Influence of Sodium Hexametaphosphate on Selected Bacteria

Sodium hexametaphosphate (HEX), the solvent of calcium alginate wool used in swabbing inanimate surfaces was studied relative to its effect on various bacterial populations, both pure cultures and wild. It was found that bacteria in wild populations were greatly inhibited, and that a percentage reduction of count was directly related to concentration of HEX. Most gram-positive bacteria were prevented from growing on a medium containing 0.1% HEX. This, or a higher concentration, occurred in the final medium when the method recommended in Standard Methods for the Examination of Dairy Products was followed. Growth of Sarcina lutea occurred on media with higher concentrations than that of inhibition (0.05%), if MgSO₄·7H₂O was incorporated in the medium. Gram-negative bacteria were capable of growing in higher concentrations, even up to 10% HEX. A large percentage of the cells of some strains (represented by Pseudomonas fluorescens) were lysed on contact with HEX. Lysis could be prevented by the addition of NaCl or MgSO₄·7H₂O. The evidence presented suggests that HEX, a phosphate-glass water-softening sequestrant, interferes with divalent cation metabolism, notably magnesium ion, and possibly others, producing cell division inhibition and loss of cell-wall integrity. The mechanism of action was not elucidated.     

Natural Transformation of Pseudomonas fluorescens and Agrobacterium tumefaciens in Soil

Little information is available concerning the occurrence of natural transformation of bacteria in soil, the frequency of such events, and the actual role of this process on bacterial evolution. This is because few bacteria are known to possess the genes required to develop competence and because the tested bacteria are unable to reach this physiological state in situ. In this study we found that two soil bacteria, Agrobacterium tumefaciens and Pseudomonas fluorescens, can undergo transformation in soil microcosms without any specific physical or chemical treatment. Moreover, P. fluorescens produced transformants in both sterile and nonsterile soil microcosms but failed to do so in the various in vitro conditions we tested. A. tumefaciens could be transformed in vitro and in sterile soil samples. These results indicate that the number of transformable bacteria could be higher than previously thought and that these bacteria could find the conditions necessary for uptake of extracellular DNA in soil.     

Biomineralization of Middle Rare Earth Element Samarium in Yeast and Bacteria Systems

Microorganisms play an important role in the mineralization of heavy metals in different environments. Previous studies have reported the phosphate mineralization of light (Ce) and heavy (Yb) rare earth elements with yeast. However, little is known about differences in the biomineralization process of middle rare earth elements (including Sm, Eu, Gd, Tb and Dy) by yeast and bacteria. We carried out a series of experiments to compare the sorption process of Sm by Saccharomyces cerevisiae (yeast), Pseudomonas fluorescens (gram-negative bacteria) and Bacillus subtilis (gram-positive bacteria) in initial pH 3, 4 and 5 solutions. The concentrations of Sm in exposure solutions decreased as a function of exposure time in all three systems, which revealed the accumulation of Sm by cells. In both yeast and bacteria systems, Sm(III) was mineralized to monazite(Sm) phase particles on cell surfaces at 5 days of exposure after a short-term adsorption process. In these three systems, nano-sized Sm phosphate formed more quickly on cell surfaces with higher pH exposure solutions. The formation of precipitation on bacterial cell surfaces was faster than in yeast. There were no significant differences in the sorption process of Sm between the two bacteria Pseudomonas fluorescens and Bacillus subtilis.     

Spectrum of biocontrol bacteria to control leaf,root and vascular diseases of dry bean

Bacterial biocontrol agents, previously selected to control bacterial blight and anthracnose, were evaluated for the control of bacterial wilt, fusarium wilt, charcoal rot and angular leaf spot of dry beans. The seeds were microbiolized with these bacteria singly or in combinations. The microbiolization resulted in reduction of severity of all four diseases, showing wide spectrum of diseases control by these bacteria. However, the severity reduction of all four diseases by combination C01 composed of isolates DFs093 and DFs769 of Bacillus cereus and DFs831 of Pseudomonas fluorescens was significantly higher than if the seeds were microbiolized by a single isolate of a biocontrol agent.     

Bacterial Ortholog of Mammalian Translocator Protein (TSPO) with Virulence Regulating Activity

The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10⁻⁵ M) adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies.     

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.     

