-pho region because it maps to an unlinked locus.

FIG. 2

Morphology of wild-type and stalk mutant cells in different media. Strains were grown in either PYE (A, D, G, J, M, and P), HIGG medium containing 10 mM phosphate (B,E,H,K,N, and Q), or 30 μM phosphate (C,F,I,L,O, and R) until saturation. Phase-contrast micrographs of NA1000 (wild type) (A to C), YB720 (phoBΩ12) (D to F), YB767 (pstS1100) (G to I), YB779 (pstC1101) (J to L), YB778 (pstA1103) (M to O), and YB777 (pstB1105) (P to R) cells are shown. Images of cells were captured with a digital camera and analyzed with the Metamorph Imaging Software package, version 3.0. The cells shown are representative of the population.

FIG. 3

Phosphate uptake in different Caulobacter strains. ³²P_i was added to a final concentration of 20 μM. Phosphate uptake was measured on cells grown in HIGG medium containing 10 mM phosphate (open symbols) or no phosphate (solid symbols) for 5 h. Results are shown for NA1000 (wild type) (squares), YB720 (phoBΩ12) (circles), YB767 (pstS1100) (triangles), and YB770 (phoBΩ12 pstS1100) (diamonds).

FIG. 4

Complementation of the phoBΩ12 mutant. Cells were grown for 48 h in HIGG medium containing 0.03 mM phosphate. (A) NA1000 (wild type). (B) YB732 (phoBΩ12/pGL10phoB). (C) YB720 (phoBΩ12). The cells shown are representative of the population.

FIG. 5

Promoter region of pstS and comparison of putative Pho box sequences. (A) Sequence upstream of the pstS gene. Pho box-like sequences are indicated by numbered arrows, with the consensus E. coli Pho box sequence shown above the Caulobacter DNA sequence for comparison. The stop codon (TGA) of the upstream PBP1A gene is shown in boldface. The predicted N-terminal amino acid sequence of PstS is indicated. The sequence shown is to the site of miniTn5lacZ insertion. (B) Comparison of Pho box-like sequences found upstream of pstS and pstC with the consensus Pho box sequence of E. coli.

FIG. 6

Construction of a nonpolar disruption in pstB. An internal fragment of the pstB gene (a and b) was cloned into pBGST18 (a′ and b′) in the same orientation as the lacZ promoter of the plasmid (Plac). Integration of the plasmid by a single crossover generates two truncated copies of pstB. pstB′ is missing C-terminal coding sequences, and ′pstB is missing N-terminal coding sequences. The Plac promoter of the plasmid ensures transcription of the downstream genes.

FIG. 7

Comparison of polar and nonpolar disruptions in pstB and a polar disruption in the unlinked pstS gene in a wild-type background and a phoB mutant background. Phase-contrast micrographs of representative cells of NA1000 (wild type) (A), YB720 (phoBΩ12) (B), YB767 (pstS1100) (C), YB770 (pstS1100 phoBΩ12) (D), YB1684 (pstB::np) (E), YB1686 (pstB::np phoBΩ12) (F), YB777 (pstB1105) (G), and YB784 (pstB1105 phoBΩ12) (H) are shown. Strains in the left panels have the phoB⁺ allele (A, C, E, and G), and strains in the right panels (B, D, F, and H) have the phoBΩ12 allele.