Iron uptake regulation in Pseudomonas aeruginosa

The Pseudomonas genus belongs to the γ division of Proteobacteria and many species produce the characteristic yellow–green siderophore pyoverdine, and often a second siderophore, of lower affinity for iron. These bacteria are known for their ability to colonize different ecological niches and for their versatile metabolism. It is therefore not surprising that they are endowed with the capacity to take up exogenous xenosiderophores via different TonB-dependent receptors. Uptake of iron is controlled by the central regulator Fur, and via extracytoplasmic sigma factors or other types of regulators (two-component systems, AraC regulators). In this review the Fur regulon (experimentally proven and/or predicted) of P. aeruginosa will be presented. An interesting feature revealed by this analysis of Fur-regulated genes is the overlap between the iron and the sulfur regulons as well with the quorum sensing system.     

Effects of iron(III) analogs on growth and pseudobactin synthesis in a chromiumtolerantPseudomonas isolate

Summary The growth and siderophore production of a fluorescentPseudomonas species isolated from soil contaminated with chromium was found to be influenced by the presence of trivalent cations. Overproduction of pseudobactin occurred when the isolate was grown in media containing 1 mM Cr(III) under ironlimited conditions but not when Fe(III) was added at 10 M Pseudobactin synthesis was derepressed in iron-limited cultures containing 1 mM Sc(III) or Y(III), examples of group III-B elements. We found that AI(III), Ga(III) or In(III), representative metals from group III-A, repressed synthesis of pseudobactin under iron-deficient conditions. Analogs of Fe(III) were found to inhibit growth of thePseudomonas isolate in iron-limited media and the trivalent metals listed in order of decreasing toxicity were as follows: Ga > In > Sc > Cr > Y > Al. The inhibition of growth by 1 mM In(III), Sc(III) and Ga(III) was greater during iron-limited growth than in media containing 10 M Fe(III). These data show that, although the metal analogs of Fe(III) have similar chemical and physical characteristics, the physiological response of the fluorescent pseudomonad when grown in the presence of these metals varied markedly.     

Ecology of a plant growth-promoting strain of Pseudomonas fluorescens colonizing the maize endorhizosphere in tropical soil

Pseudomonas fluorescens strain BR-5 stimulated the growth of maize in a natural soil and inhibited fungal root pathogens in vitro. Strain BR-5 was detected inside plant cells, indicating that it is able to colonize the endorhizosphere. No significant effect was detected on soil or ectorhizosphere microbial population after inoculation of strain BR-5 onto seeds.     

Isolation of root-associated Pseudomonas and Burkholderia spp. with biocontrol and plant growth-promoting traits

Novel, root-associated Pseudomonas and Burkholderia strains with biological control and plant growth-promoting (PGP) traits are being sought for biotechnological application in agriculture. We present a new isolation approach for recovery of rhizoplane and/or endophytic Pseudomonas and Burkholderia spp. with desirable biocontrol and PGP phenotypes. The method may enable better targeted biodiscovery of these two important genera.     

Regulation of pyrimidine synthesis in Pseudomonas resinovorans

AIMS: To investigate the regulation of de novo pyrimidine biosynthesis in the bacterium Pseudomonas resinovorans ATCC 14235. METHODS AND RESULTS: The pyrimidine biosynthetic pathway enzymes were measured in cell extracts from P. resinovorans ATCC 14235 and from an auxotroph lacking orotate phosphoribosyltransferase activity. Pyrimidine biosynthetic pathway enzyme activities in ATCC 14235 were affected by the addition of pyrimidine bases to the culture medium. The de novo enzyme activities of the phosphoribosyltransferase mutant strain increased after pyrimidine starvation indicating possible repression of the pathway by a pyrimidine-related compound. Aspartate transcarbamoylase activity in ATCC 14235 was inhibited in vitro by ATP, UTP and pyrophosphate. CONCLUSIONS: Pyrimidine biosynthesis in P. resinovorans was regulated at the level of enzyme synthesis and at the level of activity for aspartate transcarbamoylase. Its regulation of enzyme synthesis seemed to be similar to what has been observed in the taxonomically related species Pseudomonas oleovorans. SIGNIFICANCE AND IMPACT OF THE STUDY: This study found that pyrimidine biosynthesis is regulated in P. resinovorans. This could prove helpful to future studies investigating polyhydroxyalkanoate production by the bacterium.     

Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity iron transport agent of a plant growth-promoting Pseudomonas strain

A gene bank of DNA from plant growth-promoting Pseudomonas sp. strain B10 was constructed using the broad host-range conjugative cosmid pLAFR1. The recombinant cosmids contained insert DNA averaging 21.5 kilobase pairs in length. Nonfluorescent mutants of Pseudomonas sp. strain B10 were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, or UV light and were defective in the biosynthesis of its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin. No yellow-green, fluorescent mutants defective in the production of pseudobactin were identified. Nonfluorescent mutants were individually complemented by mating the gene bank en masse and identifying fluorescent transconjugants. Eight recombinant cosmids were sufficient to complement 154 nonfluorescent mutants. The pattern of complementation suggests that a minimum of 12 genes arranged in four gene clusters is required for the biosynthesis of pseudobactin. This minimum number of genes seems reasonable considering the structural complexity of pseudobactin.     

A second tonB gene in Pseudomonas aeruginosa is linked to the exbB and exbD genes

The exbBD genes of Pseudomonas aeruginosa PAO were cloned by complementation of the growth defect of an Escherichia coli exbB tolQ double mutant on iron-restricted medium. Nucleotide sequence analysis confirmed that these genes are contiguous and preceded by a second tonB gene in this organism, which we have designated tonB2. lacZ promoter fusions confirmed that expression of the tonB2-exbB-exbD genes is increased under conditions of iron limitation. Deletions within any of these genes, in contrast to deletions in the first tonB gene, tonB1, did not adversely affect growth on iron-restricted medium. On the other hand, tonB1 tonB2 double mutants were more compromised as regards growth in an iron-restricted medium than a tonB1 deletion, indicating that TonB2 could partially replace TonB1 in its role in iron acquisition. TonB1 but not TonB2 deletion strains were also compromised as regards the utilization of hemin or hemoglobin as sole iron sources, indicating that heme transport requires TonB1.     

Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267

Aims: Plant growth‐promoting Pseudomonas putida strain 267, originally isolated from the rhizosphere of black pepper, produces biosurfactants that cause lysis of zoospores of the oomycete pathogen Phytophthora capsici. The biosurfactants were characterized, the biosynthesis gene(s) partially identified, and their role in control of Phytophthora damping‐off of cucumber evaluated. Methods and Results: The biosurfactants were shown to lyse zoospores of Phy. capsici and inhibit growth of the fungal pathogens Botrytis cinerea and Rhizoctonia solani. In vitro assays further showed that the biosurfactants of strain 267 are essential in swarming motility and biofilm formation. In spite of the zoosporicidal activity, the biosurfactants did not play a significant role in control of Phytophthora damping‐off of cucumber, since both wild type strain 267 and its biosurfactant‐deficient mutant were equally effective, and addition of the biosurfactants did not provide control. Genetic characterization revealed that surfactant biosynthesis in strain 267 is governed by homologues of PsoA and PsoB, two nonribosomal peptide synthetases involved in production of the cyclic lipopeptides (CLPs) putisolvin I and II. The structural relatedness of the biosurfactants of strain 267 to putisolvins I and II was supported by LC‐MS and MS‐MS analyses. Conclusions: The biosurfactants produced by Ps. putida 267 were identified as putisolvin‐like CLPs; they are essential in swarming motility and biofilm formation, and have zoosporicidal and antifungal activities. Strain 267 provides excellent biocontrol activity against Phytophthora damping‐off of cucumber, but the lipopeptide surfactants are not involved in disease suppression. Significance and Impact of the Study: Pseudomonas putida 267 suppresses Phy. capsici damping‐off of cucumber and provides a potential supplementary strategy to control this economically important oomycete pathogen. The putisolvin‐like biosurfactants exhibit zoosporicidal and antifungal activities, yet they do not contribute to biocontrol of Phy. capsici and colonization of cucumber roots by Ps. putida 267. These results suggest that Ps. putida 267 employs other, yet uncharacterized, mechanisms to suppress Phy. capsici.     