Pseudomonas fluorescens can induce and divert the human β-defensin-2 secretion in intestinal epithelial cells to enhance its virulence

The effect of intestinal molecules produced by the host on the virulence of Pseudomonas fluorescens is poorly documented. In the present work, we evaluated the secretion of human β-defensin-2 (hBD-2) by enterocytes after infection with P. fluorescens (a species previously suggested to be involved in inflammatory bowel disease) and investigated the effect of this host-defense peptide on the bacterial virulence. The results showed that P. fluorescens can induce hBD-2 production in Caco-2/TC7 cells via P38 and ERK MAPK-dependent pathways. Surprisingly, the exposure of P. fluorescens to low doses of the antimicrobial peptide was found to enhance its cytotoxic and proinflammatory effects suggesting a potential feedback mechanism in the dialog between bacteria and the host.     

The Effect of Phylogenetically Different Bacteria on the Fitness of Pseudomonas fluorescens in Sand Microcosms

In most environments many microorganisms live in close vicinity and can interact in various ways. Recent studies suggest that bacteria are able to sense and respond to the presence of neighbouring bacteria in the environment and alter their response accordingly. This ability might be an important strategy in complex habitats such as soils, with great implications for shaping the microbial community structure. Here, we used a sand microcosm approach to investigate how Pseudomonas fluorescens Pf0-1 responds to the presence of monocultures or mixtures of two phylogenetically different bacteria, a Gram-negative (Pedobacter sp. V48) and a Gram-positive (Bacillus sp. V102) under two nutrient conditions. Results revealed that under both nutrient poor and nutrient rich conditions confrontation with the Gram-positive Bacillus sp. V102 strain led to significant lower cell numbers of Pseudomonas fluorescens Pf0-1, whereas confrontation with the Gram-negative Pedobacter sp. V48 strain did not affect the growth of Pseudomonas fluorescens Pf0-1. However, when Pseudomonas fluorescens Pf0-1 was confronted with the mixture of both strains, no significant effect on the growth of Pseudomonas fluorescens Pf0-1 was observed. Quantitative real-time PCR data showed up-regulation of genes involved in the production of a broad-spectrum antibiotic in Pseudomonas fluorescens Pf0-1 when confronted with Pedobacter sp. V48, but not in the presence of Bacillus sp. V102. The results provide evidence that the performance of bacteria in soil depends strongly on the identity of neighbouring bacteria and that inter-specific interactions are an important factor in determining microbial community structure.     

Effect of Motility on Surface Colonization and Reproductive Success of Pseudomonas fluorescens in Dual-Dilution Continuous Culture and Batch Culture Systems

The colonization of glass surfaces by motile and nonmotile strains of Pseudomonas fluorescens was evaluated by using dual-dilution continuous culture (DDCC), competitive and noncompetitive attachment assays, and continuous-flow slide culture. Both strains possessed identical growth rates whether in the attached or planktonic state. Results of attachment assays using radiolabeled bacteria indicated that both strains obeyed first-order (monolayer) adsorption kinetics in pure culture. However, the motile strain attached about four times more rapidly and achieved higher final cell densities on surfaces than did the nonmotile strain (2.03 × 10⁸ versus 5.57 × 10⁷ cells vial^-1) whether evaluated alone or in cocultures containing motile and nonmotile P. fluorescens. These kinetics were attributed to the increased transport of motile cells from the bulk aqueous phase to the hydrodynamic boundary layer where bacterial attachment, growth, and recolonization could occur. First-order attachment kinetics were also observed for both strains by using continuous-flow slide culture assays analyzed by image analysis. The DDCC system contained both aqueous and particulate phases which could be diluted independently. DDCC results indicated that when cocultures containing motile and nonmotile P. fluorescens colonized solid particles, the motile strain replaced the nonmotile strain in the system over time. Increasing the aqueous-phase rates of dilution decreased the time required for extinction of the nonmotile strain while concurrently decreasing the overall carrying capacity of the DDCC system for both strains. These results confirmed that bacterial motility conveyed a selective advantage during surface colonization even in aqueous-phase systems not dominated by laminar flow.     

Effect of electrostatic spray parameters on the viability of two bacterial biocontrol agents and their deposition on blueberry flower stigmas

