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. 2020 Feb 18;86(5):e02593-19. doi: 10.1128/AEM.02593-19

PA0335, a Gene Encoding Histidinol Phosphate Phosphatase, Mediates Histidine Auxotrophy in Pseudomonas aeruginosa

Yulu Wang a,#, Liyue Wang a,#, Jian Zhang a, Xintong Duan a, Yuqi Feng a, Shiwei Wang a,✉, Lixin Shen a,✉
Editor: Harold L Drakeb
PMCID: PMC7028973  PMID: 31862725

Histidine is a common amino acid in proteins. Because it plays critical roles in bacterial metabolism, its biosynthetic pathway in many bacteria has been elucidated. However, the pathway remains unclear in Pseudomonas aeruginosa, an important opportunistic pathogen in clinical settings; in particular, there is scant knowledge about histidinol-phosphate phosphatase (Hol-Pase), which has a complex origin and evolution. In this study, P. aeruginosa Hol-Pase was identified and characterized. Furthermore, the roles of all other predicted genes involved in histidine biosynthesis were examined. Our results illustrate the histidine synthesis pathway of P. aeruginosa. The knowledge obtained from this study may help in developing strategies to control P. aeruginosa-related infections. In addition, some enzymes of the histidine synthesis pathway from P. aeruginosa might be used as elements of histidine synthetic biology in other industrial microorganisms.

KEYWORDS: Pseudomonas aeruginosa, histidine synthesis, histidinol-phosphate phosphatase, histidine auxotrophy

ABSTRACT

The biosynthesis of histidine, a proteinogenic amino acid, has been extensively studied due to its importance in bacterial growth and survival. Histidinol-phosphate phosphatase (Hol-Pase), which is responsible for the penultimate step of histidine biosynthesis, is generally the last enzyme to be characterized in many bacteria because its origin and evolution are more complex compared to other enzymes in histidine biosynthesis. However, none of the enzymes in histidine biosynthesis, including Hol-Pase, have been characterized in Pseudomonas aeruginosa, which is an important opportunistic Gram-negative pathogen that can cause serious human infections. In our previous work, a transposon mutant of P. aeruginosa was found to display a growth defect on glucose-containing minimal solid medium. In this study, we found that the growth defect was due to incomplete histidine auxotrophy caused by PA0335 inactivation. Subsequently, PA0335 was shown to encode Hol-Pase, and its function and enzymatic activity were investigated using genetic and biochemical methods. In addition to PA0335, the roles of 12 other predicted genes involved in histidine biosynthesis in P. aeruginosa were examined. Among them, hisC2 (PA3165), hisH2 (PA3152), and hisF2 (PA3151) were found to be dispensable for histidine synthesis, whereas hisG (PA4449), hisE (PA5067), hisF1 (PA5140), hisB (PA5143), hisI (PA5066), hisC1 (PA4447), and hisA (PA5141) were essential because deletion of each resulted in complete histidine auxotrophy; similar to the case for PA0335, hisH1 (PA5142) or hisD (PA4448) deletion caused incomplete histidine auxotrophy. Taken together, our results outline the histidine synthesis pathway of P. aeruginosa.

IMPORTANCE Histidine is a common amino acid in proteins. Because it plays critical roles in bacterial metabolism, its biosynthetic pathway in many bacteria has been elucidated. However, the pathway remains unclear in Pseudomonas aeruginosa, an important opportunistic pathogen in clinical settings; in particular, there is scant knowledge about histidinol-phosphate phosphatase (Hol-Pase), which has a complex origin and evolution. In this study, P. aeruginosa Hol-Pase was identified and characterized. Furthermore, the roles of all other predicted genes involved in histidine biosynthesis were examined. Our results illustrate the histidine synthesis pathway of P. aeruginosa. The knowledge obtained from this study may help in developing strategies to control P. aeruginosa-related infections. In addition, some enzymes of the histidine synthesis pathway from P. aeruginosa might be used as elements of histidine synthetic biology in other industrial microorganisms.

