Formation of structured communities by natural and transgenic naphthalene-consuming bacteria

This study concerns the formation of structured communities by pure cultures and binary associations of Pseudomonasfluorescens transgenic strains and natural heterotrophic bacterial species in naphthalene-containing media with various osmotic pressures. It was shown that cells of P. fluorescens strain 5RL, harboring a recombinant construct in the chromosome, were more resistant to the combined action of the stress factors under study than P. fluorescens 82/pUTK21, harboring a recombinant construct within a plasmid. Natural P. fluorescens 1 strains, particularly Vibrio sp. 14, were more viable at high osmotic pressures and naphthalene concentrations. Experiments with the combined introduction of transgenic and natural bacterial strains at high osmotic pressures demonstrated the stable coexistence of bacterial associations in biofilms, independent of naphthalene concentration. Strains considered for introduction into the environment for bioremediation should be assessed with regard to their susceptibility to the combined effect of anthropogenic and natural stress factors. The design of bacterial associations for the same purpose should take into account the effect of factors important for their survival in polluted areas.     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10^?5–10^?4 per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

Diversity of genetic systems responsible for naphthalene biodegradation in <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> strains

The genetic systems that are responsible for naphthalene catabolism were analyzed in 18 naphthalene-degrading Pseudomonas fluorescens strains isolated from oil-contaminated soils in different regions of Russia. It was found that 13 strains contain plasmids, from 20 to 120 kb in size, at least 5 of which are conjugative and bear the catabolic genes responsible for the complete utilization of naphthalene and salicylate. Five plasmids belong to the P-7 incompatibility group, and two plasmids belong to the P-9 incompatibility group. The naphthalene biodegradation genes of P. fluorescens are highly homologous to each other. The study revealed a new group of the nahAc genes and two new variants of the nahG gene. The suggestion is made that the key genes of naphthalene biodegradation, nahAc and nahG, evolve independently and occur in P. fluorescens strains in different combinations.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 70–78.Original Russian Text Copyright © 2005 by Izmalkova, Sazonova, Sokolov, Kosheleva, Boronin.     

Biocontrol of Collar Rot Disease of Betelvine ( Piper betle L.) Caused by Sclerotium rolfsii by Using Rhizosphere-Competent Pseudomonas fluorescens NBRI-N6 and P. fluorescens NBRI-N

Collar rot disease of betelvine (Piper betle L.) caused by Sclerotium rolfsii is difficult to control by conventional means by use of chemicals; therefore, use of biocontrol agents is desirable. In the present study, 186 bacterial strains of different morphological types were screened for their biocontrol activity against S. rolfsii under in vitro conditions. Two strains, Pseudomonas fluorescens NBRI-N6 and P. fluorescens NBRI-N, were selected for further studies because of their ability to inhibit the mycelial growth of the pathogen significantly. Spontaneous rifampicin-resistant (Rif^r) derivatives of P. fluorescens NBRI-N6 and P. fluorescens NBRI-N showing growth rate and membrane protein composition comparable to the wild type were selected to facilitate their monitoring in the rhizosphere. Field trials demonstrated that strain P. fluorescens NBRI-N6 was better than P. fluorescens NBRI-N in increasing the yield of betelvine significantly, whereas a consortium of the two strains controlled the disease more than either of the strains. The screening method should prove useful in identifying rhizosphere bacteria with the greatest potential for controlling diseases caused by phytopathogenic fungi. RID= ID= <E5>Correspondence to:</E5> C.S. Nautiyal; <E5>email:</E5> nautiyalnbri&commat;yahoo.com Received: 5 August 2002 / Accepted: 7 October 2002     

Grazing of nonindigenous bacteria by nano-sized protozoa in a natural coastal system