The fungus Monilinia vaccinii-corymbosi infects blueberry flowers via the stigma-style ovary pathway to cause mummy berry disease. Previous laboratory experiments documented considerable activity of stigma-applied biofungicides containing the bacteria Bacillus subtilis and, to a lesser extent, Pseudomonas fluorescens against flower infection by the pathogen. However, adequate and targeted delivery of the biocontrol agents to the stigmatic surfaces of open flowers in the field has remained problematic. Here we consider the application of the biofungicides Serenade AS (containing B. subtilis QST713) and BlightBan A506 (containing P. fluorescens A506) to blueberry flowers by air-assisted electrostatic spraying. In laboratory experiments with typical field-use rates, viability of B. subtilis and P. fluorescens was unaffected by different levels of induction-charging voltage (0–1.2 kV) and atomizing pressure (138–276 kPa) applied to an electrostatic spray-charging nozzle, showing that the bacteria in both formulations readily survived exposure to the intense electrical fields and near-sonic atomizing air shear encountered during electrostatic spraying. Electrostatically charged application significantly (P<0.0001) increased deposition of B. subtilis on the stigmatic surfaces of detached blueberry flower clusters by a factor of 4.5 compared with conventional hydraulic spraying; a similar comparison showed that population densities of P. fluorescens on the stigma were increased by a factor of 2.9, but this effect was not statistically significant (P=0.1487). For Serenade, the increased coverage and/or retention on the flower stigma, along with the excellent bacterial survival, portend well for electrostatic application for mummy berry disease control in the field.     

Spatial Organization of Dual-Species Bacterial Aggregates on Leaf Surfaces

The spatial organization of cells within bacterial aggregates on leaf surfaces was determined for pair-wise mixtures of three different bacterial species commonly found on leaves, Pseudomonas syringae, Pantoea agglomerans, and Pseudomonas fluorescens. Cells were coinoculated onto bean plants and allowed to grow under moist conditions, and the resulting aggregates were examined in situ by epifluorescence microscopy. Each bacterial strain could be localized because it expressed either the green or the cyan fluorescent protein constitutively, and the viability of individual cells was assessed by propidium iodide staining. Each pair of bacterial strains that was coinoculated onto leaves formed mixed aggregates. The degree of segregation of cells in mixed aggregates differed between the different coinoculated pairs of strains and was higher in mixtures of P. fluorescens A506 and P. agglomerans 299R and mixtures of P. syringae B728a and P. agglomerans 299R than in mixtures of two isogenic strains of P. agglomerans 299R. The fractions of the total cell population that were dead in mixed and monospecific aggregates of a gfp-marked strain of P. agglomerans 299R and a cfp-marked strain of P. agglomerans 299R, or of P. fluorescens A506 and P. agglomerans 299R, were similar. However, the proportion of dead cells in mixed aggregates of P. syringae B728a and P. agglomerans 299R was significantly higher (13.2% ± 8.2%) than that in monospecific aggregates of these two strains (1.6% ± 0.7%), and it increased over time. While dead cells in such mixed aggregates were preferentially found at the interface between clusters of cells of these strains, cells of these two strains located at the interface did not exhibit equal probabilities of mortality. After 9 days of incubation, about 77% of the P. agglomerans 299R cells located at the interface were dead, while only about 24% of the P. syringae B728a cells were dead. The relevance of our results to understanding bacterial interactions on leaf surfaces and the implications for biological control of pathogenic and other deleterious microorganisms is discussed.     

Growth temperature and OprF porin affect cell surface physicochemical properties and adhesive capacities of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> MF37

Pseudomonads adapt to various ecological niches by forming biofilms, which first requires bacterial adhesion on surfaces. We studied the influence of growth temperature on surface physicochemical properties of Pseudomonas fluorescens MF37 and on its adhesive capacities onto inert surfaces. It presented a global hydrophilic character, measured by microbial adhesion to solvent (MATS), and showed a cell surface more hydrophilic at 8 and 28°C than at 17°C. Moreover, P. fluorescens MF37 was more adhesive at 17°C. This critical temperature thus should be carefully taken into account in food safety. Adhesion onto inert surfaces is thus influenced by the growth temperature, which modifies the bacteria cell wall properties through changes in the outer membrane components. Therefore, we studied the effect of the loss of OprF, the major outer membrane protein, known to act as an adhesin (root, and endothelial cells). The OprF-deficient mutant was able to adhere to surfaces, but showed the same physicochemical and adhesion properties on abiotic surfaces whatever the growth temperature. OprF is thus not essential in this adhesion process. However, we suggest that OprF is involved in the bacterial environmental temperature sensing by P. fluorescens.This work was supported by a grant from the Région Bretagne (Doctoral fellowship to G.H.).     

