The cytoplasmic heme-binding protein (PhuS) from the heme uptake system of Pseudomonas aeruginosa is an intracellular heme-trafficking protein to the delta-regioselective heme oxygenase

The uptake and utilization of heme as an iron source is a receptor-mediated process in bacterial pathogens and involves a number of proteins required for internalization and degradation of heme. In the following report we provide the first in-depth spectroscopic and functional characterization of a cytoplasmic heme-binding protein PhuS from the opportunistic pathogen Pseudomonas aeruginosa. Spectroscopic characterization of the heme-PhuS complex at neutral pH indicates that the heme is predominantly six-coordinate low spin. However, the resonance Raman spectra and global fit analysis of the UV-visible spectra show that at all pH values between 6 and 10 three distinct species are present to varying degrees. The distribution of the heme across multiple spin states and coordination number highlights the flexibility of the heme environment. We provide further evidence that the cytoplasmic heme-binding proteins, contrary to previous reports, are not heme oxygenases. The degradation of the heme-PhuS complex in the presence of a reducing agent is a result of H2O2 formed by direct reduction of molecular oxygen and does not yield biliverdin. In contrast, the heme-PhuS complex is an intracellular heme trafficking protein that specifically transfers heme to the previously characterized iron-regulated heme oxygenase pa-HO. Surface plasmon resonance experiments confirm that the transfer of heme is driven by a specific protein-protein interaction. This data taken together with the spectroscopic characterization is consistent with a protein that functions to shuttle heme within the cell.     

The mechanism of heme transfer from the cytoplasmic heme binding protein PhuS to the delta-regioselective heme oxygenase of Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa has evolved two outer membrane receptor-mediated uptake systems (encoded by the phu and has operons) by which it can utilize the hosts heme and hemeproteins as a source of iron. PhuS is a cytoplasmic heme binding protein encoded within the phu operon and has previously been shown to function in the trafficking of heme to the iron-regulated heme oxygenase (pa-HO). While the heme association rate for PhuS was similar to that of myoglobin, a markedly higher rate of heme dissociation (approximately 10(5) s(-1)) was observed, in keeping with a function in heme-trafficking. Additionally, the transfer of heme from PhuS to pa-HO was shown to be specific and unidirectional when compared to transfer to the non-iron regulated heme oxygenase (BphO), in which heme distribution between the two proteins merely reflects their relative intrinsic affinities for heme. Furthermore, the rate of transfer of heme from holo-PhuS to pa-HO of 0.11 +/- 0.01 s(-1) is 30-fold faster than that to apo-myoglobin, despite the significant higher binding affinity of apo-myoglobin for heme (kH = 1.3 x 10(-8) microM) than that of PhuS (0.2 microM). This data suggests that heme transfer to pa-HO is independent of heme affinity and is consistent with temperature dependence studies which indicate the reaction is driven by a negative entropic contribution, typical of an ordered transition state, and supports the notion that heme transfer from PhuS to pa-HO is mediated via a specific protein-protein interaction. In addition, pH studies, and reactions conducted in the presence of cyanide, suggest the involvement of spin transition during the heme transfer process, whereby the heme undergoes spin change from 6-c LS to 6-c HS either in PhuS or pa-HO. On the basis of the magnitudes of the activation parameters obtained in the presence of cyanide, whereby both complexes are maintained in a 6-c LS state, and the biphasic kinetics of heme transfer from holo-PhuS to pa-HO-wt, supports the notion that the spin-state crossover occur within holo-PhuS prior to the heme transfer step. Alternatively, the lack of the biphasic kinetic with pa-HO-G125V, 6-c LS, and with comparable rate of heme transfer as pa-HO is supportive of a mechanism in which the spin-change could occur within pa-HO. The present data suggests either or both of the two pathways proposed for heme transfer may occur under the present experimental conditions. The dissection of which pathway is physiologically relevant is the focus of ongoing studies.     

Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa

A rapid colony immunoblot screening procedure was used to demonstrate the surface localization of porin protein F on bacterial colonies of Pseudomonas aeruginosa. By this method, we demonstrated that protein F was accessible to four different specific monoclonal antibodies in a wide variety of both mucoid and nonmucoid P. aeruginosa strains. Controls were performed to demonstrate that, using this procedure, only surface-exposed epitopes bound monoclonal antibodies and that nonspecific binding of monoclonal antibodies either to cells lacking protein F or to mucoid exopolysaccharide did not occur. Monoclonal antibodies MA4-4, MA2-10, and MA4-10, specific for protein F, also interacted with colonies of Pseudomonas putida and Pseudomonas syringae, whereas the protein F specific monoclonal antibody MA5-8 interacted only with P. aeruginosa strains. Using the above-named monoclonal antibodies, we investigated the antigenic structure of protein F. Monoclonal antibodies MA4-4, MA2-10, and MA4-10 bound to 29-31 kilodalton proteolytic fragments produced after papain or trypsin digestion of purified protein F or of protein F in outer membranes or intact cells. Antibody MA5-8 did not interact with proteolytically digested protein F but did interact with two of the six fragments produced after partial cyanogen bromide cleavage of protein F. Antibodies MA4-4, MA2-10, and MA4-10 did not interact with protein F after reduction of its internal disulphide bonds with 2-mercaptoethanol; in contrast, the reactivity of MA5-8 was unaffected. This data suggests that there are at least two distinct highly conserved surface epitopes on porin protein F.     

Identification of RegA protein from Pseudomonas aeruginosa using anti-RegA antibody

The product of Pseudomonas aeruginosa regA gene acts as a positive regulator of exotoxin A expression. The protein, RegA, was overproduced in E. coli transformed with an expression vector containing the regA gene. The overproduced RegA accumulated in E. coli in the form of inclusion bodies. The latter were isolated and served as a source of antigen for raising polyclonal antibodies. The antibodies reacted specifically with a P. aeruginosa protein whose molecular weight corresponded to that predicted for RegA from its known DNA sequence, and whose response to modulating factors matched that expected for RegA. The immunodetectable RegA was localized in the membrane fraction of P. aeruginosa strain PA103.     

Identification of an estrogen-binding protein in Pseudomonas aeruginosa.

A constitutive estrogen-binding protein (EBP) has been identified in the cytosol of Pseudomonas aeruginosa, a Gram-negative bacterium. All 14 strains tested contained the EBP. Estradiol binding was rapid and maximal binding occurred by 90 min at 0 degrees C. Dissociation of estradiol from the binding protein occurred at a rate of 4.6 fmol/min with a t1/2 of 42 min. EBP binding was destroyed by protease treatment and at high temperature. Sodium molybdate had no effect on binding. The Kd determined by Scatchard analysis was 3.9 nM and the Bmax was 323 fmol/mg protein. The EBP sedimented at 8.9 S on sucrose density gradients. The presence of 0.4 M KCl increased estradiol binding 6-fold but did not cause a shift in the sedimentation value. Gel filtration of the native protein gave an estimated molecular weight of 215,000 and a Stokes radius of 50.2 A. Steroid binding specificity, in order of decreasing affinity, was estradiol, estrone, dihydrotestosterone, estriol, testosterone, progesterone and promegestone. Other steroid hormones tested did not compete for estradiol binding. Identification of an EBP in a bacterium allows a comparative analysis of other steroid-binding proteins in unicellular microorganisms.     

Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa

One of the major proteins secreted by Pseudomonas aeruginosa is a 43-kDa protein, which is cleaved by elastase into smaller fragments, including a 30-kDa and a 23-kDa fragment. The N-terminal 23-kDa fragment was previously suggested as corresponding to a staphylolytic protease and was designated LasD (S. Park and D. R. Galloway, Mol. Microbiol. 16:263-270, 1995). However, the sequence of the gene encoding this 43-kDa protein revealed that the N-terminal half of the protein is homologous to the chitin-binding proteins CHB1 of Streptomyces olivaceoviridis and CBP21 of Serratia marcescens and to the cellulose-binding protein p40 of Streptomyces halstedii. Furthermore, a short C-terminal fragment shows homology to a part of chitinase A of Vibrio harveyi. The full-length 43-kDa protein could bind chitin and was thereby protected against the proteolytic activity of elastase, whereas the degradation products did not bind chitin. The purified 43-kDa chitin-binding protein had no staphylolytic activity, and comparison of the enzymatic activities in the extracellular medium of a wild-type strain and a chitin-binding protein-deficient mutant indicated that the 43-kDa protein supports neither chitinolytic nor staphylolytic activity. We conclude that the 43-kDa protein, which was found to be produced by many clinical isolates of P. aeruginosa, is a chitin-binding protein, and we propose to name it CbpD (chitin-binding protein D).     

Identification of cleavage sites involved in proteolytic processing of Pseudomonas aeruginosa preproelastase.

The extracellular elastase (33 kDa) of Pseudomonas aeruginosa is synthesized as a 53.6 kDa preproenzyme containing a long, N-terminal propeptide. The free propeptide and the elastase precursor generated upon propeptide removal were isolated from P. aeruginosa cells and subjected to N-terminal amino acid sequence analysis. The results identified Ala-174 and Ala+1 as the amino terminal residues of the propeptide and the elastase precursor, respectively, indicating that: (1) the signal peptide consists of 23 amino acid residues and its molecular weight is 2.4 kDa, (2) the propeptide contains 174 amino acid residues and is of 18.1 kDa molecular weight, and (3) no additional N-terminal proteolytic cleavage is required for elastase maturation.     

貂源绿脓杆菌的分离鉴定及其致病性

为了揭示绿脓杆菌对水貂的致病性,本研究采用细菌分离培养方法、形态学观察、生化试验、药敏试验与16S rDNA测序等技术,对2013年在山东省诸城、文登与临沂采集的43份发病水貂肺组织进行了细菌分离鉴定。结果分离到的5株细菌均为绿脓杆菌（分别命名为F1、F5、F6、F8、F10）,分离率为11.6%。所分离的5株绿脓杆菌之间的核酸同源性为 98.9 %～99.7 %; F5、F6、F8、F10与F1在一个大的分支上。用F1株对小鼠与水貂分别进行接种攻毒,建立绿脓杆菌对小鼠与水貂的致病性研究动物模型。F1在小鼠肺部含量较高,半数致死量（LD₅₀）为 1.6×10⁶CFU/mL,对小鼠有较强的致病力;F1对水貂的LD₅₀为3.2×10⁷CFU/mL,接种3.2×10⁸CFU/mL菌液的水貂在接种后的20-44h内全部死亡,其他感染组的水貂仅表现一过性的精神不振、食欲稍有下降,这表明绿脓杆菌对水貂具有一定的致病力。     

Evidence for two flagellar stators and their role in the motility of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous bacterium capable of twitching, swimming, and swarming motility. In this study, we present evidence that P. aeruginosa has two flagellar stators, conserved in all pseudomonads as well as some other gram-negative bacteria. Either stator is sufficient for swimming, but both are necessary for swarming motility under most of the conditions tested, suggesting that these two stators may have different roles in these two types of motility.     

铜绿假单胞菌泳动能力相关新基因的筛选及鉴定

从Mu转座突变子文库中经过表型筛选,得到12株泳动(Swimming motility)能力缺陷的突变子,经Mu转座子插入位点的确认、基因克隆及测序分析发现其中10个突变子中Mu转座子分别插入到10个不同的与鞭毛运动和功能相关的基因中,2个突变子中Mu转座子插入到功能未知的新基因(PA2950和PA5022)中,电镜观察结果表明这2个突变株均具有完整的鞭毛,初步推测这2个基因可能是参与鞭毛泳动的能量代谢、趋化作用或信息传递的新基因。     

EPR of azurins from Pseudomonas aeruginosa and Alcaligenes denitrificans demonstrates pH-dependence of the copper-site geometry in Pseudomonas aeruginosa protein