Mesocosms (4.5 m³) situated in a closed bay area were used to investigate the effect of protozoan predation on nonindigenous bacteria. Pseudomonas fluorescens strain Agl was released into mesocosms as a single inoculum of 1 × 10⁵ cells ml^–1 (final concentration) or as four inocula (same concentration each) at intervals of 3 days. Mesocosms that had received growth media corresponding to the inoculum served as controls. Numbers of P. fluorescens Ag1 decreased rapidly whether released as single or multiple inocula. Direct estimation of protozoan predation using fluorescently labeled P. fluorescens from log phase and starved cultures, respectively, revealed that natural populations of heterotrophic nanoflagellates consumed substantial amounts of the nonindigenous bacterial strain. The volume of fluorescently labeled cells prepared from starved cells was 68% of log phase cell volume, but the individual clearance of the small cells was five to seven times higher than that of the log phase bacteria. The natural populations of nanoflagellates consumed 34–62% of P. fluorescens Ag1 daily if starved bacteria were offered as food, and 3–13% if the cells were in the logarithmic growth phase. This suggests that the effect of protozoan predation on nonindigenous bacterial strains is substantial because cultured bacteria are likely to starve in natural environments. The addition of P. fluorescens Ag1 and the growth medium enhanced the abundance of natural bacteria, chlorophyll a, heterotrophic nanoflagellates, and ciliates, but it did not improve the growth conditions for the released strain. The effects on the indigenous populations were more pronounced after addition of fresh medium than following inoculation with cells, which possibly was due to the lower nutrient content of spent medium. However, these results, based on direct estimation of protozoan predation on log phase and starved nonindigenous bacteria, point to the conclusion that mortality induced by bacterivorous predators is the key factor determining removal of nonindigenous bacteria introduced in natural aquatic systems. Correspondence to: K. Christoffersen.     

The EmhABC efflux pump in Pseudomonas fluorescens LP6a is involved in naphthalene tolerance but not efflux

The EmhABC efflux pump in Pseudomonas fluorescens LP6a effluxes polycyclic aromatic hydrocarbons (PAHs) such as phenanthrene and anthracene but not naphthalene. We previously showed that the presence of EmhABC decreased the efficiency of phenanthrene biodegradation. In this study, we determined whether P. fluorescens LP6a tolerance to naphthalene is a function of the EmhABC efflux pump and how its presence affects the efficiency of naphthalene biodegradation. Growth, membrane fatty acid (FA) composition, and cell morphology showed that 5-mmol?L^?1 naphthalene is inhibitory to P. fluorescens LP6a strains. The deleterious effect of naphthalene is suppressed in the presence of EmhABC, which suggests that, although naphthalene is not effluxed by EmhABC, this efflux pump is involved in tolerance of naphthalene toxicity. LP6a mutants lacking the EmhB efflux pump were unable to convert cis-unsaturated FAs to cyclopropane FAs, indicating that naphthalene interferes with the formation of cyclopropane FAs and supporting the proposal that EmhABC is involved in FA turnover in P. fluorescens LP6a strains. The EmhABC efflux pump increases the efficiency of naphthalene metabolism in strain LP6a, which may make naphthalene efflux unnecessary. Thus, the activity of hydrocarbon efflux pumps may be an important factor to consider when selecting bacterial strains for bioremediation or biocatalysis of PAHs.     

Competitive colonization betweenPseudomonas species in sterile soils

When introduced as pairs into irradiated, sterile soils, aPseudomonas fluorescens strain prevented optimal colonization by aP. putida strain. The addition ofP. putida to sterile soil already populated byP. fluorescens impeded growth ofP. putida in that soil. However, addingP. fluorescens to soil populated withR. putida did not prevent growth ofP. fluorescens and caused a decrease in the titer ofP. putida. This preemptive colonization/exclusion phenomenon was also observed between two isogenic strains. The degree of exclusion depended on the amount of time between the inoculation of the two strains and which organism was introduced first. These results suggest competition for similar niches in soils and a hierarchy of fitness among pseudomonads sharing a soil environment. Autoclaving of the soil removed the competitive colonization betweenPseudomonas spp. observed in irradiated soils. The competitive colonization of irradiated soils by these twoPseudomonas sp. was reflected in their relative survival when introduced into nonsterile soil.     

