Cross-Species GacA-Controlled Induction of Antibiosis in Pseudomonads

Signal extracts prepared from culture supernatants of Pseudomonas fluorescens CHA0 and Pseudomonas aeruginosa PAO stimulated GacA-dependent expression of small RNAs and hence of antibiotic compounds in both hosts. Pseudomonas corrugata LMG2172 and P. fluorescens SBW25 also produced signal molecules stimulating GacA-controlled antibiotic synthesis in strain CHA0, illustrating a novel, N-acyl-homoserine lactone-independent type of interspecies communication.     

Thiamine-Auxotrophic Mutants of Pseudomonas fluorescens CHA0 Are Defective in Cell-Cell Signaling and Biocontrol Factor Expression

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 × 10⁻⁸ M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.     

Gluconic acid-producing Pseudomonas sp. prevent γ-actinorhodin biosynthesis by Streptomyces coelicolor A3(2)

Streptomyces are ubiquitous soil bacteria well known for their ability to produce a wide range of secondary metabolites including antibiotics. In their natural environments, they co-exist and interact with complex microbial communities and their natural products are assumed to play a major role in mediating these interactions. Reciprocally, their secondary metabolism can be influenced by the surrounding microbial communities. Little is known about these complex interactions and the underlying molecular mechanisms. During pairwise co-culture experiments, a fluorescent Pseudomonas, Pseudomonas fluorescens BBc6R8, was shown to prevent the production of the diffusible blue pigment antibiotic γ-actinorhodin by Streptomyces coelicolor A3(2) M145 without altering the biosynthesis of the intracellular actinorhodin. A mutant of the BBc6R8 strain defective in the production of gluconic acid from glucose and consequently unable to acidify the culture medium did not show any effect on the γ-actinorhodin biosynthesis in contrast to the wild-type strain and the mutant complemented with the wild-type allele. In addition, when glucose was substituted by mannitol in the culture medium, P. fluorescens BBc6R8 was unable to acidify the medium and to prevent the biosynthesis of the antibiotic. All together, the results show that P. fluorescens BBc6R8 impairs the biosynthesis of the lactone form of actinorhodin in S. coelicolor by acidifying the medium through the production of gluconic acid. Other fluorescent Pseudomonas and the opportunistic pathogen Pseudomonas aeruginosa PAO1 also prevented the γ-actinorhodin production in a similar way. We propose some hypotheses on the ecological significance of such interaction.     

Type III Secretion System and Virulence Markers Highlight Similarities and Differences between Human- and Plant-Associated Pseudomonads Related to Pseudomonas fluorescens and P. putida

Pseudomonas fluorescens is commonly considered a saprophytic rhizobacterium devoid of pathogenic potential. Nevertheless, the recurrent isolation of strains from clinical human cases could indicate the emergence of novel strains originating from the rhizosphere reservoir, which could be particularly resistant to the immune system and clinical treatment. The importance of type three secretion systems (T3SSs) in the related Pseudomonas aeruginosa nosocomial species and the occurrence of this secretion system in plant-associated P. fluorescens raise the question of whether clinical isolates may also harbor T3SSs. In this study, isolates associated with clinical infections and identified in hospitals as belonging to P. fluorescens were compared with fluorescent pseudomonads harboring T3SSs isolated from plants. Bacterial isolates were tested for (i) their genetic relationships based on their 16S rRNA phylogeny, (ii) the presence of T3SS genes by PCR, and (iii) their infectious potential on animals and plants under environmental or physiological temperature conditions. Two groups of bacteria were delineated among the clinical isolates. The first group encompassed thermotolerant (41°C) isolates from patients suffering from blood infections; these isolates were finally found to not belong to P. fluorescens but were closely related and harbored highly conserved T3SS genes belonging to the Ysc-T3SS family, like the T3SSs from P. aeruginosa. The second group encompassed isolates from patients suffering from cystic fibrosis; these isolates belonged to P. fluorescens and harbored T3SS genes belonging to the Hrp1-T3SS family found commonly in plant-associated P. fluorescens.     

