FolX and FolM Are Essential for Tetrahydromonapterin Synthesis in Escherichia coli and Pseudomonas aeruginosa

Tetrahydromonapterin is a major pterin in Escherichia coli and is hypothesized to be the cofactor for phenylalanine hydroxylase (PhhA) in Pseudomonas aeruginosa, but neither its biosynthetic origin nor its cofactor role has been clearly demonstrated. A comparative genomics analysis implicated the enigmatic folX and folM genes in tetrahydromonapterin synthesis via their phyletic distribution and chromosomal clustering patterns. folX encodes dihydroneopterin triphosphate epimerase, which interconverts dihydroneopterin triphosphate and dihydromonapterin triphosphate. folM encodes an unusual short-chain dehydrogenase/reductase known to have dihydrofolate and dihydrobiopterin reductase activity. The roles of FolX and FolM were tested experimentally first in E. coli, which lacks PhhA and in which the expression of P. aeruginosa PhhA plus the recycling enzyme pterin 4a-carbinolamine dehydratase, PhhB, rescues tyrosine auxotrophy. This rescue was abrogated by deleting folX or folM and restored by expressing the deleted gene from a plasmid. The folX deletion selectively eliminated tetrahydromonapterin production, which far exceeded folate production. Purified FolM showed high, NADPH-dependent dihydromonapterin reductase activity. These results were substantiated in P. aeruginosa by deleting tyrA (making PhhA the sole source of tyrosine) and folX. The ΔtyrA strain was, as expected, prototrophic for tyrosine, whereas the ΔtyrA ΔfolX strain was auxotrophic. As in E. coli, the folX deletant lacked tetrahydromonapterin. Collectively, these data establish that tetrahydromonapterin formation requires both FolX and FolM, that tetrahydromonapterin is the physiological cofactor for PhhA, and that tetrahydromonapterin can outrank folate as an end product of pterin biosynthesis.Pterins contain the bicyclic pteridine ring with an amino group in the 2-position and an oxo group in the 4-position; they can be reduced through the dihydro forms to the tetrahydro forms, which are active as cofactors (Fig. ​(Fig.1A).1A). Tetrahydropterins are known to be the cofactors for phenylalanine hydroxylases from Pseudomonas and Chromatium species as well as for mammalian aromatic amino acid hydroxylases and other mammalian enzymes (13, 17, 38, 41) (Fig. ​(Fig.1B).1B). Although the identity of the mammalian tetrahydropterin cofactor, tetrahydrobiopterin (H₄-BPt), is firmly established (38), the same is not true for bacteria, and the biosynthesis of bacterial tetrahydropterins is not well understood.Open in a separate windowFIG. 1.Tetrahydropterin structure, cofactor role, and biosynthesis. (A) The pterin nucleus, its levels of reduction, and the structures of compounds relevant to this study. (B) The requirement for a tetrahydropterin (H₄-pterin) cofactor for phenylalanine hydroxylase (PAH) and the cofactor regeneration cycle involving pterin-4a-carbinolamine dehydratase (PCD) and quinonoid dihydropterin (q-H₂-pterin) reductase (q-DHPR; EC 1.5.1.34). (C) The established steps in tetrahydrobiopterin (H₄-BPt) biosynthesis and possible routes for tetrahydromonapterin (H₄-MPt) biosynthesis in relation to the folate pathway. H₄-BPt is formed by the sequential action of 6-pyruvoyltetrahydropterin (P-H₄-Pt) synthase (PTPS-II) and sepiapterin reductase (SR). H₄-MPt could originate via the FolX-catalyzed epimerization of dihydroneopterin triphosphate (H₂-NPt-P₃) to dihydromonapterin triphosphate (H₂-MPt-P₃), followed by dephosphorylation