Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils

Abstract: A total of 132 different fluorescent Pseudomonas strains were isolated from several agricultural and industrial soils. The bacteria from the two different soil environments were compared for species and biotype variation, antibiotic and heavy metal resistance profiles, ability to degrade polyaromatic hydrocarbons, and plasmid incidence. Irrespective of the soil type, the isolates belonged to Pseudomonas fluorescens biotypes I–VI and Pseudomonas putida biotype B. Except for a streptomycin resistant isolate from one of the industrial soils, all the strains had the same antibiotic resistance profile. However, there was a higher incidence of heavy metal resistance and polyaromatic hydrocarbon degradation phenotypes in the isolates from industrial soils than from the agricultural soils. Only 2 out of 68 strains from agricultural soil were found to carry plasmids, while 28 out of 64 strains from industrial soil had plasmids. A majority of the plasmids (56%) were estimated to be larger than 50 kb, indicating that they could encode transfer functions. However, transferability as indicated by the ability to mobilize an IncQ plasmid (tra⁻, mob⁺), was observed with only one plasmid. None of the plasmid(s) containing isolates hybridized to a ³²P-labelled repP probe suggesting that none of the indigenous plasmids in the soil fluorescent Pseudomonas strains was related to the IncP group of conjugative plasmids commonly associated with resistance and catabolic genes.     

Effect of long-term vineyard monoculture on rhizosphere populations of pseudomonads carrying the antimicrobial biosynthetic genes phlD and/or hcnAB

The impact of repeated culture of perennial plants (i.e. in long-term monoculture) on the ecology of plant-beneficial bacteria is unknown. Here, the influence of extremely long-term monocultures of grapevine (up to 1603 years) on rhizosphere populations of fluorescent pseudomonads carrying the biosynthetic genes phlD for 2,4-diacetylphloroglucinol and/or hcnAB for hydrogen cyanide was determined. Soils from long-term and adjacent short-term monoculture vineyards (or brushland) in four regions of Switzerland were baited with grapevine or tobacco plantlets, and rhizosphere pseudomonads were studied by most probable number (MPN)-PCR. Higher numbers and percentages of phlD ⁺ and of hcnAB ⁺ rhizosphere pseudomonads were detected on using soil from long-term vineyards. On focusing on phlD , restriction fragment length polymorphism profiling of the last phlD -positive MPN wells revealed seven phlD alleles (three exclusively on tobacco, thereof two new ones). Higher numbers of phlD alleles coincided with a lower prevalence of the allele displayed by the well-studied biocontrol strain Pseudomonas fluorescens F113. The prevalence of this allele was 35% for tobacco in long-term monoculture soils vs. >60% in the other three cases. We conclude that soils from long-term grapevine monocultures represent an untapped resource for isolating novel biocontrol Pseudomonas strains when tobacco is used as bait.     

Interactions between the soil-borne bacteriophage Fo-l and Pseudomonas spp. on sugarbeet roots

The large plaquing (24 mm²) soilborne bacteriophage, Fo-l, did not affect the colonization ability on sugarbeet roots of its host, fluorescent Pseudomonas sp. strain B26. Phage Fo-l did not increase in numbers on sugarbeet roots when seeds were coated with less than 10⁶ CFU (colony forming units) of B26 and when less than 300 PFU (plaque forming units) of phage Fo-l was added per g of soil (dry weight). Above these threshold values, phage replication occurred and up to 2 × 10⁶ PFU per root system could be recovered.     

Sequence diversity of the OprD protein of environmental Pseudomonas strains

OprD has been widely described for Pseudomonas aeruginosa at both structural and functional levels. Here, we describe the sequence diversity of the OprD proteins from other fluorescent Pseudomonads. We analysed the sequence of the oprD gene in each of the 49 Pseudomonas isolates, mostly putida and fluorescens species, obtained from various environmental sources, including soil, rhizosphere and hospitals. Phylogeny based on OprD sequences distinguished three well-separated clusters in the P. fluorescens species whereas P. putida isolates formed only one cluster. The OprD sequences were generally well conserved within each cluster whereas on the opposite, they were highly variable from one cluster to another and particularly with regards to the cluster of P. aeruginosa. Predicted secondary structures, based on the topological model elaborated for P. aeruginosa, suggest signatures in the large extracellular loops of OprD, which are linked to the OprD-based clusters. Correlations between these OprD-based clusters and ecological niches, growth on various carbon sources and antibiotic sensitivity were investigated.     