Cross-species GacA-controlled induction of antibiosis in pseudomonads

Signal extracts prepared from culture supernatants of Pseudomonas fluorescens CHA0 and Pseudomonas aeruginosa PAO stimulated GacA-dependent expression of small RNAs and hence of antibiotic compounds in both hosts. Pseudomonas corrugata LMG2172 and P. fluorescens SBW25 also produced signal molecules stimulating GacA-controlled antibiotic synthesis in strain CHA0, illustrating a novel, N-acyl-homoserine lactone-independent type of interspecies communication.     

Effect of dissolved oxygen regime on growth dynamics of Pseudomonas spp during benzene degradation

We investigated the effect of different oxygen regimes on growth patterns of Pseudomonas spp. during benzene degradation in microcosm batch studies. Benzene degradation was induced by limiting oxygen available for microbial activity, which consists of three initial-dissolved oxygen (DO) levels of oxic, hypoxic, and anoxic conditions. Batch experiments were performed for cell growth and benzene degradation by inoculating three strains of Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida) in mineral salt medium containing aqueous benzene. Results showed that all strains were capable to grow and degrade benzene under all oxygen regimes but in a different manner. The highest cell growth of P. aeruginosa and P. fluorescens was achieved under oxic and anoxic condition, respectively, but there was no substantial difference on benzene degradation between the oxygen treatments with about 25% reduction for both strains. P. putida showed a facultative process for both cell growth and benzene degradation. This reveals that care should be taken in selection of microorganisms with regard to environmental studies since they exhibit different responses for given environmental conditions such as DO levels.     

Analysis of the draft genome of Pseudomonas fluorescens ATCC17400 indicates a capacity to take up iron from a wide range of sources,including different exogenous pyoverdines

All fluorescent pseudomonads (Pseudomonas aeruginosa, P. putida, P. fluorescens, P. syringae and others) are known to produce the high-affinity peptidic yellow-green fluorescent siderophore pyoverdine. These siderophores have peptide chains that are quite diverse and more than 50 pyoverdine structures have been elucidated. In the majority of the cases, a Pseudomonas species is also able to produce a second siderophore of lower affinity for iron. Pseudomonas fluorescens ATCC 17400 has been shown to produce a unique second siderophore, (thio)quinolobactin, which has an antimicrobial activity against the phytopathogenic Oomycete Pythium debaryanum. We show that this strain has the capacity to utilize 16 different pyoverdines, suggesting the presence of several ferripyoverdine receptors. Analysis of the draft genome of P. fluorescens ATCC 17400 confirmed the presence of 55 TonB-dependent receptors, the largest so far for Pseudomonas, among which 15 are predicted to be ferripyoverdine receptors (Fpv). Phylogenetic analysis revealed the presence of two different clades containing ferripyoverdine receptors, with sequences similar to the P. aeruginosa type II FpvA forming a separate cluster. Among the other receptors we confirmed the presence of the QbsI (thio)quinolobactin receptor, an ferri-achromobactin and an ornicorrugatin receptor, several catecholate and four putative heme receptors. Twenty five of the receptors genes were found to be associated with genes encoding extracytoplasmic sigma factors (ECF σ) and transmembrane anti-σ sensors.     

Identification of Genes and Proteins Necessary for Catabolism of Acyclic Terpenes and Leucine/Isovalerate in Pseudomonas aeruginosa

Geranyl-coenzyme A (CoA)-carboxylase (GCase; AtuC/AtuF) and methylcrotonyl-CoA-carboxylase (MCase; LiuB/LiuD) are characteristic enzymes of the catabolic pathway of acyclic terpenes (citronellol and geraniol) and of saturated methyl-branched compounds, such as leucine or isovalerate, respectively. Proteins encoded by two gene clusters (atuABCDEFGH and liuRABCDE) of Pseudomonas aeruginosa PAO1 were essential for acyclic terpene utilization (Atu) and for leucine and isovalerate utilization (Liu), respectively, as revealed by phenotype analysis of 10 insertion mutants, two-dimensional gel electrophoresis, determination of GCase and MCase activities, and Western blot analysis of wild-type and mutant strains. Analysis of the genome sequences of other pseudomonads (P. putida KT2440 and P. fluorescens Pf-5) revealed candidate genes for Liu proteins for both species and candidate genes for Atu proteins in P. fluorescens. This result concurred with the finding that P. fluorescens, but not P. putida, could grow on acyclic terpenes (citronellol and citronellate), while both species were able to utilize leucine and isovalerate. A regulatory gene, atuR, was identified upstream of atuABCDEFGH and negatively regulated expression of the atu gene cluster.     