The X- and Q-band EPR spectra of Pseudomonas aeruginosa (63Cu)azurin and Alcaligenes denitrificans azurin have been measured at pH = 5.2 and 9.2, in the presence and absence of 40% glycerol. The EPR spectra of both proteins could properly be simulated by taking into account a spread in the tetrahedral angle of the copper site. The change in the EPR spectrum of Pseudomonas aeruginosa (63Cu)azurin that is observed upon an increase of the pH from 5.2 to 9.2 is consistent with a small decrease of the average tetrahedral angle from 61 degrees to 60 degrees. This geometrical change is consistent with the interpretation of earlier NMR and EXAFS observations. No pH effect is observed for Alcaligenes denitrificans azurin, in agreement with predictions based on crystallographic evidence. Glycerol has only a marginal effect on the appearance of the EPR spectra, and does not alleviate the "g-strain."     

Spectroscopic characterization of the heme-binding sites in Plasmodium falciparum histidine-rich protein 2

Proteolysis of hemoglobin provides an essential nutrient source for the malaria parasite Plasmodium falciparum during the intraerythrocytic stage of the parasite's lifecycle. Detoxification of the liberated heme occurs through a unique heme polymerization pathway, leading to the formation of hemozoin. Heme polymerization has been demonstrated in the presence of P. falciparum histidine-rich protein 2 (PfHRP2) [Sullivan, D. J., Gluzman, I. Y., and Goldberg, D. E. (1996) Science 271, 219-221]; however, the molecular role that PfHRP2 plays in this polymerization is currently unknown. PfHRP2 is a 30 kDa protein composed of several His-His-Ala-His-His-Ala-Ala-Asp repeats and is present in the parasite food vacuole, the site of hemoglobin degradation and heme polymerization. We found that, at pH 7.0, PfHRP2 forms a saturable complex with heme, with a PfHRP2 to heme stoichiometry of 1:50. Spectroscopic characterization of heme binding by electronic absorption, resonance Raman, and EPR has shown that bound hemes share remarkably similar heme environments as >95% of all bound hemes are six-coordinate, low-spin, and bis-histidyl ligated. The PfHRP2-ferric heme complex at pH 5.5 (pH of the food vacuole) has the same heme spin state and coordination as observed at pH 7.0; however, polymerization occurs as heme saturation is approached. Therefore, formation of a PfHRP2-heme complex appears to be a requisite step in the formation of hemozoin.     

Modulation of function in a minimalist heme-binding membrane protein

De novo designed heme-binding proteins have been used successfully to recapitulate features of natural hemoproteins. This approach has now been extended to membrane-soluble model proteins. Our group designed a functional hemoprotein, ME1, by engineering a bishistidine binding site into a natural membrane protein, glycophorin A (Cordova et al. in J Am Chem Soc 129:512–518, 2007). ME1 binds iron(III) protoporphyrin IX with submicromolar affinity, has a redox potential of −128 mV, and displays peroxidase activity. Here, we show the effect of aromatic residues in modulating the redox potential in the context of a membrane-soluble model system. We designed aromatic interactions with the heme through a single-point mutant, G25F, in which a phenylalanine is designed to dock against the porphyrin ring. This mutation results in roughly tenfold tighter binding to iron(III) protoporphyrin IX (K _d,app = 6.5 × 10⁻⁸ M), and lowers the redox potential of the cofactor to −172 mV. This work demonstrates that specific design features aimed at controlling the properties of bound cofactors can be introduced in a minimalist membrane hemoprotein model. The ability to modulate the redox potential of cofactors embedded in artificial membrane proteins is crucial for the design of electron transfer chains across membranes in functional photosynthetic devices.     

