A Putative ABC Transporter,HatABCDE, Is among Molecular Determinants of Pyomelanin Production in Pseudomonas aeruginosa

Pyomelanin overproduction is a common phenotype among Pseudomonas aeruginosa isolates recovered from cystic fibrosis and urinary tract infections. Its prevalence suggests that it contributes to the persistence of the producing microbial community, yet little is known about the mechanisms of its production. Using transposon mutagenesis, we identified factors that contribute to melanogenesis in a clinical isolate of P. aeruginosa. In addition to two enzymes already known to be involved in its biosynthesis (homogentisate dioxygenase and hydroxyphenylpyruvate dioxygenase), we identified 26 genes that encode regulatory, metabolic, transport, and hypothetical proteins that contribute to the production of homogentisic acid (HGA), the monomeric precursor of pyomelanin. One of these, PA14_57880, was independently identified four times and is predicted to encode the ATP-binding cassette of an ABC transporter homologous to proteins in Pseudomonas putida responsible for the extrusion of organic solvents from the cytosol. Quantification of HGA production by P. aeruginosa PA14 strains missing the predicted subcomponents of this transporter confirmed its role in HGA production: mutants unable to produce the ATP-binding cassette (PA14_57880) or the permease (PA14_57870) produced substantially less extracellular HGA after growth for 20 h than the parental strain. In these mutants, concurrent accumulation of intracellular HGA was observed. In addition, quantitative real-time PCR revealed that intracellular accumulation of HGA elicits upregulation of these transport genes. Based on their involvement in homogentisic acid transport, we rename the genes of this operon hatABCDE.Pseudomonas aeruginosa is a metabolically versatile, opportunistic pathogen that is a major cause of life-threatening infections in patients with burn wounds, compromised immunity, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) (23, 41). A major contributor to P. aeruginosa''s pathogenicity is its remarkable genomic plasticity, which often results is a wide range of phenotypic variation among isolates obtained from both acute and chronic infections. These phenotypes include small colony variant formation (24), alginate overproduction (36), hyperpigmentation (22), autoaggregation (13), and autolysis (64). Many of these phenotypes evolve as infections progress, and most have been ascribed to “loss-of-function” genome diversification that promotes long-term survival in the host environment (54). In this regard, recent studies have stimulated interest in another example of a loss-of-function phenotype, the mutation or deletion of hmgA, which encodes the homogentisate 1,2-dioxygenase enzyme. The absence of this protein leads to the accumulation and subsequent export of homogentisic acid (HGA), which ultimately aggregates into the pyomelanin polymer that manifests as a reddish brown pigmentation of P. aeruginosa colonies and their surrounding milieu (Fig. ​(Fig.1A)1A) (5, 49).Open in a separate windowFIG. 1.Pyomelanin production by the PA14 ΔhmgA and DKN343 strains. (A) Homogentisate pathway for the catabolism of chorismate and aromatic amino acids. Enzyme names are shown above the arrows for each step. A mutation or deletion of the hmgA gene (encoding homogentisate 1,2-dioxygenase) leads to the accumulation of pyomelanin. (B) Pyomelanin overproduction by the PA14 ΔhmgA mutant is abolished when complemented with an intact hmgA gene. Complementation of a melanogenic clinical P. aeruginosa isolate, DKN343, with hmgA results in no phenotypic change, indicating that other factors contribute to its pigmentation.Production of pyomelanin (and other forms of melanin) has been described to occur in a wide range of bacterial species, including Aeromonas (4), Azotobacter (51), Azospirillum (50), Bacillus (3), Legionella (8), Marinomonas (33), Micrococcus (40), Mycobacterium (45), Proteus (1), Rhizobium (12), Shewanella (61), Sinorhizobium (38), Streptomyces (67), and Vibrio (63) species. Notably, isolates of other bacterial species associated with chronic infections of the CF lung, Burkholderia cenocepacia and Stenotrophomonas maltophilia, can also be melanogenic (28, 58), suggesting a possible role for this pigment in the establishment and/or persistence of infection. Some genera produce melanin under normal conditions via polyphenol oxidases or laccases, while others synthesize the pigment only in response to specific environmental conditions (17, 35). Many species, however, including P. aeruginosa, overproduce pyomelanin as a result of a point mutation in hmgA or large chromosomal deletions of loci containing the homogentisate operon (2, 19). While these genetic variations have been frequently reported, there is little understanding of the competitive advantage, if any, that this pigment confers to the producing bacterium.Proposed roles for pyomelanin include the enhancement of bacterial surface attachment (20), extracellular electron transfer (61), iron reduction/acquisition (8), induction of virulence factor expression (63), heavy metal binding (21), and protection from environmental stress (11, 28, 32, 44, 53, 65). A protective role has also been proposed to occur in P. aeruginosa PA14, where pyomelanin was shown to contribute to the persistence of an overproducing strain in a chronic CF infection model in mice (49). However, given that melanogenic isolates have been recovered from laboratory-grown communities of P. aeruginosa PAO1 (5, 56), it is probable that pyomelanin plays other roles in addition to protection against host defense mechanisms.As a first step toward gaining a better understanding of pyomelanin function, we sought to identify the molecular determinants of its production in P. aeruginosa. By screening a library of pTnTet/minimariner transposon mutants of a pyomelanin-overproducing clinical isolate for alterations in pigmentation, we identified several genes whose products are involved in tyrosine catabolism, central metabolic pathways, nucleotide biosynthesis, regulation, and membrane transport, in addition to hypothetical proteins of unknown function. We chose to further characterize the gene identified most frequently in our screen, one annotated as encoding a putative ATP-binding cassette of an ABC-type transporter. Here, we demonstrate that this transporter is involved in HGA transport and the subsequent extracellular formation of pyomelanin.     

