Identification and characterization of alternative homologs of histidinol-phosphate phosphatase in Pseudomonas aeruginosa

Abstract

HolPase is a crucial enzyme catalyzing dephosphorylation of L-histidinol-phosphate (Hol-P) to L-histidinol, usually the last and most difficult enzyme to be identified in histidine biosynthesis due to its complex origin and evolution. We previously reported that PA0335 encode a HolPase belonging to the HAD family and inactivation resulted incomplete L-histidine auxotrophy in Pseudomonas aeruginosa, suggesting the presence of alternate HolPase enzymes. In this study, the alternative homologs of PA0335 encoded HolPase were identified and primarily characterized in P. aeruginosa. PA3255 and PA1143 code phosphatase with side HolPase activity. PA0335 play a key role in histidine biosynthesis, PA3255 respond mainly for the residual growth when PA0335 HolPase is absence. Moreover, all three genes, especially for PA3255, are required for the resistance of PA to ciprofloxacin and meropenem, while PA3255 and PA0335 are required for the full virulence but inactivation of PA1143 increased the pathogens of P. aeruginosa. PA0335 and PA3255 could be used as targets for P. aeruginosa infection treatment separately or synergistically due to their participates in histidine auxotrophy, full virulence and obvious antibiotics resistance in P. aeruginosa. The relation and regulation of the three phosphatases with HolPase or side HolPase activity need further study.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12866-025-04092-3.

Keywords: P. aeruginosa; Antibiotics resistance; Conserved residue; Histidine biosynthesis; Histidinol-phosphate phosphatase; Isoenzyme; Pathogenesis.

Fig. 1

Bioinformatic analysis for the four homologs of PA0335. A Phylogenetic tree was constructed via neighbor-joining method to compare the relationship of the four PA0335 homologs and others from various organisms. The values at the forks indicate bootstrap values; B Multiple sequence alignment of PA0335, the four homologous sequences of PA1143, PA3255, PA5547 and PA0496 as well as the others of HAD family. Threshold for shading is > 70%, similar residues are marked as gray shadow, identical residues as black shadow and the red shadow highlights the conservative amino acid residues in all motifs (I to IV) of the HAD superfamily

Fig. 2

Growth determination for the four potential complements on MM medium containing glucose or histidine at 37 °C for different hours. A Growth of the four potential complements on MM agar plates containing glucose or histidine at 37 °C for different hours. Lane1: WT (pAK1900); Lane2: ΔPA0335 (pAK1900); Lane3: ΔPA0335 (pAK0335); Lane4: ΔPA0335 (pAK3255); Lane 5: ΔPA0335 (pAK1143); Lane 6: ΔPA0335 (pAK5547); Lane 7: ΔPA0335 (pAK4960); B Growth of ΔPA0335 (pAK3255) (diamonds) and ΔPA0335 (pAK1143) (triangles) in MM broth containing glucose at 37 °C for 24 h using WT (pAK-1900) (open circle), ΔPA0335 (pAK0335) (solid circle) and ΔPA0335 (pAK1900) (open squares) as controls. The assays were independently repeated at least three times and the data shown are the averages

Fig. 3

Heterologous expression of the proteins encoded by PA1143, PA3255 and PA0335. A SDS-PAGE analysis for the fusion proteins from E. coli BL21(DE3). Lane 1–4: E. coli (pGEX-0335), E. coli (pGEX-1143), E. coli (pGEX-3255), E. coli (pGEX-4 T-1) without IPTG induction; Lane 5–8: E. coli (pGEX-0335), E. coli (pGEX-1143), E. coli (pGEX-3255), E. coli (pGEX-4 T-1) with IPTG induction. B Analysis of alkaline phosphatase activity derived from E. coli (pGEX-0335), E. coli (pGEX-1143), E. coli (pGEX-3255), E. coli (pGEX-4 T-1). The values shown are the average of three independent experiments and error bars represent the standard deviation. Statistical significance was assessed by Student’s t-tests (* p < 0.05, ** p < 0.01)

Fig. 4

Expression profiles of the HolPase coding genes on histidine- and glucose-containing MM broth. A Histidine containing MM broth; B Glucose containing MM broth. PA0335: squares; PA1143: triangles; PA3255: diamonds; pMS-402: circles, open symbols for expression and solid symbols for corresponding growth. The assays were independently repeated at least three times and the data shown are the averages

Fig. 5

Growth of the mutants on MM agar plates containing glucose or histidine. A Growth of WT, ΔPA0335, ΔPA1143 andΔPA3255 for 24 h; B Growth of WT, ΔPA0335, ΔPA0335ΔPA1143, ΔPA0335ΔPA3255 and ΔPA0335ΔPA1143ΔPA3255 on MM agar plates containing glucose for 120 h, MM agar plates containing histidine for 24 h

Fig. 6

Effect of the conserved residues on the enzymic function of PA3255, PA1143 and PA3255. A Growth of ΔPA0335 supplemented with the plasmid pAK0335 containing no or single substitution at the conserved residue on MM agar plates containing glucose or histidine; B Growth of ΔPA0335 supplemented with the plasmid pAK1143 containing no or single substitution at the conserved residue; C Growth of ΔPA0335 supplemented with the plasmid pAK3255 containing no or single substitution at the conserved residue

Fig. 7

Involvement of PA3255, PA0335 and PA1143 in Cip or Mem resistance in PA. A Expression profiles of PA0335, PA1143 and PA3255 on LB broth with or without 1/2MIC (MIC = 1.1) Cip or (MIC = 1.0) Mem. WT (pMS402) (circle), WT (pKD-0335) (square), WT (pKD-1143) (triangle), WT (pKD-3255) (diamond), solid symbols for antibiotics presence and open for antibiotics absence; B Antibiotics susceptibility of ΔPA0335, ΔPA1143and ΔPA3255. 2.5 µg Cip and 5 µg Mem were added to the filter disks; C: Biofilms formation of ΔPA0335, ΔPA1143 and ΔPA3255 in LB broth with or without (MIC = 0.6) Cip or (MIC = 0.4) Mem at 1/2 MIC using WT as a control. The assays were independently repeated at least three times and the data shown are the averages. Statistical significance in gene expression was assessed using one-way repeated-measures ANOVA and others by Student’s t-tests (ns p > 0. 05, * p < 0. 05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Fig. 8

Virulence analysis via Chinese cabbage and Drosophila melanogaster infection models. A Chinese cabbage; B Drosophila melanogaster; WT (circle), ΔPA0335 (square), ΔPA1143 (triangles), ΔPA3255 (diamond). Sucrose (5%) was used as an uninfected control (asterisk). The assays were independently repeated at least three times and the data shown are the averages. Statistical significance on survival rate was assessed by one-way repeated-measures ANOVA and others by Student’s t-tests (ns p > 0. 05, * p < 0. 05, ** p < 0.01)