Iron uptake and metabolism in pseudomonads

Pseudomonads are ubiquitous Gram-negative γ proteobacteria known for their extreme versatility and adaptability. Some are plant pathogens (Pseudomonas syringae) which have to survive on the surface of leaves while others can colonize the rhizosphere or survive in soil (Pseudomonas fluorescens, Pseudomonas putida), and one species, Pseudomonas entomophila, is an insect pathogen. The most investigated species, Pseudomonas aeruginosa, is known to be an opportunistic pathogen able to infect plants, nematodes, insects, and mammals, including humans. Like for other bacteria, iron is a key nutrient for pseudomonads. The fluorescent pseudomonads produce siderophores, the best known being the fluorescent high-affinity peptidic pyoverdines. Often diverse secondary siderophores of lower affinity are produced as well (pyochelin, pseudomonin, corrugatins and ornicorrugatins, yersiniabactin, and thioquinolobactin). Reflecting their large capacity of adaptation to changing environment and niche colonization, pseudomonads are able to obtain their iron from heme or from siderophores produced by other microorganisms (xenosiderophores) via the expression of outer membrane TonB-dependent receptors. As expected, iron uptake is exquisitely and hierarchically regulated in these bacteria. In this short review, the diversity of siderophores produced, receptors, and finally the way iron homeostasis is regulated in P. aeruginosa, P. syringae, P. putida, and P. fluorescens, will be presented and, when possible, put in relation with the lifestyle and the ecological niche.     

6:2 Fluorotelomer alcohol (6:2 FTOH) biodegradation by multiple microbial species under different physiological conditions

Factors affecting microbial aerobic biodegradation of 6:2 fluorotelomer alcohol [6:2 FTOH, F(CF₂)₆CH₂CH₂OH] were investigated using three alkane-degrading bacteria (Mycobacterium vaccae JOB5, Pseudomonas oleovorans, and Pseudomonas butanovora) and one fluoroacetate-degrading bacterium (Pseudomonas fluorescens DSM 8341). In the presence of formate (an external reducing energy source), P. fluorescens DSM 8341 produced perfluorobutanoic acid by removing three –CF₂– groups from 6:2 FTOH. Only P. fluorescens DSM 8341 transformed 5:3 acid to 4:3 acid and perfluoropentanoic acid. However, formate showed no effects on the degradation rates, patterns, or transformation products of 6:2 FTOH by M. vaccae JOB5. When dicyclopropylketone (an alkane hydroxylase inducer) or formate was added, P. oleovorans rapidly degraded 6:2 FTOH and produced PFPeA. In the presence of lactate, P. butanovora degraded 6:2 FTOH slowly but produced diverse metabolites. Our results demonstrate that the extent and mechanisms of 6:2 FTOH biotransformation are affected by strain types, enzyme inducers, and levels of reducing energy.     

The Pseudomonas fluorescens lipase has a C-terminal secretion signal and is secreted by a three-component bacterial ABC-exporter system

Both Pseudomonas aeruginosa and Pseudomonas fluorescens secrete a lipase into the extracellular medium. Unlike the lipase of P. aeruginosa, the lipase produced by P. fluorescens does not contain any N-terminal signal sequence. We show that the P. fluorescens lipase is secreted through the signal peptide-independent pathway of the alkaline protease that we previously identified in P. aeruginosa. Secretion of this protease (AprA) is dependent on the presence of three genes located adjacent to the aprA gene, aprD, aprE and aprF. The three secretion functions permit an efficient secretion of P. fluorescens lipase. Inactivation of one of them (AprE) prevented this secretion. In Escherichia coli, the three proteins AprD, AprE, AprF are necessary and sufficient for efficient secretion of lipase to the extracellular medium. The secretion signal is located within the C-terminal part of the lipase sequence and can promote efficient secretion of a passenger protein. Thus the P. fluorescens lipase secretion system belongs to the group of the three-component bacterial ABC-exporter systems.     

