Cadmium-regulated gene fusions in Pseudomonas fluorescens

To study the mechanisms soil bacteria use to cope with elevated concentrations of heavy metals in the environment, a mutagenesis with the lacZ-based reporter gene transposon Tn5B20 was performed. Random gene fusions in the genome of the common soil bacterium Pseudomonas fluorescens strain ATCC 13525 were used to create a bank of 5,000 P. fluorescens mutants. This mutant bank was screened for differential gene expression in the presence of the toxic metal cadmium. Fourteen mutants were identified that responded with increased or reduced gene expression to the presence of cadmium. The mutants were characterized with respect to their metal-dependent gene expression and their metal tolerance. Half the identified mutants reacted with differential gene expression specifically to the metal cadmium, whereas some of the other mutants also responded to elevated concentrations of copper and zinc ions. One of the mutants, strain C8, also showed increased gene expression in the presence of the solvent ethanol, but otherwise no overlap between cadmium-induced gene expression and general stress response was detected. Molecular analysis of the corresponding genetic loci was performed using arbitrary polymerase chain reaction (PCR), DNA sequencing and comparison of the deduced protein products with sequences deposited in genetic databases. Some of the genetic loci targeted by the transposon did not show any similarities to any known genes; thus, they may represent 'novel' loci. The hypothesis that genes that are differentially expressed in the presence of heavy metals play a role in metal tolerance was verified for one of the mutants. This mutant, strain C11, was hypersensitive to cadmium and zinc ions. In mutant C11, the transposon had inserted into a genetic region displaying similarity to genes encoding the sensor/regulator protein pairs of two-component systems that regulate gene expression in metal-resistant bacteria, including czcRS of Ralstonia eutropha, czrRS of Pseudomonas aeruginosa and copRS of Pseudomonas syringae. Although the P. fluorescens strain used in this study had not been isolated from a metal-rich environment, it nevertheless contained at least one genetic region enabling it to cope with elevated concentrations of heavy metals.     

A time-resolved fluorescence study of azurin and metalloazurin derivatives

Nickel and cobalt derivatives of Pseudomonas fluorescens (ATCC 13525) azurin were prepared and their steady-state fluorescence and time-resolved fluorescence monitored. Like the copper-containing native protein, the fluorescence decay of both metallo derivatives was best fit to a sum of three exponentials, whereas the apoazurin from which they were prepared obeyed single-exponential decay kinetics. However, comparison of the lifetimes and fractional of each of the components in these derivatives to those in the oxidized and reduced native proteins revealed significant differences. These results suggest that the presence of a metal center in azurin imparts a conformational heterogeneity which is strongly dependent on the nature of the metal center. Further, the results are used to comment on current ideas concerning the geometry of the active site in this redox protein.     

Effect of temperature and starvation upon survival strategies of Pseudomonas fluorescens CHA0: comparison with Escherichia coli

Microorganisms in aquatic systems are exposed to continuous modifications in their environmental conditions. In these systems, both autochthonous and allochthonous bacteria respond to adverse conditions by expressing viable but nonculturable phenotype. On the basis of this common response, the behaviour of a few species is extrapolated to others. We compared the survival strategies of Escherichia coli (allochthonous, mesophile bacterium) and Pseudomonas fluorescens CHA0 (ubiquitous, psychrotrophic bacteria) under nonoptimal temperature and nutrient deprivation. In the absence of nutrients, the effect of temperature on the loss of culturability did not show a common pattern. Whereas the survival of E. coli had an inverse relationship with temperature, whereas for P. fluorescens a direct relationship between temperature and T?? values was only established in the range 5-15°C, with an inverse relationship at higher temperatures. When the subproteome of the outer membrane of P. fluorescens was comparatively analysed, starvation was not the main source of change. The most relevant modifications were due to variations in temperature. OprF, the major surface protein of the genus Pseudomonas, showed a high expression in nonculturable as well as culturable populations under all the adverse situations analysed. We therefore propose OprF as a suitable marker for Pseudomonas detection in the environment.     

Up-regulation of pathogenesis-related proteins in the apoplast of Malus domestica after application of a non-pathogenic bacterium

The intercellular washing fluid (IWF) of Malus domestica cv. Holsteiner Cox before and after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the leaves was investigated in a comparative manner. SDS-PAGE in combination with ESI Q-ToF mass spectrometry, and homology search in relevant data bases revealed the highly up-regulated expression of several pathogenesis-related plant proteins in the apoplast of the leaves treated with P. fluorescens. These proteins were beta3-1,3-glucanase, chitinase, thaumatin-like protein, ribonuclease-like protein, and a hevein-like protein. Moreover, a 9 kDa non-specific lipid transfer protein was significantly reduced after the application of P. fluorescens. The possible relevance of a pre-treatment of apple cultivars with the non-pathogenic bacterium P. fluorescens Bk3, as an alternative method to the treatment with fungicides, for increasing the resistance of susceptible apple cultivars against an infection with the fungus Venturia inaequalis is discussed.     