to dihydromonapterin (H₂-MPt) and reduction by a dihydropterin reductase (EC 1.5.1.33), putatively FolM. H₄-MPt also could come from the FolB-mediated epimerization of dihydroneopterin (H₂-NPt) followed by reduction. FolB also mediates the side chain cleavage of H₂-NPt or H₂-MPt to give 6-hydroxymethyldihydropterin (H₂-HMPt); the H₂-MPt cleavage is omitted for simplicity. Other abbreviations: P-ase, phosphatase; H₂-HMPt-P₂, 6-hydroxymethyldihydropterin diphosphate; pABA, p-aminobenzoate; H₂-pteroate, dihydropteroate; H₂-folate, dihydrofolate; H₄-folate, tetrahydrofolate.While a few bacterial taxa, such as Cyanobacteria and Chlorobia, produce H₄-BPt, most do not, as judged directly from pterin analysis and indirectly from the rarity of H₄-BPt biosynthesis genes 6-pyruvoyltetrahydropterin synthase II (PTPS-II) and sepiapterin reductase (SR) (Fig. ​(Fig.1C)1C) among sequenced genomes (12, 25). As bacteria lacking H₄-BPt include Pseudomonas and many others with phenylalanine hydroxylase genes, it is clear that bacterial phenylalanine hydroxylases generally must use a cofactor other than H₄-BPt. The most prominent candidate is tetrahydromonapterin (H₄-MPt), which occurs in Escherichia coli (21) and almost certainly also in Pseudomonas species (11, 17). H₄-MPt could be derived from the dihydropterin intermediates of folate biosynthesis via two different routes (Fig. ​(Fig.1C).1C). These are (i) the conversion of dihydroneopterin triphosphate (H₂-NPt-P₃) to dihydromonapterin triphosphate (H₂-MPt-P₃) by H₂-NPt-P₃ epimerase (FolX) followed by dephosphorylation and reduction to the tetrahydro level, and (ii) the conversion of dihydroneopterin (H₂-NPt) to dihydromonapterin (H₂-MPt) by the epimerase action of dihydroneopterin aldolase (FolB) and then reduction. FolB is a fairly well-understood enzyme of folate synthesis (9), but FolX has no known biological role and a folX deletant has no obvious phenotype (19). folX genes apparently are confined to Gammaproteobacteria (9).Although the epimerase activities of FolX and FolB have been demonstrated amply in vitro (1, 5, 19), no genetic evidence links either enzyme to H₄-MPt formation in vivo. The situation with the reduction of H₂-MPt to H₄-MPt is even less clear, because this activity has not been investigated experimentally. A candidate enzyme for this step nevertheless can be proposed on bioinformatic grounds: the somewhat mysterious FolM protein (9). FolM belongs to a subset of the short-chain dehydrogenase/reductase (SDR) family having the characteristic motif TGX₃RXG (in place of TGX₃GXG, which typifies other SDRs). The archetype of this subset is Leishmania pteridine reductase 1 (PTR1), which reduces various dihydropterins to the tetrahydro state (15). E. coli FolM has low dihydrofolate (H₂-folate) and dihydrobiopterin (H₂-BPt) reductase activities in vitro (14), but neither of these is likely to be its physiological function, since H₂-folate reduction normally is mediated by FolA and E. coli lacks H₄-BPt. folM genes occur in many Gram-negative organisms, including Chlamdiae, Chloroflexi, Cyanobacteria, Acidobacteria, Planctomycetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria (9).We report here comparative genomic and genetic evidence that FolX and FolM are required for H₄-MPt synthesis in E. coli and P. aeruginosa, the bacteria in which H₄-MPt has been most studied, and biochemical evidence that FolM has high H₂-MPt reductase activity. We also point out gaps in the understanding of pterin metabolism that our data bring sharply into focus.     

