细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

Functional characterization of an aminotransferase required for pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa PAO1

The fluorescent dihydroxyquinoline chromophore of the pyoverdine siderophore in Pseudomonas is a condensation product of D-tyrosine and l-2,4-diaminobutyrate. Both pvdH and asd (encoding aspartate beta-semialdehyde dehydrogenase) knockout mutants of Pseudomonas aeruginosa PAO1 were unable to synthesize pyoverdine under iron-limiting conditions in the absence of l-2,4-diaminobutyrate in the culture media. The pvdH gene was subcloned, and the gene product was hyperexpressed and purified from P. aeruginosa PAO1. PvdH was found to catalyze an aminotransferase reaction, interconverting aspartate beta-semialdehyde and l-2,4-diaminobutyrate. Steady-state kinetic analysis with a novel coupled assay established that the enzyme adopts a ping-pong kinetic mechanism and has the highest specificity for alpha-ketoglutarate. The specificity of the enzyme toward the smaller keto acid pyruvate is 41-fold lower. The enzyme has negligible activity toward other keto acids tested. Homologues of PvdH were present in the genomes of other Pseudomonas spp. These homologues were found in the DNA loci of the corresponding genomes that contain other pyoverdine synthesis genes. This suggests that there is a general mechanism of l-2,4-diaminobutyrate synthesis in Pseudomonas strains that produce the pyoverdine siderophore.     

Elevated zinc induces siderophore biosynthesis genes and a zntA-like gene in Pseudomonas fluorescens

Zinc-regulated genes were analyzed in Pseudomonas fluorescens employing mutagenesis with a reporter gene transposon. Six mutants responded with increased gene expression to elevated concentrations of zinc. Genetic and biochemical analysis revealed that in four of the six mutants the transposon had inserted into genes essential for the biosynthesis of the siderophore pyoverdine. The growth of one of the mutants was severely impaired in the presence of elevated concentrations of cadmium and zinc ions. In this mutant, the transposon had inserted in a gene with high similarity to P-type ATPases involved in zinc and cadmium ion transport. Four mutants reacted with reduced gene expression to elevated concentrations of zinc. One of these mutants was sensitive to zinc, cadmium and copper ions. The genetic region targeted in this mutant did not show similarity to any known gene.     

Iron limitation by transferrin promotes simultaneous cheating of pyoverdine and exoprotease in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a primary bacterial model to study cooperative behaviors because it yields exoproducts such as siderophores and exoproteases that act as public goods and can be exploited by selfish nonproducers behaving as social cheaters. Iron-limited growth medium, mainly casamino acids medium supplemented with transferrin, is typically used to isolate and study nonproducer mutants of the siderophore pyoverdine. However, using a protein as the iron chelator could inadvertently select mutants unable to produce exoproteases, since these enzymes can degrade the transferrin to facilitate iron release. Here we investigated the evolutionary dynamics of pyoverdine and exoprotease production in media in which iron was limited by using either transferrin or a cation chelating resin. We show that concomitant loss of pyoverdine and exoprotease production readily develops in media containing transferrin, whereas only pyoverdine loss emerges in medium treated with the resin. Characterization of exoprotease- and pyoverdine-less mutants revealed loss in motility, different mutations, and large genome deletions (13–33 kb) including Quorum Sensing (lasR, rsal, and lasI) and flagellar genes. Our work shows that using transferrin as an iron chelator imposes simultaneous selective pressure for the loss of pyoverdine and exoprotease production. The unintended effect of transferrin uncovered by our experiments can help to inform the design of similar studies.Subject terms: Bacteriology, Microbial ecology     

Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of twitching motility

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important human pathogen. The production of several virulence factors by P. aeruginosa is controlled through two quorum-sensing systems, las and rhl. We have obtained evidence that both the las and rhl quorum-sensing systems are also required for type 4 pilus-dependent twitching motility and infection by the pilus-specific phage D3112cts. Mutants which lack the ability to synthesize PAI-1, PAI-2, or both autoinducers were significantly or greatly impaired in twitching motility and in susceptibility to D3112cts. Twitching motility and phage susceptibility in the autoinducer-deficient mutants were partially restored by exposure to exogenous PAI-1 and PAI-2. Both twitching motility and infection by pilus-specific phage are believed to be dependent on the extension and retraction of polar type 4 pili. Western blot analysis of whole-cell lysates and enzyme-linked immunosorbent assays of intact cells were used to measure the amounts of pilin on the cell surfaces of las and rhl mutants relative to that of the wild type. It appears that PAI-2 plays a crucial role in twitching motility and phage infection by affecting the export and assembly of surface type 4 pili. The ability of P. aeruginosa cells to adhere to human bronchial epithelial cells was also found to be dependent on the rhl quorum-sensing system. Microscopic analysis of twitching motility indicated that mutants which were unable to synthesize PAI-1 were defective in the maintenance of cellular monolayers and migrating packs of cells. Thus, PAI-1 appears to have an essential role in maintaining cell-cell spacing and associations required for effective twitching motility.     