Measurement of Glucose Utilization by Pseudomonas fluorescens That Are Free-Living and That Are Attached to Surfaces

The assimilation and respiration of glucose by attached and free-living Pseudomonas fluorescens were compared. The attachment surfaces were polyvinylidene fluoride, polyethylene, and glass. Specific uptake of [¹⁴C]glucose was determined after bacterial biomass was measured by (i) microscopic counts or (ii) prelabeling of cells by providing [³H]leucine as substrate, followed by dual-labeling scintillation counting. The glucose concentration was 1.4, 3.5, 5.5, 7.6, or 9.7 μM. Glucose assimilation by cells which became detached from the surfaces during incubation with glucose was also measured after the detached cells were collected by filtration. The composition of the substratum had no effect on the amount of glucose assimilated by attached cells. Glucose assimilation by attached cells exceeded that by free-living cells by a factor of between 2 and 5 or more, and respiration of glucose by surface-associated cells was greater than that by free-living bacteria. Glucose assimilation by detached cells was greater than that by attached bacteria. Measurements of biomass by microscopic counts gave more consistent results that those obtained with dual-labeling, but in general, results obtained by both methods were corroborative.     

Selective bactericidal activity of nanopatterned superhydrophobic cicada Psaltoda claripennis wing surfaces

The nanopattern on the surface of Clanger cicada (Psaltoda claripennis) wings represents the first example of a new class of biomaterials that can kill bacteria on contact based solely on its physical surface structure. As such, they provide a model for the development of novel functional surfaces that possess an increased resistance to bacterial contamination and infection. Their effectiveness against a wide spectrum of bacteria, however, is yet to be established. Here, the bactericidal properties of the wings were tested against several bacterial species, possessing a range of combinations of morphology and cell wall type. The tested species were primarily pathogens, and included Bacillus subtilis, Branhamella catarrhalis, Escherichia coli, Planococcus maritimus, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Staphylococcus aureus. The wings were found to consistently kill Gram-negative cells (i.e., B. catarrhalis, E. coli, P. aeruginosa, and P. fluorescens), while Gram-positive cells (B. subtilis, P. maritimus, and S. aureus) remained resistant. The morphology of the cells did not appear to play any role in determining cell susceptibility. The bactericidal activity of the wing was also found to be quite efficient; 6.1?±?1.5?×?10⁶ P. aeruginosa cells in suspension were inactivated per square centimeter of wing surface after 30-min incubation. These findings demonstrate the potential for the development of selective bactericidal surfaces incorporating cicada wing nanopatterns into the design.     

No apparent costs for facultative antibiotic production by the soil bacterium Pseudomonas fluorescens Pf0-1

Background

Many soil-inhabiting bacteria are known to produce secondary metabolites that can suppress microorganisms competing for the same resources. The production of antimicrobial compounds is expected to incur fitness costs for the producing bacteria. Such costs form the basis for models on the co-existence of antibiotic-producing and non-antibiotic producing strains. However, so far studies quantifying the costs of antibiotic production by bacteria are scarce. The current study reports on possible costs, for antibiotic production by Pseudomonas fluorescens Pf0-1, a soil bacterium that is induced to produce a broad-spectrum antibiotic when it is confronted with non-related bacterial competitors or supernatants of their cultures.

Methodology and Principal Findings

We measured the possible cost of antibiotic production for Pseudomonas fluorescens Pf0-1 by monitoring changes in growth rate with and without induction of antibiotic production by supernatant of a bacterial competitor, namely Pedobacter sp.. Experiments were performed in liquid as well as on semi-solid media under nutrient-limited conditions that are expected to most clearly reveal fitness costs. Our results did not reveal any significant costs for production of antibiotics by Pseudomonas fluorescens Pf0-1. Comparison of growth rates of the antibiotic-producing wild-type cells with those of non-antibiotic producing mutants did not reveal costs of antibiotic production either.

Significance

Based on our findings we propose that the facultative production of antibiotics might not be selected to mitigate metabolic costs, but instead might be advantageous because it limits the risk of competitors evolving resistance, or even the risk of competitors feeding on the compounds produced.     

Influence of Solid Surface, Adhesive Ability, and Inoculum Size on Bacterial Colonization in Microcosm Studies