Inducible surface proteins from some strains of Pseudomonas species bacteria while adapting to cobalt

The possibility of increasing resistance of some Pseudomonas strains to cobalt at adaptation to monotonous increasing its concentration was studied. Strains Pseudomonas fluorescens B5242 and Pseudomonas fluorescens B894 are capable to increase its resistance in such conditions via inducible synthesis of protective surface proteins. The molecular masses of such proteins were 55.0; 45.0 and 33.0 kDa for P. fluorescens B5242 strain.     

CzcR-CzcS, a two-component system involved in heavy metal and carbapenem resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an environmental bacterium involved in mineralization of organic matter. It is also an opportunistic pathogen able to cause serious infections in immunocompromised hosts. As such, it is exposed to xenobiotics including solvents, heavy metals, and antimicrobials. We studied the response of P. aeruginosa upon exposure to heavy metals or antibiotics to investigate whether common regulatory mechanisms govern resistance to both types of compounds. We showed that sublethal zinc concentrations induced resistance to zinc, cadmium, and cobalt, while lethal zinc concentrations selected mutants constitutively resistant to these heavy metals. Both zinc-induced and stable zinc-resistant strains were also resistant to the carbapenem antibiotic imipenem. On the other hand, only 20% of clones selected on imipenem were also resistant to zinc. Heavy metal resistance in the mutants could be correlated by quantitative real time PCR with increased expression of the heavy metal efflux pump CzcCBA and its cognate two-component regulator genes czcR-czcS. Western blot analysis revealed reduced expression of the basic amino acid and carbapenem-specific OprD porin in all imipenem-resistant mutants. Sequencing of the czcR-czcS DNA region in eight independent zinc- and imipenem-resistant mutants revealed the presence of the same V194L mutation in the CzcS sensor protein. Overexpression in a susceptible wild type strain of the mutated CzsS protein, but not of the wild type form, resulted in decreased oprD and increased czcC expression. We further show that zinc is released from latex urinary catheters into urine in amounts sufficient to induce carbapenem resistance in P. aeruginosa, possibly compromising treatment of urinary tract infections by this class of antibiotics.     

Wood ants use resin to protect themselves against pathogens

Social life is generally associated with an increased exposure to pathogens and parasites, due to factors such as high population density, frequent physical contact and the use of perennial nest sites. However, sociality also permits the evolution of new collective behavioural defences. Wood ants, Formica paralugubris, commonly bring back pieces of solidified coniferous resin to their nest. Many birds and a few mammals also incorporate green plant material into their nests. Collecting plant material rich in volatile compounds might be an efficient way to fight bacteria and fungi. However, no study has demonstrated that this behaviour has a positive effect on survival. Here, we provide the first experimental evidence that animals using plant compounds with antibacterial and antifungal properties survive better when exposed to detrimental micro-organisms. The presence of resin strongly improves the survival of F. paralugubris adults and larvae exposed to the bacteria Pseudomonas fluorescens, and the survival of larvae exposed to the entomopathogenic fungus Metarhizium anisopliae. These results show that wood ants capitalize on the chemical defences which have evolved in plants to collectively protect themselves against pathogens.     

Characterization of the proteins of bacterial strain isolated from contaminated site involved in heavy metal resistance--a proteomic approach

The present study describes response of a bacterial strain isolated from a polluted river to heavy metal toxicity. The bacterium was identified to be Klebsiella pneumoniae by biochemical tests using API 20E strips and 16S ribotyping. The isolate was studied for its tolerance to two heavy metals, i.e., cobalt (Co(2+)) and lead (Pb(2+)) by growing it in citrate mineral medium (CMM). Proteomic approach involving two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry (MS) was used to identify the differentially expressed proteins under heavy metal stress. Two of the differentially expressed proteins were identified to be l-isoaspartate protein carboxymethyltransferase type II and DNA gyrase A. To our knowledge, this is for the first time that K. pneumoniae has been reported to be present in metal contaminated site and l-isoaspartate protein carboxymethyltransferase type II protein to be over expressed under heavy metal stress. The role of these proteins in metal tolerance is discussed.     