INTRODUCTION

Amino acids are essential for maintaining bacterial cell morphology and growth: they are involved in protein synthesis, affect RNA synthesis, and mediate the expression of signaling molecules (1, 2). Most amino acids are synthesized from intermediates of the tricarboxylic acid cycle, the glycolytic pathway, or the pentose phosphate pathway (3–6). However, the biosynthesis of histidine originates from phosphoribosyl pyrophosphate (PRPP), an extremely important intermediate in cellular metabolism (7). Histidine biosynthesis has been thoroughly studied, and it appears to be conserved, with nine enzymes catalyzing 10 reactions in bacteria, such as Escherichia coli, Salmonella enterica serovar Typhimurium, and Corynebacterium glutamicum (8–10). For example, in C. glutamicum the histidine biosynthesis pathway is as follows (Fig. 1). First, PRPP and ATP are condensed to phosphoribosyl-ATP (PR-ATP) catalyzed by ATP phosphoribosyl transferase (HisG); PR-ATP is further hydrolyzed to generate phosphoribosyl-AMP (PR-AMP) by phosphoribose-ATP pyrophosphatase (HisE). Phosphoribosyl-AMP cyclohydrolase (HisI) then modifies PR-AMP to produce 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino) methylideneamino] imidazole-4 carboxamide (5′ProFAR), which is converted to 5-[(5-phospho-1-deoxyribulos-1-ylamino) methylideneamino]-1-(5 phosphoribosyl) imidazole-4-carboxamide (PRFAR) by 5’ProFAR isomerase (HisA). In the fifth step, PRFAR is transformed to imidazole-glycerol-phosphate (IGP) by IGP synthase (HisFH); IGP is sequentially dehydrated and transaminated by IGP dehydratase (HisB) and histidine phosphotransaminase (HisC) to form imidazole-acetol-phosphate and l-histidinol-phosphate (Hol-P), respectively. Hol-P is then dephosphorylated to l-histidinol via histidinol-phosphate phosphatase (Hol-Pase, HisN). Finally, a single enzyme, histidinol dehydrogenase (HisD), catalyzes the last two steps of histidine biosynthesis to sequentially transform l-histidinol to l-histidinal and l-histidine. In C. glutamicum, the nine enzymes are encoded by ten genes: hisGEIA(FH)BCND. However, in E. coli and S. enterica Typhimurium, the nine enzymes are encoded by nine genes, hisGEIA(FH)BCD, because the sixth and eighth steps are both catalyzed by the bifunctional enzyme HisB encoded by hisB, which possesses IGP dehydratase and Hol-Pase activity (8). It has been reported that inactivation in any of the eight enzymes (HisGEIAFBCD) in C. glutamicum or E. coli results in complete histidine auxotrophy (8, 11), though deletion of HisN in C. glutamicum results in incomplete histidine auxotrophy (12).

FIG 1.

FIG 1

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Histidine biosynthetic pathway in C. glutamicum. The abbreviations used for enzymes are indicated in boldface. The corresponding products are shown in yellow boxes.

Hol-Pase belongs to the haloacid dehalogenase (HAD) family, which catalyzes phosphoryl group transfer of a variety of substrates (13, 14). HAD family members include 2-l-haloalkanoic acid dehalogenase, azetidine hydrolase, phosphonoacetaldehyde hydrolase, phosphoserine phosphatase, phosphomannomutase, and P-type ATPase (13, 15). They all use aspartate as an active-site residue for nucleophilic catalysis and contain four highly conserved motifs, namely, hhhDxDx(T/V) (L/V)h, hhhhhh (S/T), [KR], and (G/S) (D/S)×3-4 (D/E) hhhh (where h represents a hydrophobic residue and x any amino acid) (15–18).

Pseudomonas aeruginosa is an important opportunistic Gram-negative pathogen known for its metabolic diversity and ability to cause serious human infections (19, 20). Except for the hisN gene encoding Hol-Pase, all other genes corresponding to the other nine enzymes in histidine biosynthesis have been predicted according to the KEGG pathway database (https://www.kegg.jp/kegg/pathway.html) and the P. aeruginosa genome database (http://www.pseudomonas.com) (Table 1). However, their function remains to be proven experimentally.

TABLE 1.

Proposed genes involved in the histidine biosynthesis in PAO1

Catalytic enzyme Gene Function
HisG PA4449 ATP-phosphoribosyltransferase
HisE PA5067 Phosphoribosyl-ATP pyrophosphohydrolase
HisI PA5066 Phosphoribosyl-AMP cyclohydrolase
HisA PA5141 Phosphoribosylformimino-5-aminoimidazole carboxamide
HisF1 PA5140 Imidazoleglycerol-phosphate synthase, cyclase subunit
HisF2 PA3151 Imidazoleglycerol-phosphate synthase, cyclase subunit
HisH1 PA5142 Glutamine amidotransferase
HisH2 PA3152 Glutamine amidotransferase
HisB PA5143 Imidazoleglycerol-phosphate dehydratase
HisC1 PA4447 Histidinol-phosphate aminotransferase
HisC2 PA3165 Histidinol-phosphate aminotransferase
HisD PA4448 Histidinol dehydrogenase
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In our previous study, a transposon mutant of P. aeruginosa PAO1 was found to display a growth defect on solid minimal medium (MM) containing 0.2% glucose (21). In the present study, the growth defect was found to be due to incomplete histidine auxotrophy, which was caused by PA0335 inactivation. Furthermore, PA0335 was proven to encode Hol-Pase, and the function and enzymatic activity of this protein in histidine biosynthesis of PAO1 was investigated by genetic and biochemical methods. In addition to PA0335, the roles of 12 other predicted genes involved in histidine biosynthesis in P. aeruginosa were examined. Taking all the results together with the previous literature, we outlined the histidine synthesis pathway of P. aeruginosa.

RESULTS

Inactivation of PA0335 caused incomplete auxotrophy of P. aeruginosa PAO1.

