| Amino acids are important components of proteins,which are essential for biological metabolism,cell structure and growth.Histidine is a common and unique natural amino acid,its biosynthesis pathway has already been widely studied because of its important role in the growth and survival of bacteria.Histidinol-phosphate phosphatase(Hol-Pase,His N))is involved in the eighth step of histidine biosynthesis,where Hol-P is dephosphorylated to L-histidinol by Hol-Pase.Because its origin and evolution are more complex than other enzymes in histidine biosynthesis,it is usually the last enzyme been identified in many bacteria.Pseudomonas aeruginosa(PAO1)is an important gram-negative opportunistic pathogen,which can cause serious human infection.However,there is no information about His N in the KEGG database of PAO1.In a previous study,it was found that PA0335 encodes Hol-Pase(His N)of the histidine biosynthesis pathway of Pseudomonas aeruginosa,and it was found that the protein belongs to the HAD superfamily protein,which has alkaline phosphatase activity and can catalyze the transfer of phosphate groups of the substrate.According to the analysis of amino acid spectrum,the inactivation of PA0335 will cause incomplete histidine auxotrophy of PAO1,that is,it begins to grow after 80 hours of culture on MM medium without histidine.It is suggested that there might exist other isozymes of PA0335-encoded protein.Four PA0335 homologs genes,PA1143,PA3255,PA5547 and PA4960,has been found in the genome of PAO1,sharing 42%,38%,27% and 26% identity with PA0335 respectively according to homology alignment.It is speculated that there may be isozymes of PA0335-encoded proteins.The over expression vectors p AK1143,p AK3255,p AK5547,and p AK4960 were constructed and introduced into the deletion mutant of PA0335,and the growth level determined that PA1143 and PA3255 could complement the incomplete histidine auxotrophy of PAO1(Δ0335).It shows that PA1143 and PA3255 are involved in the histidine biosynthesis of PAO1.Bioinformatics analysis showed that the proteins encoded by PA1143,PA3255 and PA0335 belong to the HAD superfamily,and the encoded protein contains the conserved domains of the HAD superfamily.By detecting the enzyme activity of the in vitro expressed proteins of PA1143 and PA3255,it was confirmed that the proteins encoded by PA1143,PA3255 and PA0335 with alkaline phosphatase activity.Site-directed mutagenesis studies confirmed the importance of several conserved amino acid residues in the conserved domains of the HAD superfamily of proteins encoded by PA1143,PA3255 and PA0335.All results show that the proteins encoded by PA1143,PA3255 are isozymes of PA0335 and have Hol-Pase activity.Simultaneously,PAO1(Δ1143),PAO1(Δ3255)two single gene deletion mutants and PAO1(Δ0335-1143),PAO1(Δ0335-3255)double gene deletion mutants and PAO1(Δ0335-1143-3255)three gene deletion mutants were constructed respectively.By detecting the growth level,it is found that in the eighth step of PAO1 histidine biosynthesis,PA0335 occupies the main position,PA3255 plays the second role,and PA1143 plays the smallest role.In addition,in PA0335 and transcription regulator PA3249 double gene deletion mutant PAO1(Δ0335-3249)and single gene deletion mutant PAO1(Δ3249),it was found that PA3249 inhibits the gene expression of PA1143 and PA3255.It shows that the transcription regulator PA3249 has a close regulatory relationship with PA1143 and PA3255.In addition to PA0335,other potential genes his G,his E,his I,his A,his F1,his F2,his H1,his H2,his B,his C1,his C2,his D in the histidine biosynthetic pathway in Pseudomonas aeruginosa were also tested.By constructing 14 deletion mutants and testing the growth,it was determined that his G(PA4449),his E(PA5067),his F1(PA5140),his B(PA5143),his I(PA5066),his C1(PA4447)and his A(PA5141)were essential because deletion of each resulted in complete histidine auxotrophy.The deletion of his H1(PA5142)or his D(PA4448)can cause incomplete histidine auxotrophy.This study found that PA1143 and PA3255 can complement the incomplete histidine auxotrophy of PA0335 deletion mutants,and both have alkaline phosphatase activity.It is confirmed that PA1143 and PA3255-encoded protein are isozymes of PA0335-encoded protein.In addition to PA0335,the role of other 12 predictive genes in the histidine biosynthesis of Pseudomonas aeruginosa was also studied,and the histidine biosynthesis pathway of Pseudomonas aeruginosa was clarify.Meanwhile,some enzymes involved in the histidine biosynthesis pathway of Pseudomonas aeruginosa can be used as elements of histidine synthesis in other industrial microorganisms. |