Identification of Virulence Genes in a Pathogenic Strain of Pseudomonas aeruginosa by Representational Difference Analysis

Pseudomonas aeruginosa is an opportunistic pathogen that may cause severe infections in humans and other vertebrates. In addition, a human clinical isolate of P. aeruginosa, strain PA14, also causes disease in a variety of nonvertebrate hosts, including plants, Caenorhabditis elegans, and the greater wax moth, Galleria mellonella. This has led to the development of a multihost pathogenesis system in which plants, nematodes, and insects have been used as adjuncts to animal models for the identification of P. aeruginosa virulence factors. Another approach to identifying virulence genes in bacteria is to take advantage of the natural differences in pathogenicity between isolates of the same species and to use a subtractive hybridization technique to recover relevant genomic differences. The sequenced strain of P. aeruginosa, strain PAO1, has substantial differences in virulence from strain PA14 in several of the multihost models of pathogenicity, and we have utilized the technique of representational difference analysis (RDA) to directly identify genomic differences between P. aeruginosa strains PA14 and PAO1. We have found that the pilC, pilA, and uvrD genes in strain PA14 differ substantially from their counterparts in strain PAO1. In addition, we have recovered a gene homologous to the ybtQ gene from Yersinia, which is specifically present in strain PA14 but absent in strain PAO1. Mutation of the ybtQ homolog in P. aeruginosa strain PA14 significantly attenuates the virulence of this strain in both G. mellonella and a burned mouse model of sepsis to levels comparable to those seen with PAO1. This suggests that the increased virulence of P. aeruginosa strain PA14 compared to PAO1 may relate to specific genomic differences identifiable by RDA.     

Crystal Structure of Dihydro-Heme d1 Dehydrogenase NirN from Pseudomonas aeruginosa Reveals Amino Acid Residues Essential for Catalysis

Many bacteria can switch from oxygen to nitrogen oxides, such as nitrate or nitrite, as terminal electron acceptors in their respiratory chain. This process is called “denitrification” and enables biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa, making it more resilient to antibiotics and highly adaptable to different habitats. The reduction of nitrite to nitric oxide is a crucial step during denitrification. It is catalyzed by the homodimeric cytochrome cd₁ nitrite reductase (NirS), which utilizes the unique isobacteriochlorin heme d₁ as its reaction center. Although the reaction mechanism of nitrite reduction is well understood, far less is known about the biosynthesis of heme d₁. The last step of its biosynthesis introduces a double bond in a propionate group of the tetrapyrrole to form an acrylate group. This conversion is catalyzed by the dehydrogenase NirN via a unique reaction mechanism. To get a more detailed insight into this reaction, the crystal structures of NirN with and without bound substrate have been determined. Similar to the homodimeric NirS, the monomeric NirN consists of an eight-bladed heme d₁-binding β-propeller and a cytochrome c domain, but their relative orientation differs with respect to NirS. His147 coordinates heme d₁ at the proximal side, whereas His323, which belongs to a flexible loop, binds at the distal position. Tyr461 and His417 are located next to the hydrogen atoms removed during dehydrogenation, suggesting an important role in catalysis. Activity assays with NirN variants revealed the essentiality of His147, His323 and Tyr461, but not of His417.     

Virulence and toxigenicity of Pseudomonas aeruginosa cultures of various origins

The virulence and toxigenicity of newly isolated P. aeruginosa strains have been studied in experiments on white mice. These biological properties have been shown to be most pronounced in P. aeruginosa strains isolated from proteins, sometimes greatly exceeding those in strains isolated from healthy persons and the environment. Virulence and the factors which determine it are definitely interrelated in microorganisms and can vary, depending on the conditions of their habitat.     