In a previous study, a transposon mutant with a growth defect on MM agar containing 0.2% glucose was found (21). The transposon insertion site was found to be located in PA0334. It is noted that two genes, PA0334 and PA0335, comprise an operon. To determine which gene(s) is related to the growth defect, a mutant of PAO1(Δ0334-5) and three complementation strains containing the gene(s) PA0334-5, PA0334, and PA0335, respectively, were constructed. The growth of the mutant and complementation strains was measured on glucose-containing MM agar. As shown in Fig. 2A, compared to the wild-type strain PAO1, the mutant PAO1(Δ0334-5) showed an obvious growth defect; the introduction of PA0334-5 and PA0335, but not PA0334, exhibited growth restoration of PAO1(Δ0334-5). To further distinguish the role of PA0334 and PA0335 in PAO1, two single gene deletion mutants, PAO1(Δ0334) and PAO1(Δ0335), and their complementation strains, PAO1(Δ0334)C and PAO1(Δ0335)C, were constructed. A growth assay using glucose-containing MM agar showed that inactivation of PA0335 resulted in the growth defect and that its complementation strain PAO1(Δ0335)C rescued the defect (Fig. 2B). However, PA0334 deletion did not affect PAO1 growth because the growth of PAO1(Δ0334) and its complementation strain was similar to that of PAO1 (Fig. 2B). In liquid MM containing 0.2% glucose, the mutant PAO1(Δ0335) displayed slow growth until 80 h compared to wild-type PAO1 and its complementation strain PAO1(Δ0335)C (Fig. 2C). Taken together, these results indicated that inactivation of PA0335 results in growth defects and incomplete auxotrophy of PAO1 on MM agar.

FIG 2.

FIG 2

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Growth of the mutants and complements on MM agar containing glucose. (A) Growth of PAO1, PAO1(Δ0334-5), PAO1(Δ0334-5)C1, PAO1(Δ0334-5)C2, and PAO1(Δ0334-5)C3 on glucose-containing MM agar at 37°C for 30 h. PAO1(Δ0334-5)C1, PAO1(Δ0334-5)C2, and PAO1(Δ0334-5)C3 were respectively complemented by the genes PA0334-5, PA0334, and PA0335. (B) Growth of PAO1, PAO1(Δ0334), PAO1(Δ0335), PAO1(Δ0334)C, and PAO1(Δ0335)C on glucose-containing MM agar at 37°C for 30 h. PAO1(Δ0334)C and PAO1(Δ0335)C were respectively complemented by the gene PA0334 and PA0335. (C) Growth of PAO1, PAO1(Δ0335), and PAO1(Δ0335)C in glucose-containing MM broth at 37°C for 120 h, PAO1 (circles), PAO1(Δ0335) (triangles), and PAO1 (Δ0335)C (squares).

PA0335 is involved in histidine biosynthesis in P. aeruginosa PAO1.

To investigate the reason for the growth defect of PAO1(Δ0335) on MM agar, different nutrient sources, including glycerol, galactose, xylose, sucrose, mannitol, vitamin B1, citrate, succinic acid salt, pyruvate, urea, and casein hydrolysate, were added, and the growth of PAO1, PAO1(Δ0335), and PAO1(Δ0335)C was examined. The results showed that only casein hydrolysate rescued the growth defect of PAO1(Δ0335) on MM agar; the growth of the three strains was similar on Luria-Bertani (LB) agar, indicating that the growth defect of PAO1(Δ0335) on MM agar was probably due to amino acid auxotrophy. To test our presumption, we examined the growth of PAO1(Δ0335) in liquid MM supplemented with a mixture of 20 amino acids or glucose. The results showed that the 20-amino-acid mixture, but not glucose, restored the growth defect of PAO1(Δ0335) in liquid MM (Fig. 3B), consistent with our hypothesis.

FIG 3.

FIG 3

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Quantification of bacterial growth of PAO1, PAO1(Δ0335), and PAO1(Δ0335)C. (A) Growth of PAO1, PAO1(Δ0335), and PAO1(Δ0335)C on MM agar containing different nutrient sources at 37°C for 30 h. (B) Growth of PAO1(Δ0335) in MM broth containing glucose (squares) or a mixture of 20 amino acids (circles).

To determine which amino acid(s) is involved in the growth defect of PAO1(Δ0335) on MM agar, the 20 amino acids were divided into six groups with different combinations and added to MM agar. PAO1(Δ0335) exhibited growth similar to that of wild-type PAO1 only in groups 2 and 6, and the overlapping amino acid between these two groups was histidine, indicating that the growth defect of PAO1(Δ0335) might be due to the failure of histidine synthesis. Furthermore, supplementation of MM agar with histidine restored the growth of PAO1(Δ0335) to the level of PAO1 (Fig. 4B). All of these results indicate that deficiency in histidine synthesis resulted in the growth defect of PAO1(Δ0335) on MM agar and that PA0335 might be involved in the histidine biosynthesis pathway.