Cloning of the gene coding for the outer membrane receptor protein for ferric pseudobactin, a siderophore from a plant growth-promoting Pseudomonas strain

Plant growth-promoting Pseudomonas B10 produces its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin under iron-limiting conditions. A structural gene encoding the 85,000-Da putative outer membrane receptor protein for ferric pseudobactin was identified in a gene bank from Pseudomonas B10 prepared with the broad host-range conjugative cosmid cloning vector pLAFR1. Transposon Tn5 mutagenesis of recombinant plasmid pJLM300 localized the functional gene to a region of approximately 2.4 kilobases consistent with the apparent molecular weight of the receptor protein. Mobilization of pJLM300 into Pseudomonas A124 and A225, whose growth was inhibited by Pseudomonas B10 or pseudobactin, rendered these strains no longer susceptible to iron starvation by pseudobactin because they were now able to transport ferric pseudobactin. Pseudobactin biosynthetic genes flanked this receptor gene on both sides and were on separate operons. Transposon Tn5 insertion mutants of Pseudomonas B10 lacking this receptor protein were generated by a marker exchange technique and were defective in ferric pseudobactin transport. Such mutants could be complemented in trans by pJLM300. The production of pseudobactin, the receptor protein, and four other outer membrane proteins in Pseudomonas B10 was coordinately regulated by the level of intracellular iron.     

Overproduction of pyoverdins by Fur mutants of Pseudomonas aeruginosa strains PAO1 and Fe10 in stirred bioreactors

Fur mutants FPA12 and FF13 of strains Pseudomonas aeruginosa PAO1 and Fe10, respectively, were prepared and their production of pyoverdin evaluated. The strains were cultivated in stirred bioreactor in iron-deficient and iron-supplemented medium containing Casamino acids (CA) or succinate as a source of carbon and energy. When the pyoverdin production rate reached its maximum, the demand of iron-depleted cultures for O₂ was decreased. Mutant FF13 overproduced pyoverdin in both iron-depleted (862 mg l^–1) and iron-supplemented (428 mg l^–1) CA medium and could also be used to produce pyoverdin when grown in a conventional stirred tank fermenter.     

Proteomics of a plant growth-promoting rhizobacterium, Pseudomonas fluorescens MSP-393, subjected to salt shock

Summary Pseudomonas fluorescens MSP-393, a plant growth-promoting rhizobacterium is an efficient biocontrol agent in rice grown in saline soils of coastal ecosystems. To understand the mechanism of salt tolerance, proteome analysis of the bacterium was carried out employing two-dimensional gel electrophoresis and MALDI-TOF. This technique was used to investigate the regulation of gene product expression of P. fluorescens MSP-393 grown under high osmolarity and used peptide mass fingerprinting and in silico investigation to identify those proteins with altered expression. Among them 15 were assigned to proteins with known functions. Their roles in response to salt stress are discussed.     

Mechanism of insoluble phosphate solubilization by Pseudomonas fluorescens RAF15 isolated from ginseng rhizosphere and its plant growth-promoting activities

Aims:  To investigate the mechanism of insoluble phosphate (P) solubilization and plant growth-promoting activity by Pseudomonas fluorescens RAF15.
Methods and Results:  We investigated the ability of Ps. fluorescens RAF15 to solubilize insoluble P via two possible mechanisms: proton excretion by ammonium assimilation and organic acid production. There were no clear differences in pH and P solubilization between glucose-ammonium and glucose-nitrate media. P solubilization was significantly promoted with glucose compared to fructose. Regardless of nitrogen sources used, Ps. fluorescens RAF15 solubilized little insoluble P with fructose. High performance liquid chromatography analysis showed that Ps. fluorescens RAF15 produced mainly gluconic and tartaric acids with small amounts of 2-ketogluconic, formic and acetic acids. During the culture, the pH was reduced with increase in gluconic acid concentration and was inversely correlated with soluble P concentration. Ps. fluorescens RAF1 showed the properties related to plant growth promotion: pectinase, protease, lipase, siderophore, hydrogen cyanide, and indoleacetic acid.
Conclusion:  This study indicated that the P solubility was directly correlated with the organic acids produced.
Significance and Impact of the Study:  Pseudomonas fluorescens RAF15 possessed different traits related to plant growth promotion. Therefore, Ps. fluorescens RAF15 could be a potential candidate for the development of biofertilizer or biocontrol agent.