Resistance of certain strains of Pseudomonas bacteria to toxic effect of heavy metal ions]

The effect of heavy metal cations (cobalt, nickel, and copper) and the anion detergent sodium dodecyl sulfate (SDS) on the barrier properties of the plasma membrane (PM) of the Co-sensitive stain Pseudomonas putida BS394 and the Co-resistant strains Pseudomonas sp. BS501 (wild-type strain) and Pseudomonas putida BS394 (pBS501) (transformed strain) was studied by high-frequency electro-orientational spectroscopy. The cations were found to rank, in order of decreasing damage inflicted on the PM, as copper > cobalt > nickel. The strains studied were found to rank, in order of increasing resistance of the PM to damage inflicted by copper and cobalt cations, as P. putida BS394 < P. putida BS394 (pBS501) < Pseudomonas sp. BS501. In order of increasing resistance to SDS, the strains ranked inversely. The strains did not differ in sensitivity to nickel cations. Investigation of the surface of intact and trypsin-treated cells by microelectrophoresis showed that the surface layers of the cell wall of wild-type and transformed cells contained increased amounts of proteins. The surface proteins of Co-resistant cells had molecular masses of 49, 40, and 32 kDa. Exposure of Co-resistant cells to trypsin considerably reduced their resistance to cobalt cations. It is assumed that the resistance of the PM of the wild-type and transformed pseudomonads to heavy metal cations is determined by plasmid pBS501 and is related to the synthesis of protective surface proteins of the cell wall.     

Effect of cadmium and zinc on attachment and detachment interactions of Pseudomonas fluorescens H2 with glass.

The physiological and physicochemical bases for the effect of 5, 10, 50, or 100 micrograms of Cd and Zn ml-1 on the attachment and detachment interactions of Pseudomonas fluorescens H2 with glass substrata were determined. Attachment and detachment varied with the type and concentration of metal and the time at which cells were exposed to the metal. The largely inhibitory effect of the metals on bacterial motility and physiological activity did not directly influence attachment. The amount of Cd or Zn accumulated by the cells increased with metal concentration and was greater for free than for attached cells. The hydrophobicity and negative and positive charges of the bacterial surfaces (measured by hydrophobic and electrostatic interaction chromatography) were increased by cell exposure to the metals, particularly after Cd treatment. Cells exposed to Cd prior to attachment showed increased adhesion. Zinc-treated cells did not. There was a positive correlation between adhesion and Cd concentration in the attachment solution. No such relationship existed for Zn. P. fluorescens H2 exposed to Cd prior to attachment desorbed similarly to untreated controls. Zinc pretreatment resulted in decreased desorption. Cells attached in 5 or 10 micrograms of Cd or Zn ml-1 detached less than those attached in 50 or 100 micrograms of Cd or Zn ml-1. The presence of Cd or Zn during detachment had little effect on desorption. The dominant influence of Cd and Zn on attachment and detachment appears to be through modification of the bacterial surface. In natural ecosystems, heavy metals may influence the distribution of bacteria between the solid and liquid phases.     

Pseudomonas putida Response to Cadmium: Changes in Membrane and Cytosolic Proteomes

Pseudomonas putida is a saprophytic bacterium with remarkable environmental adaptability and the capacity to tolerate high concentrations of heavy metals. The strain P. putida-Cd001 was isolated from soil contaminated with Cd, Zn and Pb. Membrane-associated and cytosolic proteomes were analyzed to identify proteins whose expression was modulated in response to 250 μM CdSO(4). We identified 44 protein spots in the membrane and 21 in the cytosolic fraction differentially expressed in Cd-treated samples compared to untreated controls. Outer membrane porins from the OprD and OprI families were less abundant in bacteria exposed to Cd, whereas those from the OprF and OprL, OprH and OprB families were more abundant, reflecting the increased need to acquire energy sources, the need to maintain membrane integrity and the process of adaptation. Components of the efflux system, such as the CzcB subunit of the CBA system, were also induced by Cd. Analysis of the cytosolic proteome revealed that proteins involved in protein synthesis, degradation and folding were induced along with enzymes that combat oxidative stress, showing that the entire bacterial proteome is modulated by heavy metal exposure. This analysis provides new insights into the adaptation mechanisms used by P. putida-Cd001 to survive in Cd-polluted environments.     