Microcosm studies were performed to evaluate the effect of solid surfaces, bacterial adhesive ability, and inoculum size on colonization success and persistence of Pseudomonas fluorescens and Xanthomonas maltophilia, each with a Tn5 insertion that conferred resistance to kanamycin and streptomycin. Two types of microcosms were used: (i) a simple system that was colonized by Aeromonas hydrophila and a coryneform and (ii) a complex system produced from lake water enrichment cultures. Simple microcosms contained 100 ml of peptone- and yeast extract-supplemented artificial lake water or 60 ml of peptone- and yeast extract-supplemented artificial lake water with 70 g of 3-mm glass beads. Complex microcosms contained 100 ml of lake water with no nutrient additions or 100 ml of lake water with 70 g of glass beads. The microcosms were incubated for 35 days at 20°C. In lake water enrichment microcosms, the presence of beads increased the abilities of P. fluorescens or X. maltophilia to colonize, but their numbers decreased with time in microcosms both with and without beads. The adhesiveness of the bacteria, measured in an in vitro assay, did not relate to colonization success. In simple microcosms, the inoculum size (10, 10², or 10³) of P. fluorescens did not influence colonization success. However, in complex microcosms, an inoculum of 10³ cells was insufficient to ensure colonization by P. fluorescens, while 10⁶ cells resulted in colonization of liquid and beads. Simple microcosm studies, utilizing only a few species, were poor models for complex natural systems. In complex enrichment systems, colonization of surfaces resulted in higher numbers of organisms but did not noticeably promote persistence. Adhesiveness of a particular organism may be a relatively minor factor influencing its ability to colonize solid surfaces in complex natural environments.     

Seasonal Population Changes and Characterization of Ice-Nucleating Bacteria in Farm Fields of Central Alberta

During the summer of 1983 in central Alberta, changes in the bacterial population inhabiting the leaves of field beans (Phaseolus vulgaris L.) and canola (Brassica napus L. Altex) were studied to determine if ice-nucleating bacteria were present on these plants. Three colony types (white, yellow, and peach-colored) were found on field beans and canola leaves. Approximately 25% of the isolates from the white colony group, which dominated the population, were ice-nucleating bacteria. No ice-nucleating bacteria were present on canola leaves. Out of a total of 76 ice-nucleating bacteria isolated, 5 representative cultures were characterized in detail and identified as Pseudomonas fluorescens. The fatty acid composition of these cultures was essentially identical to that of typical P. fluorescens cultures and was altered by varying the growth temperature from 10 to 30°C.     

Two-way microscale interactions between immigrant bacteria and plant leaf microbiota as revealed by live imaging

The phyllosphere – the aerial parts of plants – is an important microbial habitat that is home to diverse microbial communities. The spatial organization of bacterial cells on leaf surfaces is non-random, and correlates with leaf microscopic features. Yet, the role of microscale interactions between bacterial cells therein is not well understood. Here, we ask how interactions between immigrant bacteria and resident microbiota affect the spatial organization of the combined community. By means of live imaging in a simplified in vitro system, we studied the spatial organization, at the micrometer scale, of the biocontrol agent Pseudomonas fluorescens A506 and the plant pathogen P. syringae B728a when introduced to pear and bean leaf microbiota (the corresponding native plants of these strains). We found significant co-localization of immigrant and resident microbial cells at distances of a few micrometers, for both strains. Interestingly, this co-localization was in part due to preferential attachment of microbiota cells near newly formed P. fluorescens aggregates. Our results indicate that two-way immigrant bacteria – resident microbiota interactions affect the microscale spatial organization of leaf microbiota, and possibly that of other surface-related microbial communities.Subject terms: Microbial ecology, Microbial ecology     

Bacterial biosynthesis of 1-aminocyclopropane-1-caboxylate (ACC) deaminase,a useful trait to elongation and endophytic colonization of the roots of rice under constant flooded conditions

This study was conducted to investigate the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Pseudomonas fluorescens strain REN₁ and its ability to reduce ethylene levels produced during stress, endophytically colonize and promote the elongation of the roots of rice seedlings under gnotobiotic conditions. We isolated 80 bacteria from inside roots of rice plants grown in the farmers’ fields in Guilan, Iran. All of the isolates were characterized for plant growth promoting (PGP) traits. In addition, the colonization assay of these isolates on rice seedlings was carried out to screen for competent endophytes. The best bacterial isolate, based on ACC deaminase production, was identified and used for further study. 16S rDNA sequence analysis revealed that the endophyte was closely related to Pseudomonas fluorescens. The results of this study showed ACC deaminase containing P. fluorescens REN1 increased in vitro root elongation and endophytically colonized the root of rice seedlings significantly, as compared to control under constant flooded conditions. The trait of low amount of indole-3-acetic acid (IAA) production (<15 μg mL⁻¹) and the high production of ACC deaminase by bacteria may be main factors in colonizing rice seedling roots compared to other PGP traits (siderophore production and phosphate solubilization) in this study. Endophytic IAA and ACC deaminase-producing bacteria may be preferential selections by rice seedlings. Therefore, it may be suggested that the utilization of ACC as a nutrient gives the isolates advantages in more endophytic colonization and increase of root length of rice seedlings.