A δ-endotoxin encoded in<Emphasis Type="Italic"> Pseudomonas fluorescens</Emphasis> displays a high degree of insecticidal activity

The short field-life of Bacillus thuringiensis (Bt) insecticidal crystal protein has limited its use. When the Bt toxin is produced in Pseudomonas fluorescens it can be encapsulated and retain its effectiveness for two to three times longer than other Bt formulations. In order to improve Bt expression, we have synthesized cryIA(c) Bt -endotoxin encoding region (GenBank AF537267) according to the usage codon of P. fluorescens and transformed the Bt toxin expression cassette into P. fluorescens strains. T7 RNA polymerase and the T7 promoter system were used to control expression of Bt toxin. SDS-PAGE and Western blotting assay revealed that the -endotoxin was expressed as 8% of the total protein in P. fluorescens. In in vitro tests, release of toxin from dead bacteria was demonstrated. Supplementation of diets with Bt toxin-containing Pseudomonas bacterium resulted in high mortality of cabbage butterfly (Pieris brassicae) larvae.     

Lethality and Developmental Delay in Drosophila melanogaster Larvae after Ingestion of Selected Pseudomonas fluorescens Strains

Background

The fruit fly, Drosophila melanogaster, is a well-established model organism for probing the molecular and cellular basis of physiological and immune system responses of adults or late stage larvae to bacterial challenge. However, very little is known about the consequences of bacterial infections that occur in earlier stages of development. We have infected mid-second instar larvae with strains of Pseudomonas fluorescens to determine how infection alters the ability of larvae to survive and complete development.

Methodology/Principal Findings

We mimicked natural routes of infection using a non-invasive feeding procedure to study the toxicity of the three sequenced P. fluorescens strains (Pf0-1, SBW25, and Pf-5) to Drosophila melanogaster. Larvae fed with the three strains of P. fluorescens showed distinct differences in developmental trajectory and survival. Treatment with SBW25 caused a subset of insects to die concomitant with a systemic melanization reaction at larval, pupal or adult stages. Larvae fed with Pf-5 died in a dose-dependent manner with adult survivors showing eye and wing morphological defects. In addition, larvae in the Pf-5 treatment groups showed a dose-dependent delay in the onset of metamorphosis relative to control-, Pf0-1-, and SBW25-treated larvae. A functional gacA gene is required for the toxic properties of wild-type Pf-5 bacteria.

Conclusions/Significance

These experiments are the first to demonstrate that ingestion of P. fluorescens bacteria by D. melanogaster larvae causes both lethal and non-lethal phenotypes, including delay in the onset of metamorphosis and morphological defects in surviving adult flies, which can be decoupled.     

Potential of biotic inducers on disease severity and variation of Cucumber mosaic cucumovirus in cucumber plants

Six bacterial strains (Bacillus subtilis, Bacillus polymyxa, Bacillus circulans, Pseudomonas putida, Pseudomonas fluorescens 2 and Pseudomonas fluorescens 8) and one fungal isolate (Trichoderma harzianum) were tested for their ability to protect Cucumis sativus L. cv. Beith Alpha against the disease development of Cucumber mosaic cucumovirus (CMV). Seed treatment with individual bacterial and fungal liquid cultures significantly and consistently reduced the disease severity (DS) of infected cucumber plants, after 14 days from CMV inoculation onto cotyledons. All seven biotic inducers reduced the CMV infection at the range of 16.6–39% and 0–46.5% under sterilised and non-sterilised soils, respectively. The effect of treatment with each biotic inducer has a significant difference in the percentage of CMV DS. In sterilised soil, B. circulans has a low percentage of DS (42%), while P. fluorescens 8 has a high DS percentage (62.9%). In non-sterilised soil, the low DS percentage was 30% for T. harzianum, while P. putida had the highest DS percentage (70%). On the other hand, CMV variability on Chenopodium amaranticolor revealed that all biotic treatments differed according to the local lesion number, similarity and morphology.     