Identification and Characterization of Higher Plant Myosins Responsible for Cytoplasmic Streaming

in vitro using these myosins and of localization studies using antiserum raised against each heavy chain, we suggested that both myosins are molecular motors for generating the motive force for cytoplasmic streaming in higher plant cells. The 170-kDa myosin is expressed not only in somatic cells but also in germinating pollen. In contrast, the 175-kDa myosin is distributed only in somatic cells. In the tip region of growing pollen tubes, it has been demonstrated that a tip-focused Ca²⁺ gradient is indispensable for growth and tube orientation. Cytoplasmic streaming in this region has been shown to be inactivated by high concentrations of Ca²⁺. The motile activity in vitro of 170-kDa myosin is suppressed by low (μM) levels of Ca²⁺ through its CaM light chain, suggesting that this suppression is one of the mechanisms for inactivating cytoplasmic streaming near the tip region of pollen tubes. The motile activity in vitro of 175-kDa myosin is also inhibited by Ca²⁺ at concentrations higher than 10⁻⁶M. It has been revealed that the elevation of cytosolic Ca²⁺ concentrations causes the cessation of cytoplasmic streaming even in somatic cells. Therefore, Ca²⁺-sensitivity of the motile activity of myosin appears to be a general molecular basis for Ca²⁺-induced cessation of cytoplasmic streaming. Received 6 September 2000/ Accepted in revised form 7 October 2000     

Identification and Regulation of fusA,the Polyketide Synthase Gene Responsible for Fusarin Production in Fusarium fujikuroi

Fusarins are a class of mycotoxins of the polyketide family produced by different Fusarium species, including the gibberellin-producing fungus Fusarium fujikuroi. Based on sequence comparisons between polyketide synthase (PKS) enzymes for fusarin production in other Fusarium strains, we have identified the F. fujikuroi orthologue, called fusA. The participation of fusA in fusarin biosynthesis was demonstrated by targeted mutagenesis. Fusarin production is transiently stimulated by nitrogen availability in this fungus, a regulation paralleled by the fusA mRNA levels in the cell. Illumination of the cultures results in a reduction of the fusarin content, an effect partially explained by a high sensitivity of these compounds to light. Mutants of the fusA gene exhibit no external phenotypic alterations, including morphology and conidiation, except for a lack of the characteristic yellow and/or orange pigmentation of fusarins. Moreover, the fusA mutants are less efficient than the wild type at degrading cellophane on agar cultures, a trait associated with pathogenesis functions in Fusarium oxysporum. The fusA mutants, however, are not affected in their capacities to grow on plant tissues.     