FIG 4.

FIG 4

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Identification of histidine auxotroph. (A) Growth of PAO1(Δ0335) in six groups of amino acids with different combinations. Spot 1, mixture of Lys, Arg, Met, Cys, Leu, and Ile; spot 2, mixture of Val, Arg, Phe, Tyr, Trp, and His; spot 3, mixture of Thr, Met, Phe, Glu, Pro, and Asp; spot 4, mixture of Ala, Cys, Tyr, Glu, Gly and Ser; spot 5, mixture of Gln, Leu, Trp, Pro, Gly, and Asn; spot 6, mixture of Gln, Ile, His, Asp, and Ser. (B) Growth of PAO1, PAO1(Δ0335), and PAO1(Δ0335)C on MM agar containing histidine.

Bioinformatic analysis of the PA0335-encoded protein.

Although in the P. aeruginosa genome and KEGG pathway databases the gene product of PA0335 is described as a hypothetical protein, the KEGG pathway database also showed that it contains motifs of HAD and hydrolase. Furthermore, a BLASTP search indicated that the protein encoded by PA0335 shares various identity (24% ∼ 47%) with some proteins in other bacteria, including Bordetella pertussis Tohama, Neisseria meningitidis, Vibrio cholerae, Mycobacterium tuberculosis, Campylobacter jejuni, and E. coli. All these proteins have been predicted as HAD superfamily members (22–33). The phylogenetic tree of the homologous proteins showed that PA0335 has a close relationship with 3FVV_A of 71 and NMB1075 of 98 (Fig. 5A). It has been reported that all HAD superfamily members contain four conserved domains (18, 34, 35). Multiple sequence alignment analysis of PA0335 and PA0335-like proteins showed that the protein encoded by PA0335 possesses all four conserved motifs of the HAD superfamily, as described above (Fig. 5B). These results suggest that PA0335 likely encodes an HAD superfamily protein.

FIG 5.

FIG 5

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Characterization of PA0335 by bioinformatics analysis. (A) Phylogenetic tree constructed using the neighbor-joining method to compare the relationship between PA0335-encoded protein and other homologous proteins from various organisms. The numbers above the branches refer to bootstrap values. (B) Multiple sequence alignment of PA0335-encoded protein, 3FVV_A, NMB1075-Nme, PA1143, VC1940-Vch, Cj0733-Cje, Rv3661-Mtu, and PgpC-Eco. Identical residues are black shadowed, similar residues are gray shadowed, and the conserved amino acid residues in all motifs (I to IV) of the HAD superfamily are highlighted by red shading.

The PA0335-encoded protein shows phosphatase activity.

In the histidine biosynthesis pathway of PAO1, the only enzyme belonging to the HAD family is Hol-Pase (8), a type of alkaline phosphatase (AP) that catalyzes phosphate group transfer from Hol-P to l-histidinol. To explore whether the gene product of PA0335 has AP activity, PA0335 was cloned into the pET-28a expression vector and heterologously expressed in E. coli BL21(DE3). The produced protein was approximately 27 kDa according to SDS-PAGE (Fig. 6A), consistent with the hypothetical molecular weight. Further analysis showed that the protein produced in E. coli BL21(DE3) showed obvious AP activity compared to the strain carrying empty vector (Fig. 6B). These results strongly show that the PA0335-encoded protein is a Hol-Pase with AP activity.

FIG 6.

FIG 6

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Heterologous expression of PA0335-encoded protein. (A) SDS-PAGE analysis of PA0335-encoded protein expression in E. coli BL21(DE3). Column 1, E. coli BL21(pET-28a); column 2, E. coli BL21(pET-0335). (B) Alkaline phosphatase activity analysis of E. coli(pET-28a) and E. coli BL21(pET-0335). The values shown are the averages of three independent experiments. Error bars indicate standard deviations. Statistical analysis was performed using a Student t test. *, P < 0.05. (C) Growth of PAO1, PAO1(Δ0335), and PAO1(Δ0335)C1 on glucose-containing MM agar at 37°C for 30 h.

HisN from C. glutamicum rescues the defective growth of PAO1(Δ0335).

Because Hol-Pase encoded by PA0335 is similar to HisN from C. glutamicum (8), we hypothesized that HisN from C. glutamicum might be able to rescue the defective growth of PAO1(Δ0335). To verify whether our expectation is correct, HisN from C. glutamicum was cloned into PAO1(Δ0335) to produce the complementation strain PAO1(Δ0335)C1. Consequently, PAO1(Δ0335)C1 displayed the same growth as the wild-type strain PAO1 and PAO1(Δ0335)C on glucose-containing MM agar, whereas PA(Δ0335) could not grow under the same conditions (Fig. 6C). These results confirmed that Hol-Pase from C. glutamicum can completely complement the inactivation of PA0335 from PAO1.

Determining the roles of other genes predicted to be involved in histidine biosynthesis in P. aeruginosa PAO1.