Isolation of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> species highly resistant to pentachlorobenzene

Polychlorinated aromatic compounds, including pentachlorobenzenes and hexachlorobenzenes, are recalcitrant industrial pollutants that cause adverse effects on living cells. In this paper, the isolation of Pseudomonas fluorescens species with high resistance to pentachlorobenzene (PeCB) is reported. It was found that, in contrast to its slightly negative effect on P. fluorescens growth, PeCB readily inhibited the cell growth of Serratia spp. and Escherichia coli strains, thus indicating that inhibition of bacterial growth by PeCB is species-dependent. Analysis of a P. fluorescens isolate revealed that the exposure to PeCB induced production of reactive oxygen species and led to an increase in the level of alkyl hydroperoxide reductase C (AhpC), an important enzyme enhancing the cell tolerance to organic hydroperoxides usually accumulated under oxidative stress. The putative mechanism conferring PeCB resistance to P. fluorescens and the potential use of P. fluorescens in bioremediation are discussed.     

Changes in the Lung Microbiome following Lung Transplantation Include the Emergence of Two Distinct Pseudomonas Species with Distinct Clinical Associations

Background

Multiple independent culture-based studies have identified the presence of Pseudomonas aeruginosa in respiratory samples as a positive risk factor for bronchiolitis obliterans syndrome (BOS). Yet, culture-independent microbiological techniques have identified a negative association between Pseudomonas species and BOS. Our objective was to investigate whether there may be a unifying explanation for these apparently dichotomous results.

Methods

We performed bronchoscopies with bronchoalveolar lavage (BAL) on lung transplant recipients (46 procedures in 33 patients) and 26 non-transplant control subjects. We analyzed bacterial communities in the BAL fluid using qPCR and pyrosequencing of 16S rRNA gene amplicons and compared the culture-independent data with the clinical metadata and culture results from these subjects.

Findings

Route of bronchoscopy (via nose or via mouth) was not associated with changes in BAL microbiota (p = 0.90). Among the subjects with positive Pseudomonas bacterial culture, P. aeruginosa was also identified by culture-independent methods. In contrast, a distinct Pseudomonas species, P. fluorescens, was often identified in asymptomatic transplant subjects by pyrosequencing but not detected via standard bacterial culture. The subject populations harboring these two distinct pseudomonads differed significantly with respect to associated symptoms, BAL neutrophilia, bacterial DNA burden and microbial diversity. Despite notable differences in culturability, a global database search of UM Hospital Clinical Microbiology Laboratory records indicated that P. fluorescens is commonly isolated from respiratory specimens.

Interpretation

We have reported for the first time that two prominent and distinct Pseudomonas species (P. fluorescens and P. aeruginosa) exist within the post-transplant lung microbiome, each with unique genomic and microbiologic features and widely divergent clinical associations, including presence during acute infection.     

Numerical taxonomy of fluorescent Pseudomonas associated with tomato roots

The phenetic taxonomy of 110 fluorescent bacterial strains, isolated from the roots of tomatoes and other plants was numerically studied through 97 features including 69 assimilation tests. Thirty-two reference strains of various Pseudomonas spp. were additionally included. The strains clustered into 16 clusters at the 74% similarity level when using Jaccard similarity coefficients. Almost all field strains belonged to the P. fluorescens/P. putida-complex while none clustered with P. syringae and allied bacteria. The biovar II branch, as well as the newly described biovar VI of P. fluorescens, made up 55% and 20% respectively, of the field strains; two % were allocated to P. fluorescens biovar I and three % to biovar IV. Eleven % of the root associated strains were designated P. putida; six strains were biovar A, three strains biovar B while four strains could not be referred to any known biovar. The continuum within the P. fluorescens/P. putida-complex as well as the taxonomic status of the six biovars of P. fluorescens and the three biovars of P. putida are discussed.     