草鱼出血病混合感染的嗜水气单胞菌的分离、鉴定与理化特性

从患出血病草鱼的肝脏病灶中分离筛选出2株致病菌。取病鱼样品组织过滤液接种CIK细胞、培养, 电镜下观察到细胞质中含有草鱼呼肠孤病毒样颗粒和包涵体, 病毒颗粒大小65 nm~ 70 nm, 包涵体0.46 μm~1.81 μm。人工回归感染实验显示分离的菌株及细胞毒悬液均能使草鱼致病死亡。对分离菌株进行细胞形态学、理化特性分析及药敏试验, 初步判定所分离的2株菌均为嗜水气单胞菌。进一步对菌株进行DNA分子鉴定, 结果显示2株菌的16S rRNA基因、促旋酶亚单位蛋白(gryB)基因均与GenBank上的嗜水     

Identification and Characterization of a Periplasmic Aminoacyl-phosphatidylglycerol Hydrolase Responsible for Pseudomonas aeruginosa Lipid Homeostasis

Specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine (or with lysine) was shown to render various organisms less susceptible to antimicrobial agents and environmental stresses. In this study, we make use of the opportunistic pathogen Pseudomonas aeruginosa to decode ORF PA0919-dependent lipid homeostasis. Analysis of the polar lipid content of the deletion mutant ΔPA0919 indicated significantly enlarged levels of alanyl-PG. The resulting phenotype manifested an increased susceptibility to several antimicrobial compounds when compared with the wild type. A pH-dependent PA0919 promoter located within the upstream gene PA0920 was identified. Localization experiments demonstrated that the PA0919 protein is anchored to the periplasmic surface of the inner bacterial membrane. The recombinant overproduction of wild type and several site-directed mutant proteins in the periplasm of Escherichia coli facilitated a detailed in vitro analysis of the enzymatic PA0919 function. A series of artificial substrates (p-nitrophenyl esters of various amino acids/aliphatic acids) indicated enzymatic hydrolysis of the alanine, glycine, or lysine moiety of the respective ester substrates. Our final in vitro activity assay in the presence of radioactively labeled alanyl-PG then revealed hydrolysis of the aminoacyl linkage, resulting in the formation of alanine and PG. Consequently, PA0919 was termed alanyl-PG hydrolase. The elucidated enzymatic activity implies a new regulatory circuit for the appropriate tuning of cellular alanyl-PG concentrations.     

Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway

Riboflavin (vitamin B₂) is the precursor of the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide. In Escherichia coli and other bacteria, sequential deamination and reduction steps in riboflavin biosynthesis are catalyzed by RibD, a bifunctional protein with distinct pyrimidine deaminase and reductase domains. Plants have two diverged RibD homologs, PyrD and PyrR; PyrR proteins have an extra carboxyl-terminal domain (COG3236) of unknown function. Arabidopsis (Arabidopsis thaliana) PyrD (encoded by At4g20960) is known to be a monofunctional pyrimidine deaminase, but no pyrimidine reductase has been identified. Bioinformatic analyses indicated that plant PyrR proteins have a catalytically competent reductase domain but lack essential zinc-binding residues in the deaminase domain, and that the Arabidopsis PyrR gene (At3g47390) is coexpressed with riboflavin synthesis genes. These observations imply that PyrR is a pyrimidine reductase without deaminase activity. Consistent with this inference, Arabidopsis or maize (Zea mays) PyrR (At3g47390 or GRMZM2G090068) restored riboflavin prototrophy to an E. coli ribD deletant strain when coexpressed with the corresponding PyrD protein (At4g20960 or GRMZM2G320099) but not when expressed alone; the COG3236 domain was unnecessary for complementing activity. Furthermore, recombinant maize PyrR mediated NAD(P)H-dependent pyrimidine reduction in vitro. Import assays with pea (Pisum sativum) chloroplasts showed that PyrR and PyrD are taken up and proteolytically processed. Ablation of the maize PyrR gene caused early seed lethality. These data argue that PyrR is the missing plant pyrimidine reductase, that it is plastid localized, and that it is essential. The role of the COG3236 domain remains mysterious; no evidence was obtained for the possibility that it catalyzes the dephosphorylation that follows pyrimidine reduction.Riboflavin is the substrate for biosynthesis of the essential flavocoenzymes FMN and FAD, which occur in all kingdoms of life and have roles in diverse redox reactions as well as in other processes such as DNA repair, light sensing, and bioluminescence (Fischer and Bacher, 2005). Plants and many microorganisms can synthesize riboflavin, but humans and other animals cannot, so they must obtain it from the diet (Powers, 2003). Plant foods are important sources of riboflavin for humans, and the riboflavin pathway is a target for engineering biofortified crops (Fitzpatrick et al., 2012).Riboflavin biosynthesis proceeds via the same pathway in bacteria and plants (Fischer and Bacher, 2005; Roje, 2007). This pathway starts from GTP, which is converted by GTP cyclohydrolase II (named RibA in Escherichia coli) to the pyrimidine derivative 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-P. Deamination of the pyrimidine ring then yields 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-P, and subsequent reduction of the ribosyl moiety gives 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-P. After dephosphorylation, this product is condensed with 3,4-dihydroxy-2-butanone 4-P to give 6,7-dimethyl-8-ribityllumazine, whose dismutation yields riboflavin. Figure 1 shows the first four steps of this pathway.Open in a separate windowFigure 1.The first four steps of the riboflavin biosynthesis pathway in bacteria and plants. The enzymes involved are GTP cyclohydrolase II (RibA), pyrimidine deaminase (Deam), pyrimidine reductase (Red), and a specific phosphatase (Pase). Enzymes for which the plant genes are not known are colored red. Intermediates are as follows: 1, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-P; 2, 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-P; 3, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-P; 4, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.In E. coli, the deamination and reduction steps are catalyzed by a single bifunctional enzyme, RibD, which has N-terminal deaminase and C-terminal reductase domains that retain their respective activities when expressed separately (Richter et al., 1997; Magalhães et al., 2008). The situation in plants seems superficially similar but is in fact more complex (Gerdes et al., 2012). The bidomain bacterial RibD protein has two types of homologs in plants (Fischer et al., 2004; Chatwell et al., 2006; Chen et al., 2006), here called PyrD and PyrR, both with apparent deaminase and reductase domains (Fig. 2A). Only PyrD, represented by At4g20960, has been studied biochemically; it was found to have pyrimidine deaminase but not reductase activity (Fischer et al., 2004). The function of PyrR, represented by At3g47390, is unknown, although it has been inferred to have reductase activity (Chatwell et al., 2006; Chen et al., 2006; Ouyang et al., 2010) and perhaps to lack deaminase activity (Ouyang et al., 2010). Another mystery surrounding PyrR proteins is the presence of an extra C-terminal domain of unknown function (COG3236 in the Clusters of Orthologous Groups database; Fig. 2A); this domain occurs as a stand-alone protein in many bacteria. One possibility is that it catalyzes the dephosphorylation that follows the pyrimidine reduction step in the pathway (Fig. 1). The phosphatase involved is most likely substrate specific, but it has not been identified in plants or any other organism (Roje, 2007; Gerdes et al., 2012), and genes for enzymes in the same pathway, especially for successive steps, are quite commonly fused (Suhre, 2007). A mutation (phs1) that deleted the COG3236 domain from Arabidopsis (Arabidopsis thaliana) PyrR resulted in a photosensitive phenotype that could be rescued by supplied FAD (Ouyang et al., 2010).Open in a separate windowFigure 2.Structure and phylogeny of plant PyrD and PyrR proteins. A, Domain architectures. The examples shown are Arabidopsis At4g20960 and At3g47390; the predicted plastid targeting peptide (TP) varies in length between species. B, Phylogenetic tree of PyrD and PyrR proteins. Sequences were aligned with ClustalW; the tree was built by the neighbor-joining method with MEGA5. Bootstrap values (%) for 1,000 replicates are next to the nodes. Only the tree topology is shown. Note that the PyrD proteins of green algae (underlined) lack a reductase domain. C, Alignments showing the conservation of the zinc-binding residues (arrowheads) in the deaminase domain of PyrD but not PyrR proteins and the conservation of the predicted substrate-binding residues (asterisks) in the reductase domain of PyrR but not PyrD proteins. The deaminase sequences correspond to residues 45 to 85 of B. subtilis RibD (synonym RibG); the reductase sequences correspond to residues 150 to 210 and (separated by dots) 288 to 292 of B. subtilis RibD. Identical zinc- or substrate-binding residues are black, and conservative replacements are gray. Dashes indicate gaps that maximize the alignment.The plant riboflavin synthesis pathway is considered to be plastidial (Roje, 2007), but this location is based almost solely on bioinformatics and high-throughput proteome analyses (Gerdes et al., 2012). In only one case is there more definitive experimental support: in vitro chloroplast import data for the pathway’s penultimate enzyme, 6,7-dimethyl-8-ribityllumazine synthase (Jordan et al., 1999). Similarly, clear genetic support for the function of most plant riboflavin synthesis enzymes is lacking (Gerdes et al., 2012), the exception being an Arabidopsis RibA homolog (Hedtke and Grimm, 2009).The work reported here established, using maize (Zea mays) and Arabidopsis, that PyrR is indeed the missing pyrimidine reductase, that it lacks deaminase activity, and that its COG3236 domain is not essential for pyrimidine reductase activity and most likely lacks phosphatase activity. We also demonstrated the import of PyrR and PyrD into chloroplasts in vitro and confirmed that the gene for PyrR is essential.     