In addition to PA0335, 12 other genes have been predicted to encode the eight remaining enzymes involved in histidine biosynthesis in PAO1, according to the KEGG database. Histidinol-phosphate aminotransferase (HisC) is the assigned function for PA3165 (hisC1) or PA4447 (hisC2), and IGP synthase with 2 subunits (HisF and HisH) is proposed to be encoded by four different genes, namely, hisF1 (PA5140), hisF2 (PA3151), hisH1 (PA5142), and hisH2 (PA3152). The six other enzymes—HisG, HisE, HisA, HisI, HisB, and HisD—are predicted to be encoded by single genes, namely, hisG (PA4449), hisE (PA5067), hisA (PA5141), hisI (PA5066), hisB (PA5143), and hisD (PA4448), respectively.

To experimentally verify the roles of these 12 genes in the histidine synthesis of PAO1, 14 deletion mutants, including 12 single-gene mutants and 2 double-gene mutants [PAO1(ΔhisF1H1) and PAO1(ΔhisF2*H2)], were constructed, and their growth on LB and MM agar containing glucose or histidine was examined (Fig. 7). According to the results, PAO1(ΔhisG), PAO1(ΔhisE), PAO1(ΔhisI), PAO1(ΔhisA), PAO1(ΔhisC1), PAO1(ΔhisF1), PAO1(ΔhisB), and PAO1(ΔhisF1H1) could not grow on glucose-containing MM agar; in contrast, PAO1(ΔhisD) and PAO1(ΔhisH1) displayed inhibited growth, and PAO1(ΔhisC2), PAO1(ΔhisF2), PAO1(ΔhisH2), and PAO1(ΔhisF2H2) showed growth similar to that of the wild-type strain PAO1. Moreover, all mutants displayed growth similar to that of the wild-type strain PAO1 on histidine-containing MM and LB plates (data not shown). These results confirmed that the proposed genes hisG, hisE, hisH1, hisF1, hisB, hisI, hisC1, hisA, and hisD are involved in histidine synthesis. In summary, HisC2, HisH2, and HisF2 are dispensable for histidine synthesis in P. aeruginosa PAO1; conversely, inactivation of HisD or HisN results in incomplete histidine auxotrophy, and inactivation of any of the eight other genes, including hisG, hisE, hisH1, hisF1, hisB, hisI, hisC1 and hisA, leads to complete histidine auxotrophy in P. aeruginosa PAO1.

FIG 7.

FIG 7

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Growth analysis of PAO1, PAO1(ΔhisG), PAO1(ΔhisE), PAO1(ΔhisA), PAO1(ΔhisF1), PAO1(ΔhisH1F1), PAO1(ΔhisC1), PAO1(ΔhisC2), and PAO1(ΔhisD). The dilutions were spotted on MM agar containing glucose (A) or histidine (B) or LB agar plates (C). The plates were cultivated at 37°C for 30 h.

DISCUSSION

Amino acids play important roles in protein synthesis and gene expression in microorganisms to maintain normal growth and reproduction. Bacteria can synthesize all 20 proteinogenic amino acids involved in protein synthesis (35, 36). The biosynthesis of many amino acids has been extensively studied, including the metabolic pathways, enzymatic activity, structure, gene expression, and regulation (37). Histidine biosynthesis was first studied in S. enterica Typhimurium and then thoroughly in other organisms, such as E. coli and C. glutamicum (9). Histidine biosynthesis displays high conservation and is catalyzed by nine enzymes in 10 reactions, though there are differences in gene organization among microorganisms (8).

Although histidine biosynthesis is important in bacteria, knowledge is lacking about what occurs in P. aeruginosa, an important opportunistic pathogen involved in many nosocomial infections. In addition to the Hol-Pase-encoding gene, which catalyzes the eighth step, the other corresponding genes of histidine biosynthesis have been predicted according to KEGG pathway analysis but not yet investigated by experimentation. In this study, PA0335 was first identified to be involved in histidine biosynthesis in P. aeruginosa PAO1. Bioinformatics analysis suggested that PA0335 belongs to the HAD superfamily (Fig. 5), which contains phosphatases, phosphoglucomutases, phosphonatases, and dehalogenases (38). In P. aeruginosa histidine biosynthesis, the only enzyme belonging to the HAD family is Hol-Pase, a type of AP (8). Indeed, we found lower AP activity in the extract of PAO1(Δ0335) than in the extract of PAO1, though the difference was not significant (data not shown). When the PA0335-encoded protein was heterologously expressed in E. coli BL21, obvious AP activity was observed (Fig. 6B). In addition, the growth defect of PA0335 mutant was also rescued by expressing hisN from C. glutamicum (Fig. 6C), the enzymatic function of which have been confirmed before (11, 37). All these results indicated the AP function of the PA0335-encoded protein.