Effect of soil type and plant species on the fluorescent pseudomonads nitrate dissimilating community

The distribution of nitrogen dissimilative abilities among 618 isolates of fluorescent pseudomonads was studied. These strains were isolated from two uncultivated soils (C and D; collected at Chateaurenard and Dijon, France, respectively) and from rhizosphere, rhizoplane and root tissue of two plant species (flax and tomato) cultivated on these two soils. According to their ability to dissimilate nitrogen, the isolates have been distributed into three metabolic types: non-dissimilators, NO₂ ^- accumulators and denitrifiers. While the three metabolic types were recovered in all the compartments of soil D experiments, only two (non-dissimilators and denitrifiers) were recovered in all the compartments of soil C experiments. Even under the contrasting conditions of the two soil types, both plants were able to select the nitrate dissimilating community among the total community of fluorescent Pseudomonas, but the mode of this selection seems to be dependent on both plant and soil type. The soil type appears to be unable to significantly modulate the strong selective effect of tomato. Indeed, similar dissimilator to non-dissimilator ratios were found in the root tissue of this plant species cultivated in both soils. In contrast, the different dissimilator to non-dissimilator ratios observed in flax roots between soils C and D suggest that the selective effect of flax was modulated by the soil type. Taxonomic identifications showed that the 618 isolates were distributed among three species (P. chlororaphis, P. fluorescens, P. putida) plus an intermediate type between P. fluorescens and P. putida. However, no clear relationship between the distribution of the metabolic types (functional diversity) and the distribution of bacterial species has been found. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fluorescent pseudomonads in the rhizosphere of plants and their relation to root exudates

Fluorescent pseudomonads were present in chernozem soil not influenced by plant roots (10(3)-10(4) per g dry soil) in the rhizosphere soil of various plants (10(4)-10(5) per g soil) and on roots (10(3) to 10(7) per g fresh roots), depending on the species and age of the plant. Relative species representation of fluorescent pseudomonads changed on the roots and in the plant rhizosphere as compared with free soil. Pseudomonas fluorescens, representing 60-93% of the population of fluorescent pseudomonads predominated on the roots of all plants investigated. Somewhat different results were obtained in rhizosphere soil. Relatively higher numbers of P. fluorescens were detected in the rhizosphere soil of cucumber and maize, numbers in the rhizosphere soil of wheat were practically the same as in free soil and higher numbers of P. putida were found in the rhizosphere soil of barley. Almost all components contained in the root exudates of the plants studied, including beta-pyrazolylalanine from the root exudates of cucumbers were utilized as carbon and energy sources. Root exudates of wheat and maize were utilized by the strain P. putida K2 with an efficiency of 73-91%, depending on species and age of the plant.     

Survival of genetically modified Pseudomonas fluorescens introduced into subtropical soil microcosms

Abstract A genetically modified strain of Pseudomonas fluorescens and its parent showed grossly similar decline rates following introduction into subtropical clay and sandy soils. In unplanted clay soit at pH 6.9 and 25°C, population densities declined progressively from about 10⁸ to 10³ colony forming units (cfu) g⁻¹ dry soil over 75 days, but in unplanted sandy soil the introduced populations could not be detected after 25 days. In clay soil at pH 8.7 or 4.7, or at environmental temperature, decay rates were enhanced as compared to those at pH 6.9 and 25°C. Counts of introduced strains in clay bulk soil and in rhizosphere and rhizoplane of maize suggested that the introduced bacteria competed well with the native bacteria, and colonized the roots at about 10⁶ cfu g⁻¹ dry root at 25°C, over 20 days. However, rhizoplane colonization was lower at environmental temperature. The decay rate of both strains was slower in planted than in unplanted sandy soil. The population densities in the rhizosphere and rhizoplane in the sandy soil were significantly lower than those in the clay soil. Both introduced strains colonized the maize roots in both soils, using seeds coated with bacteria in 1% carboxymethyl cellulose. Introduced cells were localized at different sites along the roots of plants developing in clay soil, with higher densities in the original (near the seeds) and root hair zones as compared to the intermediate zones. No significant difference was observed between the extent of root colonization of the genetically modified strain and its parent.     

Postweaning diarrhoea in pigs: an interaction between change to dry food and colibacillosis

Abstract Production of pyochelin by Pseudomonas species was measured in ethyl acetate extracts of culture supernatants. Pyochelin was purified by paper chromatography, followed by two-dimensional, thin-layer chromatography. Strains of P. cepacia , P. multivorans , and P. fluorescens produced pyochelin, while P. stutzeri , P. putida and P. maltophila were negative for pyochelin production. Outer membranes prepared from these strains had a protein of M _r 14000 which was shown to bind [⁵⁹Fe]pyochelin.     

