An intracellular phosphorus-starvation signal activates the PhoB/PhoR two-component system in Salmonella enterica

Bacteria acquire P primarily as inorganic orthophosphate (Pi, PO43−). Once internalized, Pi is rapidly assimilated into biomass during the synthesis of ATP. Because Pi is essential, but excessive ATP is toxic, the acquisition of environmental Pi is tightly regulated. In the bacterium Salmonella enterica (Salmonella), growth in Pi-limiting environments activates the membrane sensor histidine kinase PhoR, leading to the phosphorylation of its cognate transcriptional regulator PhoB and subsequent transcription of genes involved in adaptations to low Pi. Pi limitation is thought to promote PhoR kinase activity by altering the conformation of a membrane signaling complex comprised by PhoR, the multicomponent Pi transporter system PstSACB and the regulatory protein PhoU. However, the identity of the low Pi signal and how it controls PhoR activity remain unknown. Here we characterize the PhoB-dependent and independent transcriptional changes elicited by Salmonella in response to P starvation, and identify PhoB-independent genes that are required for the utilization of several organic-P sources. We use this knowledge to identify the cellular compartment where the PhoR signaling complex senses the Pi-limiting signal. We demonstrate that the PhoB and PhoR signal transduction proteins can be maintained in an inactive state even when Salmonella is grown in media lacking Pi. Our results establish that PhoR activity is controlled by an intracellular signal resulting from P insufficiency.


Supporting Information
. RNA-Seq summary.  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   Putative Pho boxes on the positive and negative strands were predicted with MEME Suit (11).
Primary and secondary transcription start sites have been experimentally inferred (12,13).
Putative translation start/stop sites were obtained from the genome annotation (14).
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Figure S1. (A) Fluorescence from wild-type (14028s) and phoB (EG9054) Salmonella carrying pPphoB-gfpAAV or pPpstS-gfpAAV. Cultures were propagated to mid-logarithmic phase in MOPS medium containing 1 mM K2HPO4. Subsequently, cultures were either subjected to a nutritional downshift to MOPS medium lacking K2HPO4 or any other alternative P source (-P) or maintained in the same medium containing 1 mM K2HPO4 (+Pi) as control (see Materials and Methods for further experimental details). The maximum GFP expression from phoB and pstS transcriptional fusions occurs at 35 min following the removal of P, and is indicated with a «. (B) Growth curve of wild-type (14028s) or phoB (EG9054) Salmonella harboring pPpstS-gfpAAV subjected to the same nutritional shifts described in (A). (C) Viable cell counts of wild-type (14028s) and phoB (EG9054) Salmonella at the beginning (t0) and the end (t35) of Pi downshift. *** P < 0.001, paired two-tailed t test. In all cases, means ± SDs of at least three independent experiments are shown.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 23, 2023. ; https://doi.org/10.1101/2023.03.23.533958 doi: bioRxiv preprint Figure S2. Salmonella PhoB-independent response to P starvation. Heatmaps depicting fold changes in transcript levels between -P and +Pi treatments for wild-type (14028s) and phoB (EG9054) Salmonella. Graphs show selected transcripts from genes that show no significant changes between wild-type and phoB during P starvation (Table  S1). Displayed genes are organized by COG categories. Note that gene RS07905 corresponds to NCBI gene locus tag STM14_RS07905).
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 23, 2023. ; https://doi.org/10.1101/2023.03.23.533958 doi: bioRxiv preprint Figure S3. Salmonella PhoB binding motif. Sequence logo of the PhoB binding sites. The logo was generated by the MEME algorithm using as input 450-bp upstream of the translation start site of the canonical PhoB-activated operons listed in Table S3 (6). . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Plates were incubated at 37°C during 14-18 h before being imaged. Images are representative of three independent experiments. Dashed lines separate non-contiguous sections of the same plate. Note the presence of P-compound(s) in the noble agar that allows the formation of small Salmonella colonies. This is not observed during growth in liquid medium (A).
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   Fig. 6. Prior to performing the readings, cultures were grown to mid-logarithmic phase in MOPS medium containing 1 mM Pi (K2HPO4), washed in MOPS medium lacking a P source and resuspended in MOPS medium containing 1 mM of the indicated organic P source (G6-P: glucose 6-P; F6-P: fructose 6-P; PGA: 3phosphoglyceric acid; ATP: adenosine triphosphate), 1 mM Pi (K2HPO4) or lacking P. Means ± SDs of at least three independent experiments are shown. (F) (Left) growth curve . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 23, 2023. ; https://doi.org/10.1101/2023.03.23.533958 doi: bioRxiv preprint and (right) corresponding fluorescence of wild-type (14028s) and argH (EG6537) Salmonella harboring pPpstS-gfp or the vector control (pVector, pFPV25). Cultures were grown to mid-logarithmic phase in MOPS medium containing 1.6 mM arginine and 1 mM Pi (K2HPO4), washed in MOPS medium lacking arginine and a P source. Cells were subsequently resuspended in either MOPS medium containing or lacking 1.6 mM arginine (+Arg; -Arg) and 1 mM K2HPO4 (+Pi; -Pi). Growth and green fluorescence were then monitored for 8 h. (G) (Left) growth curve and (right) corresponding fluorescence of wild-type (14028s) and manA (MP50) Salmonella harboring pPpstS-gfp or the vector control (pVector, pFPV25). Cultures were grown to mid-logarithmic phase in MOPS medium containing 22 mM glucose and 1 mM K2HPO4. Cells were washed in MOPS medium lacking carbon and P and then resuspended in MOPS medium containing either 22 mM mannose (+Man) or 22 mM glucose (+Glc) as C sources, and either with or without 1 mM K2HPO4 (+Pi; -Pi). Growth and green fluorescence were monitored for the next 8 h. Graphs depicted in F-G show the means ± SDs of at least three biological replicates are shown and are representatives of at least 3 experiments.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 23, 2023. ; https://doi.org/10.1101/2023.03.23.533958 doi: bioRxiv preprint