Identification and Molecular Characterization of Parkin in Clonorchis sinensis

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn²⁺ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.     

Identification and Characterisation of the Murine Homologue of the Gene Responsible for Cystinosis, Ctns

Background  

Cystinosis is an autosomal recessive disorder characterised by an intralysosomal accumulation of cystine, and affected individuals progress to end-stage renal failure before the age of ten. The causative gene, CTNS, was cloned in 1998 and the encoded protein, cystinosin, was predicted to be a lysosomal membrane protein.     

Identification and Characterization of Brassinosteroid Biosynthesis and Signaling Pathway Genes in Solanum tuberosum

Russian Journal of Plant Physiology - Brassinosteroids (BRs) are plant hormones which promote plant growth and development. Their biosynthetic pathway and signal transduction pathway have been well...     

Identification and Characterization of the Major Proteins of Mammalian Brain Synaptic Vesicles

Highly purified rat and cow brain synaptic vesicles contain major proteins with molecular weights of approximately 74,000, 60,000, 57,000, 40,000, 38,000, and 34,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of the major proteins on synaptic vesicles was confirmed by immunoprecipitation of intact rat brain synaptic vesicles with a synaptic vesicle-specific monoclonal antibody. The 40,000-Mr protein appeared to be identical to the 38,000-Mr integral membrane glycoprotein, p38 or synaptophysin, previously identified as a major component of mammalian synaptic vesicles. The isoelectric point of the 75,000-Mr proteins from either rat or cow brain synaptic vesicles is 5.0, and the pI of the 57,000-Mr protein is approximately 5.1 in both species. The similarity in size and charge of several major proteins in rat and cow synaptic vesicles suggests a high degree of structure conservation of these proteins in diverse mammalian species and raises the possibility that a set of functions common to most or all mammalian synaptic vesicles is mediated by these proteins.     

Identification and Molecular Characterization of Two Acetylcholinesterases from the Salmon Louse,Lepeophtheirus salmonis

Acetylcholinesterase (AChE) is an important enzyme in cholinergic synapses. Most arthropods have two genes (ace1 and ace2), but only one encodes the predominant synaptic AChE, the main target for organophosphates. Resistance towards organophosphates is widespread in the marine arthropod Lepeophtheirus salmonis. To understand this trait, it is essential to characterize the gene(s) coding for AChE(s). The full length cDNA sequences encoding two AChEs in L. salmonis were molecularly characterized in this study. The two ace genes were highly similar (83.5% similarity at protein level). Alignment to the L. salmonis genome revealed that both genes were located close to each other (separated by just 26.4 kbp on the L. salmonis genome), resulting from a recent gene duplication. Both proteins had all the typical features of functional AChE and clustered together with AChE-type 1 proteins in other species, an observation that has not been described in other arthropods. We therefore concluded the presence of two versions of ace1 gene in L. salmonis, named ace1a and ace1b. Ace1a was predominantly expressed in different developmental stages compared to ace1b and was possibly active in the cephalothorax, indicating that ace1a is more likely to play the major role in cholinergic synaptic transmission. The study is essential to understand the role of AChEs in resistance against organophosphates in L. salmonis.