Influence of an arbuscular mycorrhizal fungus on Pseudomonas fluorescens DF57 in rhizosphere and hyphosphere soil

The influence of Glomus intraradices (BEG87) on Pseudomonas fluorescens DF57 in hyphosphere and rhizosphere soil was examined. Cucumis sativus (Aminex, F₁ hybrid) was grown in symbiosis with the arbuscular mycorrhizal fungus G. intraradices in PVC tubes, consisting of a central root compartment and two lateral root-free compartments. Two Tn 5 - lux AB-marked strains of P. fluorescens DF57 were used. Strain DF57-P2, which has an insertion of Tn 5::lux AB in a phosphate starvation-inducible locus, was used as a phosphate starvation reporter. Another lux -tagged strain DF57-40E7, which carries a constitutively expressed lux AB fusion, was used as control for strain DF57-P2 and for measuring the metabolic activity of P. fluorescens DF57. A strain of P. fluorescens DF57, which carries a constitutively expressed gfp gene, was used in studies of attachment between the bacteria and the hyphae. G. intraradices decreased the culturability of P. fluorescens DF57 significantly, both in rhizosphere and hyphosphere soil, whereas the total number of P. fluorescens DF57 measured by immunofluorescence microscopy was decreased in hyphosphere soil only. G. intraradices did not induce a phosphorus starvation response in P. fluorescens DF57, and the metabolic activity of the bacteria was not affected by the fungus after 48 h. P. fluorescens DF57 did not attach to G. intraradices hyphae and was not able to use the hyphae as carbon substrate. The negative effect of G. intraradices on culturability and on number of P. fluorescens DF57 in hyphosphere soil is discussed.     

Development of a molecular detection method for naphthalene degrading pseudomonads

Abstract: A combined polymerase chain reaction amplification and reverse dot blot assay was designed for the detection of bacterial genes from soil and sediments. Total nucleic acids were directly extracted from soil using a lysozyme/sodium dodecyl sulfate/freeze-thaw method followed by rapid purification through gel electrophoresis. DNA was amplified using a highly stringent polymerase chain reaction with primers directed against the nahR regulatory gene present in plasmid NAH7 of Pseudomonas putida G7. The resulting amplification product was detected colorimetrically by reverse dot blot with an improved sensitivity ten-fold greater than traditional ethidium bromide staining after gel electrophersis. A lower limit of 10³, P. putida G7 cfu (g soil)⁻¹ was detected. This method was successfully employed to detect indigenous naphthalene-degrading bacteria from subsurface sediment collected from different locations of a naphthalene-contaminated site. Similar approaches could be developed for other soil-borne genetic markers.     

Stability and conjugal transfer kinetics of a TOL plasmid in Pseudomonas aeruginosa PAO 1162

Abstract Batch mating experiments were employed to study the kinetics of the conjugal transfer of a TOL plasmid, using the transconjugant strain Pseudomonas aeruginosa PAO 1162 (TOL) as the plasmid donor and Pseudomonas putida PB 2442 and Pseudomonas aeruginosa PAO 1162N as the plasmid recipients. Transfer rates from PAO 1162 (TOL) to PAO 1162N and PB 2442 measured for exponentially grown PAO 1162 (TOL) were 1.81 × 10⁻¹⁴ (standard error (S.E.) 1.25 × 10⁻¹⁵) ml·cell⁻¹min⁻¹ and 3.32 × 10⁻¹³ (S.E. 4.42 × 10⁻¹⁴) ml·cell⁻¹min⁻¹, respectively. The instability of the TOL plasmid in PAO 1162 (TOL) was evaluated under conditions that were non-selective for maintenance of the TOL catabolic functions. The measured rates of instability were 6.7 10⁻⁶ to 8.3 10⁻⁶ min⁻¹, and the loss of the catabolic functions was mainly caused by structural instability of the plasmid.     

Influence of Two Plant Species (Flax and Tomato) on the Distribution of Nitrogen Dissimilative Abilities within Fluorescent Pseudomonas spp