Cross-reactivity of antibodies raised to Pseudomonas fluorescens protease with extracellular proteins produced by meat-spoiling pseudomonads

The cross-reactivity patterns of antibodies to Pseudomonas fluorescens protease with the extracellular proteins produced by a number of meat-spoiling pseudomonads were studied. Immunoblotting studies showed that purified IgG to Ps. fluorescens protease cross-reacted with extracellular proteins in the cell culture supernatant fluids of Pseudomonas spp., including Ps. fragi and Ps. lundensis. In the case of Ps. lundensis and Pseudomonas spp. 11390, the cross-reactive moieties were of similar molecular weight to the Ps. fluorescens protease (46 kDa). However, in Ps. fragi the cross-reactive moiety was a lower molecular weight protein (8 kDa). This may represent a fragment of the active enzyme. These results indicate the presence of common antigenic determinants among the proteases of meat spoiling pseudomonads.     

Outer membrane proteins of Pseudomonas

In this review, we describe the outer membrane proteins of Pseudomonas aeruginosa and related strains from the Pseudomonas fluorescens rRNA homology group of the Pseudomonadaceae, with emphasis on the physiological function and biochemical characteristics of these proteins. The use of opr (for outer membrane protein) is proposed as the genetic designation for the P. aeruginosa outer membrane proteins and letters are assigned, in conjunction with this designation, to known outer membrane proteins. Proteins whose primary functions involve pore formation, transport of specific substrates, cell structure determination and membrane stabilization are discussed. The conservation of selected proteins in the above Pseudomonas species is also examined.     

Pseudomonas fluorescens as a potential pathogen: adherence to nerve cells

In order to determine the infectious potential of the psychrotrophic bacterium Pseudomonas fluorescens, a species closely related to the opportunistic pathogen P. aeruginosa, we investigated the binding activity of this bacterium on primary cultures of rat neonate cortical neurons and glial cells, adrenal paraneurons and NG108-15 neuroblastoma cells. Incubated at concentrations of 10(6) and 10(8) CFU/mL, P. fluorescens MF37 exhibited a high binding activity on neurons in the same range as that of P. aeruginosa PAO1. A significant, but lower, adherence of P. fluorescens was also detected on glial cells and adrenal paraneurons. In contrast, when P. fluorescens MF37 or P. aeruginosa PAO1 were incubated with neuroblastoma cells, no binding was observed. In neurons, the association of P. fluorescens with the plasma membrane occurred both on neurites and cell body. Leakage of the cytoplasmic content was frequently noted. Studies performed using the fluorescent probe Hoechst 33258 revealed that in 10% of neurons, P. fluorescens induced the appearance of densely stained clusters of DNA that was typical of an early step of apoptosis. In glial cells exposed to P. fluorescens, marked changes in the morphology of the nucleus, including fragmentation into lobular structures and aggregation of DNA, were also reminiscent of the existence of a possible apoptotic mechanism. Taken together, these results reveal that P. fluorescens can bind to nerve cells and affect their physiology and, in agreement with recent clinical observations, suggest that P. fluorescens could behave as a pathogen.     

Inhibition of proteasome activity is involved in cobalt-induced apoptosis of human alveolar macrophages

Inhalation of particulate cobalt has been known to induce interstitial lung disease. There is growing evidence that apoptosis plays a crucial role in physiological and pathological settings and that the ubiquitin-proteasome system is involved in the regulation of apoptosis. Cadmium, the same transitional heavy metal as cobalt, has been reported to accumulate ubiquitinated proteins in neuronal cells. On the basis of these findings, we hypothesized that cobalt would induce apoptosis in the lung by disturbance of the ubiquitin-proteasome pathway. To evaluate this, we exposed U-937 cells and human alveolar macrophages (AMs) to cobalt chloride (CoCl(2)) and examined their apoptosis by DNA fragmentation assay, 4',6-diamidino-2'-phenylindol dihydrochloride staining, and Western blot analysis. CoCl(2) induced apoptosis and accumulated ubiquitinated proteins. Exposure to CoCl(2) inhibited proteasome activity in U-937 cells. Cobalt-induced apoptosis was mediated via mitochondrial pathway because CoCl(2) released cytochrome c from mitochondria. These results suggest that cobalt-induced apoptosis of AMs may be one of the mechanisms for cobalt-induced lung injury and that the accumulation of ubiquitinated proteins might be involved in this apoptotic process.     