Assessment of a chemostat-coupled modified Robbins device to study biofilms

The combination of a modified Robbins device (MRD) attached to the effluent line of a continuous cultivation vessel was assessed by the adhesion of planktonic bacteria maintained at a controlled growth rate. This combination of a chemostat and an MRD provides a large number of sample surfaces for monitoring both the formation and control of biofilms over extended periods of time. This apparatus was used to monitor the colonization of two soil isolates,Pseudomonas fluorescens (EX101) andPseudomonas putida (EX102) onto silastic rubber surfaces. At a similar _rel, both bacteria attached to the silastic, howeverP. fluorescens formed confluent, dense biofilms in less than 24 h, whereasP. putida adhered as single cells or microcolonies after the same period. The metabolic activity, measured by INT-formazan formation, was similar for both organisms with a peak at 6 h of colonization and a subsequent decrease after 24 h. Long term colonization studies ofP. fluorescens produced a population of greater than 9.5 log cfu cm^–2 at 28 days demonstrating the advantages of the chemostat-MRD association. This technique proved to be successful for studying bacterial adhesion and biofilm formation in tubular devices by bacterial populations at controlled and low growth rates.     

Conditions that influence bacterial luminescence in the presence of blood serum

Conditions that influence the luminescence of natural and recombinant luminescent bacteria in the presence of blood serum were studied. In general, blood serum quenched the luminescence of the marine Photobacterium phosphoreum and the recombinant Escherichia coli strains harboring the luminescent system genes of Photobacterium leiognathi, but enhanced the luminescence of the soil bacterium Photorhabdus luminescens Zm1 and the recombinant E. coli strain harboring the lux operon of P. luminescens Zm1. The quenching effect of blood serum increased with its concentration and the time and temperature of incubation. The components of blood serum that determine the degree and specificity of its action on bacterial luminescence were identified.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 191–197.Original Russian Text Copyright © 2005 by Deryabin, Polyakov.     

Gentamicin-resistantPseudomonas aeruginosa: Concepts regarding their evolution and attenuated virulence

Pseudomonas aeruginosa, a free-living bacterial species, is a major nosocomial pathogen, especially of compromised patients within medical facilities. Numerous factors contribute to the ecological selection of this bacterial species within the hospital environment, among which the expression of newly acquired or quiescent enzymatic capability seems par-amount. The emergence of pathogenic strains ofP. aeruginosa appears to be gradual, embodying a transition of strains from their natural aquatic environment, to establishing inanimate (hospital) and animate (human) reservoirs. In this stepwise transition, subsets ofP. aeruginosa may evolve which express a survival trait, for example, gentamicin resistance, but concomitantly suffer a loss of invasive potential. In this study,P. aeruginosa strains from natural [22], hospital [11], and stool [17] sources were evaluated for their physiological and exoenzymatic activity and compared with gentamicin-resistantP. aeruginosa (GRPA) strains [49] of clinical origin. As a whole, environmental and hospital isolates showed reduced enzymatic potential, for example, frequency of production of elastase, lipase, deoxyribonuclease, and pyocyanin production. Human fecal isolates most closely resembled the prototype of human invasiveP. aeruginosa in their gentamicin susceptibility (95%) and increased frequencies of exoenzymes, including elastase production. On the other hand, GRPA were frequently apyocyanogenic (9/49), lacked extracellular enzymes correlated with pathogenicity, and were rarely isolated from systemic sites. When encountered, these strains appeared to represent colonization of a body site rather than incitants of overt infection. As a subset ofP. aeruginosa, gentamicin resistance was seen predominantly among serotype 11 strains, and encountered most frequently from patients with localized urinary tract infections.     