The distribution of nitrogen-dissimilative abilities among 317 isolates of fluorescent pseudomonads was studied. These strains were isolated from an uncultivated soil and from the rhizosphere, rhizoplane, and root tissue of two plant species (flax and tomato) cultivated on this same soil. The isolates were distributed into two species, Pseudomonas fluorescens (45.1%) and Pseudomonas putida (40.4%), plus an intermediate type (14.5%). P. fluorescens was the species with the greatest proportion of isolates in the root compartments and the greatest proportion of dissimilatory and denitrifying strains. According to their ability to dissimilate nitrogen, the isolates have been distributed into nondissimilatory and dissimilatory strains, nitrate reducers and true denitrifiers with or without N(inf2)O reductase. The proportion of dissimilatory isolates was significantly enhanced in the compartments affected by flax and tomato roots (55% in uncultivated soil and 90 and 82% in the root tissue of flax and tomato, respectively). Among these strains, the proportion of denitrifiers gradually and significantly increased in the root vicinity of tomato (44, 68, 75, and 94% in uncultivated soil, rhizosphere, rhizoplane, and root tissue, respectively) and was higher in the flax rhizoplane (66%) than in the uncultivated soil. A higher proportion of N(inf2)O reducers was also found in the root compartments. This result was particularly clear for tomato. It is hypothesized that denitrification could be a selective advantage for the denitrifiers in the root environment and that this process could contribute to modify the specific composition of the bacterial communities in the rhizosphere.     

Effect of Two Plant Species, Flax (Linum usitatissinum L.) and Tomato (Lycopersicon esculentum Mill.), on the Diversity of Soilborne Populations of Fluorescent Pseudomonads

Suppression of soilborne disease by fluorescent pseudomonads may be inconsistent. Inefficient root colonization by the introduced bacteria is often responsible for this inconsistency. To better understand the bacterial traits involved in root colonization, the effect of two plant species, flax (Linum usitatissinum L.) and tomato (Lycopersicon esculentum Mill.), on the diversity of soilborne populations was assessed. Fluorescent pseudomonads were isolated from an uncultivated soil and from rhizosphere, rhizoplane, and root tissue of flax and tomato cultivated in the same soil. Species and biovars were identified by classical biochemical and physiological tests. The ability of bacterial isolates to assimilate 147 different organic compounds and to show three different enzyme activities was assessed to determine their intraspecific phenotypic diversity. Numerical analysis of these characteristics allowed the clustering of isolates showing a high level (87.8%) of similarity. On the whole, the populations isolated from soil were different from those isolated from plants with respect to their phenotypic characteristics. The difference in bacteria isolated from uncultivated soil and from root tissue of flax was particularly marked. The intensity of plant selection was more strongly expressed with flax than with tomato plants. The selection was, at least partly, plant specific. The use of 10 different substrates allowed us to discriminate between flax and tomato isolates. Pseudomonas fluorescens biovars II, III, and V and Pseudomonas putida biovar A and intermediate type were well distributed among the isolates from soil, rhizosphere, and rhizoplane. Most isolates from root tissue of flax and tomato belonged to P. putida bv. A and to P. fluorescens bv. II, respectively. Phenotypic characterization of bacterial isolates was well correlated with genotypic characterization based on repetitive extragenic palindromic PCR fingerprinting.     

Improved phosphate biosorption by bacterial surface display of phosphate-binding protein utilizing ice nucleation protein

The conventional enhanced biological phosphorus removal (EBPR) system often deteriorates at low chemical oxygen demand (COD) or under aeration conditions. A new approach that incorporates phosphate-eutrophic wastewater remediation was introduced through immobilization of an intracellular phosphate-binding protein (PBP) onto the surface of Pseudomonas putida or Escherichia coli , using the N-terminal anchor (InaQ-N) of a newly identified ice nucleation protein from Pseudomonas syringae . A green fluorescent protein-fusion protein was expressed and used to confirm surface localization. The PBP was then targeted to the surface of E. coli JM109 and P. putida AB92019. The engineered P. putida and E. coli microorganisms were capable of absolute biosorption of total phosphates at rates of 60 and 80 mg L⁻¹, respectively, over 5 h. In the recombinant P. putida cells, a surface-immobilized PBP fusion that had three tandemly repeated InaQ-Ns exhibited the maximum increment in phosphate biosorption, at sixfold compared with the control strain. Even heat-killed recombinant cells of either P. putida or E. coli retained substantial biosorptive activities. The current study demonstrates that the bacterial surface display of PBP should be considered as a strong contender for generating organisms capable of functioning in EBPR systems under low COD, resulting in improved removal of eutrophic phosphorus from wastewaters.     

Detection of Pseudomonas putida B in the rhizosphere by RAPD

A method was developed for the detection of Pseudomonas putida B MM12 released into the rhizosphere of non-sterile barley, using a Random Amplified Polymorphic DNA (RAPD)-generated probe for hybridization with RAPD products generated from DNA extracted from the rhizosphere. The detection procedure involves extraction of rhizosphere bacteria by sonication, extraction of DNA by boiling, RAPD and Southern hybridization with RAPD products and the selected probe. The level of detection of MM12 was at least 1·9×10⁴ cells g⁻¹ barley root. MM12 was detected in rhizosphere when it constituted as little as 0·5% of the culturable population.     

