Study of the Relationship between Bacterial Motility and Inorganic Phosphate Metabolism Regulation

Abstract

Phosphorus is the essential nutrient for cellular function including energy metabolism and the maintenance of genome stability. Cells acquire phosphorous in the form of inorganic phosphate, and this uptake is regulated by the bacterial Pst system, a high-inorganic phosphate affinity transporting system. This transport system manages bacterial phosphate metabolism to maintain optimum levels of inorganic phosphate within the cell. The most of bacterial Pst systems are encoded by the pst operon, cosisting of pstS, pstC, pstA, pstB and phoU genes. They encode the PstS periplasmic binding protein, PstC and PstA membrane-spanning proteins forming the permease complex, PstB ATP-binding protein, and the regulatory protein PhoU. According to E. coli model, the regulation of this system expression is mediated by histidine kinase PhoR and response regulator PhoB two component siganl transduction system. In this regulatory mechanism, under conditions of high extracellular phosphate concentration, PhoU interacts with PhoR, inhibiting its autophosphorylation activity. This interaction prevents phosphorylation of PhoB, thus inhibiting the transcription of genes in PhoB regulon, including the pst operon. Conversely, when the extracellular phosphate concentrations are low, the decrease in PhoU and PhoR interactions allow PhoR to autophosphorylate and subsequently phosphorylate PhoB. This study found by chance that phoU mutation abrogates bacterial motility in an enteric pathogen Salmonella enterica serovar Typhimurium. Functions related to bacterial motility, adhesiveness, and cell invasion depend on flagella. In S. Typhimurium, flagella formation follows a hierarchical activation of Class I, II, and III genes for flagella biogenesis. Additionally, bacteria cause changes in gene expression of flagellins FliC (H1 antigen) and FljB (H2 antigen) through flagellar phase variation. The effect of regulation of inorganic phosphate metabolism on bacterial motility remains not explored. Based on the finding of PhoU-dependent Salmonella motility, this study has studied the relationship between the regulation of inorganic phosphate metabolism and bacterial motility. The phoU mutant strain significantly inhibited bacterial growth, while there was no significant difference in bacterial growth of phoB mutant, compared to WT strain. Additionally, in contrast to phoU mutant, phoB mutant strain showed similar motility to WT. Considering the fact that, in E. coli phoU mutant, activated PhoB increased the expression of the inorganic phosphate transport genes, which resulted in an increase in intracellular inorganic phosphate, these results suggest that activated PhoB due to phoU mutation can reduce bacterial growth and motility. In an analysis observing Hin-mediated flagellar phase variation, there were changes in the orientation of Hin-specific DNA segment in chromosomes of both phoU and phoB mutants, suggesting that phosphate metabolism may be involved in Hin-dependent phase variation. However, analysis on mRNA levels of genes for flagellar biogenesis were not consistent with the results from Hin-dependent phase variation, rather exhibited decrease in transcription of flagella class II genes, as well as class III genes. This suggest that there are the phosphate metabolism-related regulatory mechanisms for flagella biogenesis at the transcriptional level in addition to Hin-mediated phase variation. Additionally, phoU mutant could not invade to epithelial cells, suggesting that PhoU-dependent motility may affect bacterial invasion ability. This study demonstrates important roles of PhoU regulator in relating inorganic phosphate metabolism to flagella biogenesis. Further study is necessary to identify new genes regulated by PhoB to understand in more detail their effects on flagellar biogenesis and bacterial motility.