The heme oxygenase(s)-phytochrome system of Pseudomonas aeruginosa

For many pathogenic bacteria like Pseudomonas aeruginosa heme is an essential source of iron. After uptake, the heme molecule is degraded by heme oxygenases to yield iron, carbon monoxide, and biliverdin. The heme oxygenase PigA is only induced under iron-limiting conditions and produces the unusual biliverdin isomers IXbeta and IXdelta. The gene for a second putative heme oxygenase in P. aeruginosa, bphO, occurs in an operon with the gene bphP encoding a bacterial phytochrome. Here we provide biochemical evidence that bphO encodes for a second heme oxygenase in P. aeruginosa. HPLC, (1)H, and (13)C NMR studies indicate that BphO is a "classic" heme oxygenase in that it produces biliverdin IXalpha. The data also suggest that the overall fold of BphO is likely to be the same as that reported for other alpha-hydroxylating heme oxygenases. Recombinant BphO was shown to prefer ferredoxins or ascorbate as a source of reducing equivalents in vitro and the rate-limiting step for the oxidation of heme to biliverdin is the release of product. In eukaryotes, the release of biliverdin is driven by biliverdin reductase, the subsequent enzyme in heme catabolism. Because P. aeruginosa lacks a biliverdin reductase homologue, data are presented indicating an involvement of the bacterial phytochrome BphP in biliverdin release from BphO and possibly from PigA.     

Complex formation between heme oxygenase and phytochrome during biosynthesis in Pseudomonas syringae pv. tomato

The plant pathogen Pseudomonas syringae pv. tomato carries two genes encoding bacterial phytochromes. Sequence motifs identify both proteins (PstBphP1 and PstBphP2, respectively) as biliverdin IXα (BV)-binding phytochromes. PstbphP1 is arranged in an operon with a heme oxygenase (PstBphO)-encoding gene (PstbphO), whereas PstbphP2 is flanked downstream by a gene encoding a CheY-type response regulator. Expression of the heme oxygenase PstBphO yielded a green protein (λ(max) = 650 nm), indicative for bound BV. Heterologous expression of PstbphP1 and PstbphP2 and in vitro assembly with BV IXα yielded the apoproteins for both phytochromes, but only in the case of PstBphP1 a light-inducible chromoprotein. Attempts to express the endogenous heme oxygenase BphO and either of the two phytochromes from two plasmids yielded only holo-PstBphP1. Relatively small amounts of soluble holo-PstBphP2 were just obtained upon co-expression with BphO from P. aeruginosa. Expression of the operon containing PstbphO:PstbphP1 led to an improved yield and better photoreactivity for PstBphP1, whereas an identical construct, exchanging PstbphP1 for PstbphP2 (PstbphO:PstbphP2), again yielded only minute amounts of chromophore-loaded BphP2-holoprotein. The improved yield for PstBphP1 from the PstbphO:PstbphP1 operon expression is apparently caused by complex formation between both proteins during biosynthesis as affinity chromatography of either protein using two different tags always co-purified the reaction partner. These results support the importance of protein-protein interactions during tetrapyrrole metabolism and phytochrome assembly.     

Variable composition of heme oxygenases with different regiospecificities in Pseudomonas species

Heme oxygenases (HO) degrade heme yielding iron, carbon monoxide and one of four possible biliverdin (BV) isomers. Pseudomonas aeruginosa PAO1 is thus far the only organism to contain two HOs with different regiospecificities: BphO and PigA. While BphO cleaves heme to exclusively yield BV IXα, PigA produces the BV isomers IXβ and IXδ. We bioinformatically identified putative HOs in diverse Pseudomonas strains, tested their enzymatic functionality and determined their regiospecificity. Surprisingly, even high amino acid sequence identities to the P. aeruginosa HOs were not sufficient to correctly predict the HO regiospecificity in all cases. Based on our results, Pseudomonas strains differ in their HO composition containing either BphO or PigA or both HO types. Concomitantly with the existence of bphO is the occurrence of at least one gene encoding a bacterial phytochrome implying that only BV IXα is the sufficient phytochrome chromophore. In contrast, pigA genes are organized in gene clusters associated with iron utilization implying a role of PigA in iron acquisition. However, at least in strains containing no PigA this function maybe fulfilled by BphO. Only a combination of homology searches and analyses of genetic environments is appropriate for a reliable prediction of the regiospecificity of Pseudomonas HOs.     

Biochemical and spectroscopic characterization of the bacterial phytochrome of Pseudomonas aeruginosa

Phytochromes are photochromic biliproteins found in plants as well as in some cyanotrophic, photoautotrophic and heterotrophic bacteria. In many bacteria, their function is largely unknown. Here we describe the biochemical and spectroscopic characterization of recombinant bacterial phytochrome from the opportunistic pathogen Pseudomonas aeruginosa (PaBphP). The recombinant protein displays all the characteristic features of a bonafide phytochrome. In contrast with cyanobacteria and plants, the chromophore of this bacterial phytochrome is biliverdin IXalpha, which is produced by the heme oxygenase BphO in P. aeruginosa. This chromophore was shown to be covalently attached via its A-ring endo-vinyl group to a cysteine residue outside the defined bilin lyase domain of plant and cyanobacterial phytochromes. Site-directed mutagenesis identified Cys12 and His247 as being important for chromophore binding and photoreversibility, respectively. PaBphP is synthesized in the dark in the red-light-absorbing Pr form and immediately converted into a far-red-light-absorbing Pfr-enriched form. It shows the characteristic red/far-red-light-induced photoreversibility of phytochromes. A chromophore analog that lacks the C15/16 double bond was used to show that this photoreversibility is due to a 15Z/15E isomerization of the biliverdin chromophore. Autophosphorylation of PaBphP was demonstrated, confirming its role as a sensor kinase of a bacterial two-component signaling system.     