The Effect of Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense on Induction of Defense Enzymes and Phenolics in Banana

The effect of Pseudomonas fluorescens treatment and Fusarium oxysporum f. sp. cubense inoculation on induction of phenylalanine ammonia-lyase (PAL), peroxidase (POX), chitinase, -1,3-glucanase and accumulation of phenolics in banana (Musa sp.) was studied. When banana roots were treated with P. fluorescens strain Pf10, a two-fold increase in phenolic content in leaf tissues was recorded 3 – 6 d after treatment. Challenge inoculation with F. oxysporum, the wilt pathogen, steeply increased the phenolic content in P. fluorescens-treated banana plants. Significant increase in POX activity was detected 6 – 9 d after P. fluorescens treatment. PAL, chitinase and -1,3-glucanase activities increased significantly from 3 d after P. fluorescens treatment and reached the maximum 6 d after treatment. Challenge inoculation with F. oxysporum further increased the enzyme activities. These results suggest that the enhanced activities of defense enzymes and elevated content of phenolics may contribute to bioprotection of banana plants against F. oxysporum.     

The Microbial Transformation of Phenanthrene and Anthracene

The transformation of phenanthrene and anthracene by Rhodococcus rhodnii 135, Pseudomonas fluorescens 26K, and Arthrobacter sp. K3 is studied. Twenty-one intermediates of phenanthrene and anthracene transformation are identified by HPLC, mass spectrometry, and NMR spectroscopy. P. fluorescens 26K and Arthrobacter sp. K3 are found to produce a wide range of intermediates, whereas R. rhodnii 135 oxidizes phenanthrene, resulting in the formation of a sole product, 3-hydroxyphenanthrene. Putative transformation pathways of phenanthrene and anthracene are proposed for the three bacterial strains studied. These strains can be used to obtain valuable compounds (such as hydroxylated polycyclic aromatic hydrocarbons) that are difficult to produce by chemical synthesis.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 357–364.Original Russian Text Copyright © 2005 by Baboshin, Baskunov, Finkelstein, Golovlev, Golovleva.     

A suite of recombinant luminescent bacterial strains for the quantification of bioavailable heavy metals and toxicity testing

Background  

Recombinant whole-cell sensors have already proven useful in the assessment of the bioavailability of environmental pollutants like heavy metals and organic compounds. In this work 19 recombinant bacterial strains representing various Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas fluorescens) bacteria were constructed to express the luminescence encoding genes luxCDABE (from Photorhabdus luminescens) as a response to bioavailable heavy metals ("lights-on" metal sensors containing metal-response elements, 13 strains) or in a constitutive manner ("lights-off" constructs, 6 strains).     

Biological control of rice root-knot nematode,Meloidogyne graminicola through mixture of Pseudomonas fluorescens strains

The plant growth promoting rhizobacterium, Pseudomonas fluorescens strains PF1, TDK1, and PY15 were evaluated individually and in combinations for their efficacy against root-knot nematode, Meloidogyne graminicola, in rice plants under in vitro, glass house and field conditions. Culture filtrates of these strains either individually or as mixture inhibited egg hatching and caused mortality of juveniles of M. graminicola in vitro. The efficacy was more pronounced when filtrates of the strain were used as mixtures than as individual strains. Mixtures of P. fluorescens strains signficantly reduced M. graminicola infestation when applied as bacterial suspensions through seed treatment. The higher activity of peroxidase and chitinase enzymes was observed in plants treated with P. fluorescens mixtures than the plants treated with individual strains, two strain mixtures and untreated control. In field trials on rice, talc formulations of the P. fluorescens strains individually as well as mixtures were evaluated as seed treatment, soil treatment and combination of both. A mixture of the three strains was the most effective when applied either as seed + soil treatment or as seed treatment alone. The introduced P. fluorescens strains survived endophytically on rice roots. The application of the P. fluorescens mixture PF1 + TDK1 + PY15 in seed + soil treatment resulted in higher grain yield which provided a 27.3% increase over the control followed by P. fluorescens mixture PF1 + TDK1 + PY15 in seed treatment alone, which increased the grain yield of rice by 24.7% compared to the control.