In general, Hol-Pase-encoding genes are the last gene in bacterial histidine biosynthesis to be characterized due to the complexity of their origin and evolution (39, 40). It has been reported that there are at least two typical types of Hol-Pases, DDDD and PHP families and that their enzymatic activity depends on different catalytic residues. In E. coli and S. enterica Typhimurium, the activity of Hol-Pase is associated with the N-terminal domain of HisB, a bifunctional enzyme with Hol-Pase and IGP dehydratase enzymatic activities belonging to the DDDD superfamily. Hol-Pase in B. subtilis and S. cerevisiae is assigned to HisN, acting as a monofunctional enzyme belonging to the PHP superfamily (8, 39, 41). By screening a random mutagenesis library, Mormann et al. (37) identified a Hol-Pase-encoding gene in C. glutamicum, representing s a new class of Hol-Pase (11, 37). When the Hol-Pase corresponding genes of hisB and hisN from E. coli and C. glutamicum were expressed in PAO1(Δ0335), it was found that HisN, a monofunctional enzyme from C. glutamicum (39), was able to complement dysfunctional PA0335 of PAO1(Δ0335) (Fig. 6C), but HisB from E. coli did not complement (data not shown. These results indicate that P. aeruginosa Hol-Pase might belong to the same class of Hol-Pase in C. glutamicum. However, their difference should be investigated in the future.

Although PAO1(Δ0335) showed a growth defect compared to the wild-type strain PAO1, it could still grow very slowly, indicating that other isozymes of PA0335-encoded proteins might be present. Indeed, we found that four genes—PA1143, PA3255, PA5547, and PA4960—and their amino acid sequences share 42, 38, 27, and 26% identities, respectively, with PA0335. According to the P. aeruginosa genome database (http://www.pseudomonas.com/), PA1143, PA3255, and PA5547 encode hypothetical proteins, and PA4960 probably encodes a phosphoserine phosphatase. These genes might be active during extended cultivation, whereby complementation of PA0335 dysfunction results in incomplete histidine auxotrophy of PAO1(Δ0335). According to Kulis-Horn et al. (8), there is more than one Hol-Pase-encoding gene in C. glutamicum because the hisN mutant did not show complete histidine auxotrophy in the media tested. One or more of the four hisN homologs—cg0911, cg2090, cg2298, and cg0967—may partially complement the hisN deletion in C. glutamicum (8). However, this speculation regarding the complementation of PA0335 by PA1143, PA3255, PA5547, PA4960, and/or other unknown gene(s) needs further investigation.

In addition to PA0355, twelve other genes have been proposed to be involved in histidine biosynthesis in P. aeruginosa according to the KEGG database. The growth of all the deletion mutants in our study was similar to that in C. glutamicum, except for PAO1 (ΔhisD) (8). The growth difference of PAO1 (ΔhisD) may be due to the different genetic backgrounds. In addition, it is worth mentioning that HisC2, HisH2, and HisF2 do not play predominant roles in histidine synthesis in PAO1 because their inactivation did not change bacterial growth in the media tested. Similarly, the hisH deletion mutant of C. glutamicum did not display a histidine auxotrophy phenotype (8). The actual role of hisC2, hisH2, and hisF2 in P. aeruginosa PAO1 histidine biosynthesis requires further research. Based on our results, we outlined the histidine biosynthetic pathway of P. aeruginosa (Fig. 8).

FIG 8.

FIG 8

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Histidine biosynthetic pathway in PAO1. A solid line frame indicates enzymes definitely involved in histidine biosynthesis, and a dotted line frame indicates enzymes that do not play a predominant role in histidine biosynthesis of PAO1.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table 2. P. aeruginosa and E. coli were both routinely cultivated at 37°C with MM, LB agar or broth. When necessary, tetracycline (Tc; 15 μg/ml), kanamycin (Kan; 50 μg/ml), or gentamicin (Gm; 15 μg/ml) was added to LB medium for E. coli growth, and Tc (300 μg/ml), Gm (150 μg/ml), or carbenicillin (Cb; 250 μg/ml) was added to Pseudomonas isolation agar (PIA) (Difco) for P. aeruginosa growth. All antibiotics used were purchased from Amresco (Solon).

TABLE 2.