Cloning and characterisation of the rpoS gene from plant growth-promoting Pseudomonas putida WCS358: RpoS is not involved in siderophore and homoserine lactone production

The rpoS gene which encodes a stationary phase sigma factor has been identified and characterised from the rhizosphere-colonising plant growth-promoting Pseudomonas putida strain WCS358. The predicted protein sequence has extensive homologies with the RpoS proteins form other bacteria, in particular with the RpoS sigma factors of the fluorescent pseudomonads. A genomic transposon insertion in the rpoS gene was constructed, these mutants were analysed for their ability to produce siderophore (iron-transport agent) and the autoinducer quorum-sensing molecules called homoserine lactones (AHL). It was determined that RpoS was not involved in the regulation of siderophore and AHL production, synthesis of these molecules is important for gene expression at stationary phase. P. putida WCS358 produces at least three different AHL molecules.     

Identification of a novel regulator of the quorum-sensing systems in Pseudomonas aeruginosa

Quorum sensing is a global gene-regulatory mechanism in bacteria that enables individual bacterial cells to communicate and coordinate their population behaviors. Quorum sensing is central to the pathogenesis of many bacterial pathogens including Pseudomonas aeruginosa and therefore has been exploited as a target for developing novel antipathogenic drugs. In P. aeruginosa , three intertwined quorum-sensing systems, las, rhl , and the 2-alkyl-4(1 H )-quinolone system, which includes the Pseudomonas quinolone signal (PQS), control virulence factor production, and pathogenesis processes. Previously, we obtained a mutant with diminished expression of the phzA1B1C1D1E1F1G1 operon that is involved in the production of virulence factor phenazine compounds. In this study, the mutant was further characterized, and evidence indicating that the disrupted gene PA1196 in the mutant is a potential regulator of the rhl and PQS systems is presented. PA1196 positively controls the expression of the rhl and PQS systems and affects bacterial motility and multiple virulence factor expression via the quorum-sensing systems. This adds an important new player in the complex quorum-sensing network in P. aeruginosa .     

The pvc Gene Cluster of Pseudomonas aeruginosa: Role in Synthesis of the Pyoverdine Chromophore and Regulation by PtxR and PvdS

A putative operon of four genes implicated in the synthesis of the chromophore moiety of the Pseudomonas aeruginosa siderophore pyoverdine, dubbed pvcABCD (where pvc stands for pyoverdine chromophore), was cloned and sequenced. Mutational inactivation of the pvc genes abrogated pyoverdine biosynthesis, consistent with their involvement in the biosynthesis of this siderophore. pvcABCD expression was negatively regulated by iron and positively regulated by both PvdS, the alternate sigma factor required for pyoverdine biosynthesis, and PtxR, a LysR family activator previously implicated in exotoxin A regulation.     

Role for rpoS gene of Pseudomonas aeruginosa in antibiotic tolerance

The alternative sigma factor, RpoS has been described as a central regulator of many stationary phase-inducible genes and a master stress-response regulator under various stress conditions. We constructed an rpoS mutant in Pseudomonas aeruginosa and investigated the role of rpoS gene in antibiotic tolerance. The survival of the rpoS mutant cells in stationary phase was approximately 70 times lower when compared with that of the parental strain at 37 degrees C for 2 h after the addition of biapenem. For imipenem, the survival was approximately 40 times lower. Heat stress promoted an increase in the survival of the parental strain to biapenem, but the same was not found to be the case for the rpoS mutant. Our results indicate that rpoS gene is involved in tolerance to antibiotics in P. aeruginosa during the stationary phase and heat stress. However, under osmotic stress, tolerance to biapenem was not dependent on the rpoS gene.     

铜绿假单胞菌PA1550基因对泳动及蹭动的影响

[目的]:研究与铜绿假单胞菌运动能力相关的基因.[方法]:以一株临床分离的铜绿假单胞菌PA68做受体菌,应用人工Mu转座技术建立了库容为2000的突变子文库,从中筛选出泳动能力和蹭动能力丧失或减弱的突变子,通过基因克隆、测序,GenBankBLAST比对测序结果,互补基因表达确定与铜绿假单胞菌运动能力相关的基因.[结果]:突变子Y46在丧失了泳动运动能力的同时,蹭动能力也发生了减弱.在Y46突变子中,Mu转座子插入到功能完全未知的基因PA1550中.对极性效应及PA1550所在操纵子的分析表明,Mu转座子对插入点下游的基因的转录并不造成影响.[结论]:PA1550与铜绿假单胞菌的泳动及蹭动能力有关.