1H, 13C and 15N assignments of the holo-acyl carrier protein of Pseudomonas aeruginosa

Acyl carrier proteins (ACPs) are a group of highly conserved and abundant proteins in bacteria. ACPs play a central role as the acyl group carriers in bacterial fatty acid biosynthesis, providing building blocks for membrane biogenesis and the production of secondary metabolites. In the versatile human pathogen Pseudomonas aeruginosa, three ACP homologs have been identified. One homolog, AcpP, exhibits the strongest sequence homology to the canonical Escherichia coli ACP. Here we report the ¹H, ¹³C and ¹⁵N assignments of the holo-AcpP of P. aeruginosa.     

Degradation of n-alkanes and polycyclic aromatic hydrocarbons in petroleum by a newly isolated Pseudomonas aeruginosa DQ8

A bacterial isolate, designated as DQ8, was found capable of degrading diesel, crude oil, n-alkanes and polycyclic aromatic hydrocarbons (PAHs) in petroleum. Strain DQ8 was assigned to the genus Pseudomonas aeruginosa based on biochemical and genetic data. The metabolites identified from n-docosane as substrate suggested that P. aeruginosa DQ8 could oxidize n-alkanes via a terminal oxidation pathway. P. aeruginosa DQ8 could also degrade PAHs of three or four aromatic rings. The metabolites identified from fluorene as substrate suggested that P. aeruginosa DQ8 may degrade fluorene via two pathways. One is monooxygenation at C-9 of fluorene, and the other is initiated by dioxygenation at C-3 and C-4 of fluorene. P. aeruginosa DQ8 should be of great practical significance both in bioremediation of oil-contaminated soils and biotreatment of oil wastewater.     

Adaptation of Iron Homeostasis Pathways by a Pseudomonas aeruginosa Pyoverdine Mutant in the Cystic Fibrosis Lung

Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections, most notably by Pseudomonas aeruginosa, which persists for decades in the lungs and undergoes extensive evolution. P. aeruginosa requires iron for virulence and uses the fluorescent siderophore pyoverdine to scavenge and solubilize ferric iron during acute infections. Pyoverdine mutants accumulate in the lungs of some CF patients, however, suggesting that the heme and ferrous iron acquisition pathways of P. aeruginosa are more important in this environment. Here, we sought to determine how evolution of P. aeruginosa in the CF lung affects iron acquisition and regulatory pathways through the use of longitudinal CF isolates. These analyses demonstrated a significant reduction of siderophore production during the course of CF lung infection in nearly all strains tested. Mass spectrometry analysis of one of these strains showed that the later CF isolate has streamlined the metabolic flux of extracellular heme through the HemO heme oxygenase, resulting in more-efficient heme utilization. Moreover, gene expression analysis shows that iron regulation via the PrrF small RNAs (sRNAs) is enhanced in the later CF isolate. Finally, analysis of P. aeruginosa gene expression in the lungs of various CF patients demonstrates that both PrrF and HemO are consistently expressed in the CF lung environment. Combined, these results suggest that heme is a critical source of iron during prolonged infection of the CF lung and that changes in iron and heme regulatory pathways play a crucial role in adaptation of P. aeruginosa to this ever-changing host environment.     

Inactivation of the Regulatory Gene algU or gacA Can Affect the Ability of Biocontrol Pseudomonas fluorescens CHA0 To Persist as Culturable Cells in Nonsterile Soil

Rifampin-resistant Pseudomonas fluorescens CHA0-Rif and mutants in which the regulatory gene algU (encoding sigma factor σ^E) or gacA (encoding a global regulator of secondary metabolism) was inactivated were compared for persistence in three nonsterile soils. Functional algU and (particularly) gacA were needed for CHA0-Rif to maintain cell culturability in soil.     