Plasmid-determined resistance to chromate in Pseudomonas aeruginosa

Abstract Resistance to chromate in five independent Pseudomonas aeruginosa clinical isolates was transferred by conjugation to P. aeruginosa strain PU21. All chromate-resistant transconjugants contained large plasmids that also conferred resistance to inorganic mercury. One of these plasmids, pUM505, increased the resistance to CrO₄²⁻ and decreased the accumulation of intracellular ⁵¹CrO₄²⁻ by the host cells as compared to the plasmidless strain PU21.     

Physiological role of phosphatidylcholine in the Pseudomonas putida A ATCC 12633 response to tetradecyltrimethylammonium bromide and aluminium

Aims:  To evaluate the effect of tetradecyltrimethylammonium bromide (TTAB) and aluminium stresses on the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633.
Methods and Results:  Pseudomonas putida were grown with TTAB in the presence or absence of AlCl₃, and the PL composition was analysed. The presence of TTAB resulted in an increase in phosphatidylglycerol and phosphatidic acid levels (6- and 20-fold, respectively) with respect to the levels in cells grown without the surfactant. With AlCl₃, phosphatidylcholine (PC) increased (threefold) and cell-free extracts contained approximately threefold more phosphatidylcholine synthase activities than extracts without AlCl₃, indicating that the PC level is dependent upon activation of this enzyme.
Conclusions:  The negative charges of the headgroups of PL are the primary membrane-associated factors for the response to TTAB. PC are involved in cellular responses to binding Al³⁺ and should be viewed as a temporary reservoir of available Al³⁺ to allow a more efficient utilization of TTAB by Ps. putida .
Significance and Impact of the Study:  The changes in the PL of Ps. putida in the presence of TTAB and AlCl₃ indicate that different responses are utilized by bacteria to maintain optimal PL composition in the presence of such environmental pollutants.     

A new pathway for bacterial catabolism of 3-hydroxybenzoic acid

Abstract This paper is the first to describe the transformation of 3-hydroxybenzoate (3-HBA) by Pseudomonas putida BS893 by a new pathway via 2,3-dihydroxybenzoate (2,3-DBA) and catechol. We have compared the intermediates and appropriate enzyme activities in P. putida BS893 (pBS241) and in a cured derivative BS662 (Bph⁻) thereof, for the ascertainment of plasmid or chromosomal genetic control over 3-HBA-catabolism. The results presented show that catabolism of 3-HBA in P. putida BS893 (pBS241) is controlled by chromosomal genes.     

Lysine decarboxylase in Pseudomonas aeruginosa]

The research of lysine, ornithine and arginine decarboxylases has been made for 50 strains of fluorescent Pseudomonas (P. aeruginosa, P. fluorescens, P. putida). By thin layer chromatography, all the strains of Pseudomonas aeruginosa and the fifth of the strains of P. putida had lysine decarboxylase activity at alcaline pH (optimal pH 8) ; Pseudomonas fluorescens did not produce this decarboxylase. Arginine and ornithine decarboxylase are absent for all the strains of fluorescent Pseudomonas.     

Rhizosphere competence of fluorescent Pseudomonas sp. B24 genetically modified to utilise additional ferric siderophores

Abstract: The ability to utilise additional siderophores may increase the ecological fitness of biocontrol inoculants of Pseudomonas in the rhizosphere. Plasmid pCUP2 carries a copy of the gene pbu A coding for the membrane receptor of ferric pseudobactin M114. Pseudomonas sp. B24Rif containing pCUP2 can utilise ferric pseudobactin of P. fluorescens M114 in addition to its own siderophore. A larger fraction of the culturable resident fluorescent pseudomonads in the rhizosphere of sugarbeet grown in a low-iron sandy loam soil could supply siderophore-complexed iron to B24Rif(pCUP2) rather than to B24Rif. However, B24Rif and B24Rif(pCUP2) were found at similar population levels in the rhizosphere for 21 days after their inoculation on seeds. A total of 25 of 43 isolates of resident fluorescent Pseudomonas unable to cross-feed iron to B24Rif could cross-feed B24Rif(pCUP2) and they were subdivided into seven different strains by arbitrary-primed PCR fingerprinting. The siderophores produced by 11 of them were typed by HPLC and they were similar to pseudobactin M114. However, the ability to utilise ferric pseudobactin M114 did not improve the ecological fitness of B24Rif in the rhizosphere of sugarbeet although a larger fraction of the culturable resident fluorescent pseudomonads could supply pseudobactin M114-complexed iron to B24Rif(pCUP2) than to B24Rif.