The sequence and function of the recA gene and its protein in Pseudomonas aeruginosa PAO

Summary The recA gene of Pseudomonas aeruginosa has been isolated and its nucleotide sequence has been determined. The coding region of the recA gene has 1038 bp specifying 346 amino acids. The recA protein of P. aeruginosa showed a striking homology with that of Escherichia coli except for the carboxy-terminal region both at the nucleotide and amino acid level. The recA ⁺-carrying plasmids restored the UV sensitivity and recombination ability of several rec mutants of P. aeruginosa. The precise location of the recA gene on the chromosome was deduced from the analysis of R plasmids.     

Identification of an additional member of the secretin superfamily of proteins in Pseudomonas aeruginosa that is able to function in type II protein secretion

Pseudomonas aeruginosa is a prolific exporter of virulence factors and contains three of the four protein secretion systems that have been described in Gram-negative bacteria. The P. aeruginosa type II general secretory pathway (GSP) is used to export the largest number of proteins from this organism, including lipase, phospholipase C, alkaline phosphatase, exotoxin A, elastase and LasA. Although these exoproteins contain no sequence similarity, they are specifically and efficiently transported by the secretion apparatus. Bacterial homologues of XcpQ (GspD), the only outer membrane component of this system, have been proposed to play the role of gatekeeper, by presumably interacting and recognizing the exported substrates to allow their passage through the outer membrane. While determining the phenotype of non-polar deletions in each of the xcp genes, we have shown that a deletion of the P. aeruginosa strain K xcpQ does not completely abolish protein secretion. As the proposed function of XcpQ should be requisite for secretion, we searched for additional factors that could carry out this role. A cosmid DNA library from a PAK strain deleted for xcpP-Z was tested for its ability to increase protein secretion by screening for enhanced growth on lipid agar, a medium that selects for the secretion of lipase. In this manner, we have identified an XcpQ homologue, XqhA, that is solely responsible for the residual export observed in a Δ xcpQ strain, although it is not required for efficient secretion in wild-type P. aeruginosa . We have also demonstrated that this protein is capable of recognizing all of the exoproteins of P. aeruginosa , arguing against the proposed role of members of the secretin family as determinants of specificity.     

Identification of the Aedes aegypti peritrophic matrix protein AeIMUCI as a heme-binding protein

The gene Aedes aegypti intestinal mucin 1 (AeIMUC1) encodes a putative peritrophic matrix (PM) protein that is expressed in the midgut of mosquito larvae and adults and is upregulated in response to exposure to heavy metals. The AeIMUC1 protein has a predicted secretory signal peptide and three putative chitin-binding domains (CBDs) with an intervening mucin-like domain. Immunofluorescence and immunoelectron microscopy experiments established that AeIMUC1 is a bona fide PM protein, and binding of the recombinant protein to chitin was demonstrated in vitro. Previous experiments suggested that the Ae. aegypti PM can bind toxic heme molecules generated during blood digestion. However, the identity of the binding molecule(s) was unknown. Using of heme-agarose beads and spectrophotometric and microcalorimetric titrations, we show that recombinant AeIMUC1 can bind large amounts of heme in vitro, suggesting for the first time a role for a PM protein in heme detoxification during blood digestion. Binding of heme to AeIMUC1 was accompanied by an altered circular dichroism spectrum indicating a change in protein conformation, consistent with an increase in secondary structure. Heme-binding activity was mapped to the AeIMUC1 CBDs, suggesting that these domains possess dual chitin- and heme-binding activity.     

Separation of the cytoplasmic and outer membrane of Pseudomonas aeruginosa PAQ.

A method is described for the isolation of the cytoplasmic and outer membranes of $\text{Pseudomonas aeruginosa}$ PAO.1. The cytoplasmic membrane exhibits nicotinamide adenine dinucleotide oxidoreductase, lactate dehydrogenase DD-carboxypeptidase and succinate dehydrogenase activities. The outer membrane is rich in 2-keto-3-deoxyoctonate and exhibits phospholipase A and DD-carboxypeptidase activity. At least 25 protein species have been detected in the cytoplasmic membrane by polyacrylamide gel electrophoresis. Using the same technique, the outer membrane contains only five protein species of molecular weights, 56,000, 53,000, 38,000, 21,000 and 16,000.