Strains and plasmids used in this study

Strain or plasmid Genotype or phenotype Source or reference
Strains
    E. coli
        DH10B F− mcrAΔ(mrr-hsdRMS-mcrBC)ϕ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara-leu)7697 galU galK λ− rpsL nupG/pMON14272/pMON7124 This lab
        DH5α fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Δ80lacZΔM15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 This lab
    P. aeruginosa This lab
        PAO1 Wild type
        PAO1(Δ0335) PA0335 deletion mutant in PAO1 This lab
        PAO1(Δ0334-5) PA0334 and PA0335 double-deletion mutant in PAO1 This study
        PAO1(ΔhisG) PA4449 deletion mutant in PAO1 This study
        PAO1(ΔhisE) PA5067 deletion mutant in PAO1 This study
        PAO1(ΔhisI) PA5066 deletion mutant in PAO1 This study
        PAO1(ΔhisA) PA5141 deletion mutant in PAO1 This study
        PAO1(ΔhisF1) PA5140 deletion mutant in PAO1 This study
        PAO1(ΔhisF2) PA3151 deletion mutant in PAO1 This study
        PAO1(ΔhisH1) PA5142 deletion mutant in PAO1 This study
        PAO1(ΔhisH2) PA3152 deletion mutant in PAO1 This study
        PAO1(ΔhisB) PA5143 deletion mutant in PAO1 This study
        PAO1(ΔhisC1) PA4447 deletion mutant in PAO1 This study
        PAO1(ΔhisC2) PA3165 deletion mutant in PAO1 This study
        PAO1(ΔhisD) PA4448 deletion mutant in PAO1 This study
        PAO1(ΔhisF1H1) PA5140 and PA5142 double-deletion mutant in PAO1 This study
        PAO1(ΔhisF2H2) PA3151 and PA3152 double-deletion mutant in PAO1 This study
        PAO1(Δ0334-5)C1 PAO1(Δ0334-5) containing PA0334 and PA0335 This study
        PAO1(Δ0334-5)C2 PAO1(Δ0334-5) containing PA0334 This study
        PAO1(Δ0334-5)C3 PAO1(Δ0334-5) containing PA0335 This study
        PAO1(Δ0335)C PAO1(Δ0335) containing PA0335 This study
        PAO1(Δ0335)C1 PAO1(Δ0335) containing hisN from C. glutamicum This study
Plasmids
    pEX18Tc Broad-host-range gene replacement vector; sacB; Tcr This lab
    pRK2013 Broad-host-range helper vector; Tra+ Kanr 45
    pAK1900 Multicopy E. coli-P. aeruginosa shuttle vector, MCS within lacZ fragment; Abr Cbr 46
    pBT20 The plasmids were used to construct transposon mutants; Gmr Apr; mariner transposon This lab
    pET-28a Expression vector carrying 6×N/C-His, N-thrombin, N-T7; Kanr This lab
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Determination of the location of transposon insertion.

The transposon insertion location of the mutant was determined by arbitrary-primed PCR, as described previously (42, 43), which was performed with minor modifications as below. Briefly, the specific primer P7-1 (5′-CTAACAATTCGTTCAAGCCG-3′) for the transposon sequence was paired with the semidegenerate primer ARB1 (5′-GGCCACGCGTCGACTAGTACNNNNNNNNNNGATAT-3′) for the first round of PCR using chromosomal DNA of the mutant as the template. Two microliters of the first-round PCR product was used as the template for the second round of PCR with the specific primer P7-2 (5′-GGATGCGTCTAAAAGCCTGC-3′) paired with the primer ARB12 (5′-GGCCACGCGTCGACTAGTAC-3′). The PCR product was purified using the BioSpin gel extraction kit and then sequenced by the company TSINGKE Biological Technology (Beijing, China). The resulting sequences were compared to P. aeruginosa chromosome sequences to localize the transposon insertion site (44).

Construction of mutants.

Mutant construction was carried out by allelic exchange using sucrose counterselection as described previously (45). To construct the PA0335-knockout mutant, the upstream fragment of PA0335 was amplified using the forward primer PA0335-D-A and the reverse primer PA0335-D-S with the PstI and BamHI restriction sites, respectively. The downstream region of PA0335 was generated with the forward primer PA0335-UP-A and the reverse primer PA0335-UP-S containing the BamHI and EcoRI restriction sites, respectively. The digested PCR fragments were ligated to the pEX18-Tc vector using the same restriction enzymes, yielding the plasmid pEX18Tc-0335.

The PA0335-knockout mutant was obtained by means of triparental mating according to a previous report (46). The donor strain E. coli containing pEX18Tc-0335 and the helper strain containing pRK2013 were cultured overnight in 50 ml of LB broth with Tc and Kan, respectively. The recipient PAO1 was cultured in LB broth. The cells were collected and then resuspended in 1 ml of phosphate-buffered saline (PBS). The three bacteria were mixed at a ratio of 1:1:1, and the mixture was spotted onto LB agar plates. After overnight cultivation at 37°C, the mixture was collected and resuspended in 0.5 ml of LB medium. The liquid was applied to PIA plates containing 300 μg/ml Tc and cultivated at 37°C for 24 to 32 h. The clones were then assessed on 10% sucrose-containing PIA agar. The resulting PA0335-knockout mutant was verified by PCR and named PAO1(Δ0335).

The mutants PAO1(Δ0334-5), PAO1(ΔhisG), PAO1(ΔhisE), PAO1(ΔhisD), PAO1(ΔhisC1), PAO1(ΔhisC2), PAO1(ΔhisH1), PAO1(ΔhisH2), PAO1(ΔhisF1), PAO1(ΔhisF2), PAO1(ΔhisI), PAO1(ΔhisB), PAO1(ΔhisA), PAO1(ΔhisF1H1), and PAO1(ΔhisF2H2) were constructed using the same method with different primers (see Table S1 in the supplemental material).