Suppression of bacterial wilt in Eucalyptus urophylla by fluorescent Pseudomonas spp. in China

Bacterial wilt caused by Ralstonia solanacearum race 1, biovar III has become a severe problem in Eucalyptus plantations in south China. The disease mainly attacks young eucalypt trees, and no effective control measures are available yet. To explore possibilities to develop biological control of the disease, strains of fluorescent Pseudomonas spp. that are effective in suppressing plant diseases by known mechanisms, were tested for their potential to control bacterial wilt in Eucalyptus. Pseudomonas putida WCS358r, Pseudomonas fluorescens WCS374r, P. fluorescens WCS417r, and Pseudomonas aeruginosa 7NSK2 antagonize R. solanacearum in vitro by siderophore-mediated competition for iron, whereas inhibition of pathogen growth by P. fluorescens CHA0r is antibiosis-based. No correlations were found between antagonistic activities of these Pseudomonas spp. in vitro and biocontrol of bacterial wilt in Eucalyptus in vivo. None of the strains suppressed disease when mixed together with the pathogen through the soil or when seeds or seedlings were treated with the strains one to four weeks before transfer into soil infested with R. solanacearum. However, when the seedlings were dipped with their roots in a bacterial suspension before transplanting into infested soil, P. fluorescens WCS417r significantly suppressed bacterial wilt. P. putida WCS358r was marginally effective, whereas its siderophore-minus mutant had no effect at all, indicating that siderophore-mediated competition for iron can contribute but is not effective enough to suppress bacterial wilt in Eucalyptus. A derivative of P. putida WCS358r, constitutively producing 2,4-diacetylphloroglucinol (WCS358::phl) reduced disease. Combined treatment with P. fluorescens WCS417r and P. putida WCS358::phl did not improve suppression of bacterial wilt.     

Characterisation ofPseudomonas spp. isolated from foods

PutativePseudomonas spp. (102 isolates) from different foods were first characterised by API 20NE and then tested for some enzymatic activities (lipase and lecithinase production, starch hydrolysis and proteolytic activity). However subsequent molecular tests did not always confirm the results obtained, thus highlighting the limits of API 20NE. Instead RFLP ITS1 and the sequencing of 16S rRNA gene grouped the isolates into 6 clusters:Pseudomonas fluorescens (cluster I),Pseudomonas fragi (duster II and V)Pseudomonas migulae (cluster III),Pseudomonas aeruginosa (cluster IV) andPseudomonas chicorii (cluster VI). The pectinolytic activity was typical of species isolated from vegetable products, especiallyPseudomonas fluorescens. InsteadPseudomonas fragi, predominantly isolated from meat was characterised by proteolytic and lipolytic activities.     

Molecular basis for coordinating secondary metabolite production by bacterial and plant signaling molecules

The production of secondary metabolites is a major mechanism used by beneficial rhizobacteria to antagonize plant pathogens. These bacteria have evolved to coordinate the production of different secondary metabolites due to the heavy metabolic burden imposed by secondary metabolism. However, for most secondary metabolites produced by bacteria, it is not known how their biosynthesis is coordinated. Here, we showed that PhlH from the rhizobacterium Pseudomonas fluorescens is a TetR-family regulator coordinating the expression of enzymes related to the biosynthesis of several secondary metabolites, including 2,4-diacetylphloroglucinol (2,4-DAPG), mupirocin, and pyoverdine. We present structures of PhlH in both its apo form and 2,4-DAPG-bound form and elucidate its ligand-recognizing and allosteric switching mechanisms. Moreover, we found that dissociation of 2,4-DAPG from the ligand-binding domain of PhlH was sufficient to allosterically trigger a pendulum-like movement of the DNA-binding domains within the PhlH dimer, leading to a closed-to-open conformational transition. Finally, molecular dynamics simulations confirmed that two distinct conformational states were stabilized by specific hydrogen bonding interactions and that disruption of these hydrogen bonds had profound effects on the conformational transition. Our findings not only reveal a well-conserved route of allosteric signal transduction in TetR-family regulators but also provide novel mechanistic insights into bacterial metabolic coregulation.