铜绿假单胞菌噬菌体K5中DNA聚合酶等基因在宿主中的表达

本研究分析了铜绿假单胞菌噬菌体K5基因在宿主中的表达及其影响因素. 通过测定融合报告基因dnaP-lacZ、capP-lacZ、bapP-lacZ和rdr-lacZ编码的β 半乳糖苷酶活力,分析了噬菌体K5相关基因的表达水平,发现噬菌体K5的不同基因在宿主细胞内表达水平存在较大差异,其中噬菌体K5的DNA聚合酶基因dnaP的表达水平最高,而主要衣壳蛋白基因capP的表达水平最低. 加入噬菌体后,除二磷酸核糖核苷酸还原酶基因rnr外,其它基因的表达水平均有明显提高,说明噬菌体自身因子能够调控噬菌体部分基因在宿主细胞中的表达. 进一步分析显示,噬菌体基因在对数生长前期细胞中的表达水平显著高于平衡期. 同时,噬菌体感染对数生长前期的宿主菌,其释放量为12.8 PFU/感染中心,是平衡期释放量的9.2倍. 噬菌体以对数生长期宿主为指示菌时噬菌体的滴度为4.7×108 PFU/mL,而以平衡期宿主菌为指示菌噬菌体K5滴度仅能达到2.5×104 PFU/mL,噬菌体K5的裂解能力显著降低. 这些结果对研究噬菌体与宿主细胞的相互作用机制具有重要作用.     

Pyocyanin Facilitates Extracellular DNA Binding to Pseudomonas aeruginosa Influencing Cell Surface Properties and Aggregation

Pyocyanin is an electrochemically active metabolite produced by the human pathogen Pseudomonas aeruginosa. It is a recognized virulence factor and is involved in a variety of significant biological activities including gene expression, maintaining fitness of bacterial cells and biofilm formation. It is also recognized as an electron shuttle for bacterial respiration and as an antibacterial and antifungal agent. eDNA has also been demonstrated to be a major component in establishing P. aeruginosa biofilms. In this study we discovered that production of pyocyanin influences the binding of eDNA to P. aeruginosa PA14 cells, mediated through intercalation of pyocyanin with eDNA. P. aeruginosa cell surface properties including cell size (hydrodynamic diameter), hydrophobicity and attractive surface energies were influenced by eDNA in the presence of pyocyanin, affecting physico-chemical interactions and promoting aggregation. A ΔphzA-G PA14 mutant, deficient in pyocynain production, could not bind with eDNA resulting in a reduction in hydrodynamic diameter, a decrease in hydrophobicity, repulsive physico-chemical interactions and reduction in aggregation in comparison to the wildtype strain. Removal of eDNA by DNase I treatment on the PA14 wildtype strain resulted in significant reduction in aggregation, cell surface hydrophobicity and size and an increase in repulsive physico-chemical interactions, similar to the level of the ΔphzA-G mutant. The cell surface properties of the ΔphzA-G mutant were not affected by DNase I treatment. Based on these findings we propose that pyocyanin intercalation with eDNA promotes cell-to-cell interactions in P. aeruginosa cells by influencing their cell surface properties and physico-chemical interactions.     

Clustering of Functionally Related Genes in Pseudomonas aeruginosa

Genes for the mandelate and benzoate pathways in Pseudomonas aeruginosa are clustered to a greater degree than that predicted on the basis of the induction pattern.     

铜绿假单胞杆菌QS相关基因差异表达的研究

选取100个与铜绿假单胞杆菌（Pseudomonas aeruginosa）群感效应（quorum-sensing,QS）相关的基因,克隆这些基因片段于pMD-18T载体,测序鉴定,点样制备cDNA基因芯片。制备cy3-dCTP／cy5-dCTP标记的探针,与芯片杂交。初步研究了处于不同生长期的铜绿假单胞杆菌基因的表达差异。指数中期和平台初期相比,有9个QS基因表达量最著增加,有6个基因表达量显著下降。利用芯片做针对铜绿菌假单胞杆菌药物的筛选：妥布霉素（Tobramycin）给药后细菌基因发生差异表达。证明了该cDNA芯片用于药物筛选的可行性。在国内首次研制开发了QS相关基因的cDNA芯片。应用基因芯片技术建立的铜绿假单胞杆菌QS相关基因研究平台,为找到能较好抑制铜绿假单胞杆菌正常生长的药物研究提出新的解决方法。