Induction of laccase activity in Rhizoctonia solani by antagonistic Pseudomonas fluorescens strains and a range of chemical treatments

Fungi often produce the phenoloxidase enzyme laccase during interactions with other organisms, an observation relevant to the development of biocontrols. By incorporating the laccase substrate 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) into agar, we analyzed laccase induction in the plant-pathogenic fungus Rhizoctonia solani when paired against isolates of the soil bacterium Pseudomonas fluorescens. Substantial induction of R. solani laccase was seen only in pairings with strains of P. fluorescens known to produce antifungal metabolites. To study laccase induction further, a range of chemical treatments was applied to R. solani liquid cultures. p-Anisidine, copper(II), manganese(II), calcium ionophore A23187, lithium chloride, calcium chloride, cyclic AMP (cAMP), caffeine, amphotericin B, paraquat, ethanol, and isopropanol were all found to induce laccase; however, the P. fluorescens metabolite viscosinamide did not do so at the concentrations tested. The stress caused by these treatments was assessed by measuring changes in lipid peroxidation levels and dry weight. The results indicated that the laccase induction seen in pairing plate experiments was most likely due to calcium or heat shock signaling in response to the effects of bacterial metabolites, but that heavy metal and cAMP-driven laccase induction was involved in sclerotization.     

Studies on the behaviour of Pseudomonas fluorescens biofilms after Ortho-phthalaldehyde treatment

A relatively novel biocide, ortho-phthalaldehyde (OPA), was tested to control biofilms formed by Pseudomonas fluorescens on stainless steel surfaces. The toxic action of OPA was assessed in terms of inactivation and removal of the biofilm by means of, respectively, the determination of the respiratory activity and the variation in the dry weight of the biofilms. For comparison, the activity of OPA against suspended bacteria was also evaluated. The results showed that higher concentrations of OPA and longer exposure times are needed to inactivate P. fluorescens biofilms than planktonic populations, thus denoting that sessile bacteria have a reduced susceptibility to OPA. This appears to be associated with the reaction with the proteins of the matrix, as demonstrated by the reduction of the antimicrobial action of OPA in the presence of a protein (bovine serum albumin). The application of OPA appeared to cause little effect in the removal of biofilms from the metal slides since the mass of biofilm that remained on the surfaces, after biocide treatment, was within the same range as those observed in the control tests. These results suggest that, with OPA application, biofilms can be inactivated but stay attached to the surfaces, decreasing thereby the success of the chemical treatment.     

Maintenance of the supercooled state in the gut fluid of overwintering pyrochroid beetle larvae, Dendroides canadensis : role of ice nucleators and antifreeze proteins

  The effect of gut fluid ice nucleators and antifreeze proteins on maintenance of supercooling was explored in fire-colored beetle larvae, Dendroides canadensis, via seasonal monitoring of supercooling points, antifreeze protein activity and ice nucleator activity of gut fluid and/or larvae. During cold hardening in the field, freeze-avoiding larvae evacuated their guts and depressed larval supercooling points. Analysis of gut fluid indicated supercooling points and ice nucleator activity decreased, whereas antifreeze protein activity increased as winter approached. Suspensions of bacteria isolated from guts of feeding larvae collected in spring/summer had higher supercooling points than those from midwinter-collected non-feeding larvae, suggesting bacterial ice nucleators are removed from midwinter gut fluid. The ice nucleation active bacterium Pseudomonas fluorescens was isolated from gut fluid of feeding larvae but was absent in winter. When mixed with purified D.␣canadensis hemolymph antifreeze proteins (structurally similar and/or identical to those in gut fluid), the cumulative ice nucleus spectra of P. fluorescens suspensions were shifted to lower temperatures indicating an inhibitory effect on the bacteria's ice-nucleating phenotype. By extending larval supercooling capacity, both gut clearing and masking of bacterial ice nucleators by antifreeze proteins may contribute to overwintering survival in supercooled insects. Accepted: 8 August 1996     

Cloning and characterization of the Pseudomonas fluorescens ATP-binding cassette exporter, HasDEF, for the heme acquisition protein HasA

Two ATP-binding cassette (ABC) exporters are present in Pseudomonas fluorescens no. 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA. The hasDEF genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the Serratia marcescens ABC exporter Lip system. P. fluorescens HasA showed sequence identity of 40 to 49% with HasA proteins from Pseudomonas aeruginosa and Serratia marcescens. The P. fluorescens Has exporter secreted HasA proteins from P. fluorescens and P. aeruginosa but not S. marcescens HasA in Escherichia coli, whereas the Has exporter from S. marcescens allowed secretion of all three HasA proteins. The P. fluorescens HasDEF system also promoted the secretion of the lipase and alkaline protease of P. fluorescens. Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of export substrates. Chimeric HasA proteins containing both P. fluorescens and S. marcescens sequences were produced and tested for secretion through the Has exporters. The C-terminal region of HasA was shown to be involved in the secretion specificity of the P. fluorescens Has exporter.