Complementation of mutants.

To complement the knockout mutant of PA0335, the multicopy-number E. coli-P. aeruginosa shuttle vector pAK1900 was used (47). Gene PA0335 with its promoter region was PCR amplified using the forward primer pAK-PA0335-S and the reverse primer pAK-PA0335-A with restriction sites. The digested PCR product was ligated to pAK1900 to produce the plasmid pAK-0335. pAK-0335 was then transformed into PAO1(Δ0335) by electroporation. Integrants were selected on PIA plates containing 250 μg/ml Cb. The resulting strain was designated PAO1(Δ0335)C. PAO1(Δ0334-5)C1, PAO1(Δ0334-5)C2, and PAO1(Δ0334-5)C3, PAO1(Δ0335)C, and PAO1(Δ0335)C1 were constructed similarly.

Bacterial quantification.

Five milliliters of overnight culture was diluted to an optical density at 600 nm (OD600) of ∼0.8, and 5 μl of a serial dilution was transferred to MM agar supplemented with different nutrients, including glucose, xylose, glycerol, sucrose, galactose, mannitol, vitamin B1, urea, citrate, succinic acid salt, pyruvate, and casein hydrolysate. Bacterial growth was examined after incubation overnight at 37°C.

Auxanography assay.

PAO1(Δ0335) and PAO1 were cultivated in 5 ml of LB broth overnight at 37°C; 1 ml of the culture was centrifuged for 10 min at 3,000 × g, and the harvested cells were washed and resuspended in 1 ml of PBS. Next, sterile filter disks were spotted with six various groups of amino acid nutrient mixtures, as shown in Table 3 , and placed on top of MM agar containing 20 ml of the suspensions (the concentration of each amino acid in the nutrient solution was 2 mM). The plates were incubated at 37°C for 12 h. Auxotrophy was identified by the growth of bacteria around each filter disk.

TABLE 3.

Six groups of amino acid mixture

Group Amino acid mixture
1 Lys-Arg-Met-Cys-Leu-Ile
2 Val-Arg-Phe-Tyr-Trp-His
3 Thr-Met-Phe-Glu-Pro-Asp
4 Ala-Cys-Tyr-Glu-Gly-Ser
5 Gln-Leu-Trp-Pro-Gly-Asn
6 Gln-Ile-His-Asp-Ser
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Bioinformatic analysis.

Clustal X was used for multiple alignment of homologous HisN protein sequences from different bacteria (48). MEGA 7.0 was used for phylogenetic tree construction of these homologous HisN proteins (49).

Protein expression.

The pET-28a vector was used to express the PA0335-encoded protein in E. coli BL21(DE3). The PA0335 gene was amplified by PCR using the primers pET-0335-S and pET-0335-A containing BamHI and HindIII restriction sites, respectively. The digested PCR product was ligated to pET-28a to obtain the recombinant plasmid pET28a-0335.

pET28a-0335 and pET-28a were electroporated into E. coli BL21(DE3) to produce E. coli(pET-0335) and E. coli(pET-28a), respectively. The resulting strains were cultivated in LB broth supplemented with 50 μg/ml Kan at 37°C for 16 h. The culture was diluted to an OD600 of ∼0.6 with LB medium, and the PA0335-encoded protein was induced at 30°C for 3 h with 0.2 mM IPTG (isopropyl-β-d-thiogalactopyranoside). The cells were sonicated, the lysate was centrifuged at 4°C and 13,000 rpm for 10 min, and the supernatant was examined by SDS-PAGE.

AP assay.

An AP assay kit (Beyotime, Shanghai, China) was used to determine the activity of the PA0335-encoded protein according to the manufacturer’s instructions. Overnight cultures were washed three times with PBS. The resuspended cells were sonicated on ice for 30 min and centrifuged at 4°C and 12,000 rpm for 10 min. AP activity in the supernatant was analyzed based on the conversion of colorless p-nitrophenyl phosphate (pNPP) to colored p-nitrophenol after coincubation at 37°C for 30 min. The results were normalized to the total intracellular protein content determined by the bicinchoninic acid protein assay (Beyotime); one enzyme activity unit (U) is defined as the amount of AP required for releasing 1/3 μmol of p-nitrophenol per min according to the manufacturer’s instructions (Beyotime).

Statistical analysis.

Student t test was used to determine significant difference in AP activity of the PA0335-encoded protein and the control. All results are expressed as means ± standard deviations. All statements of significance are based on probabilities of P < 0.05 (*) and P < 0.01 (**).

Supplementary Material

Supplemental file 1
AEM.02593-19-s0001.pdf (258.8KB, pdf)

ACKNOWLEDGMENTS

This project was supported by grants from the Provincial Natural Science Foundation of Shaanxi Province (2018ZDXM-SF-004) and the NSFC (31570131, 31770152, and 31400094). The authors declare no conflict of interest.

Footnotes

Supplemental material is available online only.

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Supplemental file 1
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