Las-like quorum-sensing system negatively regulates both pyoluteorin and phenazine-1-carboxylic acid production in Pseudomonas sp. M18

A las-like quorum-sensing system in Pseudomonas sp. M18 was identified, which consisted of lasI and lasR genes encoding LuxI-LuxR type regulator. Several functions of the las system from strain M18 were investigated in this study. The chromosomal inactivation of either lasI or lasR by recombination increased the production of both pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA) by 4-5 fold and 2-3 fold over that of the wild type strain of M18, respectively. Production of both antibiotics was restored to wild-type levels after in trans complementation with the wild-type lasI or lasR gene. Expression of the translational fusions pltA'-'lacZ and phzA'-'lacZ further confirmed the negative effect of lasI or lasR on both biosynthetic operons, and it was also demonstrated that the las system was related to the ability of swarming motility and the inhibition of cell growth.     

Mapping of mutations affecting pyoverdine production in Pseudomonas aeruginosa

Abstract Pyoverdine, the yellow-green fluorescent pigment produced by Pseudomonas aeruginosa , is a highly efficient siderophore. Pyoverdine-deficient ( pvd ) mutants of P. aeruginosa PAO isolated after mutagenesis were non-fluorescent and unable to grow in the presence of 2.8 mM ethylenediamine-di-( o -hydroxyphenylacetate) (EDDHA). Addition of purified pyoverdine to media containing EDDHA restored growth of pvd mutants. 6 pvd mutations were mapped between catA and mtu -9002 (at 65–70 min on the chromosome map) by R68.45-mediated conjugation. 2 slightly leaky pvd mutations were localised between argC and strA (at 35 min) by transduction. Thus, we have identified at least 2 genes or gene clusters required for pyoverdine production in P. aeruginosa .     

Quorum-sensing antagonist (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone influences siderophore biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa

Siderophore synthesis of Pseudomonas putida F1 was found to be regulated by quorum sensing since normalized siderophore production (per cell) increased 4.2-fold with cell density after the cells entered middle exponential phase; similarly, normalized siderophore concentrations in Pseudomonas aeruginosa JB2 increased 28-fold, and a 5.5-fold increase was seen for P. aeruginosa PAO1. Further evidence of the link between quorum sensing and siderophore synthesis of P. putida F1 was that the quorum-sensing-disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the marine red alga Delisea pulchra was found to inhibit the formation of the siderophore produced by P. putida F1 in a concentration-dependent manner, with 57% siderophore synthesis repressed by 100 g/ml furanone. In contrast, this furanone did not affect the siderophore synthesis of Burkholderia cepacia G4 at 20–40 g/ml, and stimulated siderophore synthesis of P. aeruginosa JB2 2.5- to 3.7-fold at 20–100 g/ml. Similarly, 100 g/ml furanone stimulated siderophore synthesis in P. aeruginosa PAO1 about 3.5-fold. The furanone appears to interact with the quorum-sensing machinery of P. aeruginosa PAO1 since it stimulates less siderophore synthesis in the P. aeruginosa qscR quorum-sensing mutant (QscR is a negative regulator of LasI, an acylated homoserine lactone synthase).     

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.     

The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues

Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-enterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of siderophore receptor redundancy in P. aeruginosa.     

Thioquinolobactin, a Pseudomonas siderophore with antifungal and anti-Pythium activity

Under conditions of iron limitation Pseudomonas fluorescens ATCC 17400 produces two siderophores, pyoverdine, and a second siderophore quinolobactin, which itself results from the hydrolysis of the unstable molecule 8-hydroxy-4-methoxy-2-quinoline thiocarboxylic acid (thioquinolobactin). Pseudomonas fluorescens ATCC 17400 also displays a strong in vitro antagonism against the Oomycete Pythium, which is repressed by iron, suggesting the involvement of a siderophore(s). While a pyoverdine-negative mutant retains most of its antagonism, a thioquinolobactin-negative mutant only slowed-down Pythium growth, and a double pyoverdine-, thioquinolobactin-negative mutant, which does not produce any siderophore, totally lost its antagonism against Pythium. The siderophore thioquinolobactin could be purified and identified from spent medium and showed anti-Pythium activity, but it was quickly hydrolysed to quinolobactin, which we showed has no antimicrobial activity. Analysis of antagonism-affected transposon mutants revealed that genes involved in haem biosynthesis and sulfur assimilation are important for the production of thioquinolobactin and the expression of antagonism.     

Temperature and pyoverdine-mediated iron acquisition control surface motility of Pseudomonas putida

Pseudomonas putida KT2440 is unable to swarm at its common temperature of growth in the laboratory (30 degrees C) but exhibits surface motility similar to swarming patterns in other Pseudomonas between 18 degrees C and 28 degrees C. These motile cells show differentiation, consisting on elongation and the presence of surface appendages. Analysis of a collection of mutants to define the molecular determinants of this type of surface movement in KT2440 shows that while type IV pili and lipopolysaccharide O-antigen are requisites flagella are not. Although surface motility of flagellar mutants was macroscopically undistinguishable from that of the wild type, microscopy analysis revealed that these mutants move using a distinct mechanism to that of the wild-type strain. Mutants either in the siderophore pyoverdine (ppsD) or in the FpvA siderophore receptor were also unable to spread on surfaces. Motility in the ppsD strain was totally restored with pyoverdine and partially with the wild-type ppsD allele. Phenotype of the fpvA strain was not complemented by this siderophore. We discuss that iron influences surface motility and that it can be an environmental cue for swarming-like movement in P. putida. This study constitutes the first report assigning an important role to pyoverdine iron acquisition in en masse bacterial surface movement.